Keloids and Hypertrophic Scars: Treatment With

1. FRONTISPIECE

بسم ہللا الرح ٰمن الرحیم

Bismillah hir Rahman nir Raheem

In the name of Allah, the Most Beneficent, the Most Merciful

KELOIDS AND HYPERTROPHIC SCARS: TREATMENT WITH

INTRALESIONAL INTERFERON GAMMA VERSUS TRIAMCINOLONE

ACETONIDE

Keloids production in a New Zealand White Rabbit using Mannan & Hannan Technique

2. TITLE

KELOIDS AND HYPERTROPHIC SCARS: TREATMENT

WITH INTRALESIONAL INTERFERON GAMMA VERSUS

TRIAMCINOLONE ACETONIDE

A THESIS SUBMITTED FOR DEGREE OF

DOCTOR OF PHILOSOPHY (SURGERY)

PROF. DR. ABDUL MANNAN BABAR

MBBS, FCPS, FRCS

UNIVERSITY OF HEALTH SCIENCES

LAHORE, PAKISTAN

22 DEC 2015 (Final)

3. DEDICATION

This Study is dedicated to

AL-AWAAM-UN-NAAS

The Common Man

4. FOREWORD

Prof. Dr. Abdul Mannan Babar s/o Ali Akbar, MBBS,

FCPS, FRCS, date of birth 07.05.1960, of Pakistani

Nationality, with National Identity Card No. 31202-0323879-

3, Passport No. AG3028792, and Pakistan Medical and

Dental Council (PMDC) Registration No: 9124-P, belonging to Postgraduate Medical Institute, Ameer-ud-Din Medical

College, Lahore General Hospital, Lahore Pakistan, having Telephone No. +92-333-2622227, and Email address [email protected], University of Health Sciences (UHS), Lahore

Pakistan Registration No. 2008-PGE-9901-UHS,.performed this Research Work under my direct supervision at Department of Pathology and Experimental Research Laboratory,

University of Health Sciences, Lahore Pakistan, for his Doctor of Philosophy (PhD) Degree in Surgery, from 23.12.2008 to 22.12.2015.

Dr. Babar did all this work with his both hands, under my direct guidance. Starting from topic selection, literature search, synopsis preparation, synopsis printing and binding, acquiring funds, acquiring materials, pilot studies, animal husbandry, animal anaesthesia, operative work, post-operative care, data collection, data analysis, statistical tests, use of software, photography, information & communication technology work, thesis preparation, and thesis printing and binding. He worked day and night for this project; he was granted special permission by the University authorities to use facilities of Experimental Research

Laboratory and Pathology Laboratory 24 hours a day, including holidays. The work done is original, novel, and latest. It includes both basic and applied research. Many permanent facilities were created at the University for this Project only; these facilities are now available

5 for future researchers both in basic and clinical disciplines.

My guidance was available to him 24/7.

This Thesis has been prepared according to latest standard International Guidelines, keeping in view the recommendations of the Grey Literature Network Service

(GreyNet, 1999 [Bib]). The Guidelines of Higher Education Commission (HEC), PMDC, and

UHS are also followed. The Thesis is based on the results of Experiments carried out by the candidate at Department of Pathology and Experimental Research Laboratory, University of

Health Sciences, Lahore Pakistan, and this Research has not been previously presented for any Degree. I have read the Thesis and found it to be upto the mark for grant of Doctor of

Philosophy degree in Surgery. According to my best knowledge and belief no such study has been carried anywhere in the world before. The Thesis has been checked by the University for Plagiarism using Turnitin plagiarism detection software, and is found to be free from plagiarism. Approval from Ethical Review Committee was taken before start of this study.

The candidate has fulfilled all statutory requirements, and is qualified to submit this Thesis for Doctor of Philosophy Degree in Surgery.

Prof. Dr. Abdul Hannan Nagi

MBBS, FCPS, FRCP, FRCPath, PhD

Head of the Department of Pathology

Incharge Experimental Research Laboratory

University of Health Sciences, Lahore, Pakistan

Dated 22nd December 2015

5. PREFACE

Becoming a student again when you are Professor for as long as 20 years is a wonderful experience. I had been trying to start PhD Programme in Clinical Medical Sciences at various institutions of postgraduate medical education in

Pakistan for 11 years before was successful to start one such

Programme at University of Health Sciences Lahore. That D-

Day was 23rd of December 2008 when Advanced Studies & Research Board of UHS approved my Synopsis for PhD in Surgery, and I was registered as PhD Scholar.

A lot of teething problems occurred. The main problem to start with was of funding, amounting to Pakistan Rupees (PKR) 5.5 million. Luckily Government of the Punjab

Province had allocated PKR 10 million for research to each Government Sector Medical

College in Punjab, including my institution ie Postgraduate Medical Institute Lahore. I was a strong contestant for that, but mysteriously that fund disappeared. Then I looked for second option ie Higher Education Commission (HEC), Government of Pakistan. It took my

Supervisor and me about two years to get the Project approved and money released.

The second problem was procurement of equipment and consumables. As most of these things had to be imported, it took almost six months for one consignment to be delivered. There were many such consignments. Sometimes the problem occurred that the items supplied were not according to our specifications, and so another six months lapsed.

For some of these materials no importer was available, and things had to be procured using personal connections.

The third problem was establishment of Research facility. There was a rudimentary Animal House available at

University of Health Sciences, having only Rats, and one self-trained animal-keeper. After the project started, this animal house was converted into full-fledged Experimental

Research Laboratory. Mice, Rats, Guinea Pigs, and Rabbits keeping and breeding facilities were started. A full time Veterinary Officer and a Veterinary

Assistant were hired. Necessary equipment was purchased to establish full-fledged Animal

Operation Theatre. Now this Laboratory has become a resource centre for replication all over

Pakistan.

First two years (2009-2010) were spent in Course Work. It was a one-on-one coaching given to me by my Supervisor, 2-4 pm daily, 6 days a week. My supervisor taught me basics of Experimental Research. Professors of Anatomy, Physiology, and Biostatics also taught me in this course work. There was a Comprehensive Examination at the end of this

Course Work. I also did a lot of self-study related to Anatomy, Physiology, Pathology,

Pharmacology, Surgery, Plastic Surgery, Dermatology, Zoology, Experimental Research,

Mathematics, Biostatistics, Computer Software, and Internet Resources. During these 2 years,

I was also continuously refining my research project.

At the start of third year (2011), was ready to start my research project. First, I had to do a few pilot studies, as exact guidelines about my project were not available in literature.

So third year of my course was spent in these Pilot Projects. These included production of

Keloid animal model in different animals, dosing of Transforming Growth Factor Beta 1 for production of Keloid animal model, multiple Keloid production in one animal, production of

8 transparent solution of Triamcinolone Acetonide, and dosing of Interferon Gamma for treatment of Keloids in rabbits.

Fourth year of my course (2012) was spent on further studies, as new findings had come up based on pilot studies.

So further literature search as well as self-reflection was done to make this project fool proof. Moreover, more materials were to be procured, based on findings of these pilot studies. These procurements were done during this period.

Fifth & sixth years (2013-2014) were spent in data collection and data analysis.

Whole data collection was done by my own hands, and no research assistant was hired. Data analysis was also done by me personally, as I had learned biostatistics myself, including biostatistics software. The study was triple blinded, and de-blinding was done after calculation of p-value. Data collection and data analysis was completed before the final day of sixth year ie 22.12.2014.

The last portion of my work was Thesis writing, which took me further one year

(2015). This Thesis consists of 100,000 words and 200 Figures and Tables. I wrote my first

Thesis in 1983 (Babar, 1983A [Bib]), and second again in 1983 (Babar, 1983B [Bib]), during my final year MBBS. That was the era of manual typewriter, and manual literature search. I wrote my third Thesis in 1987 for Fellowship (Babar, 1987 [Bib]). The computer still had not reached us, but electric typewriter was available. Literature search was again manual, but some institutions had reference retrieval service available, which I used. I also got assistance from National Library of Medicine USA. Now we are in a digital era, with both advantages and disadvantages.

In this Thesis, I have used a liberal format keeping in view modern Information & Communication Technology

(ICT) developments. For example, I have omitted Roman numerals, and used Arabic numerals starting from

Frontispiece. This is in line with the ICT requirements, as two types of numerals cause confusion during downloading/printing. I have also followed recommendations of the Grey Literature Network

Service. Grey Literature is that which is created by governments, universities, businesses and industries, whether printed or digital, and is not owned by publishing industry (GreyNet,

1999 [Bib]). It consists of Bibliographies, Conference Proceedings, Non-Commercial

Translations, Official Documents, Reports, Technical Specifications & Standards, and Theses

(Alberani, 1990).

Microsoft (MS) and other software companies have stopped differentiating between

Figures and Tables; they have put them in one category ie Object. In MS Windows/Word, they appear under one heading ie Table of Figures. Therefore, Caption of Tables has been placed below the Tables, as they are formatted just like Figures. Further in statistical software

International Business Machines-Statistical Package for Social Sciences (IBM-SPSS) it is only possible to export a Table as an Object, which can only be formatted like a camera picture. Therefore, I have put some Figures (eg histogram) under Tables. The Figures and

Tables have been placed at the end of chapters/sub-chapters/sections, as there is no feature in

MS Word to keep Figure/Table on a separate page. There is no option available to put

Landscape pages in between Portrait pages. Therefore, all Landscape pages have first been changed to Portable Document Format (PDF) using Adobe Acrobat Reader, and then saved as Object using Joint Photographic Experts Group (JPEG) format; they have been labelled in

Portrait orientation. Most of the Photos have been shot by me personally; rest are taken from Public Domain/Open Access sources. Photos have been edited using Adobe Photoshop software.

Harvard Referencing System has been used in this

Dissertation. Unfortunately, nobody owns Harvard System, not even Harvard University from which it originated. This is unlike other referencing systems, eg Vancouver System, which is owned and controlled by a specific organization. Consequently, different institutions and universities have their own versions of Harvard System. However, basic format of

Author-Date is common in all these, I have used this system according to my convenience, but consistency is maintained throughout.

EndNote has been used to sort references. References section includes only Journal articles, while all Books and other materials have been included in Bibliography section; in in-text citations, they have been identified by the term [Bib] just after the date. Not all books and other materials mentioned in Bibliography section have been referred to in-text. A total of 111 sources have been included in Bibliography. UK English is used throughout, except in

Citations, References, and Bibliography, where original spellings are preserved.

Now something about the topic. Keloid is a disease of black and coloured people.

Therefore, it is primarily a duty of the black and the coloured to do research on this. Keloid is a disease with which one has to live with, and cannot hope to die with. Unfortunately, it is an incurable disease, but there are 111 treatments available. With current treatment modalities,

Recurrence Rate may be as high as 100%, and Remission/Response Rate may be as low as -

100%. The problem about Keloid research is that this disease does not occur in animals (although one reference is available about Keloid in horse and one about keloid in dogs)

(Marcenac, 1951; Mikaelian & Gross, 2002).

Keloid is thought to be a snap diagnosis, requiring no investigation at all; unfortunately this is not true. Many studies have shown pitfalls of the snap diagnosis. Even sometimes malignant tumours have been confused with Keloids. I have done a detailed study of differential diagnoses of Keloid, and have found 151 diseases mimicking Keloids. Therefore I recommend that every Keloid should be investigated, at least by histopathology. Other investigations like immunohistochemistry, scanning electron microscopy, transmission electron microscopy, and genomic profiling should be done whenever required.

Different techniques have been used to make animal models of Keloid, but success has been limited. I have invented a new technique, with which I have been able to produce

Keloid animal model.

Treatment of Keloid with Interferon Gamma has already been done in the past.

However, these are only anecdotal case reports or small studies. No double blind study has been done so far. Now that my study has produced highly significant efficacy of Interferon

Gamma as compared to current gold standard ie Triamcinolone Acetonide, I have already started replicating this study in humans.

I consulted 5555 references for this study, mostly original articles, but some review

12 articles. These spans over a period of around 5555 years

(3500 BC-2015 AD). I have only included only 555 in my

Thesis, due to lack of space. These articles have mostly been written by dermatologists, plastic surgeons, pathologists, and general surgeons, in that order. I have seen many examples of plagiarism and self-plagiarism in these articles. Further many authors have referred to Introduction/Review of other articles without going to primary source. This has led to many mistakes, glaring example of which is wrong assumption that

Keloids are mentioned in Edwin Smith Surgical Papyrus.

I have included 111 Books and other resources references in Bibliography. The books have written only one to two pages about keloids, and that are very superficial and outdated.

Only a few dermatology books have a separate chapter on keloids. Further, during my literature search for Keloid I found out that not a single Book has been written on Keloids.

Therefore, I have already started writing a Book on this subject.

That is all for today. Let us call it a day.

PROF. DR. ABDUL MANNAN BABAR MBBS, FCPS, FRCS Glasgow (UK) Head of the Department of Surgery Postgraduate Medical Institute Ameer-ud-Din Medical College Lahore General Hospital Lahore, Pakistan Dated 22nd December 2015

6. ACKNOWLEDGEMENTS

All Thanks belong to Allah. We praise Him, and ask

Him for guidance and forgiveness. We believe in Him, and put our trust in Him. We seek protection in Allah from the malice of our own souls, and the evil of our actions. Whom

Allah guides, no one can lead him astray, and whom He makes astray, no one can lead him back to the right path. I bear witness that there is no other deity but Allah, by Himself, no associate to Him, and I bear witness that Mohammad is His bondman and Prophet, whom He sent with truth, as a giver of glad tidings and as a warner, before Hour of Judgment. Whoso obeyed Allah and His

Prophet, he got the right guidance, and whoso disobeyed any or both of them, he will not harm anybody except his own soul, and he will not harm Allah at all. After that, I take refuge with Allah from the accursed Satan. In the name of Allah, the Most Beneficent, the Most

Merciful.

Allah! There is no God but He; the Living, the Self-subsisting. Neither slumber can seize Him nor sleep. His are all things in the heavens and on earth. Who is there to intercede in His presence except as he permits? He knows what is before them, and what is behind them. People shall not encompass aught of His knowledge except as He wills. His throne does extend over the heavens and on earth and He feels no fatigue in guarding them, for He is the Most High, the Supreme (Allah, 632 AD [Bib])

Mohammad is the Messenger of Allah, and those with him are forceful against the disbelievers, merciful among themselves. You see them bowing and prostrating in prayers,

14 seeking bounty from Allah, and His pleasure. Their mark is on their faces from the trace of prostration. That is their description in the Torah. And their description in the Gospel is as a plant, which produces its offshoots and strengthens them so they grow firm, and stand upon their stalks, delighting the sowers, so that Allah may enrage by them the disbelievers. Allah has promised those who believe and do righteous deeds among them forgiveness and a great reward (Allah, 632 AD [Bib]).

Allah, Lord of the worlds, has given me faith in Him and faith in myself. I therefore strive in Allah’s cause He has advanced me in knowledge, and gave me motivation to undertake the profession of a healer, as whoso saved one life, he saved the life of all mankind. When I ail, He heals me. Mohammad, the last Prophet, has brought the message of

Allah to me to reflect in myself and my surrounding, and foster Attributes of Allah in myself.

Mohammad has directed us to seek knowledge from cradle to grave. He also told us that there is no ailment for which remedy does not exist; we have only to discover this remedy

(Mohammad, 632 AD [Bib]). Knowledge is one of the Attributes of Allah. Seeking and generating knowledge is part of my Jihad in Allah’s Cause. May Allah save me and my people from Hell-Fire. Amen.

Prof. Dr. Abdul Hannan Nagi, my supervisor, taught me the basics of Experimental

Research for this study, and also guided and practically helped me in all matters related to this research, be it professional, managerial, financial, or lay.

Prof. Dr. Mohammad Tahir, MBBS, MPhil, PhD, Professor of Anatomy UHS

15 provided me support, especially about ethical aspects of this study. Prof. Dr. Khalid Pervez Lone, MSc, PhD, Professor of

Physiology UHS provided me help, especially regarding

Triamcinolone Acetonide. Prof. Dr. Shakila Zaman, MBBS,

PhD, Professor of Preventive Paediatrics provided me guidance, especially regarding biostatistics. Prof. Dr. Irshad

Ahmad. Naveed, Director Medical Education UHS provided me support during this project.

Dr. Ali Ahmad, DVM, Veterinary Officer UHS assisted me regarding animal anaesthesia, and provided me office space. Mr. Asif Munir, Librarian UHS helped me in literature search.

Prof. Dr. Malik Hussain Mubashar, and Prof. Dr. Mohammad Aslam, past and present

Vice Chancellors of UHS, provided full support for this project. Staff of Department of

Pathology assisted me during my study. Staff of Experimental Research Laboratory was very helpful throughout this research. Staff of Immunology Department, Medical Education

Department, Registrar Office, Examination Department, Administration & Coordination

Department, Information Technology Department, Library, Biostatistics Section, Finance

Department, Purchase Cell, and many other Departments of UHS were also helpful. Higher

Education Commission, Government of Pakistan, Islamabad provided full financial support for this study.

Last, but not the least, my parents, my better half Farah, and my kids Hanzla, Huzaifa, and Ayesha helped me by tolerating my absence from home until late night during this study.

My father helped me in translation of Arabic prayers given in the beginning and at the end of this dissertation. My kids actually provided me support regarding Information &

Communication Technology during this undertaking. I am thankful to all mentioned above.

7. TABLE OF CONTENTS

1. FRONTISPIECE ...... 1

2. TITLE ...... 2

3. DEDICATION ...... 3

4. FOREWORD ...... 5

5. PREFACE ...... 7

6. ACKNOWLEDGEMENTS ...... 14

7. TABLE OF CONTENTS ...... 17

8. TABLE OF FIGURES ...... 21

9. TABLE OF TABLES ...... 28

10. TABLE OF ABBREVIATIONS ...... 30

11. ABSTRACT ...... 35

12. INTRODUCTION ...... 37

13. REVIEW OF LITERATURE ...... 45

14. History of Keloids & Hypertrophic Scars ...... 47

15. Ancient Era ...... 49

16. Medieval Era ...... 62

17. Modern Era ...... 75

18. Present Era ...... 125

19. Future Era ...... 136

20. Anatomy of Keloids & Hypertrophic Scars ...... 144

21. Histology ...... 146

22. Embryology ...... 150

23. Physiology of Keloids & Hypertrophic Scars ...... 153

24. Biochemistry ...... 157

25. Incidence & Prevalence of Keloids & Hypertrophic Scars ...... 161

26. Aetiology of Keloids & Hypertrophic Scars ...... 163

27. Pathogenesis of Keloids & Hypertrophic Scars ...... 169

28. Pathogenesis (Abnormal) ...... 179

29. Pathogenesis Theories ...... 184

30. Pathology of Keloids & Hypertrophic Scars ...... 187

31. Microscopic Pathology ...... 189

32. Classifications of Keloids & Hypertrophic Scars ...... 192

33. Symptoms & Signs of Keloids & Hypertrophic Scars ...... 196

34. Investigations of Keloids & Hypertrophic Scars ...... 198

35. Diagnosis of Keloids & Hypertrophic Scars ...... 200

36. Microscopic Diagnosis ...... 208

37. Differentiation of Keloids & Hypertrophic Scars ...... 214

38. Prophylaxis of Keloids & Hypertrophic Scars ...... 220

39. Treatment of Keloids & Hypertrophic Scars ...... 222

40. Surgery ...... 229

41. Steroids, Intralesional ...... 233

42. Silicone Sheet ...... 236

43. Pressure Garments ...... 239

44. Laser Surgery ...... 242

45. Cryosurgery ...... 245

46. Radiotherapy ...... 248

47. Cytotoxics ...... 251

48. Immunomodulators, Topical ...... 254

49. Calcineurin Inhibitors, Topical ...... 256

50. Verapamil, Intralesional ...... Error! Bookmark not defined.

51. Botulinum Toxins, Intralesional ...... 260

52. Cytokines & Anti-Cytokines ...... 262

53. Growth Factors & Anti-Growth Factors ...... 265

54. Gene Therapy ...... 268

55. Stem Cell Therapy ...... 272

56. Nano Medicine ...... 276

57. Miscellaneous Treatments...... 279

58. Symptomatic Treatments ...... 286

59. Historical Treatments ...... 289

60. Multi-Modal Therapy ...... 294

61. Complications of Keloids & Hypertrophic Scars ...... 296

62. Prognosis & Follow-Up of Keloids & Hypertrophic Scars ...... 298

63. Animal Model of Keloids & Hypertrophic Scars ...... 300

64. Role of TGFB 1 in Keloids & Hypertrophic Scars ...... 304

65. Role of DMSO in Keloids & Hypertrophic Scars ...... 307

66. Role of Triamcinolone Acetonide in Keloids & Hypertrophic Scars ...... 309

67. Role of Interferon Gamma in Keloids & Hypertrophic Scars ...... 312

68. Technique of Intralesional Injection in Keloids & Hypertrophic Scars ...... 316

69. OBJECTIVE ...... 319

70. HYPOTHESIS ...... 319

71. OPERATIONAL DEFINITION ...... 319

72. PILOT STUDIES ...... 320

73. MATERIALS & METHODS ...... 321

74. RESULTS ...... 383

75. DISCUSSION ...... 425

76. CONCLUSION ...... 432

77. RECOMMENDATION ...... 432

78. PATENT ...... 433

79. GLOSSARY ...... 434

80. BIOGRAPHIES ...... 442

15. BIBLIOGRAPHY ...... 444

16. REFERENCES ...... 453

83. PUBLICATIONS ...... 505

84. Surgical Adjuvant Cytokines versus Steroids for Keloids (Report) ...... 506

85. Keloids: Treatment with Interferon Gamma versus Triamcinolone (Thesis) ...... 508

86. Keloids and Hypertrophic Scars (Review Article) ...... 510

87. Keloids & Hypertrophic Scars: Epidemiology and Management (Book) ...... 512

88. Keloids: Comparison of Treatment with Interferon µ vs Triamcinolone (OA) ..... 514

89. History of Keloids & Hypertrophic Scars: From 3500 BC to 2015 AD (Review) .. 516

90. Production of Keloid Animal Model (Original Article) ...... 518

91. Development of New Techniques for Rabbit Ear Keloid Model (OA) ...... 520

92. Mannan & Hannan Technique for Production of Rabbit Ear Keloid Model (O) .. 522

93. Production of Multiple Keloids in one Animal (Original Article) ...... 524

94. Dosing of Transforming Growth Factor Beta for Keloid Animal Model (OA) ...... 526

95. Dosing of Interferon Gamma for Treatment of Keloid Animal Model OA) ...... 528

96. Production of Transparent Solution of Triamcinolone Acetonide (OA)...... 530

97. Estimation of Hypothesized Difference and Population Variance for Keloids (O) 532

98. APPENDIX I: RESEARCH PROTOCOL ...... 534

99. APPENDIX II: MANUAL CALCULATIONS ...... 540

100. APPENDIX III: ABOUT THE AUTHOR ...... 541

101. ENDNOTE ...... 547

102. INDEX ...... 549

103. BACKISPIECE ...... 554

8. TABLE OF FIGURES

Figure 1: Imhotep, the Author of Edwin Smith Surgical Papyrus (3000 BC) ...... 44

Figure 2: PubMed (Medline) Literature Search Portal (2015 [Bib]) of NCBI of NLM of

NIH of USA. This Online Database was used for Literature Search for this Treatise .... 46

Figure 3: An Ancient Era Artifact: Medical Cuneiform Writings from Mesopotamia; the earliest known Book of Medicine (Anonymous, 3500 BC [Bib]) ...... 48

Figure 4: The Edwin Smith Surgical Papyrus Translation Case No. 46, Page No. 463

(3000 BC) (Breasted, 1930 [Bib]) ...... 52

Figure 5: The Edwin Smith Surgical Papyrus Original Manuscript Column No. XV,

Cases No. 43-46(Imhotep, 3000 BC [Bib]) ...... 53

Figure 6: Ancient American Aztec god Xipe Totec, displaying linear Keloids on his face

(Xipe Totec, 2600 BC [Bib]) ...... 54

Figure 7: Code of Hammurabi from Babylonia. It contained Law on Treatment of

Wounds as well (Hammurabi, 1775 BC [Bib]) ...... 55

Figure 8: Pharaoh Horemheb’s mummy (1323 BC-1295 BC) showing many Abnormal

Scars and a Keloid on face (chin) ...... 56

Figure 9: Pharaoh Horemheb’s statue (1323 BC-1295 BC) showing many Abnormal

Scars, and a Keloid on face (chin) ...... 57

Figure 10: An unknown Egyptian Pharaoh with Ornamental Scarification Keloid on

Right Shoulder and a true Keloid on Right Cheek (1300 BC) ...... 58

Figure 11: Nubian Ornamental Scars on cheeks of prisoners from the Saqqara Tomb of

Horemheb (1300 BC) ...... 59

Figure 12: ‘Seated Boxer' from Greece showing Abnormal Scars on face (330 BC) ...... 60

Figure 13:B; ‘Dying Gladiator’ from Rome; the First Graphic of Wound (230 BC)...... 61

Figure 14: Quran; the latest Divine book Allah, 632 AD [Bib] ...... 65

Figure 15: Az-Zahrawi (936-1013 AD). He wrote about Treatment of Wounds ...... 66

Figure 16: An illustration from Al-Zahrawi’s Kitab al-Tasrif (1000 AD [Bib]) ...... 67

Figure 17:B Al-Zahrawi’s BookKitab al-Tasrif (Liber Theoricae) (1000 AD [Bib]) ...... 68

Figure 18:B; Roger of Salerno (1140 AD-1195 AD); wrote about Wound Treatment .... 69

Figure 19: Roger of Salerno’s Chirurgia showing Wound Treatment (1180 AD [Bib]) .. 70

Figure 20: King of Ife Empire of Yoruba with Ornamental Keloids (1300 AD Bib]) ..... 71

Figure 21: Queen of Ife Empire of Yoruba with Ornamental Keloids (1300 AD [Bib]) . 72

Figure 22:B; Facial Ornamental Keloids on a statue from Ife Empire of Yoruba, Nigeria

(Anonymous, 1300 AD [Bib]) ...... 73

Figure 23:B; Ornamental Keloids are still common in Africa. Photograph of a Modern

Negro Boy showing Multiple Keloids on Face (Anonymous, n.d. [Bib]) ...... 74

Figure 24:B; Ambroise Pare's Ten Books of Surgery (1564 AD [Bib]) showing first illustration of Suturing a Wound ...... 115

Figure 25:B; J. Scultetus' Book Armamentarium Chirurgicum (1655 AD [Bib]) showing

Curved Needle ...... 116

Figure 26:B; Benjamin Bell’s Book A System of Surgery (1791 AD [Bib]) showing straight and curved Needles for suturing Wounds ...... 117

Figure 27:B; Retz’ Book Second Edition (1786 AD [Bib]) ...... 118

Figure 28:B; Alibert’s Book First Edition (1806 AD [Bib]) ...... 119

Figure 29:B; Alibert Book Second Edition (1825 AD [Bib]) ...... 120

Figure 30:B; Alibert Book Third Edition (1833 AD [Bib]) ...... 121

Figure 31: First Drawing of Keloid in second Edition of Alibert’s Book (1825 AD [Bib])

...... 122

Figure 32:B; Gordon (slave), also called Whipped Peter, photographed with Keloids from being whipped (McPherson and Oliver, 1863 AD [Bib]) ...... 123

Figure 33:B; Ulcerated Keloid case presented by MacCormac (1934) ...... 124

Figure 34:B; Scar Classification by Mustoe et al (2002) ...... 133

Figure 35:B; Scar Prevention Algorithm by Mustoe et al (2002) ...... 134

Figure 36:B; Scar Treatment Algorithm by Mustoe et al (2002) ...... 135

Figure 37: Cell and ECM interactions. Binding of growth factors or adhesion molecules to matrix or cell receptors initiates cell-signalling mechanisms (Mustsaers et al 1997) 140

Figure 38:B; Lactate concentration in wound fluids. A stable increase in lactate concentration causes increased collagen deposition (Trabold et al, 2003) ...... 141

Figure 39: RNA Microarray on fibroblasts of keloids and normal scars with & without

Hydrocortisone (HC), showing multiple fibrosis-related pathways (Smith et al, 2008) 142

Figure 40:B; Flowchart of steps & findings of Keloid gene biomarking done on biopsies from Keloid margins and Normal skin (Shih et al, 2010) ...... 143

Figure 41: Anatomy of Skin (Google, 2015 [Bib]) ...... 145

Figure 42: Histology of Skin (Flores, 2015 [Bib]) ...... 149

Figure 43: Embryology of Skin: A; 5 weeks; B; 7 weeks; C; 4 months (Google, 2015

[Bib])...... 152

Figure 44: Functions of different Constituents of Skin (Venus et al, 2010)...... 156

Figure 45: Keratin molecular structure (Stanley, 2015 [Bib]) ...... 160

Figure 46:B; Age of onset of Keloid among 1075 patients (Tirgan, 2015 [Bib]) ...... 162

Figure 47: Fitzpatrick Scale of Skin Colour (Fitzpatrick, 1988) ...... 168

Figure 48: Phases of Normal Wound Healing (Townsend et al, 2012 [Bib]) ...... 178

Figure 49: Abnormal Wound Healing(Townsend et al, 2012 [Bib]) ...... 183

Figure 50: Wound healing in pathological scars has prolonged inflammation phase, inappropriate cytokines & delayed healing response (Huang et al 2013) ...... 186

Figure 51: Gross Pathology of Keloid ...... 188

Figure 52: Microscopic Pathology of Keloid ...... 191

Figure 53: Butterfly Keloid (Tirgan, 2015 [Bib]) ...... 195

Figure 54: Keloid on Sole of Foot (Google, 2015 [Bib]) ...... 197

Figure 55:B; SEM of Keloid (Kemble, 1976) ...... 199

Figure 56: Morphea (Google, 2015 [Bib]) ...... 207

Figure 57: Acne Keloidosis Nuchae ...... 213

Figure 58: Hypertrophic Scar ...... 219

Figure 59: Keloid formation after Facelift Operation ...... 221

Figure 60:B; Thomas Addison ...... 228

Figure 61: Surgical Excision of a Pedunculated Keloid (Google, 2015)...... 232

Figure 62: Intralesional Steroid Injection ...... 235

Figure 63: Silicone Sheet ...... 238

Figure 64: Pressure Garments ...... 241

Figure 65: Laser Treatment of Keloid ...... 244

Figure 66: Cryosurgery for Keloid ...... 247

Figure 67:B; Radiotherapy Machine ...... 250

Figure 68:B; 5- Fluorouracil ...... 253

Figure 69:B; Imiquimod ...... 255

Figure 70:B; Tacrolimus ...... 257

Figure 71:B; Verapamil ...... 259

Figure 72: Botulinum Toxin ...... 261

Figure 73: Effects of Cytokines on Fibroblasts to promote or prevent fibrosis (Backovic et al, 2010) ...... 264

Figure 74: TGFB3 versus Placebo for the Treatment of Scars (Ferguson et al, 2009) .. 267

Figure 75: Gene Therapy (Google, 2015 [Bib]) ...... 271

Figure 76: Stem Cell Therapy (Google, 2015 [Bib] ...... 275

Figure 77: Different structures of nanomedicines and their sizes (BSNM, 2015) ...... 278

Figure 78: Camouflage of Keloid: above; before, below; after (Tirgan, 2015 [Bib]) .... 285

Figure 79: White Petroleum Jelly ...... 288

Figure 80:B; Chloroquine ...... 293

Figure 81: Multimodal Therapy (Surgery+Radiotherapy) (Jones et al, 2015) ...... 295

Figure 82: Malignant Transformation in a Keloid ...... 297

Figure 83:B; Vancouver Scar Scale (VSS)...... 299

Figure 84: Animal Model of Keloid ...... 303

Figure 85: TGF Beta effects on wound healing (Werner & Grose, 2003) ...... 306

Figure 86:B; DMSO ...... 308

Figure 87:B; Triamcinolone Acetonide ...... 311

Figure 88: Interferon Gamma commercial preparation ...... 315

Figure 89: Dermojet ...... 318

Figure 90: Experimental Research Laboratory UHS, Outer View ...... 328

Figure 91: Experimental Research Laboratory UHS, Inner View...... 329

Figure 92: Animal Operation Theatre UHS ...... 330

Figure 93: Pathology Department UHS ...... 331

Figure 94: Sample Size Calculation (Select-Statistics, 2015 [Bib]) ...... 332

Figure 95: The Author with Universal Protection ...... 333

Figure 96: Rabbit Cage Numbering ...... 334

Figure 97: Rabbits in Cages with Air-Conditioner and Exhaust Fan ...... 335

Figure 98: Animal Breeding Facility UHS...... 336

Figure 99: Rabbits in a Cage...... 337

Figure 100: Animal Keeping ...... 338

Figure 101: Rabbit Coding with Eosin...... 339

Figure 102: TGFB1 Injection in Micro Burette in Freezer Compartment ...... 340

Figure 103: Surgical Instruments ...... 341

Figure 104: Surgical Instruments Cupboard ...... 342

Figure 105: Preparations for Operation ...... 343

Figure 106: Anaesthetizing Rabbit ...... 344

Figure 107: Disinfecting Rabbit Ear ...... 345

Figure 108: Keloid Sites...... 346

Figure 109: Marking Keloid Sites with Gentian Violet & Graduated Caliper ...... 347

Figure 110: Measuring Thickness of ear with Digital Micrometre Gauge ...... 348

Figure 111: Injecting TGF Beta 1 at Keloid Points with Insulin Syringe ...... 349

Figure 112: Dorsal Surface of Ear showing swellings at Sites of TGFB 1 Injections .... 350

Figure 113: Skin Punch ...... 351

Figure 114: Punch of ear Skin and Cartilage has been cut ...... 352

Figure 115: Punch area seen through Magnifying Lamp ...... 353

Figure 116: Four Punches of Skin and Cartilage excised ...... 354

Figure 117: Bleeding being controlled with Artery Forceps ...... 355

Figure 118: Wounds being dried with Hot Air Dryer ...... 356

Figure 119: Wounds dressed with Hydrocolloid Dressing ...... 357

Figure 120: Post-operative Case ...... 358

Figure 121: Keloids developed on Rabbit Ear ...... 359

Figure 122: Random Numbers Generation (Code::Blocks, 2015 [Bib]) ...... 360

Figure 123:B; Random Number Table ...... 361

Figure 124: Interferon Gamma ...... 362

Figure 125: Triamcinolone Acetonide ...... 363

Figure 126: Syringe Filter for Sterilization of Triamcinolone Acetonide ...... 364

Figure 127: Blinded Drugs in Insulin Syringes ...... 365

Figure 128: Triple Blinding Documents ...... 366

Figure 129: Keloids on Rabbit Ear ...... 367

Figure 130: Keloids Marked for Measurement ...... 368

Figure 131: Keloid Thickness Measurement ...... 369

Figure 132: Keloid Diameter Measurement ...... 370

Figure 133: Spherical Cap Volume Calculation (Wolfram-MathWorld, 2015 [Bib]) ... 371

Figure 134: Drug 1 or 2 being injected in Keloids ...... 372

Figure 135:B; Blank Performa ...... 373

Figure 136:B; Filled Performa ...... 374

Figure 137:B; Data Sheet ...... 375

Figure 138:B; Filled Data Sheet...... 376

Figure 139:B; Products and Manufacturers’ Directory 1...... 377

Figure 140:B; Products and Manufacturers’ Directory 2...... 378

Figure 141:B; Products and Manufacturers’ Directory 3...... 379

Figure 142:B; Products and Manufacturers’ Directory 4...... 380

Figure 143:B; Products and Manufacturers’ Directory 5...... 381

Figure 144:B; Products and Manufacturers’ Directory 6...... 382

9. TABLE OF TABLES

Table 1:B; Group 1 (TAC) Raw Data (1-1) ...... 385

Table 2:B; Group 1 (TAC) Raw Data (1-2) ...... 386

Table 3:B; Group 1 (TAC) Raw Data (1-3) ...... 387

Table 4:B; Group 1 (TAC) Raw Data (1-4) ...... 388

Table 5:B; Group 2 (IFNG) Raw Data (2-1) ...... 389

Table 6:B; Group 2 (IFNG) Raw Data (2-2) ...... 390

Table 7:B; Group 2 (IFNG) Raw Data (2-3) ...... 391

Table 8:B; Group 2 (IFNG) Raw Data (2-4) ...... 392

Table 9:B; Comparative Statistics of all Groups ...... 393

Table 10:B; Group 1 Detailed Statistics...... 394

Table 11:B; Group 1 Frequencies & Percentages ...... 395

Table 12:B; Group 1 P-P Plot ...... 396

Table 13:B; Group 1 Q-Q Plot ...... 397

Table 14: Group 1 Histogram with Normal Curve...... 398

Table 15: Group 1 Bar Chart ...... 399

Table 16: Group 1 Boxplot ...... 400

Table 17 Group 1 Pie Chart ...... 401

Table 18:B; Group 2 Detailed Statistics...... 402

Table 19:B; Group 2 Frequencies & Percentages ...... 403

Table 20:B; Group 2 P-P Plot ...... 404

Table 21:B; Group 2 Q-Q Plot ...... 405

Table 22: Group 2 Histogram with Normal Curve...... 406

Table 23: Group 2 Bar Chart ...... 407

Table 24: Group 2 Boxplot ...... 408

Table 25: Group2 Pie Chart...... 409

Table 26:B; Group 1+2 Detailed Statistics ...... 410

Table 27:B; Group 1+2 Frequencies & Percentages ...... 411

Table 28:B; Group 1+2 P-P Plot ...... 412

Table 29:B; Group 1+2 Q-Q Plot ...... 413

Table 30: Group 1+2 Histogram with Normal Curve ...... 414

Table 31: Group 1+2 Bar Chart ...... 415

Table 32: Group 1+2 Boxplot ...... 416

Table 33: Group 1+2 Pie Chart ...... 417

Table 34: Z-Test Calculation (In-Silico, 2015 [Bib])...... 418

Table 35:B; Z Distribution Table (1) (University-of Wisconsin, 2015 [Bib]) ...... 419

Table 36:B; Z Distribution Table (2) (University-of-Wisconsin, 2015 [Bib]) ...... 420

Table 37: P-Value Calculation (Social-Science-Statistics, 2015 [Bib]) ...... 421

Table 38: Normal Rabbit Skin, H & E Stain ...... 422

Table 39: Keloid in Rabbit, H & E Stain ...... 423

Table 40: Keloid in Rabbit, Periostin Stain ...... 424

10. TABLE OF ABBREVIATIONS

1. ACE Angiotensin Converting Enzyme

2. AD Anno Domini(Year of Lord[Christ])

3. Ad Adenovirus

4. ADSC Adipose Derived Stem Cell

5. AKT A (code) Kinase (Thymoma)

6. AMC Ameer-ud-din Medical College

7. ATP Adenosine TriPhosphate

8. BAPN Beta AminoPropioNitrile fumarate

9. BAX Bcl-2 Associated X-protein

10. BC Before Christ

11. BCG Bacillus Calmette–Guérin

12. Bcl 2 B cell lymphoma 2

13. BED Biologically Effective Dose

14. bFGF basic Fibroblast Growth Factor

15. Bib Bibliography

16. BMSC Bone Marrow Stem Cell

17. BMZ Basement Membrane Zone

18. CD Cluster of Differentiation

19. CDC Centre for Diseases Control & prevention

20. cDNA complementary DeoxyriboNucleic Acid

21. CGF Control Gel Formula

22. CM Conditioned Media

23. COX Cyclo-OXygenase

24. CTGF Connective Tissue Growth Factor

25. DMSO DiMethyl SulfOxide

26. DNA DeoxyriboNucleic Acid

27. Dr Doctor

28. dUTP deoxyUridine TriPhosphate

29. DVM Doctor of Veterinary Medicine

30. ECM ExtraCellular Matrix

31. EGF Epidermal Growth Factor

32. ESC Epidermal Stem Cell

33. Fas Fatty acid synthase

34. FCPS Fellow of College of Physicians & Surgeons

35. FGF Fibroblast Growth Factor

36. FRCP Fellow of Royal College of Physicians

37. FRCPath Fellow of Royal College of Pathologists

38. FRCS Fellow of Royal College of Surgeons

39. FU FluoroUracil

40. GAG GlycosAminoGlycan

41. GFP Gene Fluorescence Protein

42. H0 Null Hypothesis

43. HA Alternate Hypothesis

44. H & E Haematoxylin & Eosin

45. HDF Human Dermal Fibroblast

46. HEC Higher Education Commission

47. HGF Hepatocyte Growth Factor

48. HLA Human Leukocyte Antigen

49. HSP Heat Shock Protein

50. hWJSC human Wharton Jelly Stem Cell

51. IBM International Business Machines

52. ICD International Classification of Diseases

53. ICT Information & Communication Technology

54. IFNG Interferon Gamma

55. IGF Insulin-like Growth Factor

56. IHC ImmunoHistoChemistry

57. IL Interleukin

58. iPSC induced Pluripotent Stem Cell

59. IU International Unit

60. JPEG Joint Photographic Experts Group

61. KGF Keratinocyte Growth Factor

62. KLC Keratinocyte Like Cell

63. LASER Light Amplification by Stimulated Emission of Radiation

64. LASH Laser Assisted Skin Healing

65. LGH Lahore General Hospital

66. MBBS Bachelor of Medicine & Bachelor of Surgery

67. MeSH Medical Subject Headings

68. MMP Matrix MetalloProteinases

69. MPhil Master of Philosophy

70. mRNA messenger RiboNucleic Acid

71. miRNA micro RiboNucleic Acid

72. MS Microsoft

73. MSc Master of Science

74. MSC Mesenchymal Stem Cell

75. mTOR mammalian Target Of Rapamycin

76. MTT Microculture TeTrazolium

77. N Number

78. NCBI National Center for Biotechnology Information

79. Nd:YAG Neodymium-doped Yttrium Aluminium Garnet

80. NGF Nerve Growth Factor

81. NIH National Institutes of Health

82. NLM National Library of Medicine

83. NNP Novel Nuclear Protein

84. NOS Not Otherwise Specified

85. NZW New Zealand White

86. P Probability

87. P1CP Procollagen type 1 Carboxyterminal Propeptide

88. PCNA Proliferating Cell Nuclear Antigen

89. PCR Polymerase Chain Reaction

90. PDF Portable Document Format

91. PDGF Platelet Derived Growth Factor

92. PG ProstaGlandin

93. PGMI Postgraduate Medical Institute

94. PhD Doctor of Philosophy

95. PKR PaKistan Rupees

96. PLGA Poly Lactic-co-Glycolic Acid

97. PMDC Pakistan Medical & Dental Council

98. PMN PolyMorphoNuclear

99. P-P Probability-Probability

100. Prof Professor

101. Q-Q Quantile-Quantile

102. RNA RiboNucleic Acid

103. RT Reverse Transcription

104. SMA Smooth Muscle Actin

105. SMAD SMA (SMAll body size)

+MAD (Mothers Against Decapentaplegic)

106. SEM Scanning Electron Microscopy

107. SPSS Statistical Package for Social Sciences

108. TAC Triamcinolone Acetonide

109. TEM Transmission Electron Microscopy

110. TGFB Transforming Growth Factor Beta

111. TIMP Tissue Inhibitor of MetalloProteinase

112. TLP TRAP-1 Like Protein

113. TLR Toll Like Receptor

114. TNF Tumour Necrosis Factor

115. TUNEL Terminal Utp Nick End Labelling

116. UHS University of Health Sciences

117. UK United Kingdom

118. URL Uniform Resource Locator

119. USA United States of America

120. VEGF Vascular Endothelial Growth Factor

121. VSS Vancouver Scar Scale

122. WHO World Health Organization

11. ABSTRACT

Introduction: Keloids & Hypertrophic Scars are basically an overabundance of fibrosis in cutaneous healing process (27.1). Presently there is no single effective management protocol for Keloids & Hypertrophic Scars (39.1). But surgical excision followed by intralesional steroid injection is the most popular (39.1). Profibrotic growth factors like Transforming

Growth Factor Beta cause formation of exuberant fibrous tissue during wound healing process (39.2). Cytokines like Interferon Gamma which inhibit profibrotic growth factors may be future treatment of Keloids & Hypertrophic Scars (39.2). At present there is no randomized controlled trial available evaluating the efficacy of Interferon Gamma in the treatment of Keloids & Hypertrophic Scars (39.2).

Objective: To compare the efficacy of intralesional Interferon Gamma and Triamcinolone

Acetonide for the treatment of Keloids & Hypertrophic Scars (69.1).

Hypothesis (HA): Intralesional Interferon Gamma is more efficacious than Triamcinolone

Acetonide for the treatment of Keloids & Hypertrophic Scars (70.2).

Operational Definition: Efficacy; Decrease in volume of Keloids & Hypertrophic Scars

(71.1).

Main Outcome Measure: Decrease in volume of Keloid & Hypertrophic Scar (73.1).

Design: Prospective Randomized Triple Blind Active Controlled Trial (73.2).

Setting: Department of Pathology and Experimental Research Laboratory, University of

Health Sciences, Lahore, Pakistan (73.3).

Duration: Two years (73.4).

Sample Size: Gross = 240; Net = 187 subjects (Keloids & Hypertrophic Scars) divided into control and interventional groups (73.5).

Sampling Technique: Simple Random Selection (three stages), and Allocation (three stages)

(73.6).

Data Collection Procedure: Animal models of Keloids & Hypertrophic Scars were produced by injecting Transforming Growth Factor Beta 1 on ventral surface of ear of randomly selected New Zealand White Rabbits, and excising a punch of skin and cartilage.

Subjects (Keloids & Hypertrophic Scars) were allocated randomly to control (Triamcinolone

Acetonide), and interventional (Interferon Gamma) groups (73.16). Keloids & Hypertrophic

Scars were treated with intralesional injection of Triamcinolone Acetonide or Interferon

Gamma, and decrease in volume was noted (73.23).

Data Analysis Procedure: Data was analyzed manually and also using Microsoft Excel,

International Business Machines Statistical Package for Social Sciences and other softwares.

Z-Test was applied to measure statistical significance. A p-value of ≤ 0.05 was considered statistically significant (73.27).

Results: Out of net 187 subjects, 93 were in control (Triamcinolone Acetonide), and 94 in interventional (Interferon Gamma) group (74.1). Mean reduction in volume of Keloids &

Hypertrophic Scars in Triamcinolone Acetonide group was 6.56661 mm3 (range 43.763 to -

22.959), and in Interferon Gamma group 13.49791 mm3 (range 44.504 to -16.519) (74.2).

The p-value was 0.000016 which is highly significant (74.3).

Conclusion: Treatment of Keloids & Hypertrophic Scars with Interferon Gamma is significantly more efficacious than with Triamcinolone Acetonide (76.1).

Recommendation: This study should be replicated in human beings (77.1).

Key Words: Cicatrix, Hypertrophic; Cytokines; Injections, Intralesional; Interferon Gamma;

Keloid; Model, Animal; Steroids; Transforming Growth Factor Beta; Triamcinolone

Acetonide.

12. INTRODUCTION

Wounds are as ancient as history of life on this earth. Every Homo sapiens and also

:which give rise to Wounds (Urdu ,(چوٹ، أذی :other species suffer from Trauma (Urdu

some of which become ,(نشا ِن زخم، ندب :which ultimately give rise to Scars (Urdu ,(زخم،جرح

is the most significant abnormal (ندبۂ ضخامی بیش نشا ِن زخم،:abnormal. Hypertrophic Scar (Urdu

Different.(الجدرةابھا ِر زخم، :scar, which sometimes grows further to become Keloid (Urdu

Wound Trains are given below. The whole Keloid Train is included in this treatise.

1. Keloid Train Trauma  Wound  Scar  Hypertrophic Scar  Keloid

2. Ulcer Train 1 Trauma  Wound  Ulcer  Hypotrophic Scar

3. Ulcer Train 2 Trauma  Wound  Ulcer  Contracture

4. Normal Train Trauma  Wound  Normal Scar

5. Foetal Train Trauma  Wound  Regeneration No Scar (14.1)

Abnormal scarring, which is now called Keloid, is said to have been first described in

Edwin Smith Surgical Papyrus (Imhotep, 3000 BC [Bib]). It is claimed that Case No. 45 titled Bulging Tumours of the Breast is actually Keloid (Breasted, 1930 [Bib]) (Figure 4, 5).

But in whole description, there is no mention of an earlier wound. To the Author it seems to be a case of benign breast tumour like gynaecomastia (Nagi & Babar, 2015 [Bib]). Most of authors have not mentioned which case they are referring to in this Papyrus; they have referred to each other’s work only. (15.4)

37 is again ornamental, on a metal coin or medal, on face of a king, found in the relics of same civilization. In Aztec civilization ritual Scarification was usually limited to gods or priestly caste. Boys devoted to the religious life received scars at young age (Marshall, 1929 [Bib]).

(15.5)

Reference to Keloid is given in Medieval Era writings of a Nigerian tribe Yoruba, in language of same name. Accurate descriptions of these lesions are mentioned in their proverbs as well as traditional medicine. Bronze statue of King of Ife Empire (1300 AD

[Bib]) of Yoruba in Nigeria was discovered with Ornamental Keloids on face (Figure 20).

Similarly statue of Queen of Ife Empire (1300 AD [Bib]) also depicted Ornamental

Scarification Keloids (Figure 21). Facial Ornamental Scarifications Keloids was also discovered in a common man from Ife (Anonymous, 1300 AD [Bib]) (Figure 22).

Ornamental Scarification Keloids are still found in modern Africa (Anonymous, n.d.

[Bib]).Sara and Hausa tribes of central Africa also perform ritual scarifications.In Ethiopia's

Karo tribe, people make scar on chests to show killing of people of enemy tribes. Women having scarred breasts are thought sensual and attractive (Omo-Dare, 1972 [Bib]) (Figure 23).

(16.8)

The first scientific description of Keloid came in 1785 AD from Noel Retz. In his book in French titled Des maladies de la peau, particulierement de celles du visage, et des affections morales qui les accompagnent; leur origine, leur description, leur traitment (The diseases of the skin, particularly those of the face, and moral affections accompanying them; their origin, their description, their treatment) (first edition, 1785 AD [Bib]; second edition,

1786 AD [Bib]) (Figure 27). He described them lésions rouges sombre donnant l'apparence d'être comprimé par une substance dense qui, avec le temps de former les fosses (lesions dark

38 red giving the appearance of being compressed by a dense substance which with the time form the pits). He thought its cause was hernie du tissu adipeux sous la peau (herniation of fat tissue under the skin). He also used the term dartre de graisse (dry patch of fat) for this condition. He however did not use the term Keloid. Third edition of his book (1790 AD

[Bib]) contained greater detail of the subject. (17.4)

Jean Louis Marie Alibert (1806 AD [Bib]) in his book in French titled Description des maladies de la peau, observées a l'Hôpital Saint-Louis et exposition des meilleures methodes suivies pour leur traitement (Description of diseases of the skin, observed at the Saint-Louis

Hospital, and exhibition of the best methods used in their treatment), used the term Cancroide for these lesions (Figure 28). The term Keloide was first used by Alibert (1816 AD) in his article titled Note sur la keloide (Note on the Keloide) published in Journal Universel des

Sciences Medicales (International Journal of Medical Sciences). He wrote another article

(1817 AD) titled Quelques recherches sur la cheloide (Some researches on the keloid), published in Mem de la Soc Med d'Emul (Proceedings of the Medical Society of Emul). In second edition of his book (1825 AD [Bib]) he used the terms Cancroide or Keloide to avoid confusion with cancer as well as syphilis (Figure 29). In third edition of this book titled

Clinique de l'Hopital Saint-Louis, ou traite complet des maladies de la peau, contenany la description de ces maladies et leurs meilleurs de traitment(Clinics of the Saint-Louis

Hospital, or complete treatment of the diseases of the skin, containing description of these diseases and their best treatment) (1835 AD [Bib]) (Figure 30), he used many synonyms for

Keloids: Keloide, Kelodes, Kelos, Cheloide, Cancroide, Tubercules Durs Cancelli,

Cancroma, Cancre Blanc, and Le Crebe. Alibert described two types of keloids: Vraie keloide (kelodes genuina) or true keloids or spontaneous keloids; and Fausse keloide (kelodes spuria) or false keloids, due to extreme inflammation. Vraie keloide is what is now called

Keloid, and Fausse keloide is now called Hypertrophic Scar. Second and third editions also showed paintings of Keloids (Figure 31). (17.5)

Keloids are found only in human beings (although some cases have been reported in horses and dogs). Keloids are found on average in 6% (range 4.5-16%) of world population.

Prevalence of Keloids is 10% (range 15-20%) in non-White population. Keloids are 15 times more common in Blacks than in Whites (Niessen, 1999). Incidence of Keloids is 10-15% in all types of wound taken together, irrespective of any other factor. Even in coloured races, people with dark complexion are more prone to form Keloids as compared to those with fair complexion. Albinos are least affected by this disease, suggesting some role of melanocytes or melanin in production of this disease. Most common age of development of Keloids is 20 years (range 10 to 30 years). Assi et al (2009) from West Africa reported average age of 28 years. Tirgan (2015 [Bib]) has reported peak age of development of Keloid as 26 years

(Figure 46). Keloids are more common in women than men; this may be an artefact, as women more often get earlobe piercing, the commonest cause and site of Keloid. Sun et al

(2014) reported that Incidence of keloid in ethnic Chinese population was 0.15%, male to female ratio 1:1.33, and women with fibroid of uterus had a 2.25 fold risk of Keloids. (25.1)

Keloids & Hypertrophic Scars are generally sequelae of wounds, whether surgical or traumatic. They can also follow minor abrasions, or needle wound, like injection, or may be spontaneous. They form 3 months to many years after injury. They are very variable in size; small injuries may cause big lesions. Exact aetiology of Keloids & Hypertrophic Scars is not known. However many factors have been labelled as aetiological/risk factors These can be intrinsic ie Race, Genetics, Age, Sex, Site, Tension, Direction, Hormones, Syndromes,

Hypertension, Autoimmunity, and Spontaneous; or extrinsic ie Piercing, Tattooing, Epilation,

Buns, Bites, Vaccination, Infection, Irritation, Inflammation, Sutures, Foreign Body, and

Drug Induced. (26.1)

Keloids & Hypertrophic Scars are basically an overabundance of fibrosis in cutaneous healing process. Keloids are benign cutaneous fibro-proliferative tumours. Hypertrophic scars are similar in nature, but do not grow into normal tissue. The Author suggests that Keloids and Hypertrophic Scars may be considered variation of same theme (Nagi & Babar, 2015

[Bib]). Further, Hypertrophy is a misnomer here, as in pathology hypertrophy means increase in size of cell. In hypertrophic scars there is no increase in size of cells; rather there is increase in collagen fibres. Scar is not a medical term, and MeSH uses the term cicatrix instead. But the term Scar has become so common in medical literature that this term is retained in this treatise. However to avoid unnecessary confusion, the Author recommends that the term Hypertrophic Scar should be discontinued, and any scar which remain elevated from surface of surrounding skin three months after initial insult (or healing of wound) should be considered Keloid. Keloidosis is the term coined by the Author for the disorder of formation of Keloids & Hypertrophic Scars (Nagi & Babar, 2015 [Bib]). (27.1)

Classic theory is that Keloids and Hypertrophic Scars are considered different entities.

Hypertrophic scars grow above skin, yet remain in limits of primary scar and frequently regress spontaneously. Keloids grow above skin too, yet spread outside borders of primary scar and seldom regress spontaneously. Both Keloids & Hypertrophic Scars form after trauma to skin, but in rare cases Keloids can occur spontaneously as well (Brunicardi, 2015

[Bib]; Kumar, 2014 [Bib]; Patterson, 2015 [Bib]; Townsend, 2012 [Bib]). (28.1)

41 weave pattern. In Normal Scars, collagen bundles are placed parallel to each other, in a sheet pattern. In Hypertrophic Scars, collagen bundles are more flat, more stretched, more random, like a wavy pattern, with intervening fibroblasts. In Keloids, collagen bundles are almost absent, and collagen fibres are placed haphazardly in disarrayed pattern. Collagen fibres are much large and thick, and fibroblasts are missing from centre of Keloid. (28.2)

Different scientists have described pathogenesis of Keloids, from different perspectives; genetics, mechanics, endocrinology, metabolism, circulation, immunology, and nutrition. These pathogenesis theories are perceptions or speculations of learned people about mechanisms of keloid formation. At best, these can be considered as educated guess. Further research is needed to find out the truth (Huang et al, 2013) (Figure 50). (29.1)

Keloids and Hypertrophic Scars usually present after 3 to 12 months of original event.

There is as swelling on skin after healing of a wound, or infection. Size and shape depend on those of initial injury. It can become several cm in height. It grows slowly taking months and even years to enlarge. It extends beyond initial wound. It rarely invades hypodermis. It ultimately become quiescent and stable, or may regress to some extent. Swelling is accompanied by pruritus/itching, which is more marked in Keloids, and occurs especially in summer seasons, and in people living in tropical areas. Some patients complain of burning sensation or stinging. There is only mild pain or discomfort. Sometimes hyperaesthesia also occurs. Keloid is inconvenient and unsightly, and many patients present for cosmetic reasons only. If Keloid is over a joint, it can also restrict movement, or can cause contracture. (33.1)

Examination shows that Keloid swelling is usually irregularly shaped, corresponding to shape of initial injury, and is linear after a surgical scar. It may be heaped-up, ridged, raised, dome-shaped, exuberant, or sharply elevated. Its surface is smooth and shiny. It is of skin colour; colour changes from pink to red to brown to pale. It may occasionally be pigmented. It is devoid of hair, and is dry due to lack of sweat and sebum. It is soft, firm,

42 tough, doughy, rubbery, hard, indurated, or nodular. It may be mobile, or tender. It enlarges slowly, in a claw-like fashion (Figure 54). (33.2)

Thomas Addison wrote in his article on Keloids (1854 AD): ‘In regard to treatment little can be said’ (Figure 60). Unfortunately, this is still true even in 2015 AD. Presently there is no single effective management protocol for Keloids & Hypertrophic Scars.

Treatment at present is aimed at restoration of normal function, control of symptoms, cosmesis, and prevention of relapse. List of therapies includes surgical excision, intralesional steroids, radiotherapy, silicone sheets, pressure garments, laser, cryosurgery, intralesional chemotherapeutic drugs, and topical retinoids etc. Often two, three, four, or even more modalities of treatment are combined to achieve acceptable results. Unfortunately a universal approach in Keloids & Hypertrophic Scars treatment is yet to be identified. But surgical excision followed by intralesional steroids is the most popular. However, with current treatment modalities, average recurrence rate is 75%. Treatment is more effective on younger

Keloids & Hypertrophic Scars. (39.1)

Because exact cause of Keloids & Hypertrophic Scars is still not known, many modalities of treatment are being used without much success. Profibrotic growth factors like

Transforming Growth Factor Beta cause formation of exuberant fibrous tissue during wound healing process. Cytokines like Interferon Gamma which inhibit profibrotic growth factors may be future treatment of Keloids & Hypertrophic Scars. At present there is no randomized controlled trial available evaluating the efficacy of Interferon Gamma in the treatment of

Keloids & Hypertrophic Scars. As the treatment of Keloids & Hypertrophic Scars is a challenging problem, development of new and better therapies is need of the hour. (39.2)

The objective of this study is: To compare the efficacy of intralesional Interferon

Gamma and Triamcinolone Acetonide for the treatment of Keloids & Hypertrophic Scars.

(69.1)

Figure 1: Imhotep, the Author of Edwin Smith Surgical Papyrus (3000 BC)

13. REVIEW OF LITERATURE

Review of Literature of Keloids was done mainly with PubMed (Figure 2), as follows:

1. History of Keloids & Hypertrophic Scars

2. Anatomy of Keloids & Hypertrophic Scars

3. Physiology of Keloids & Hypertrophic Scars

4. Biochemistry of Keloids & Hypertrophic Scars

5. Incidence &Prevalence of Keloids & Hypertrophic Scars

6. Aetiology of Keloids & Hypertrophic Scars

7. Pathogenesis of Keloids & Hypertrophic Scars

8. Pathology of Keloids & Hypertrophic Scars

9. Classifications of Keloids & Hypertrophic Scars

10. Symptoms & Signs of Keloids & Hypertrophic Scars

11. Investigations of Keloids & Hypertrophic Scars

12. Diagnosis of Keloids & Hypertrophic Scars

13. Differentiation of Keloids & Hypertrophic Scars

14. Prophylaxis of Keloids & Hypertrophic Scars

15. Treatment of Keloids & Hypertrophic Scars

16. Complications of Keloids & Hypertrophic Scars

17. Prognosis & Follow-Up of Keloids & Hypertrophic Scars

18. Animal Model of Keloids & Hypertrophic Scars

19. Role of TGFB 1in Keloids & Hypertrophic Scars

20. Role of DMSO in Keloids & Hypertrophic Scars

21. Role of Triamcinolone Acetonide in Keloids & Hypertrophic Scars

22. Role of Interferon Gamma in Keloids & Hypertrophic Scars

23. Technique of Intralesional Injection in Keloids & Hypertrophic Scars

Figure 2: PubMed (Medline) Literature Search Portal (2015 [Bib]) of NCBI of NLM of NIH of USA. This Online Database was used for Literature Search for this Treatise 46

14. History of Keloids & Hypertrophic Scars

Wounds are as ancient as history of life on this earth. Every Homo sapiens and also

:which give rise to Wounds (Urdu ,(چوٹ، أذی :other species suffer from Trauma (Urdu

some of which become ,(نشا ِن زخم، ندب :which ultimately give rise to Scars (Urdu ,(زخم،جرح

is the most significant abnormal (ندبۂ ضخامی بیش نشا ِن زخم،:abnormal. Hypertrophic Scar (Urdu

Different.(الجدرةابھا ِر زخم، :scar, which sometimes grows further to become Keloid (Urdu

Wound Trains are given below. The whole Keloid Train is included in this treatise.

6. Keloid Train Trauma  Wound  Scar  Hypertrophic Scar  Keloid

7. Ulcer Train 1 Trauma  Wound  Ulcer  Hypotrophic Scar

8. Ulcer Train 2 Trauma  Wound  Ulcer  Contracture

9. Normal Train Trauma  Wound  Normal Scar

10. Foetal Train Trauma  Wound  Regeneration No Scar

History of these trains is divided in five Era. These eras are described in pages that follow. Very little work was done on keloids in Ancient and Medieval era. Most of the work was done in Modern and Present era, and this accounts for main bulk of History section.

Future era includes ongoing research which is going to give us new insight into Keloid pathology. Future treatments are described in Treatment section.

1. Ancient Era upto 475 AD (Figure 3)

2. Medieval Era 476 AD to 1492 AD

3. Modern Era 1493 AD to 1999 AD

4. Present Era 2000 AD to 2015 AD

5. Future Era 2016 AD onwards

Figure 3: An Ancient Era Artifact: Medical Cuneiform Writings from Mesopotamia; the earliest known Book of Medicine (Anonymous, 3500 BC [Bib]) 48

15. Ancient Era

In Ancient Era (Adam to 475 AD), many major civilizations have flourished in this world: Start of Civilization (10000 BC); First Walled City ie Jericho (9000 BC); Start of

Agriculture (8000 BC); Start of Copper Age (6000 BC); Birth of Adam & Eve (3760

BC);Mesopotamia (Sumer) (3500 BC); Egypt (3400 BC); India/Indus (3300 BC); Persia

(3200 BC); China (2800 BC); Ancient America (Aztec, Maya, Inca, Olmec) (2600 BC);

Babylonia (2200 BC); Greece (2000 BC); Rome (750 BC); and others. All these civilizations had their system of medicine and healing of wounds, but very patchy record was preserved.

Writing started at 3500 BC in Sumer, a city-state of Mesopotamia (now Iraq). This writing was called Cuneiform, which is world’s first written language. It consisted of drawing of original thing (Pictogram), which overtime became simpler, and eventually wedge-shaped ie cuneiform. It was written on clay tablets, and then baked in a kiln. Oldest known medical bookies Sumerian physician’s collection of empiric prescriptions, written on clay tablets, called Medical Cuneiform Writings (3500 BC [Bib]). Unfortunately, no Cuneiform clay tablet devoted exclusively to wounds or surgery is available.

Papyri of Egypt were written and re-written from 3000 BC to 1700 BC. These contain important medical records of that time. Name of exact author is not known, but it is thought that Imhotep, chief physician of Pharaoh Djoser has written them. Edwin Smith Surgical

Papyrus describes 48 cases, out of which 45 deal with injuries and wounds. Other medical papyri ie Georg Ebers Papyrus, Berlin Medical Papyrus, London Medical Papyrus, Hearst

Papyrus, Kahun Papyrus, and Chester Beatty Papyrus, rarely deal with injuries or wounds.

Abnormal scarring, which is now called Keloid, is said to have been first described in

Edwin Smith Surgical Papyrus (Imhotep, 3000 BC [Bib]). It is claimed that Case No. 45 titled Bulging Tumours of the Breast is actually Keloid (Breasted, 1930 [Bib]) (Figure 4, 5).

Ancient American civilization flourished in Central America from 2600 BC upto colonization. The oldest known graphic representation of Keloids is ornamental, on a statue, on face of Aztec god Xipe Totec (2600 BC [Bib]) (Figure 6). Another graphic representation is again ornamental, on a metal coin or medal, on face of a king, found in the relics of same civilization. In Aztec civilization ritual Scarification was usually limited to gods or priestly caste. Boys devoted to the religious life received scars at young age (Marshall, 1929 [Bib]).

Babylonia was next major civilization, and its most famous ruler was Hammurabi, whose Code was the most prominent writing of that period (Figure 7). Almost all medical rules in Code of Hammurabi (1775 BC [Bib]) are concerned with wounds and operations. It says: ‘If a doctor has treated a freeman with a metal knife for a severe wound, and he cured the freeman, ---, then he shall receive ten shekels of silver. If a doctor has treated a freeman with a metal knife for a severe wound, and caused the freeman to die, ---, his hands should be cut off’. It is the first written record of treatment of wounds by a doctor.

Pharaoh Horemheb (1323-1295 BC) had many Hypertrophic Scars on face, and a

Keloid on chin, as depicted in his statue. This was also found on his mummy (Figure 8, 9).

This is the oldest Keloid found on a human body. Another unknown Pharaoh statue showed

Ornamental Keloid on Shoulder and a true Keloid on Cheek (Figure 10).Nubian Ornamental

Scars on cheeks are found in a prisoner scene from Saqqara Tomb of Horemheb (Figure 11).

Wounds and Scars are also mentioned in Torah of Moses (1300 BC [Bib]), Psalms of

David (1000 BC [Bib]), and Gospel of Jesus (30 AD [Bib]).

1. Torah:

1. Exodus 21:25: Burn for burn, wound for wound, bruise for bruise.

2. Deuteronomy 32:39: See now that am He, and there is no god besides Me. It is I who put

to death and give life. I have wounded and it is I who heal, and there is no one who can

deliver from My hand.

3. Job 5:18: For He inflicts pain, and gives relief; He wounds, and His hands also heal.

4. Isaiah 1:6: From the sole of the foot even to the head there is nothing sound in it; only

bruises, welts and raw wounds, not pressed out or bandaged, nor softened with oil.

5. Jeremiah 10:19: Woe is me, because of my injury! My wound is incurable.

2. Psalms:

1. Psalm 38:5: My wounds grow foul and fester because of my folly.

3. Gospel:

1. Luke 10:34:And came to him and bandaged up his wounds, pouring oil and wine on

them; and he put him on his own beast, and brought him to an inn and took care of him.

2. Galatians 6:17: From now on let no one cause trouble for me, for I bear on my body the

scar-marks of Jesus.

Statue of Seated Boxer (330 BC) form Rome is first graphic illustration of multiple scars (Figure 12). The boxer has scarred and bruised face, broken nose, and broken teeth.

First graphic illustration of wound is sculpture of Dying Gladiator (230 BC) (Figure 13). The sculpture depicts a wounded, slumping gladiator (or Gaul). A bleeding sword puncture is visible in his lower right chest.

Figure 4: The Edwin Smith Surgical Papyrus Translation Case No. 46, Page No. 463 (3000 BC) (Breasted, 1930 [Bib]) 52

Figure 5: The Edwin Smith Surgical Papyrus Original Manuscript Column No. XV, Cases No. 43-46(Imhotep, 3000 BC [Bib]) 53

Figure 6: Ancient American Aztec god Xipe Totec, displaying linear Keloids on his face (Xipe Totec, 2600 BC [Bib]) 54

Figure 7: Code of Hammurabi from Babylonia. It contained Law on Treatment of Wounds as well (Hammurabi, 1775 BC [Bib]) 55

Figure 8: Pharaoh Horemheb’s mummy (1323 BC-1295 BC) showing many Abnormal Scars and a Keloid on face (chin) 56

Figure 9: Pharaoh Horemheb’s statue (1323 BC-1295 BC) showing many Abnormal Scars, and a Keloid on face (chin) 57

Figure 10: An unknown Egyptian Pharaoh with Ornamental Scarification Keloid on Right Shoulder and a true Keloid on Right Cheek (1300 BC) 58

Figure 11: Nubian Ornamental Scars on cheeks of prisoners from the Saqqara Tomb of Horemheb (1300 BC) 59

Figure 12: ‘Seated Boxer' from Greece showing Abnormal Scars on face (330 BC)

Figure 13:B; ‘Dying Gladiator’ from Rome; the First Graphic of Wound (230 BC)

16. Medieval Era

In Medieval Era (476 AD to 1492 AD), wounds and injuries are mentioned in Quran

(632 AD [Bib]) (Figure 14):

1. Quran 3:140: If a wound has touched you, then (opposing) people have already been

touched with a wound similar to it. And these days (of varying conditions) We alternate

among the people so that Allah may make known those who believe, and (may) take for

Himself from among you witnesses, and Allah does not like the wrongdoers.

2. Quran 3:172: Those (are believers) who respond to Allah and the Messenger after injury

has struck them. For those who do good amongst them and fear Allah, is a great reward.

3. Quran 5:45: In the Torah We made mandatory for the Jews these rules of retaliation:

Capital punishment for the murder of a person; an eye for an eye, a nose for a nose, an ear

for an ear, a tooth for a tooth, and a just compensation for a wound.

Abū al-Qāsim Khalaf ibn al-Abbās az-Zahrāwī (936–1013 AD), commonly called Az-

Zahrawi, Latinized as Abulcasis or Albucasis (Figure 15), was a Muslim physician and surgeon belonging to Hispania (Spain). He is the most accomplished surgeons of Islamic Era, and is considered father of surgery. He stressed on sewing-up wounds, and invented catgut for stitching, which is still used today. He was also first to use cautery for bleeding wounds.

He used opium and hashish for anaesthesia. His book titled Kitab al-Tasrif was published in

Arabic (1000AD [Bib]). It contained illustrations of many instruments for treatment of wounds (Figure 16). Later it was translated into Latin as Liber Theoricae (Figure 17).

From Muslims, especially of Hispania and Sicily, medicine and surgery passed to their next-door neighbours; southern peninsula of Europe, mainly Iberian and Italian peninsula. It should be remembered that southern France was also part of Muslim Andalusia, which helped pass medical (and other) knowledge to main France. French were pioneers in learning from Muslims of all subjects in whole of Europe. In fact, Modern Era and

Renaissance started from France. France remained the leader in knowledge and learning for next four centuries when Germany, and later UK (mainly Scotland), took over. Scots took this knowledge with them to the new world (America). The third southern peninsula of

Europe ie Balkan peninsula had just started coming in contact with Muslim knowledge at the end of Medieval Era. Roger of Salerno, Italy (1140-1195 AD) was pioneer of surgery in

Europe (Figure 18). His book titled Chirurgia (1180 AD [Bib]) showed illustrations of various treatments of wounds (Figure 19)

Looking from another angle, Keloids are intimately linked to indigenous culture of

Africa in Ancient and Medieval Eras. Ornamental Scarification has been a tradition in a lot of tribes since ancient times. In African and other dark-skinned equatorial ethnic groups, scarification was more favoured than tattooing because it was easy to create scarification designs. Further, scars looked more prominent on black skin. Scarification was usually done in those races in which a lot of melanin in skin made it difficult to appreciate a tattoo.

Scarification is generally a part of ceremonial rituals in these races, like puberty and marriage. Tribal people are often seen as imperfect if they do not undergo scarification; being scarified helps people identify with his tribe and enhance his social status. As no two designs were same, raised facial tattoos were noticeable as identity of the person. Further, when tribal women became ready to conceive, they scarified their abdomens to show their readiness.

[Bib]) of Yoruba in Nigeria was discovered with Ornamental Keloids on face (Figure 20).

Similarly statue of Queen of Ife Empire (1300 AD [Bib]) also depicted Ornamental

Scarification Keloids (Figure 21). Facial Ornamental Scarifications Keloids was also discovered in a common man from Ife (Anonymous, 1300 AD [Bib]) (Figure 22).

Ornamental Scarification Keloids are still found in modern Africa (Anonymous, n.d.

[Bib]).Sara and Hausa tribes of central Africa also perform ritual scarifications.In Ethiopia's

Karo tribe, people make scar on chests to show killing of people of enemy tribes. Women having scarred breasts are thought sensual and attractive (Omo-Dare, 1972 [Bib]) (Figure 23).

Scarification was also done by Polynesian Maori tribe of New Zealand (1400 AD).

They are excellent wood carvers, and so utilize this talent to make complex and distinctive scarifications on their skin, called moko. These artistic Keloids help identify people, tell their social standing and tribal membership, follow their ancestry, show their conquests, and indicate many other significant life occasions. Maori men think that engraving their faces makes them appear ferocious in battle and fascinating to women (Guynup, 2010 [Bib]).

Scarification has emerged as a component of modern body modification movement.

Over last decade, scarification has become extraordinarily prevalent in USA, Australia, and

Europe.Body modification movement is not an abnormal phenomenon in a society where identity is usually shown through external appearance. This in centre of society means fashion, cosmetic surgery, & botox, and on periphery of society, tattooing, scarification, and piercing (Guynup, 2010 [Bib]).

Figure 14: Quran; the latest Divine book Allah, 632 AD [Bib] 65

Figure 15: Az-Zahrawi (936-1013 AD). He wrote about Treatment of Wounds

Figure 16: An illustration from Al-Zahrawi’s Kitab al-Tasrif (1000 AD [Bib]) 67

Figure 17:B Al-Zahrawi’s BookKitab al-Tasrif (Liber Theoricae) (1000 AD [Bib])

Figure 18:B; Roger of Salerno (1140 AD-1195 AD); wrote about Wound Treatment

Figure 19: Roger of Salerno’s Chirurgia showing Wound Treatment (1180 AD [Bib])

Figure 20: King of Ife Empire of Yoruba with Ornamental Keloids (1300 AD Bib])

Figure 21: Queen of Ife Empire of Yoruba with Ornamental Keloids (1300 AD [Bib])

Figure 22:B; Facial Ornamental Keloids on a statue from Ife Empire of Yoruba, Nigeria (Anonymous, 1300 AD [Bib]) 73

Figure 23:B; Ornamental Keloids are still common in Africa. Photograph of a Modern Negro Boy showing Multiple Keloids on Face (Anonymous, n.d. [Bib]) 74

17. Modern Era

In Modern Era (1493 AD to 1999 AD), Ambroise Pare (1517-1590 AD), barber- surgeon of France wrote the first book in 1560 AD [Bib] on wounds titled Le procédé de durcissement blessures par balle faite aussi par des flèches et des fléchettes, avec leurs accidents (The method of curing wounds made by gun-shot, also by arrows and darts, with their accidents) (in French, because he did not know Latin or Greek). Another book of him

Dix livres de la chirurgie, avec le magasin des instrumens nécessaires à icelle (Ten books of surgery, with the magazine of the instruments necessary for it) in French (1564 AD [Bib]) contains the first illustration of suturing of a wound using a straight needle (Figure 24).

Johannes Scultetus (or Sculteti) of Germany in 1655 AD [Bib] wrote a book titled

Armamentarium chirurgicum (Surgical arsenal) in Latin. It contained a complete catalogue of all known surgical instruments, methods of bandaging wounds and splinting fractures, and many operations, illustrated in detail. It showed curved needle for the first time (Figure 25).

In 1783 AD [Bib], Scottish Surgeon Benjamin Bell (1749-1806 AD) of Edinburgh, who later moved to USA, wrote a book titled: A system of surgery in six volumes. It was first book of surgery in English. Chapter XXXVI of this book, in Volume V, was on Wounds, and it contained XIV Sections devoted to different types of wounds, spanning over 350 pages. It depicted illustration of straight and curved needles for suturing wounds (Figure 26).

75 diseases of the skin, particularly those of the face, and moral affections accompanying them; their origin, their description, their treatment) (first edition, 1785 AD [Bib]; second edition,

1786 AD [Bib]) (Figure 27). He described them lésions rouges sombre donnant l'apparence d'être comprimé par une substance dense qui, avec le temps de former les fosses (lesions dark red giving the appearance of being compressed by a dense substance which with the time form the pits). He thought its cause was hernie du tissu adipeux sous la peau (herniation of fat tissue under the skin). He also used the term dartre de graisse (dry patch of fat) for this condition. He however did not use the term Keloid. Third edition of his book (1790 AD

[Bib]) contained greater detail of the subject.

Sciences Medicales (International Journal of Medical Sciences). He wrote another article

Clinique de l'Hopital Saint-Louis, ou traite complet des maladies de la peau, contenany la description de ces maladies et leurs meilleurs de traitment(Clinics of the Saint-Louis

Hospital, or complete treatment of the diseases of the skin, containing description of these diseases and their best treatment) (1835 AD [Bib]) (Figure 30), he used many synonyms for

Keloids: Keloide, Kelodes, Kelos, Cheloide, Cancroide, Tubercules Durs Cancelli,

Keloid, and Fausse keloide is now called Hypertrophic Scar. Second and third editions also showed paintings of Keloids (Figure 31).

P. F. O. Rayer in 1826 AD [Bib] in his book in French on skin diseases titled Traite theorique et pratique des maladies de la peau, fonde sur de nouvelles recherches d'anatomie et de physiologie pathologiques(Theoretical and practical treatment of the diseases of the skin, based on new research in anatomy and pathological physiology) described Keloids in detail. He incorporated basic sciences research into clinical sciences (Figure 34).

Biett in 1832 AD in French reported several cases of Keloid. He reported a young woman who had eight Keloids in her neck and anterior chest. She had deep pain, especially after eating. The progress of Keloids was slow; it absorbed in some cases, and rarely ulcerated. He advised against extirpation or cauterization, and advised sulphur douches.

Hawkins in 1833 AD published an article about Keloids titled: On warty tumours in cicatrices, in London Medical Gazette. He wrote: ‘There appears in the first place a little wart, or warty tumour, in the cicatrix, which is dry, and covered with a thin cuticle, but which soon becomes moist and partially ulcerated, like the warts of mucous membranes, from which a thin and offensive, and semi-purulent, fluid is secreted. In the second stage of the disease the growth of the tumour becomes more rapid, the warty appearance being in some measure lost, a more solid substance projecting from the diseased skin, which bears more

77 resemblance to the fungus of fungus haematodes; the formation of fresh warts being still seen around the tumour, and preceding the change which has been alluded to. After the tumour has become solid and prominent, a new action takes place in it, and the tumour ulcerates and sloughs alternately, with a great deal of pain and suffering; and the tumour is destroyed down to its basis, so as to present the appearance of a foul excavated ulcer, except in its circumference, where the skin is raised, thickened, and everted; and from time to time warts are generated, which again ulcerate and slough, till the patient becomes gradually worn out by suffering, but without having at all the sallow and peculiar aspect of a person dying of a malignant disease; and on examination of the body no disease of the absorbent glands is found, nor is there any sign of malignant disease in the interior of the body.’ The term

Hawkins’ Keloid is still present in World Health Organization’s ICD.

Coley in 1839 AD wrote a detailed article about Keloids, published in The Lancet.

Velpeau in 1845, in French, published an article on Keloids in Gazette des Hopitaux

(The hospital gazette) of Hôpital de la charité (Charity Hospital), Paris. Cazenave in 1845

AD [Bib], in French, wrote a book titled Leçons sur les maladies de la peau (Lessons on the diseases of the skin), in which described Keloids in detail.

Labert in 1851 AD [Bib] in his book in French titled Traite pratique des Maladies

Cancereuses, et des affections curables, confondues avec le Cancer (Practical treatment of cancerous diseases, and curable conditions, associated with cancer) described spontaneous keloids. He wrote: ‘Among the cases of spontaneous and multiplied keloid that we have observed, there were two especially curious, in consequence of their multiplicity and extent.

In one case, under M. Velpeau, at La Charite, the whole pectoral region of one side was

78 covered with these tumours; many of which were sufficiently large to have reddened and eroded the surface of the skin at their borders. In the second case, a child age 10½, had a very great number of keloid tumours, developed upon his back, red on their surfaces, and which had formed in the cicatrices which were consecutive to numerous applications of caustic potash, applied to the poor child by a charlatan, who promised to cure, by this method, a scrofulous disease under which the child laboured.’

Dieburg of Deutsche Klinik, writing in journal of same name in 1852 AD, in German, for the first time, gave a description of histopathology of Keloid. He wrote: ‘On section we observe a dull white colour, a dense tissue in which fibrous structure is visible to the naked eye and a creaking sound is produced by the knife. On pressure, no fluid exudes in most cases; in a few, a watery fluid is seen, sometimes reddened by blood. This is characteristic, as different from the tumores verrucosi cicatricum of C. Hawkins, from which a peculiar fluid may generally be expressed. Microscopical examination shows the different stages of development of the cells and fibres. We distinguish: 1. More or less rounded bodies, the largest 0 05 of a millimetre; in their interior, we see a nucleus, and frequently other molecules. 2. Cells elongated in the direction of one of their diameters, in great numbers: they seem to constitute a characteristic element of all the tumours of cicatrix-keloid (spurious keloid of Alibert). These cells, called by Follin elliptical bodies, are rounded at their extremities, and their sides present central bulging. These cells are about 0.01 millimetre in breadth, and 0.06 in length. They contain a nucleus easily distinguishable by its brightness from the dull surrounding parts. 3. Spindle-shaped bodies, bulging in their centre, and having long, waving appendages. 4. Fibres of cellular tissue and elastic fibres. The fibres of cellular tissue are formed into bundles, which cross each other, and constitute a pretty dense web. The elastic fibres are less numerous and larger than the latter, and are not easily seen without

79 immersion in acetic acid. When a slice of keloid in an early stage of development is placed under the microscope, it is found to consist almost entirely of the spindle-shaped bodies; at a somewhat later period these are seen to have lost their nuclei, and assumed a fibrous appearance: this is most frequent. At a still later period, we see distinct fibrous bundles, crossing each other, and by immersion in acetic acid, the elastic fibres become visible. The whole is nourished by a comparatively small number of blood-vessels. The surface is covered by a very thin layer of epidermis, consisting of tessellated cells, very closely pressed together, which require softening before they become visible under the microscope.’

Addison in 1854 AD wrote treatise on Keloids, titled ‘On the Keloid of Alibert, and on True Keloid’ and published in Medico-Chirurgical Transactions. Addison called Alibert’s

Keloid as False Keloid and his Keloid (which is now called Morphea) as True Keloid. Here confusion was produced due to mistake of Addison, one of the ‘great men’ of Guy’s. This misconception continued for a long time. In a wider perspective, this battle of ‘True and

False’ may be seen in the background of historical Anglo-French rivalry. Addison writes in the said article: ‘The term keloid, or keloide, the name given to the singular affections of the integument about to be described, has been variously interpreted; some deriving it from κηλη

(kele), a tumour; others, in reference to certain supposed resemblances, from χηλη (chele), a crab's claw; or from χελυς (chelys), a tortoise; whilst others, apparently with much greater propriety, derive it from κηλις (kelis),quasi ustione facta macula (like a burn spot), the disease in every instance presenting a greater or less resemblance to some one of the diversified effects left by a burn. In order, however, to illustrate and confirm this proposition, it will be necessary to give a description of both diseases; and in so doing, I will, as far as possible, avoid trespassing too much upon the time and attention of the Society. I propose distinguishing the two diseases in question by the terms Keloid of Alibert and the True

Keloid. I have given the name Keloid of Alibert to this form of disease, because I believe

Alibert to have been the first to discriminate and accurately describe it. In his celebrated work, Description des maladies de la peau will be found a very accurate representation of it, executed with all the artistic skill, and perhaps a little of the exaggeration of colouring, for which that work is so remarkable. He there suggests its holding a middle place between what he so vaguely and indiscriminately calls dartre (dry patch) and cancer, and was led in consequence to give it the name of cancroide like cancer; further justifying the appellation, however, by comparing, as others have done, the claw-like rays or processes of the extending disease to the claws of a crab. Since the period of Alibert's original publication, several other writers have furnished cases and commentaries to illustrate the character, progress, or pathology of the disease. Amongst these we find the names of Biett, Velpeau, Cazenave,

Coley, and others; but by far the most complete and elaborate essay on the subject has only lately been written by Dr. Dieburg, of Dorpt, and published in the Deutsche Klinik at Berlin, and for a knowledge of which am indebted to my colleague Mr. Birkett and Dr. Whitley.’ He further wrote: ‘It [Keloid] has its original seat in the subcutaneous areolar tissue’.

In 1855 AD, Slade published an article titled On Keloides in The Boston Medical and

Surgical Journal. He wrote: ‘The term keloid, or keloide, is applied to a singular affection of the skin, extremely rare, and not even described by many writers on cutaneous diseases. The origin of the word is involved in doubt; some deriving it from chele, a crab’s claw, while others, with more propriety, suppose that it was derived from kelis, a burn, owing to the resemblance of the affection to the cicatrix left by such an injury.’ He favoured the stance of

Addison, and reproduced much of his article.

In 1863 AD, Gordon was a black slave in American plantations in Mississippi. He

81 was beaten by his master with a whip, due to which he developed multiple Keloids. He is known in history as Whipped Peter. His was the first Keloid photograph, taken by McPherson

& Oliver on April 2, 1863 AD, at Baton Rouge, Louisiana, USA. Original photo caption was:

‘Overseer Artayou Carrier whipped me. I was two months in bed sore from the whipping. My master come [sic] after I was whipped; he discharged the overseer. The very words of poor

Peter, taken as he sat for his picture.’ (McPherson & Oliver, 1863AD [Bib]) (Figure 32).

Langhans in 1867 AD, in German, published a case report titled Ein fall von keloid (A case of keloid) in Archiv für pathologische Anatomie und Physiologie und für klinische

Medicin (Archives for pathological anatomy and physiology and for clinical medicine).

Fagge in 1868 AD wrote an article titled: On keloid, sclerosis, morphea and some allied affections, in Guy’s Hospital reports. He tried to differentiate between these diseases.

This was an effort to clarify a long lasting confusion arising out of a mistake of Addison.

Lawrence in 1898 AD from Australia treated a Keloid by scarification. With a five- bladed scarifier, he mince-meated keloid by making vertical and horizontal incisions on it, dividing it in 400blocks per inch2. He applied continuous pressure on operation site by a piece of solid India-rubber tubing. Pressure was continued for several months.

Kiliani in 1901 in his article in Annals of Surgery described traumatic keloid of median nerve, which is now called neuroma.

Tiley in 1908 described a Keloid following mastoid operation in Proceedings of the

Royal Society of Medicine.

Morris & Dore in 1909 in their article titled Extensive Acne with Scars and Keloid, wrote: ‘On examination of the patient, the whole of the skin of his chest, back, and shoulders was seen to be a mass of scar-tissue, honeycombed with deep, irregular cavities, and interspersed with indolent deep-red or bluish infiltrations and sub-epidermic abscesses. Here and there a few large comedones were visible. His axillae, upper arms, abdomen, and thighs were similarly but less extensively and less deeply affected, and there was more normal skin between the scars. On his arms and legs there were only a few scars, some of these being large and circular, some raised in the form of bands, and others surrounded by projecting processes of cicatricial tissue, There were also a few comparatively recent lesions on his scalp. Bacteriological investigation of early pustular lesions from the wrist and elbow gave cultures of Staphylococcus aureus. Streptococci and a small motile Gram-negative bacillus were obtained from a sub-epidermic abscess on the chest. It was proposed to treat him with a staphylococcus vaccine prepared from his own lesions. He had previously been treated with five injections of a stock vaccine of staphylococcus in the Newcastle Infirmary, with some temporary improvement. He had also had various ointments and a few applications of X- rays.’ This article mentions X-rays radiotherapy for treatment of Keloids for the first time.

Porter in 1909 described Massive Keloid of face and hands, in Annals of Surgery. He wrote: ‘This remarkable case of keloid of the face and hands is reported chiefly to put the photographs upon record, and to show the present result after numerous plastic operations, skin grafts, and spontaneous healing. Although the X-rays were conscientiously tried over several periods there was no benefit. This patient entered the Massachusetts General Hospital, on March 10, I905, in the service of Dr. A. T. Cabot, whose assistant I was at that time. Dr.

Cabot performed the first operation on March 20, I905; since then Dr. F. G. Balch has operated five times and I myself six times. X-rays seem to have been of no value.’ This is

83 first mention of skin grafting the treatment of wounds/keloids. There is also mention of photograph of keloid, which the Author could not find. Sequeira in 1909 described Effects of

X-rays on Scar-Keloid: ‘The patient, a boy aged 6, had been attending the Orthopaedic

Department at the London Hospital under Mr. Openshaw, and was sent by him to Dr.

Sequeira for treatment of extensive keloid of the scar of a burn of the face. The burn occurred two years ago and took four months to heal. The chin and both cheeks in their lower parts were the seat of extensive scars with great thickening. At Mr. Openshaw's request only one half was treated by the X-rays. The parts were exposed at intervals of a fortnight to pastille doses of the rays. Each part has had from eight to ten pastille doses. There has been no inflammatory reaction whatever, and the areas are now quite soft and supple and show a marked contrast to the similar lesions on the untreated side. Dr. Sequeira remarked on the better results obtained by the X-rays than by the injection of thiosinamine and fibrolysin, which he had tried in similar cases.’ This article shows radiotherapy in a child which is effective, but with complications. This article also shows intralesional treatment of keloid for the first time.

Adamson in 1910 published an article titled Multiple Keloid associated with Neurotic

Excoriations of the Dug-out Type of Colcott Fox, in Proceedings of the Royal Society of

Medicine. Little in 1910 also published a case on Acne Vulgaris with Keloids.

MacLeod in 1911 presented a case of Keloids forming on the Cicatrices resulting from Ulcerating Syphilitic Lesions In the discussion of this case differentiation was made between Keloids and Hypertrophic Scars: ‘The President (Dr. T. Colcott Fox) said it was a nice point in the case as to whether the condition was true keloid or hypertrophic scar-tissue.

In his own experience the multiple hypertrophic scars of a generalized syphilide tended to

84 resolve spontaneously, and were not true keloids. Dr. Pernet said he regarded the case as one of hypertrophic scarring, not as true keloid. He had now seen a number of cases of hypertrophied scarring in syphilis. It was the result of secondary staphylococcal infection.

Mr. McDonagh said he showed a case before the Section with keloids all over the body, many of which had disappeared spontaneously. Dr. Graham Little recalled a case shown by

Mr. Arthur Shillitoe to the Dermatological Society of Great Britain in 1903, in which the patient, a man of middle age, had developed hundreds of apparently keloid growths on the site of rupial sores. The tumour was the size of a Barcelona nut. Dr. Little had obtained a section from one of these, and found histologically the appearances characteristic of scar- keloid, namely, thinned epidermis, obliteration of the glands and hair-follicles, and immediately below the epidermis masses of fibrous tissue arranged in bundles parallel with the line of the epidermis. Nevertheless, this case had also spontaneously recovered, the tumours all disappearing some months later.’ McGavin in 1911 presented a patient of injury of fifth and sixth cervical nerve roots, with Keloid formation in scars.

MacLeod in 1912 published an article about Keloid formation following ear-piercing.

In this article tendency of keloid formation was mentioned: ‘The patient had shown no tendency to keloidal formation previously.’

Adamson in 1913 called the acne keloid as Dermatitis Papillaris Capillitii (Kaposi).

Nixon in 1914 presented a case at Royal society of Medicine titled, Case for

Diagnosis (? Sclerodermia and Keloid) present at the back of a nurse. Majority of participants called it artefaction.

Dore in 1918 presented a case of keloid following dog bite on face in a child. In this

85 article radium is mentioned as treatment of keloid. A differentiation is also made between keloid and hypertrophic scar. He told: ‘My own feeling was that radium should be applied at once, but Dr. Whitfield is of the opinion that cases of this type, which he regards as one of hypertrophic scar tissue, and not true keloid, improve spontaneously, and should not be treated for several months.’ Dore again remarked in another case in same year (1918): ‘With regard to treatment, I suggest radium, but it has just been suggested that the growth should be excised and microscoped. If it is a keloid, however, I doubt the advisability of excision, although I have heard it stated that if a keloid is sufficiently widely excised it does not recur.’

Little in 1918 published a case of Acne Keloid (now called Acne Keloidalis Nuchae).

Little in 1923 published a case of Keloid after burns. Taussig in 1923 published an article on The Treatment of Keloids with Radium, in California State Medical Journal.

Castle in 1926 presented a case of Hypertrophic Scar in a 6 months old child following impetigo.

Kisch in 1932 presented a case of post-aural scar, treated and with radium, and cured.

Hudson in 1934 presented a case of adjuvant radium needle therapy for keloid of face before Royal Society of Medicine. It was followed by a lively discussion on adjuvant radiotherapy. He told: ‘Skin flap from forehead inserted in area of keloid formation between eyelids and nose. Contraction occurred at the base of the skin flap brought down at the last operation, causing it to assume an almost cylindrical shape. Consequent on radium treatment by Dr. Fildes this contraction has receded and the interpolated skin has become correspondingly flattened out. Three radium treatments with 0.5 mm. platinum screenage

86 were given, using 5 mgm. 1 in. active needles, with five needles for 30 minutes and 40 minutes on November 27 and December 2, 1931 respectively, and with six needles for 45 minutes on February 2, 1932. The President said it was not very rare for burns to be followed by keloid formation between the side of the nose and the inner part of the lower lid. He would like to know whether in these cases it would be good treatment to give the keloid an exposure to radium, for weeks or months, before doing a plastic operation. His own view was that this might be desirable. Another point of interest in this case was, that on one, and one only, of the several occasions on which a skin graft was taken from the arm, repair of the denuded area was associated with keloid formation. He was unable to say whether the graft was cut with the same knife as was used on the face. Mr. John Foster said that the general practice in cosmetic surgery of this area would be to get rid of as much fibrous tissue as possible before grafting. This would indicate that radium should be employed before operation, rather than after. This, however, might cause difficulty in that the tissues were devitalized for six weeks after exposure to radium, and a graft would not take, as he had found to his cost in attempting to graft a raw area left after an evisceration of the orbit which had been exposed to radium to prevent recurrence of a sarcoma. It might, therefore, be well to wait a few weeks after the radium treatment before grafting in the President's case.’

MacCormac in 1934 presented a patient of Ulcerating Keloid in an Infant (Figure 33).

This was published along with photograph, which seems to be the first photograph of a

Keloid ever published. He wrote: ‘The edges of the ulcer are raised, hard and of a brown colour, resembling keloid tissue. A small typical keloid is present at the side of the main lesion. A microscopic section taken at the edge of the ulcer, shows an inflammatory cell reaction which is mainly subepidermal below which are dense bands of fibrous tissue, appearances consistent with a keloid, but inconsistent with a simple granuloma.’ With the

87 present state of knowledge, The Author doubts whether it should be called a Keloid (Nagi &

Babar, 2015).

McKenzie in 1935 presented a case of large keloids in mastoid scars.

Matthews in 1940 presented a case of Congenital Keloid on leg; it is now called congenital band. Smith in 1940 presented a case of Keloid of the Cornea.

Vargas in 1943 attempted to induce fibroids with oestrogen in female castrated

Rhesus monkey.

Levine in 1945 presented a case of folliculitis keloidalis (now called Acne Keloidalis

Nuchae). Marquez-Villegas in 1945 wrote on Keloids in Spanish in Anales Sociedad de

Biologia de Bogota (Annals of Society of Biology of Bogota) titled Algunas consideraciones sobre el tratamiento de los queloides (Some considerations on the treatment of keloids).

Lewis in 1946 presented a case of Keloid in syphilis lesion. Merino Eujercios in 1946 published an article in Spanish in Actas dermo-sifiliograficas (Proceedings of dermo- syphillography) titled Queloides primitivos de remision espontanea (Keloid early spontaneous remission). Olivier and Barasch in 1946 presented a case in French titled: Un cas de cheloides generalisees traite par parathyroidectomye (A case of generalized keloids treated by parathyroidectomy). Thomas in 1946 presented a case of multiple keloids following varicella. Weaver in 1946 presented a case of Keloid formation in both ear lobes, thereby signifying constitutional factors in Keloid formation.

Bloom in 1947 presented a case of multiple keloids in twin sisters, pointing to genetic

88 predisposition of Keloids. Lutz in 1947 published an article in German on Zur radiumbehandlung der narbenkeloide (On radium treatment of keloid-scars). Niedelman in

1947 published a review article on Keloids.

Jacobsson in 1948 published a 20 year review of treatment of keloids with radium.

Pfahler and Keefer in 1948 wrote on treatment of keloids by irradiation and electrosurgery.

Ricciardi in 1948 in Italian presented a case of Scleroedema of Buschke, and compared it with keloid. Tusler& Baur in 1948 wrote a review article on Keloids and hypertrophic scars.

Numis in 1949 published an article in Italian on Contributo allo studio della cheloidosi con ipercalcemia (Contribution to the study of keloids with hypercalcemia). He described hypercalcaemia as one of the aetiological factor for keloid formation.

Gougerot in 1950 in French presented a case of Foliaceus Keloid. Marshall and

Schadeberg in 1950 wrote a review on Keloids. Piera in 1950 in French reported a case of post-burn keloids treated by Charpy’s method. Pierre in 1950 described treatment of keloids by excision, skin grafting, and adjuvant radiotherapy in his article in French titled Cheloides etendues; traitement par excision et freffe suivies de radiotherapie (Extensive keloids; treatment by excision and graft followed by radiotherapy). Vilanova et al in 1950 for the first time used prophylactic radiotherapy after excision of Keloid, and found it of no benefit.

Report was published in Spanish as Fracaso de la irradiacion preventiva despues de la extirpacion quirurgica de un queloides (Failure of preventive irradiation following surgical excision of a keloid). Wallace in 1950 presented a case of acne keloid in a 27 years old girl, treated by surgery &radiotherapy. He used the term keloidal tendency in his paper. During discussion Dr. Hugh Gordon gave a concept which is now called Neoadjuvant Radiotherapy.

Braun-Falco and Weber in 1951 in their article in German titled Zur Lokalbehandlung von Keloiden mit Hyaluronidase (Local treatment of keloid with hyaluronidase) described treatment of keloids with local injection of hyaluronidase. Edgerton et al in 1951 described treatment of keloids with vitamin E for the first time. Ellerbroek in 1951 in German, described a case of Spontaneous Keloid (Spontankeloide).Glucksmann in 1951 wrote about local factors in development of keloids. Lewis in 1951 described keloid of conjunctiva. It was first time that keloid of mucosa was described. Marcenac in 1951 in French described Keloid in horse; first case of keloid in an animal, in an article titled Cheloides cicatricielles du cheval (Cicatricial keloids of the horse), published in Recueil de medecine veterinaire

(Veterinary medicine collection).Minissale in 1951 published his experience of parathyroidectomy for treatment of Keloids, in his paper in Italian titled Sui risultati della paratiroidectomia funzionale nel trattamento del cheloide (Results of functional parathyroidectomy in keloid therapy). Robinsonet al in 1951 published a report on use of tissue culture technique to study keloid pathology. Tamagawa et al from Japan in 1951 published a study of keloids caused by atomic bomb in Hiroshima.

Hofs in 1952 published an article in German titled Zur Frage der Keloidenststehung

(Pathogenesis of Keloids). This was first article ever published on pathogenesis of keloids.

Keining and Braun-Falco in 1952 in German wrote an article titled Vergleichende

Betrachtungen uber das Keloid, Sklerodem und myxodematose Hautveranderungen

(Comparison of the keloid, scleroderma, and myxoedematous skin lesions). Kunzfeld in 1952 published an article in German titled Uber Keloide nach Vakzination und Revakzination (On keloids after vaccination and revaccination), in which he described development of keloids after vaccination. Pankova in 1952 published an article in Russian titled Применение

подкожных вдувании кислорода при лечении келоидных рубцов (The use of subcutaneous injection of oxygen in the treatment of keloid scars).

Horton et al in 1953 reported a case of malignant change in keloids. Maggio in 1953 published an article in Italian titled Il cortisone nella cura dei cheloidi (Cortisone in the treatment of keloids), in which he described for the first time treatment of keloids with local steroids. Robinson &Hamilton in 1953 studied role of heparin in fibroproliferative disorders.

They found enhanced heparin tolerance in keloid patients. This was the first time that molecular basis of keloid formation was described. Spina et al in 1953 from Brazil published a review article in Portuguese titled Tratamento do queloide (Treatment of Keloid).

Mancini in 1954 from Argentina published an article in Spanish titled Inhibicion de la fibroplasia del queloide por accion local de hidrocortisona (Inhibition of the fibroplasia of keloid by local action of hydrocortisone). Pedoya in 1954 from France presented a case of spontaneous keloids (Un cas de cheloides spontanees). Vasileva from Russia in 1954 wrote an article titled К вопросу о лечении келоидных рубцов сверхмягкими рентгеновыми

лучами (On the question of the treatment of keloid scars with superficial X-rays). Whitehill in 1954 proposed prophylactic measures in keloid prone patients undergoing surgery.

Asboe-Hansen in 1955 in Danish wrote an article titled Behandling af keloider med lokale injektioner af hydrocortison-acetate (Treatment of keloids with local injections of hydrocortisone acetate). Hu et al in 1955 in Chinese wrote an article in Chinese Medical

Journal titled 對瘢痕疙瘩與舊中國的偏方治療的初步報告 (Preliminary report on the treatment of keloids with old Chinese remedies), in which they used traditional Chinese remedies for treatment of keloids. Keller in 1955 in German published a case report on local

91 treatment of keloid with heparin titled Kasuistischer Beitrag zur lokalen Keloidbehandlung mit Heparin. Kuiter et al in 1955 presented two case reports of treatment of keloids with ultrasonic energy. Lambranzi in Italian in 1955 published an article on use of combination therapy of X-rays and radio waves in treatment of burn keloids (La Marconi e la roentgenterapia associate nel trattamento dei cheloidi da ustione).

Bloom in 1956 published a review article on hereditary nature of keloids, and also described a family with keloids in five generations. Lagrot et al from Africa in 1956 wrote a review article on treatment of Keloids. This was first article on keloids from Africa; the

Homeland of Keloids. Luikart et al in 1956 published an article on use of dermabrasion for treatment of keloids for the first time. But they made a mistake about pathology of keloid, and called it an idiosyncratic response. Malbec and Quaife in 1956 in Spanish tried to give a classification of keloids in their article titled Algunas consideraciones de orden medico- quirurgico relacionadas con las cicatrices queloideas, fibroqueloideas y fibrohialinas; ensayo de clasificacion (Some medico-surgical considerations related to keloid scars, fibrokeloids and fibrohyalins; classification attempt). Meyer in 1956 in German warned cosmetic surgeons to be aware of tendency to keloid formation in certain patients, which could ruin outcome of cosmetic operations. Title of his article was Neigung zur keloidbildung und kosmetische operationen (Tendency to keloid formation and cosmetic operations). Miceli and Moronic in 1956, in Italian, published a trial of low voltage x-rays treatment for keloids, under title Considerazioni sui risultati della plesioroentgenterapia dei cheloidi

(Considerations on the results of radiotherapy for keloids).

Humby in 1957 from West Indies wrote a review article on keloids, and correlated leiomyoma of uterus with keloids. Kollner and Stein in 1957 in German reported successful

92 treatment of keloids with thiosinamine in their article titled Erfolgreiche Behandlung der

Narbenkeloide mit Thiosinamin (Successful treatment of keloid-scars with Thiosinamine).

Zimmer in 1957 from France described use of paper chromatography in diagnosis of keloid in his article titled Essais comparatifs de chromatographie sur papier du derme normal et du tissu cheloidien (Comparative assay of normal skin and keloid tissue with paper chromatography).In another article titled L' eponge cheloidienne du point de vue physique

(Keloid from physical point of view), Zimmer described physical characteristics of keloids.

Hummel in 1958 in German described histochemistry technique to study pathogenesis of keloid in his article titled Beitrag zur genese des keloids im hinblick auf seine histochemie

(Contribution to the genesis of keloids in terms of its histochemistry). Kelly and Pinkus in

1958 described use of oral quinones for keloid treatment. Lucchelli and Maderna in 1958 in

Italian wrote an article on perineo-vaginal keloids titled Considerazioni a proposito di tre osservazioni di manifestazioni cheloidee perineo-vaginali (Considerations about observations of three perineo-vaginal manifestations of keloid).

Arnold and Grauer in 1959, in German, in their article titled Keloide: Ätiologie und

Management durch Ausschneiden und intensive prophylaktische strahlung (Keloids: aetiology, and management by excision and intensive prophylactic radiation), described management of keloid by surgery followed by prophylactic full-dose radiotherapy. Jakob and

Balz in 1959 in French described treatment of keloids with radioactive strontium and yttrium in their article titled A propos du traitement des angiomes et des cheloides avec le radiostrontium et le radio-yttrium (About the treatment of haemangiomas and keloids with Radio-

Strontium and Radio-Yttrium). Leyh et al in 1959, in German, in his article titled Keloide entwicklung in hauttransplantation nach der Reverdin verfahren (Keloid development in skin

93 transplantation according to the Reverdin method) described keloid development in Reverdin skin graft (pinch graft). Noble in 1959 described keloid of fundus of uterus. Shell and Inmon in 1959 described use of cortisone ointment to prevent keloid formation. Velazco and

Rosenthal in 1959 described use of intralesional dexamethasone for treatment of keloids.

Lisbonne in 1960, in French, in his article titled Sur la méthode de Gate sur le traitement et la prévention des chéloïdes (On Gate's method on the treatment and prevention of keloids), described Gate’s method for treatment and prevention of keloid.

Humphries in 1961 from Africa presented a case of post-operative intestinal obstruction in a patient with keloid scars. He correlated intraperitoneal fibrosis with keloids.

Michel et al in 1961 in French in two journals reported simultaneously (now it is called plagiarism) use of Vitamin B, Vitamin C, and Iron for treatment of keloids in 20 cases.

Onizuka in 1961 in Japanese published a case report titled 術後のケロイド形成に対する放

射線の著しい抑制効果を示した興味深い事例 (An interesting case showing remarkable preventing effect of radiation on the postoperative keloid formation) showing prevention of keloid formation by post-operative radiotherapy in a keloid prone patient. This report was published in a journal titled 日本医学歩車線学会雑誌(Japan radiological society magazine). This was first article on Keloids from Japan. Rutenberg and Bookman in 1961 reported a case of keloid formation at site of insulin injections. Sherik in 1961 published an article about keloid of vocal cords in an article published in Czech titled Klinicke aspekty a lecba papilom hrtanu, zejmena u dospelych (Clinical aspects and treatment of laryngeal papilloma, particularly in adult). Szava et al in 1961 published an article in Hungarian titled

Kísérleti vizsgálatok kapcsán változások reaktív kapacitás a kötőszövet után égések: Adatok a problémát a fejlődés egy tendencia, hogy keloids (Experimental studies in connection with

94 changes in reactive capacity of the connective tissue after burns: Data on the problem of the development of a tendency to keloids). In this they reported animal studies of burns leading to pathological changes in connective tissue. Vilcek and Babor in 1961 in Czech reported on use of phototherapy with ultraviolet rays for treatment of keloid.

Mancini and Quaife in 1962 in French reported that they produced keloids in experimentally in human volunteers. Meyer and Lepinay in 1962 described role of physiotherapy in treatment of keloids. Moschella in 1962 described tendency of keloid formation in cousins, suggesting genetic basis. Plaschkes and Kaplan in 1962 in Hebrew described keloid formation after BCG vaccination in an article titled

BCG (Keloids in the acromial region following BCGקלואידיםבאזורבאנטומיההבאחיסון vaccination). Pruemers in 1962 in German described keloid of common bile duct causing stricture. Wernsdoefer in 1962 in German described use of hyaluronoglucosaminidase in treatment of keloids.

Amante et al in 1963 in Italian described histochemistry of keloid collagen using chromatography. Bairti and Depetris in 1963 in Italian described ultrastructure of keloid collagen using electron microscopy and crystallography. Kuhn in 1963 described use of steroid-vitamin A-oestrogen lotion for keloids in women. Mienicki and Kossakowska in 1963 in French described use of vitamin K in treatment of keloids. Montgomery & Portnoy;

Murray; and Thivolet & Pellerat, all in 1963, simultaneously described, for the first time, use of Triamcinolone Acetonide injection for treatment of keloids. This injection is still the most popular treatment even after lapse of 52 years. Schirren et al in 1963 in German described treatment with histaminase as prophylaxis against keloid formation in burn patients.

Viglioglia in 1963 in Spanish from Argentina described keloid treatment with betamethasone

95 injection. He used the term intralesional for the first time.

Julesz et al in 1964 from Hungary studied effects of thyrotropic hormone on granulation tissue and keloid formation. Kelly in 1964 reported effects of quinones on polyvinyl sponge implanted in albino rat. Koonin et al in 1964 published a review on aetiology of keloid. Rasmussen et al in 1964 studied isotonic and isometric heat contraction of patient’s dermis in relation to scleroderma and keloid. Saipt in 1964 in German described treatment of keloids with haparinoids. Thorek and Pandit in 1964 from Canada studied role of proteolytic enzyme in wound care leading to decrease in incidence of keloid formation.

Wechsler in 1964 in French published his experience of 40 cases of keloids.

Sibeleva et al in 1965 in Russian published article on biochemistry of keloid titled

Мукополисахариды и коллаген в келоидах человека (Mucopolysaccharides and collagen in human keloids). Wilson et al in 1965 presented his series of 500 cases in which he injected dexamethasone in post-operative wounds as prophylaxis against keloid formation.

Engel in 1966 described use of dimethyl sulfoxide in treatment of keloids. Lintilhac et al in 1966 described role of prolonged elastic compression in prevention of retractile and hypertrophic scars. Nicoletis in 1966 in French described use of aminocaproates in treatment of keloids. Parsons in 1966 presented a case of keloid of penis. Sandberg and Sandberg in

1966 from Sweden studied role of chorionic gonadotropin in collagen formation during wound healing. Sirsat and Sampat in 1966 from India published an article on keloids in India.

Bureau et al in 1967 in French described treatment of keloids with chymotrypsin.

Ketchum et al in 1967 performed animal studies to see effect of steroids on mature collagen.

Konuralp et al in 1967 in Turkish published a review on Keloids in Tip Fak Mecm journal.

Nicoletis et al in 1967 described interstitial radiotherapy with iridium after excision of keloid.

Opolski in 1967 in Polish presented a case of keloid of nose treated with excision and full thickness skin grafting in his article titled Chirurgiczne leczenie bliznowców nosa z przeszczepem skóry pełnej grubości (Surgical treatment of keloid of the nose with a full thickness skin graft). Plaksin in 1967 in Russian described development of keloids on face in victims of napalm bomb. Schmid in 1967 in German described treatment of keloids with

Volon A tincture (salicylic acid & triamcinolone acetonide). Sugawara & Hashimoto in 1967 in Japanese described hollowing-out method for treatment of keloids; this method is now called intralesional excision. Swartz in 1967 described correlation between keloid formation and idiopathic gingival hyperplasia. Vallis in 1967 used a mechanical injecting device

(Dermo-Jet) for intralesional injections in keloids.

Geominne in 1968 from Italy described a syndrome of torticollis, keloids, undescended testes, and kidney dysplasia. He thought it X-linked inheritance. Pfeiffer in

1968 in German described use of glycerol and urea in treatment of keloids. Schumann in

1968 in German (from East Germany) described radiotherapy of keloids in children.

Hakanson et al in 1969 from Switzerland described presence of histamine in mast cell in keloids. Kadanoff in 1969 in German studied pathophysiology of re-innervation in keloids.

Merkureva et al in 1969 in Russian studied hexosamine and protein-associated hexose contents of keloids. Schoppa in 1969 in German described use of allantoin and heparin for prevention of keloids in childhood burns. Serena in 1969 in Italian described homotransplant of meshed skin from mother to child for prevention of burn keloid in children. Wrong in 1969 from Canada proposed that all keloids should be treated, and preferred treatment was

97 intralesional triamcinolone acetonide.

Bazin et al in 1970 in French compared biochemistry of collagen, mucopolysaccharides, and water in skin, scar and keloid. Pavlova and Bolkhovitinova in

1970 in Russian described treatment of keloids with pyrogens. Picaurd in 1970 in French described suture-less skin closure with adhesive strips as prophylaxis against development of keloids. Ratzer in 1970 described flurandrenolone tape (steroid impregnated polyethylene tape) as occlusive dressing for treatment of keloids.

Hoopes et al in 1971 studied presence of different enzymes in keloids. Kolokolova et al in 1971 in Russian, reported use of procaine block to treat pruritus of keloids. Mate et al in

1971 in German reported use of tissue adhesives instead of sutures for wound closure in order to prevent keloid formation. Matheis in 1971 in German from Switzerland reported keloid as complication of chickenpox scar. Psillakis et al in 1971 studied electrolyte (calcium, copper) content of keloids. Shetlar et al in 1971 studied glycoproteins contents of hypertrophic scars.

Szabo et al in 1971 in German reported explanation of human keloid tissue in in chick embryo. They also described use of Vitamin C in treatment of keloids.

Balaba in 1972 in Russian reported estimation of neuraminic acid in keloid and in body fluids of keloid patients. Izadpanah in 1972 in German reported that open-air management of burns and scalds reduced incidence of keloids. Jaworski in 1972 in Polish described effect of corticoids on keloid fibroblasts. Linares et al in 1972 described electron microscopic features of keloids. Nylen and Wahlin in 1972, from Saigon (South Vietnam), in

Swedish described development (and treatment) of keloids in children following American bombing in an article titled Erfarenheter fran sjukhus i Saigon for piediatrisk rekonstruktiv

98 kirurgi (Observations from a Saigon hospital for reconstructive paediatric surgery).

Achten et al in1973 in French described use of bufexamac for treatment of keloids.

Ashley in 1973 reported skin atrophy following treatment of hypertrophic scar with cortisone injection. Kapulan in 1973 in Russian described use of bee venom for treatment of keloids.

Lapiere in 1973 described molecular biology of keloids. Leroy in 1973 in Rumanian described use of benzene derivatives as tissue adhesives for prophylaxis against keloids, in his article titled Utilizarea benzilor adezive la inchiderea plagilor (The use of benzene adhesives in the closure of wounds). Martinez et al in 1973 in French from Canada reported a meningeal keloid following discectomy (They also published same report in another journal one year later; now-a-days it is called self-plagiarism). Milsom and Craig in 1973 studied metabolism of collagen in keloid fibroblasts. Reuter in 1973 from East Germany reported treatment of keloids with lemon juice. Sharapova and Khamaganova in 1973 in Russian reported use of intralesional injection of chloroquine in treatment of keloid. Vogt in 1973 in

German reported formation of keloid after electro-epilation.

British Medical Journal in 1974 published an editorial on Keloids and x-rays. Crocket in 1974 wrote a beautiful article on scars, including keloids, in the same journal. Inalsingh in

1974 from Johns Hopkins Hospital published his experience of 501 cases of keloid; the biggest series published till that time. Kik et al in 1974 in Polish published a report on side effects of radiotherapy of keloids in children; before that radiotherapy was used indiscriminately for treatment of keloids in children. Kischer in 1974 performed scanning and transmission electron microscopy of fibroblast from normal skin and keloids. He, along with

Shetlar, in the same year, also studied histochemistry of normal skin and keloids. Mitinskaia and Kushnikova in 1974 in Russian reported keloid formation in adolescents re-vaccinated by

99 intravenous BCG. Moyanhan in 1974 reported use of penicillamine in treatment of keloids in children. Muhlbauer in 1974 in German reported use of magnetics to prevent keloid formation. Oluwasanmi in 1974 from Ibadan, Nigeria reported his 10 years’ experience about keloids. He suggested damaged collagen an allergen, and keloid as immune reaction to it. It is now known that this so-called allergen is actually cytokine(s).Pierce in 1974 described treatment of keloids with cryosurgery, for the first time. Polednak in 1974 noted that connective tissue of Negroes had different response to pathological challenges as compared to whites, thereby suggesting racial aetiology of keloids. Ramkrishnan et al in 1974 from

India presented their series of 1000 cases of keloids. Snyder in 1974 described button compression therapy for keloids of ear-lobule. WHO in 1974 (Weis) published classification of soft (mesenchymal) tissues, in which Keloids & Hypertrophic Scars were included in

Tumour-like lesions.

Bailey et al in 1975 studied characteristics of collagen of normal and hypertrophic scars. They maintained that hypertrophic scar collagen was like embryonic collagen. Baur et al in 1975 observed presence of myofibroblasts in hypertrophic scars, and called them contractile fibroblasts. Bazin et al in 1975 produced an animal model in rats for this purpose; it was the first animal model of wound healing and hypertrophic scars. Kik et al in 1975 in

Polish reported analysis of side effects of radiotherapy for keloids. Kischer et al in 1975 studied mechanism of action of compression therapy using electronic microscopy. Lennihan and MacKereth in 1975 reported decreased incidence of keloid formation in wounds closed by metallic staples as compared to nylon sutures. Motegi et al in 1975 from Japan emphasized mucosal cleavage lines similar to skin cleavage lines, and advised that incision in mucosa should also be placed parallel to these lines in order to reduce incidence of hypertrophic scar formation. Moustafa et al in 1975 presented a keloid case in pregnant lady,

100 suggesting role of oestrogens in keloid formation. Raskin in 1975 suggested possible association of osteopoikilosis with keloid.

Abdel-Fattah in 1976 reported local necrosis and Cushing syndrome in a keloid case treated with intralesional triamcinolone acetonide. Botella et al in 1976 in Spanish reported development of keloid in two cases of hydroa vacciniforme, a rare skin disease. Gay et al in

1976 in German described polymorphism of collagen in different tissues. Hassenstein and

Nusslin in 1976 in German emphasized protection of gonads while irradiating for keloids.

Kemble and Brown in 1976 reported that activities of enzymes like lactate dehydrogenase were increased in hypertrophic scars but there was no difference in alkaline phosphatase.

Levy et al in 1976 reported that post-operative low dose superficial x-irradiation after keloid excision prevented recurrence. Longacre et al in 1976 noted changes in biochemistry of scar after Z-plasty without excision. Malaker et al in1976 reported 30 cases of keloid treated with excision and post-operative irradiation delivered to sutured edge of scar by an iridium wire.

Mostafa and Abdel-Fattah in 1976 recommended shaving and skin grafting for hypertrophic scars after burns. Oluwasanmi et al in 1976 studied effect of immune response noncollagen synthesis around implanted polyvinyl sponges in rats. Russel and Witt in 1976 reported no differences in appearance between fibroblasts of skin, scar, or keloid.

Kiil et al in 1977 from Sweden did a trial comparing results of triamcinolone alone versus excision plus triamcinolone, and concluded that combined treatment gave no better results. Knapp et al in 1977 performed morphologic and biochemical analyses to differentiate skin and mature scars from keloids. Laurentaci and Dioguardi in 1977 studied histocompatibility antigens (HLA) in keloids and hypertrophic scars. Roenigk in 1977 recommended dermabrasion for keloids. Ruiz-Maldonaldo and Baker in 1977 in Spanish

101 from Mexico reported decreased incidence of keloid formation from BCG vaccination given in between spine and scapula. Russel et al in 1977 studied effect of histamine on growth of skin and keloid fibroblasts.

Gunter in 1978 from Canada proposed camouflage of keloids and hypertrophic scars in exposed areas. Kelly in 1978 noted: ‘Keloids are medically benign, but often psychologically and cosmetically malignant lesions.’ Kischer et al in 1978 performed mast cell study in granulation tissue, hypertrophic scars, pressure treated hypertrophic scars, and mature scars. They found that mast cell number was increased in hypertrophic scars, granulation tissue, and after pressure therapy. Onwukwe in 1978 presented his classification of keloids. He also reported a type of keloid which he called suppurative keloid. Peacock in

1978 proposed beta-aminopropionitrile and colchicine for control scar tissue. Sloan et al in

1978 measured partial pressures of oxygen and carbon dioxide in hypertrophic scars in burned patients, using mass spectroscopy. Tempongkol in 1978 in Thai published an article on radiotherapy of keloids titled การรักษาด้วยรังสีในการรักษาแผลเป็น (Radiation therapy in the treatment of keloids) in โชติเชียงใหม่มากกว่าบันไดแพด (Journal of the Medical

Association of Thailand). Weber et al in 1978 studied collagen polymorphism in keloids.

Babin and Ceilley in 1979 combined cryotherapy with intralesional triamcinolone, and reported several advantages of this. They called it freeze-injection method. Interestingly this was reported by them in two different journals simultaneously (now-a-days it is called self-plagiarism). Baur et al in 1979 reported presence of fibroclasts and myofibroclasts in keloids. They found them similar to osteoclasts. Bosse et al in 1979 reported use of oral

Madecassol (Asiaticoside), a glycoside of plant origin, for prevention and treatment of keloid.

Hansen in 1979 reported use of retinoic acid (tretinoin) for treatment of hypertrophic scars.

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Kischer and Shetlar in 1979 studied connective tissue repair after myocardial injury in dogs.

They also studied, in the same year, effect of pressure on microvasculature in hypertrophic scar. Kloti and Pochon in 1979 from Sweden reported usefulness of Jobst compression suits in prevention and treatment of post-burn keloids in children. Moriguchi and Fujimoto in 1979 from Japan studied crosslinks of collagen in hypertrophic scars. Yagi et al in 1979 suggested that immune reaction to sebum could cause keloids; they recommended desensitization.

James et al in 1980 reported a case of keloid comprising entirely of myofibroblasts, and no fibroblasts. Janssen de Limpens in 1980 reported treatment of keloids with 0.05% retinoic acid applied locally. He reported favourable result in 77-79% cases. Onwukwe in

1980 reported, for the first time, treatment of keloids with antineoplastics ie methotrexate.

Reilly et al in 1980 reported a case of intraoral keloid formed on forehead flap inserted in oral cavity for reconstruction after excision of squamous cell carcinoma. Sharma from Zambia reported a series of 57 cases of keloids treated by different methods. Weidauer in 1980 in

German reported tracheal stenosis after endotracheal intubation in patients of keloid tendency

Brody et al in 1981 presented an alternate theory of pathogenesis of hypertrophic scars and keloids. They proposed that mucopolysaccharides acted as glue locking buckled collagen in contracted position. They postulated a different role for myofibroblasts. Ehrilch and White in 1981 identified alpha A and alpha B collagens in keloids. Kerl et al in 1981 in

German reported treatment of keloids with calmurid, a combination of lactic acid and urea.

Kischer and Brody in 1981 studied ultra-structure of keloid collagen nodule. Kroner et al in

1981 in German reported multiple ischaemic infarcts in retina and uvea due to crystalline corticosteroid emboli after injections for keloids in face. This complication is probably due to subcutaneous rather than intralesional injection. Landthaler et al in 1981 in German reported,

103 for the first time, use of lasers in treatment of hypertrophic scars and keloids. They also proposed that low energy laser light had stimulating effects on wound healing. Mahboubi et al in 1981 reported keloid-like lesion of bowel wall in a post-nephrectomy case. Santa Cruz and Ulbright in 1981 reported accumulation of mucinous material in keloids treated with steroids. Serbrennikov in 1981 in Russian wrote on forensic examination of keloids. Topol et al in 1981recommended antihistamines to inhibit proliferation of fibroblasts of skin, scar, and keloid.

Janssen de Limpens and Cormane in 1982 in two simultaneous papers (plagiarism?) reported that keloid patients had antinuclear antibodies against fibroblasts, while patients with hypertrophic scars and healthy controls had no antinuclear antibodies. Mandl in 1982 described use microbial collagenase from Clostridium histolyticum for intralesional injection for keloid treatment. Russel et al in 1982 studied fibroblast heterogeneity in relation to steroid regulation of collagen metabolism. Shakespeare and Strange in 1982 reported that there was deficiency of fatty acids in epidermis over keloid, as compared to skin. Shen in 1982 reported gluteus maximus muscle contracture in 200 cases, treated by surgery, and followed by keloid formation in all cases. He suggested that this muscle contracture might be due to keloidal tendency in these patients. Shepherd and Dawber in 1982 managed 17 cases of keloids using cryosurgery, out of which only 2 responded. Sodenberg et al in 1982 from Sweden reported keloids and hypertrophic scar treatment with zinc tape. Valenta et al in 1982 reported a case of parathyroid adenoma as a complication of radiotherapy of keloid.

Aleksic et al in 1983 in Bosnian presented a case report titled Rana nekrektomija, homo i autotransplaticija koze kod duboke opekotine deteta od 19 meseci sa 45% opecene povrsine (Early necrectomy, homo- and autotransplantation of skin in a 19-month-old child

104 with deep burns over 45% of the body). In this they reported skin graft taken form child’s father, and there was no keloid formation. Kischer and Hendrix in 1983 studied fibronectin in keloids. Linares in 1983 reported enhanced collagen-proteoglycan interaction in hypertrophic scars in comparison with normal scars, normal skin, and granulation tissues. Minet et al in

1983 in French from Belgium suggested immunological basis of keloid formation. Muti and

Ponzio in 1983 in Italian and English emphasized the use of nitrogen protoxide cryotherapy for keloids. Interestingly they published English translation of this article simultaneously in another journal (self-plagiarism?).Ng et al in 1983 from Singapore reported their series of

280 patients treated with pressure garments. Redmond and Baker in 1983 reported a case of keloidal calcification. Richard and Boulnois in 1983 in Italian reported that Helio-Neon laser was best for keloids. Troielli in 1983 in Spanish from Portugal reported good therapeutic results of pressure-treated hypertrophic scars.

Ehrlich and Buttle in 1984 concluded from an ex-vivo study that epidermis somehow controlled production of hypertrophic scar collagenase. Grosser et al in 1984 in German reported treatment of hypertrophic scars and keloids with orgotein (superoxide dismutase).

James et al in 1984 reported Keloids in two cases of systemic sclerosis. Kischer in 1984 compared ultrastructure of keloids and hypertrophic scars. He found quiescent fibroblasts in keloids, and active in hypertrophic scars. Levi-Alfonso in 1984 from Cuba in Spanish reported keloid formation in children after facial injuries. Moulton-Levy in 1984 reported that keloids had a multicellular origin and that they did not develop clonally as did most neoplasms. Sela and Taicher in 1984 made ear clip prosthesis for pressure on earlobe keloids.

Berry et al in 1985 reported that skin oxygen tension was index of maturity of keloids treated with compression. Hurtado and Crowther in 1985 reported methyl methacrylate stent

105 for treatment of ear keloids. Kantor et al in 1985 reported treatment of ear keloids with CO2 laser. Katz et al in 1985 described objective method of measurement of hypertrophic burn scar using tonometry and ultrasonography. Kolchina and Shakhnes in 1985 in Russian reported use of dibunol (butylated hydroxytoluene) in treatment of keloid. Le Coultre and

Graber in 1985 used plastic face mask, and silicone gloves &stockings for treatment of keloids. Lenz in 1985 reported use of punctiform argon ion laser for treatment of keloids.

Quinn et al in 1985 for the first time reported use of silicone gel for keloid treatment.

Shafranov et al in 1985 in Russian reported use of microwaves for treatment of keloids.

Shakespeare and van Renterghem in 1985 observed structure of collagen in hypertrophic scars. Shetlar et al in 1985 reported, for the first time, animal model for study of keloid.

Cabbabe and Korock in 1986 studied role of vitamin C deficiency in wound healing in

Guinea pigs. They found that there was relationship between vitamin C deficiency and hypertrophic scar formation. Hagerty and Zubowitz in 1986 reported use of tissue expander in treatment of hypertrophic scars. Hosoda et al in 1986 used laser Doppler flowmetery for diagnosis of hypertrophic scars. Hulsbergen Henning et al in 1986 reported treatment of keloids with argon laser. They reported disappointing results. Mikhailova et al in 1986 in

Russian reported use of microbial collagenase in treatment of keloids.

Chadzynska in 1987 reported treatment of keloid with allantoin, heparin and extractum cepae ointment. Robb et al in 1987 developed an animal model for study of keloid formation. However Shetlar, in his letter to the editor, asserted prior claim on this model

(Shetlar 1985). Smith et al in 1987 described role of mast cells information of keloids. Tsuji and Sawabe in 1987 reported presence of large number of elastic fibres in hypertrophic scars, while they were absent from normal dermis.

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Bartell et al in 1988 reported use of elastometer to measure elasticity of normal skin and keloid; elasticity of keloid was less than normal skin. Bilbey et al in 1988 reported mesothelioma of pleura and carcinoma of breast 23 years after radiation therapy for treatment of a chest wall keloid. Eddy et al in 1988 proved that myofibroblasts were of non-muscle origin, and therefore had no role in scar contraction. Engrav et al in 1988 compared of intramarginal and extramarginal excision of keloid. They concluded that intramarginal excision was better than extramarginal excision. Golladay in 1988 reported use of single intraoperative perilesional pre-excision injection of betamethasone sodium phosphate and betamethasone acetate suspension for keloids. Jemec in 1988 reported perilymphatic subcutaneous fat atrophy due to intralesional steroid injection. Jimbow et al in 1988 reported keloidal lesions with an unusual accumulation of type IV collagen in progeria. Kucharz in

1988 from Romania reviewed role of glucocorticoids in collagen metabolism. Larrabee et al in 1988 studied role of myofibroblasts in scar contraction. They reported that myofibroblasts in granulation tissue had a lot of features of smooth muscle cells, and might be the cause of wound contraction. This report is in contradiction to Eddy et al (1988), who proved that myofibroblasts were not responsible for wound contraction. Matsouka et al in 1988 reported that myofibroblasts were present in granulation tissue, but not in keloids, which only contained fibroblasts. Russell et al in 1988 reported similarities between keloid fibroblasts and foetus fibroblasts. Sherman and Rosenfeld in 1988 reported use of Nd:YAG laser in management of keloids.

Berman and Duncan in 1989, for the first time, used an Interferon, Alfa-2b, for treatment of keloids. Castro et al in 1989 studied photodynamic therapy with Nd:YAG laser using fibroblast culture sensitized to Q-switch II dye. Edwards et al in 1989 reported use of

A-scan ultrasound for assessment of thickness, regularity, density, and other features of

107 keloids. Kreig and Heckmann in 1989 from Italy described, for the first time, role of cytokines, through stimulation of fibroblasts, in formation of keloids. Ow in 1989 from

Australia reported keloid of floor of mouth, and its association with keloid formation in skin.

Sagher in 1989 recommended use of dental syringe for intralesional steroid injection in keloids and hypertrophic scars. Waseda in 1989 reported use of Tranilast, an antiallergic drug having anti-proliferative properties, in treatment of keloids. Wester et al in 1989 in Danish mentioned use of prostaglandin inhibitors in treatment of keloids

Berman and Duncan in 1990 reported that Pentoxifylline, an analogue of methylxanthine theobromide inhibited growth of keloid fibroblasts. Datubo-Brown in 1990 in a review article reported use of systemic chemotherapy in treatment protocol of keloids.

Granstein et al in 1990, for the first time reported effect of Interferons alpha, beta, and gamma on collagen synthesis. They wrote that IFNs alpha, beta, and gamma reduced collagen production by skin fibroblasts. IFN gamma also inhibited collagen production by myofibroblasts and synovial fibroblast-like cells. Granstein et al also performed first trial of use of Interferon Gamma for keloids. Patients were treated with 0.01 or 0.1 mg of recombinant human IFNG injection in one lesion and placebo in other lesion, thrice weekly for 3 weeks. Three days after last injection, biopsies were done on experimental and control lesions. Six out of eight patients who completed treatment course showed decrease in height at treated site, with mean decrease of 30.4% in treated lesion versus 1.1% in control lesion.

Keloids treated with Interferon Gamma showed changes in both epidermis and dermis.

Epidermis developed thinning of suprapapillary plates, compact hyperkeratosis, focal or diffuse parakeratosis, exocytosis of lymphocytes, and enhanced amount of mucin. Dermis had a decreased amount of thicken collagen bundles and active fibroblasts and enhanced number of inflammatory cells and amount of mucin. They recommended that Interferon

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Gamma was feasible in treatment of Keloids. Ikard and Wahl in 1990 reported a case of umbilical stump keloid in a neonate. Larrabee Jr et al (1990) studied efficacy and toxicity of intralesional Interferon Gamma injection in management of Keloids & Hypertrophic Scars in

10 patients. All lesions reduced in width and height. Five of 10 keloids reduced by 50% in width. They recommended that IFNG should be given intralesionally once weekly, upto 0.05 mg per injection, for 10 weeks. There were no serious side effects. Most common side effect was minor headache. Lee and Ping in 1990 studied use of calcium antagonists to retard extracellular matrix production. Pinol et al in 1990 in Spanish from Portugal proposed use of minoxidil in keloids, as it inhibited fibroblasts. Stuttgen et al in 1990 from Germany compared topical heparin with mucopolysaccharide polysulfuric acid ester (Hirudoid). Teepe et al in 1990 described, for the first time, cultured autologous epidermis for treatment of major burns. They reported decreased incidence of keloid formation with it.

Busch et al in 1991 treated vaginal keloids (formed after reconstruction for agenesis with intra-cavity radiotherapy. Kozlov et al in 1991 in Russian reported excretion of oxyproline, a metabolite of collagen in cases of keloids and hypertrophic scars. They wrote that formation of postburn pathological scars caused rise in excretion of collagen degradation products; rate of increase depended on area and age of scar. Lee et al in 1991 studied estimation of mRNA level of keloids using in-situ hybridization. LeFlore and Antonie in

1991 reported a case of keloid of palm. Peltonen et al in 1991 studied co-localization of collagen type I & VI and TGF beta 1 mRNA. They noted, for the first time, that TGF beta 1 was responsible for fibrosis. Perez-Aebej et al in 1991 in Spanish proposed that in cases of bladder neck stenosis after prostatectomy, surgery appeared to be the triggering mechanism in these patients who were likely to be predisposed to developing this condition.

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Babu et al in 1992 reported altered response to TGF-beta 1 by keloid fibroblasts.

Hambleton et al in 1992 wrote about observing progress of keloid using ultrasound for measurement of thickness. McCauley et al in 1992 reported altered cytokines production by keloid patients. Suzawa et al in 1992 from Japan studied mechanism of action of tranilast in keloids. Suzawa et al published four articles on this in 1992.

Bleacher et al in 1993 compared foetal tissue repair with adult wound healing, and role of TGF beta. Castgnoli et al in 1993 reported that TNF alpha might be significant in normal wound healing, and keloid formation might be a result of a low level of TNF alpha.

Garner et al in 1993 studied dissimilarities in cytokine sensitivity of keloid and skin fibroblasts. They concluded that formation of collagen, and not non-collagenous proteins, was enhanced in keloid as compared to skin cells, both with and without stimulation by low dose TGF beta 1.’ Ghahary et al in 1993 reported increased expression of mRNA of TGF beta I and III procollagen in keloids. Ghahary et al in the same year, reported in another article, of overexpression of fibronectin messenger RNA in keloids. Hughes and Buckley in

1993 reported that in contrast to Keloids, desmoid tumours, and fibromatosis, there was abundance of macrophages in retroperitoneal fibrosis. Ntiri in 1993 from Somalia reported vaginal keloids after ritual female circumcision. Sahara et al in 1993 reported decrease of in vitro keloid fibroblast contraction when treated with interferon alfa-2b. Shafikov et al in 1993 in Russian reported use of Xymedone, a pyrimidine derivative for prevention of keloids in burn patients. Takahashi et al in 1993 in Japanese reported calcification of heart and tricuspid valve 23 years after radiotherapy for chest wall keloid. Tan et al in 1993 reported role of fibroblast growth factor (FGF) in regulation of various extracellular matrix proteins.

Astler in 1994, for the first time, reported treatment of keloids by pulsed dye laser.

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Ehlrich et al in 1994 described morphological and immunochemical dissimilarities of keloids versus hypertrophic scars. Fedorova et al in 1994, in Russian reported use of acoustic apparatus, which measures propagation rates of superficial acoustic waves in skin, to study mechanical parameters of scars. Lee K. S. et al in 1994 from Ireland studied effect of radiotherapy on extracellular matrix gene expression of keloid fibroblasts. Lee R. C. et al in

1994 used intralesional verapamil in post-burn scar patients and found it effective.

McGrouther in 1994 suggested not to differentiate between hypertrophic scars and keloids, and to invest our time and resources in research on their treatment. Messadi et al in 1994 examined TGF beta-1 synthesis by normal and fibrotic cutaneous tissues fibroblasts. Paradisi et al in 1994 reported spontaneous keloids in a seven years old boy suffering from Dubowitz syndrome. Pittet et al (1994) investigated therapeutic effects of intralesional IFN gamma on human hypertrophic scars in four patients. IFN gamma decreased symptoms and size of lesions; immunofluorescence study revealed that alpha-SMA level was reduced in myofibroblasts. Further, fibroblast culture showed that IFN gamma reduced replication and expression of alpha-SMA. They suggested that IFN gamma could be a useful treatment of hypertrophic scars. Resnik and Capland in 1994 reported a case of keloids formed due to lightning strike. Schmitt-Gräff in 1994 wrote that fibroblasts were undifferentiated cells and could take any phenotype based on local needs and stimuli. Schwartz et al in 1994 reported keloids in Kaposi’s sarcoma lesions. Scot et al in 1994 reported linear keloids due to anabolic androgenic steroids abuse in athletes. Shigematsu in 1994 form Netherlands reported that magnesium lithospermate extracted from Chinese medicinal herb Salviae Miltorrhizae Radix inhibited collagen synthesis by skin fibroblasts without affecting DNA or noncollagen protein synthesis. Stark et al in 1994 in German reported treatment of keloids with intralesional

Lipotalon, a microsomal corticoid. Younani et al in 1994 studied changes in collagen synthesis by TGF beta in keloid fibroblasts.

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Garner et al in 1995 studied scar contraction by using keloid and skin fibroblast- populated collagen lattices. Ghahary et al in 1995 reported that IFN γ & IFN α 2b regulated level of collagenase and TIMP-1 mRNA in keloid & skin fibroblasts differently. Ghahary et al in the same year studied immunolocalization of TGF-beta 1 in keloids and skin. Harrop et al in 1995 investigated action of IFN gamma on cell growth, collagen production, and expression of types I & III procollagen mRNA in postburn keloid fibroblasts. They reported

55 and 36% reductions (P < 0.05 for each) in type I & III procollagen mRNA expression respectively, after treatment for 12 hours with IFN-gamma (1000 u/ml). Kikuchi et al (1995) studied actions of many growth factors on [3H]thymidine incorporation and procollagen type

I carboxyterminal propeptide (P1CP) formation, which reflected type I collagen metabolism, in Keloid and normal fibroblasts. Six fibroblast cell strains, which were taken from Keloid or normal skin, showed same growth responses to PDGF, TGF Beta 1, IFN Gamma, and histamine. In contrast, Keloid fibroblasts revealed markedly increased growth response to

EGF than normal fibroblasts. P1CP production was 4.4 times higher in six strains of Keloid fibroblasts than in six controls. Treatment with IFNG (100 U/ml) decreased P1CP production in both groups; effect was significantly greater in Keloid fibroblasts. TGFB1 treatment upregulated P1CP production in both groups. They concluded that Interferon Gamma decreased production of type 1 collagen, more so in Keloids. Reiffel in 1995 reported prevention of keloids by long-term paper tape application. Russel et al in 1995 studied steroid regulation of elastin synthesis in fibroblasts.

Arakawa et al in 1996 reported decreased collagenase gene expression in keloid fibroblasts. Berman and Bieley in 1996 in a review article reported a recurrence rate, with surgery alone for keloids, ranging from 45% to 100%; this statistic is still found in majority of the textbooks even after 20 years. Broker et al (1996) treated keloids in nine patients with

112 excision followed by Interferon Gamma or placebo injections. Three weeks after excision, each patient received 10 weekly injections of Interferon Gamma in one wound, and normal saline placebo in other. Out of nine, patients, only three completed complete course of 10 injections. Seven patients were available evaluation at 12 weeks, and only four at one year.

Statistical test was not done due to low number, but three out of four patients felt that they had significant recurrence. Dolynchuk in 1996 proposed use of Putrescine

(tetramethylenediamine) for treatment of hypertrophic scars. Requena et al in 1996 reported two cases of nodular keloids on face, with no history of trauma. Histology showed basaloid cells of basal cell carcinoma, interspersed with keloidal collagen bundles. They proposed the name keloidal basal cell carcinoma for it. Ricketts et al in 1996 reported use of non-silicone hydrogel dressing for keloids. Snyder et al in 1996 reported association of keloids with hypertension. Wolfort et al in 1996 proposed treatment of keloids with antibody-targeted photolysis of myofibroblasts.

Berman and Flores in 1997 reported therapeutic advantage of postoperative interferon alpha 2b over triamcinolone acetonide. Giele et al in 1997 developed a technique of measurement of intradermal pressure created by pressure garments, and reported it to be 9 to

90 mmHg. Koch et al in 1997 described role of serum-free keloid fibroblast culture in research on keloids. Mann et al in 1997 studied scar/garment interface pressure of custom- fitted pressure garments, and found it quite variable; it was less than 22 mm Hg at soft areas, and more than 28 mm Hg at hard areas. Morris et al in 1997, for the first time developed rabbit ear hypertrophic scar model. Sheridan et al in 1997 used pulsed dye laser as prophylaxis against hypertrophic scar formation in burn cases.

Garner et al in 1998 reported that epidermis regulated dermal fibroblast activity, and

113 lack of epidermis led to excess collagen production. Giele et al in 1998 studied variations in pressure produced by pressure garments in different regions of body. Hu et al in 1998 suggested topical tamoxifen for treatment of excessive scarring. Krötzsch-Gómez in 1998 reported different cytokines expression in hypertrophic scars. Ladin et al in 1998 studied p53 and apoptosis changes in keloids lesions as well as keloid fibroblasts. They found that Keloid lesions fibroblasts had low apoptosis as compared to controls, and Keloid fibroblasts showed increased apoptosis when treated with steroids, IFN gamma, and hypoxia. Liu et al in 1998 in

Chinese reported inhibitory effect of tetrandrine, on growth of human skin fibroblast. Schmid et al in 1998 reported increased level of TGF beta I & II receptors in granulation tissue & keloid. Shetlar et al in 1998 reported treatment of keloid implants with Pirfenidone.

Bláha et al in 1999 reported use of citalopram for prophylaxis of hypertrophic scars in burn patients. Cen and Yan in 1999 studied effect of IFN gamma on fibroblasts of keloids.

They reported in Chinese that apoptotic ratio was 17.7% in IFN gamma treated group, and

10.9% in control group; difference was statistically significant. Costa et al in 1999 studied mechanism of action of pressure for keloid treatment. They concluded that pressure accelerated remodelling phase of repair. Lee et al in 1999 studied level of TGF beta 1, 2, and

3 in keloids. Ono in 1999 from Sweden reported use of electric syringe pump for intralesional injection of triamcinolone acetonide. Sandler in 1999 reported a case of plantar keloid.

Venugopal et al in 1999 reported that chlorphenaramine maleate decreased proliferation rate and DNA synthesis in fibroblasts. Wan and Evans in 1999 reported increase in free radicals in hypertrophic scars. Wang et al in 1999 reported that addition of TGF beta2 resulted in enhanced collagen production in keloids, and anti-TGF beta2 antibody reduced collagen production. Wu et al in 1999 reported that intralesional collagenase caused collagen degradation in explanted hypertrophic scars.

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Figure 24:B; Ambroise Pare's Ten Books of Surgery (1564 AD [Bib]) showing first illustration of Suturing a Wound 115

Figure 25:B; J. Scultetus' Book Armamentarium Chirurgicum (1655 AD [Bib]) showing Curved Needle 116

Figure 26:B; Benjamin Bell’s Book A System of Surgery (1791 AD [Bib]) showing straight and curved Needles for suturing Wounds 117

Figure 27:B; Retz’ Book Second Edition (1786 AD [Bib])

118

Figure 28:B; Alibert’s Book First Edition (1806 AD [Bib])

119

Figure 29:B; Alibert Book Second Edition (1825 AD [Bib]) 120

Figure 30:B; Alibert Book Third Edition (1833 AD [Bib]) 121

Figure 31: First Drawing of Keloid in second Edition of Alibert’s Book (1825 AD [Bib])

122

Figure 32:B; Gordon (slave), also called Whipped Peter, photographed with Keloids from being whipped (McPherson and Oliver, 1863 AD [Bib]) 123

Figure 33:B; Ulcerated Keloid case presented by MacCormac (1934)

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18. Present Era

The period from 2000 to 2015 is included in the Present Era. Feng et al in 2000 reported that TNF-alpha had different biological effects on fibroblasts derived from hypertrophic scar or normal dermis; it decreased cytoactivity of fibroblasts of hypertrophic scar, and increased that of fibroblasts of skin. Lu et al in 2000 analyzed protein p53 expression and Fas gene mutation in Keloids. Qi et al in 2000 reported positive effects of

Asiaticoside on hypertrophic scars, and suggested it to be related to its inhibition of fibroblast proliferation. Tian et al in 2000 reported that TGF beta 3 had action opposite to that of TGF beta1 and 2 ie it helps maturation of scars. They suggested that TGF beta 3 would be beneficial for prevention of keloids. Tredget et al (2000), using three sets of keloid and normal fibroblasts from same patients, treated nonconfluent and near confluent fibroblasts with TGF beta, and cell growth and collagen synthesis were measured using cell counting

18 and O2 isotopic uptake into hydroxyproline before analysis by gas chromatography-mass spectrometry. Keloid and normal fibroblasts were treated with IFN alpha 2b or IFN gamma or both for 96 hours. Quantitative RT-PCR and Northern blot analysis were done using internal standards for human TGF beta 1. They concluded that TGF-beta stimulated both keloid and normal fibroblast growth. Collagen production was higher in keloid than in normal fibroblasts and was maximally increased at 75 pM TGF-beta. TGF beta stimulated collagen production was blocked by IFN alpha or IFN gamma or both in an additive fashion. They supported use of IFN alpha and IFN gamma for treatment of keloids. Tsou et al in 2000 used cDNA microarrays technique to analyze keloid and normal scar gene expression. Wu et al in

2000 studied level of mRNA of TGF-beta and TIMP-1 in keloid. They concluded: ‘The average integral optical density of mRNA for TGF-beta and TIMP-1 increased by 8 and 7 folds (P < 0.001) related to normal skin and the expression of them showed highly positive

125 correlation.’ Zhang et al in 2000 in Chinese reported that hypertrophic scar and keloid could be differentiated by intracellular actin, F actin and F/G ratio; they were increased in hypertrophic scars than in keloids.

Espana et al in 2001 from Spain described use of intralesional Bleomycin for management of keloids. Gulec in 2001 reported a case of Noonan syndrome with tendency for keloid formation. Humbert et al in 2001 in French presented a case of Hoigne's syndrome

(pseudo-anaphylactic reaction) with intralesional triamcinolone acetonide injection. Kim et al in 2001reported elevated level of gli-1 oncogene in keloids, and recommended topical use of rapamycin analogues, including tacrolimus, for keloids. Kuhn et al in 2001 proved in an in vitro study that silicone sheet acted by decreasing fibrogenic cytokines like TGF beta2. Li et al in 2001 described production of rabbit ear hypertrophic scar model by making round and rectangular wounds. Lim et al in 2001 from Singapore investigated effect of keloid keratinocytes on fibroblast proliferation. Liu et al in 2001 in Chinese reported high level of

Bcl-2, but normal level of Fas gene in keloids. Renò et al in 2001 reported that mechanism of action of mechanical compression on keloids might be due to prostaglandin E2 release.

Tyring in 2001 from Canada suggested use of topical Imiquimod, an immune response modifier, for prevention and treatment of keloids. Uppal et al in 2001 reported use of 5- fluorouracil for wound irrigation after extralesional excision of keloids. Yavuzer et al in 2001 reported plantar keloid in a case of Proteus syndrome.

Aksoy et al in 2002 produced hypertrophic scar animal model in guinea pigs by skin excision and coal tar application. Mikaelian and Gross in 2002 described Keloidal fibromas and fibrosarcomas in dog. There is controversy whether these lesions should be called keloids. International Advisory Panel on Scar Management in 2002 made proposals for

126 evidence based treatment of keloids (Mustoe et al, 2002) (Figures 34-36).

Atiyeh et al in 2003 reported significantly superior scar quality in wound treated with

Moist Exposed Burn Ointment (MEBO). Ha et al in 2003 from China reported gene therapy for keloids. They used HGF facilitated with recombinant adenovirus vector. Hasegawa et al in 2003 reported that Interferon gamma did not inhibit TGF beta induced fibrosis in hypertrophic scar fibroblasts. Hyakusoku and Ogawa in 2003 from Japan described what they called small-wave incision, a modified form of intralesional W-plasty for treatment of long linear keloids. Jacob et al in 2003 reported that 5% imiquimod applied topically to keloids altered apoptosis genes. Leake et al in 2003 reported that urokinase-mediated plasminogen activation system was involved in spread of keloids beyond wound margins due to degradation of extracellular matrix. Luo et al in 2003 in Chinese reported apoptosis of keloid- derived fibroblasts induced by Fas gene transfection. Ma et al in 2003 studied gene transfer in human keloid using adeno-associated virus vector. Ogawa et al in 2003 studied relapse rates in keloids managed by surgery and adjuvant electron-beam radiotherapy. Paddock et al in

2003 analyzed gene expression patterns in postburn keloids. Renò et al in 2003 reported that mechanical compression induced apoptosis in hypertrophic scar. Sharquie and Al-Dhalimi in

2003 from Iraq reported epidemiology of Keloids in war-torn Iraq. Simman et al in 2003 showed that Mitomycin C slowed proliferation of Keloid fibroblasts.

Lee et al in 2004 gave histological differentiation of keloid and hypertrophic scar. Liu et al in 2004 suggested targeting TGF beta, using gene therapy, for treatment of keloids. Lu et al in 2004 in Chinese described level of TGFB and its receptors in keloids. Marneros et al in

2004 performed genome scans in two different keloid-prone families, and found keloid predisposition loci on chromosomes 2q23 and 7p11. Matthews et al in 2004 reported that

127 osteoarthritis induced by cruciate ligament transection in animal models produced a scar-like mass on medial side of knee joint, which might be used as model of hypertrophic scars.

Moulin et al in 2004 compared Fas antibody induced apoptosis of skin and hypertrophic scar myofibroblasts. Na et al in 2004 investigated dissimilarities of gene expression between keloids and contiguous skin, and concluded that nine genes, especially NNP-1, contributed to keloid production. Roseborough et al in 2004 complained about confusing terminology of scars. They wrote; ‘advances in scar management have been hampered by the confusing or ambiguous terminology. There is no consensus on what amount of post-traumatic skin scar formation is normal and what should be considered hypertrophic. In the World Health

Organization's ICD-9, there is no diagnostic code for hypertrophic scar--only keloid is listed.

Yet, the medical and scientific literature distinguishes them as different conditions.’ Zhang et al in 2004 studied mechanism of action of Interferon gamma. They concluded: ‘IFN-gamma transiently increased proliferation and collagen synthesis of hypertrophic scar fibroblasts and normal skin fibroblasts by activation of protein kinase C and subsequently inhibited proliferation and collagen synthesis by activation of protein kinase A.’

Derderian et al in 2005 studied effect of mechanical strain on gene expression in a hypertrophic scarring model. Hochman et al in 2005 from Brazil reported Keloid heterograft in hamster cheek pouch as a new animal model. Kal and Veen in 2005 performed meta- analysis of biologically effective doses (BED) for adjuvant radiation in prophylaxis of keloids. Meshkinpor et al in 2005 treated hypertrophic scars and keloids with radiofrequency waves, but found no clinical improvement. Miller and Nanchahal in 2005 from New Zealand suggested enhancing relative ratio of TGF beta-3 to TGF beta-1 &TG F beta-2 to decrease scar formation. Phan et al in 2005 reported that Smad3 signalling played a significant role in keloid formation through epithelial-mesenchymal interactions, and suggested inhibition of

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Smad signalling for keloid treatment. Wang D. et al in 2005 in Chinese reported formation of recombinant Smad 7 adenoviral vector. Wang H. et al in 2005 in Chinese reported research on assembling animal models of keloid employing the method of tissue engineering.

Cubison et al in 2006 studied correlation between healing time and formation of keloids in children due to scalds. Iannello et al in 2006 reported use of angiotensin converting enzyme inhibitor enalapril for treatment of keloid. Kang et al in 2006 reported that management of keloids using intralesional collagenase was not effective. Leppert et al in

2006 studied molecular similarities between fibroids and keloids. Murakami et al in 2006 suggested that there was a close relationship between subcutaneous ischaemia and hypertrophic scar formation. Naeini et al in 2006 reported Bleomycin tattooing in management of large keloids. Neiburger in 2006 reported a case of tongue keloid secondary to piercing. Onuigbo in 2006 reported a case of squamous cell carcinoma developed within long standing keloids due to burns (without irradiation). Song et al in 2006 established immortal lymphoblastoid cell bank of keloids pedigree.

Hosalkar et al in 2007 reported increased incidence of keloids in patients of multiple hereditary exostoses. Hosnuter et al in 2007reported that onion extract gel improved hypertrophic scars and keloids, especially scar colour. Li (or Lu) and Gao in 2007 in Chinese reported gene therapy of keloid in athymic nude mice by recombinant adenovirus coding for

Fas gene. Louw in 2007 proposed use of fatty acids in prevention and management of keloids. Luo et al in 2007 did genomic and proteomic profiling to compare gene expression in keloids, surgical scars, and leiomyomas. Ogawa et al in 2007 demonstrated that dose of postoperative radiotherapy for keloids should be customized by site, form 10 to 20 Gy.

Oittinen in 2007 reported multiple nonsyndromic spontaneous keloids in a patient suffering

129 from allergic diseases. Ozdol et al in 2007 reported increased incidence of coronary stent stenosis in in patients with keloids and hypertrophic scars. Shivaswamy and Thappa in 2007 reported a case of extensive keloids due to cyclosporin treatment in a pemphigus vulgaris case. Wang et al in 2007 in Chinese, studied expression of periostin in keloids, and its relation to TGF-beta1 and its receptors. Yang et al in 2007 reported development of a new hypertrophic scar model by transplanting full thickness human skin on backs of nude mice.

Burd in 2008 in an editorial titled, So what is a keloid scar? questioned attitude toward keloids. He wrote that doctors had individual concepts about keloid, but it was not considered a well-defined category. Kose and Waseem in 2008, in an article titled Keloids and hypertrophic scars: are they two different sides of the same coin? observed that keloids and hypertrophic scars were two separate phases of one process. Occleston et al in 2008 reported prevention of keloid by TGF beta 3. Rei Ogawa in a letter to editor in 2008 described Keloids a serious disease like cancer. Parikh et al in 2008 reported treatment of keloids with suture banding. Ramos et al in 2008 in an article titled, Is there an ideal animal model to study hypertrophic scarring? described five different types of animal models. They stressed need of research for an ideal animal model. Zhibo & Miaobo in 2008 suggested use of botulinum toxin A in management of keloid. Zhu et al in 2008 suggested need of research for correlation between anatomy of rabbit ear and creation of hypertrophic scar.

Ardekani et al in 2009 reported treatment of keloids with topical captopril. Capon et al in 2009 reported laser assisted skin healing (LASH) for treatment of keloids. Ferguson et al in 2009 compared TGFB3 with placebo for the treatment of scars, and found it significantly better. Lau et al in 2009, for the first time, explored use of stem cells in wound repair and keloid prevention. Liu et al (2009) studied effects of IFN Gamma on TGF Beta/Smad

130 pathway in Keloid fibroblasts, and its mechanism of action in treatment of pathological scar.

Fibroblasts from Keloid tissue of 3 patients were separated and cultured in vitro. They were divided into control group, TGF Beta group, Interferon IFN group, and TGF Beta+IFN

Gamma group. Expression of mRNA and protein of CTGF, and alpha SMA protein were detected by different techniques. They reported that Interferon Gamma could down-regulate level of Smad 3, and up-regulate level of Smad 7 in a time- and dose-dependent manner, and reduce level of CTGF and alpha SMA in basic state or stimulated by TGF Beta. Mroweitz and Seifert in 2009 reported use of recombinant TGF-B3, interleukin 10, and imatinib mesylate for treatment of keloids. Ogawa et al in 2009 performed a literature search about risk of malignancy due to radiotherapy of keloids. In another article, Ogawa et al presented differential diagnosis of diseases resembling keloids.

Akaishi et al in 2010 reported male to female ratio of keloid as roughly 1:2. Campbell et al in 2010 studied effects of aminolevulinic acid/methyl aminolevulinic acid photodynamic therapy on keloids. Ma et al in 2010 reported reconstruction of large hypertrophic scar on thigh with liposuction. Mao et al in 2010 reported preparation of various sizes of ethosomes encapsulating 5-FU using nanotechnology, and their permeability in keloids. They concluded:

‘three different sizes of 5-FU ethosomes were prepared successfully, the ethosomes of 65 nm in diameter with 5-FU can penetrate scar highly efficiently, which has potential in application such as anti-scar drug carriers in scar therapy in near future.’ Scuderi et al in 2010 studied efficacy of topical cyanoacrylates in treatment of hypertrophic scars. They concluded that cyanoacrylates had positive effect on hypertrophic scars.

Hunter in 2011 reported treatment of keloid with acupuncture. Ogawa et al in 2011 suggested clinical uses of basic studies which showed that decreasing skin tension might

131 prevent and manage keloids. They highlighted tension reduction sutures for treatment of keloid. Ogawa in another article also stressed importance of mechanobiology of scarring.

Han et al in 2012 reported about anti-motility signalling mechanism of TGFβ3 which controlled cell traffic during wound healing. Lee et al in 2012 reported that Relaxin- expressing adenovirus decreased collagen formation and up-regulated MMP level in keloid fibroblasts. Zhang et al in 2012 performed a study of ethosome penetration in normal skin and keloid. They established that ethosomes were highly efficient carriers in hypertrophic scars.

Babalola et al in 2013 described role of microRNA in skin fibrosis. They concluded that miRNA-based therapies were important and might change management of keloids.

Mohammadzadeh in 2013 recommended Nano silver for keloid therapy.

Huang et al in 2014 studied the question: Are keloid and hypertrophic scar different forms of the same disorder? They concluded that hypertrophic scar and keloid could be thought as stages of the same fibrotic disease, with different grades of severity. Huang and

Ogawa in 2014 studied connection among hypertension and keloid formation. They proposed hypertension as an aggravating influence for keloids. Mseddi et al in 2014 reported treatment of keloids with 40% topical phenol. Sun et al in 2014 from Taiwan reported that incidence of keloid in ethnic Chinese population was 0.15%, male to female ratio 1:1.33, and women with fibroid of uterus had a 2.25 fold risk of keloids.

Abdel-Meguid et al in 2015 compared intralesional and contact cryosurgery for keloids, and found intralesional cryosurgery superior regarding efficacy and safety. Arima et al in 2015 studied correlation of hypertension and keloid. Sako and Worwick in 2015 reported extensive keloids in a patient suffering from pemphigus vulgaris. van Leeuwen et al in 2015 did a meta-analysis of intralesional cryosurgery for Keloids. Zhang et al in 2015 studied role of adipose stem cells in reducing scarring in rabbit ear

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Figure 34:B; Scar Classification by Mustoe et al (2002) 133

Figure 35:B; Scar Prevention Algorithm by Mustoe et al (2002) 134

Figure 36:B; Scar Treatment Algorithm by Mustoe et al (2002) 135

19. Future Era

Future Era is 2016 onwards. New research is going on about aetiology, pathogenesis, prevention, and treatment of Keloids. Future research on Keloids revolves around following:

1. Mechanisms of Wound Healing

2. Genomic Profiling of Keloids

3. Cytokines & Anti-Cytokines Therapy

4. Growth Factors & Anti-Growth Factors Therapy

5. Gene Therapy

6. Stem Cell Therapy

1. Mechanisms of Wound Healing: Interaction of cells and extracellular matrix through

adhesive molecules and participation of cytokines in repair are main topics of research for

future. Role of cytokines, growth factors & their soluble receptors are under investigation

in order to change healing process. Contrarily, fibrotic process is being prevented by use

of antibodies, antagonists or proteins targeting the cytokines. Antisense nucleotides which

bind cell DNA or mRNA are also under investigation as blockers. These methods, and

other new cellular and molecular methods, are helpful in making quick advancement in

studying mechanisms of such processes. Better knowledge of mechanisms of start,

progress and end of tissue repair will lead us to novel treatments of fibroproliferative

disorders. Research in these areas has significant clinical consequences. But many

problems require solution prior to use of growth factor, and growth factor inhibition in

routine treatment. Such problems comprise safety, dose, regimens and delivery. Besides,

main focus of investigation is on solitary agents. It is possible that addition or subtraction

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of multiple growth factors, or other mediators like protease inhibitors, may prove better in

certain situations. In spite of these limitations, control of normal and pathological wound

repair using growth factor treatment has much role in future, and will lead to new

treatment protocols (Mutsaers et al, 1997) (Figure 37).

Cytokines and growth factors are main signalling agents in tissue repair, but there is

very little data available regarding mechanisms of their own regulation. Trauma causes

interruption of microcirculation, followed by change in local milieu due to inflammation.

Hence primary signals for growth factor/cytokine secretion are from inside wound’s

extracellular milieu. Tissue hypoxia is thought the chief signal as it provokes VEGF, and

collagen gene expression which are required for wound repair. Increased lactate level

may produce same effects. In contrast, angiogenesis and collagen formation are decreased

by hypoxia and their formation increases when level of oxygen rises. But lactate increases

in wounds irrespective of oxygen level, and it also increases VEGF formation and

collagen secretion. Continuous rise in lactate level along with normal wound oxygen

levels enhances level of cytokines taking part in wound repair mechanisms like

angiogenesis and collagen formation (Trabold et al, 2003) (Figure 38).

2. Genomic Profiling of Keloids: Main gene alterations responsible for keloid formation

have not been identified so far. Lu et al (2000) analyzed protein p53 expression and Fas

gene mutation in Keloids. Tsou et al (2000) used cDNA microarrays technique to analyze

keloid and normal scar gene expression. Kim et al (2001) reported elevated level of gli-1

oncogene in keloids. Liu et al (2001) reported high level of Bcl-2, but normal level of Fas

gene in keloids. Paddock et al (2003) analyzed gene expression patterns in postburn

keloids. Marneros et al in 2004 performed genome scans in two different keloid-prone

137 families, and found keloid predisposition loci on chromosomes 2q23 and 7p11. Derderian et al (2005) studied effect of mechanical strain on gene expression in a hypertrophic scarring model. Luo et al (2007) did genomic and proteomic profiling to compare gene expression in keloids, surgical scars, leiomyomas, and peritoneal adhesions, all fibrotic conditions, using microarray and PCR. Analysis showed 3 to 12% of genes as differentially expressed in these tissues at p> 0.005. Among such genes, 400 were differentially expressed in leiomyomas in comparison to keloids/surgical scars, and 85 in comparison to peritoneal adhesions. Functional analysis showed that most of these genes are regulators of cell growth, tissue turnover, transcription factors and signal transduction.

Wang et al in 2007 studied expression of periostin in keloids, and its relation to TGF- beta1 and its receptors. They reported increased periostin's expression in keloids.

Periostin is a cicatrix specific gene, which performs a significant role in the formation of keloids, which is related to TGF-beta1 closely.

In a research, Affymetrix-based microarray was done on RNA of normal scars and keloids fibroblasts cultured with and without hydrocortisone. It was noted that 500 genes out of 38,000 present on Affymetrix chip were differentially regulated. Most interesting of these was rise in level of many IGF-binding and IGF-binding-related proteins and reduced level of Wnt blockers and IL-1-inducible genes. Enhanced level of CTGF and

IGF binding protein-3 was seen only in keloid fibroblasts treated with hydrocortisone.

This study confirms that there are many fibrosis-related paths in formation of hypertrophic scars (Smith et al, 2008) (Figure 39).

Shih et al in 2010 studied keloid gene bio-marking. Keloid margins and normal skin were harvested from keloid cases. RNA was extracted from these as well as second-

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passage cultures. Reverse-transcriptase quantitative PCR was used to see gene expression.

Ten genes were upregulated in keloid margin samples. Top-5 showed variations 10 - 175

times, containing aggrecan, asporin, inhibin beta A, TNF-α inducible protein 6, and

chromosome 5 open reading frame 13 (Figure 40).

Jones et al in 2015 studied methylation profile of keloid genome. Genome profiling

was done using InfiniumHumanMethylation450 BeadChip to investigate DNA from six

keloids and six uninjured skin specimen from 12 volunteers. Genes highly differently

methylated amongst keloid and uninjured skin were detected by 3-tier method. Of 685

differently methylated CpGs at Tier 3, 510 were hypomethylated and 175 were

hypermethylated, with 190 CpGs in promoter and 495 in nonpromoter regions in keloids

in comparison to uninjured skin. The 190 promoter region CpGs matched to 152 genes:

96 (63%) were hypomethylated and 56 (37%) hypermethylated. This study of keloid

methylome signifies a preponderance of hypomethylated genome, favouring nonpromoter

regions. DNA methylation, a supplementary method for gene regulation in keloid

formation, can lead to new therapies to reverse deleterious epigenetic alterations.

Cytokines & anti-cytokines, growth factors & anti-growth factors, gene therapy, and stem cell therapy will be discussed in Treatment sections.

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Figure 37: Cell and ECM interactions. Binding of growth factors or adhesion molecules to matrix or cell receptors initiates cell-signalling mechanisms (Mustsaers et al 1997) 140

Figure 38:B; Lactate concentration in wound fluids. A stable increase in lactate concentration causes increased collagen deposition (Trabold et al, 2003) 141

Figure 39: RNA Microarray on fibroblasts of keloids and normal scars with & without Hydrocortisone (HC), showing multiple fibrosis-related pathways (Smith et al, 2008) 142

Figure 40:B; Flowchart of steps & findings of Keloid gene biomarking done on biopsies from Keloid margins and Normal skin (Shih et al, 2010) 143

20. Anatomy of Keloids & Hypertrophic Scars

Skin covers whole of external surface of body; internal surfaces of body are covered by mucosa, which is akin to skin. It is the biggest organ of body. It consists of 8% of whole body weight, and its weight in a normal adult is about 6 kg. Its surface area in a normal adult is 2 m2. Its average thickness is 2.5 mm (range 0.5–4.5 mm); out of this epidermis is 0.10-

0.15 mm, and rest is dermis. Thickness of skin depends upon its state of maturation, age of person and region of body. Thick skin is found at palm of hand, palmer surface of digits, and sole of foot; epidermis is mainly responsible for thickness of skin at these sites. Thin skin is found at face; skin around eyes is the thinnest. Skin forms a crossing point between body and environment, and is the main place of communication between them. It is an ever-changing entity, and continuously sheds its cells. It contains many specialized structures; hair, nails, sweat glands, and sebaceous glands are part of the skin (Standring, 2008 [Bib]).

Apart from body surface, skin covers external auditory meatus, outer surface of tympanic membrane, and vestibule of nose. It is continuous with mucosae of alimentary, respiratory, and urogenital tracts; there special muco-cutaneous junction skin is found. It is continuous with conjunctiva as well, at eyelid edges, and with lining of lachrymal canaliculi at lachrymal puncta (Sinnatamby, 2011 [Bib]).

Colour of skin depends upon the amount of blood in skin, thickness of outer layer of epidermis, and number and state of melanocytes producing melanin. Racial variances in skin colour are primarily because of variances in quantity, type, and dissemination of melanin.

Skin features are also affected by some other things, eg hair and glands. Health of man is mirrored by look of skin, and signs of many systemic diseases are found in skin (Figure 41).

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Figure 41: Anatomy of Skin (Google, 2015 [Bib])

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21. Histology

Epidermis: Epidermis consists of keratinized, stratified squamous epithelium, and its main cells are keratinocytes. Other cells include melanocytes, Langerhans cells, and lymphocytes.

Merkel cells, which are basically mechanoreceptors, are connected to nerve endings. Non- keratinocytes and Merkel cells appear as clear cells. Their cytoplasm lacks prominent filament bundles. Keratinocytes undergo continuous regeneration throughout life; basal layer regenerates, and replaces cells of superficial layers. Keratinocytes undergo continuous changes while moving from base to surface. Epidermis is divided into five layers: basal layer

(stratum basale), spinous or prickle cell layer (stratum spinosum), granular layer (stratum granulosum), clear layer (stratum lucidum) and cornified layer (stratum corneum). First three strata are metabolically alive, via which keratinocytes travel and differentiate further when they go to superficial two layers. Two superficial layers undergo keratinization. The epidermal appendages (hair, nails, sebaceous and sweat glands) are created by invagination of epidermis, which is called interfollicular epidermis (Standring, 2008 [Bib]; Young et al, 2013

[Bib]).

Dermis: It is also called Corium. Dermis is composed of irregular connective tissue. Its milieu comprises collagen & elastic fibres, alongwith amorphous ground substance comprising of glycoproteins, glycosaminoglycans, and water. It contains nerves, vessels, lymphatics, epidermal appendages, and cells. Dermis provides mechanical strength to skin due to its collagen and elastic fibres. Dermis is important for existence of epidermis, and signals are exchanged at border between them. Dermis is divided into two layers; superficial papillary layer and deep reticular layer: interface amongst them is not clear. Adult skin collagen is mostly of types I (80-85%), and III (15-20%). Coarse fibred type I is mainly

146 present in reticular layer, and fine type III is there in papillary layer. Elastic fibres are present amongst collagen bundles in the form of a network. Two main types of cell are present in dermis: permanent and migratory. Permanent cells are of organized structures like nerves, vessels and arrector pili muscles, and fibroblasts, which secrete ECM. The migratory cells arise from bone marrow and consist of macrophages, eosinophils, neutrophils, mast cells, T and B lymphocytes, and dendritic cells (Figure 42).

Hypodermis: It is also called superficial fascia or subcutaneous tissue. It consists of loose connective tissue, which joins with under surface of dermis. Histologically speaking, it is not a part of skin, but is anatomically intimately associated with it. Structure and function of skin relies a lot on hypodermis. It contains a lot of adipose tissue, particularly between dermis and muscles. It is responsible for enhanced mobility of skin, and adipose portion helps in heat insulation, shock absorption, and energy store, which is consumed during starvation.

Subcutaneous nerves, vessels, and lymphatics lie in hypodermis, and their major trunks lie deep, where fat is less.

Blood Supply: Blood supply of skin comes from three major systems: direct cutaneous, musculocutaneous, and fasciocutaneous. Direct cutaneous vessels come from main arteries and veins eg dorsalis pedis artery. Musculocutaneous perforators come from intramuscular vessels. Fasciocutaneous system comprises perforating branches from vessels lying under deep fascia. Direct cutaneous vessels, musculocutaneous perforators, and fasciocutaneous perforators take part in six anastomosing horizontal reticular plexi of arterioles, which in the end give blood supply to dermis. Three plexi are found in skin proper and provide blood to all components like sweat and sebaceous glands, and hair. They are subpapillary plexus, reticular dermal plexus, and deep dermal plexus. Remaining three plexi are subcutaneous

147 plexus, and plexuses on superficial and deep surfaces of deep fascia.

Lymphatic Drainage: Lymphatics collect interstitial fluid and macromolecules, and return them to veins. These carry lymphocytes, Langerhans cells, and macrophages to regional lymph nodes. They are blind at distal end, and lie deep to papillary dermis. They drain in superficial lymphatic plexus deep to subpapillary venous plexus, and then through collecting vessels into a deeper plexus at juncture of reticular dermis and hypodermis, which drains into larger subcutaneous channels.

Nerve Supply: Pacinian corpuscles transmit deep pressure and vibration sensation, and are found deep in corium or in subcutis. Meissner's corpuscles are present in papillary dermis, and are sensitive to touch. Main input is transmitted by neurons whose cells are present in spinal and cranial ganglia. Efferent autonomic fibres innervate arterioles, arrector pili muscles, and myoepithelial cells of sweat and sebaceous glands. In corium, nerve fasciculi divide into branches widely to make deep reticular plexus, which supplies most of corium, including hair follicles, sweat glands, and larger arterioles. A lot of minor fasciculi travel from this plexus to ramify in an additional superficial papillary plexus at juncture of reticular and papillary dermis (Young et al, 2013 [Bib]).

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Figure 42: Histology of Skin (Flores, 2015 [Bib])

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22. Embryology

Skin develops from ectoderm and mesenchyme deep to it. Surface ectoderm forms general surface epidermis, pilosebaceous units, sweat glands and nail units. Main descendants of ectodermal cells are keratinocytes. Melanocytes and Merkel cells develop from neural crest, and Langerhans cells from bone-marrow. Dermis is developed from mesenchyme, and from neural crest (in head). Angiogenic mesenchyme forms blood vessels of corium. Nerves and Schwann cells originate form neural crest, and then go into corium (Sadler, 2014 [Bib];

Standring, 2008 [Bib]) (Figure 43).

In the initial 4–5 weeks, embryo’s skin is a solitary layer of ectodermal cells, which lies over mesenchyme. This basal germinative layer later forms postnatal epidermis. A transient layer forms superficial to epidermis, called periderm. Periderm grows by mitosis of its own cells, free of germinative layer. Proliferation of germinative layer gives rise to stratified epithelium, forming contiguous layers of cells amid it and periderm. Pre- implantation keratins (K8 and K18) are substituted by keratinocyte basal cell keratins (K5 and K14), and then by high molecular weight keratins (K1 and K10) at 10 to 12 weeks. After this there is formation of profilaggrin and filaggrin, and later keratohyalin granules at about

20 weeks. First fully formed keratinocytes appear immediately after this. At 24 to 26 weeks a distinct keratinized layer appears at some sites, and at 30 weeks, interfollicular epidermis becomes similar to its postnatal form.

Embryonic dermis is more cellular than adult dermis. The mesenchymal cells are responsible for cellular signalling that controls ectodermal differentiation. Mesenchymal cells under ectoderm communicate with one another by thin elongations, thereby forming a

150 network. Later there is formation of different types of cells, including fibroblasts, endothelial cells, mast cells, and association of matrix constituents to form collagen fibres and elastic fibres. Collagens type I, III, V, and VI are spread evenly irrespective of foetal age. In some areas levels of type III and V are more than adult skin. Collagens type IV and VII are present mainly in BMZ. Further differentiation of corium separates it from hypodermis at 12 weeks.

There are also alterations in configuration and dimensions of collagen fibrils, as well as their formation of bundles, among which cell become less. Papillary and reticular layers are distinct at 14 weeks, but overall development of dermis continues even after birth.

Epidermal–dermal interactions at BMZ take place during foetal life, and even in whole life. At ectodermal stage, BMZ comprises of basal plasma membrane of an ectodermal cell; on its cytoplasmic side lie different cytoskeletal elements, and deep to it, a layer of microfibrillar-amorphous matter secreted by cell. At bilaminar stage, a distinct lamina densa is there, segregated from basal plasma membrane by a lamina lucida containing loose fibrillar matter. Alike strands travel from lamina densa to mesenchymal matrix (Sadler, 2014 [Bib];

Standring, 2008 [Bib]).

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Figure 43: Embryology of Skin: A; 5 weeks; B; 7 weeks; C; 4 months (Google, 2015 [Bib]) 152

23. Physiology of Keloids & Hypertrophic Scars

Skin comprises two major layers: epidermis performs barrier functions; and dermis gives tensile strength to skin, and maintains body temperature. Skin performs several important functions (Venus et al. 2010; Barrett et al, 2015 [Bib]; Hall, 2016 [Bib]):

1. Sensory Function: Skin contains approximately one million nerve fibres, mostly in face

and extremities. Sensations comprise pain, touch, temperature, pressure, vibration, and

itch. Many receptors are present in skin, which detect these stimuli. Meissner’s corpuscles

perceive light touch and vibration. Merkel cell receptors also sense light touch, and also

continued pressure. Pacinian corpuscles perceive sudden pressure and vibration, and

Ruffini receptors sense skin stretch. Pain receptors are free nerve endings, not true

receptors. They detect a number of stimuli simultaneously, like pain, heat, cold,

corrosives etc (Figure 44).

2. Thermoregulatory Function: Thermoregulatory mechanisms occurring in skin comprise

insulation, sweating, and blood flow regulation. Humans are insulated by hypodermal fat.

Sweat glands are stimulated to secrete sweat when body temperature is raised above

37˚C. Sweat then cools body by evaporation. Skin has a rich blood supply, which helps in

temperature control. When body temperature is high, body loses heat by enhancing blood

flow to skin. Heat is also lost from body by radiation, conduction, and convection. If body

becomes cold, heat loss is decreased by vasoconstriction, which decreases flow of warm

blood to limbs from body’s core.

3. Barrier Function: As physical barrier, skin protects body from mechanical stress due to

desmosomes in epidermis, and collagen, elastin, and reticulin in dermis. As radiological

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barrier, it protects body against ultraviolet rays, thanks to melanocytes, which synthesize

melanin. Melanin is inserted into melanosomes, which are transported to other epidermal

cells. In these cells, melanin is present at superficial to nucleus and it protects DNA from

ultraviolet light. Chemical barrier is created by fats in extracellular area in keratinized

layer. The lipid-rich matrix is arranged in a layered arrangement, which provides

waterproofing barrier. As biological barrier, acidic skin secretions inhibit colonization by

pathological microorganisms. Skin is the first line of defence against harmful

microorganisms. Skin has an acidic pH (4 to 5.6), which kills bacteria.

4. Fluid Balance Function: Dermis is very permeable to liquids, but epidermis is not. Outer

layer of epidermis is almost impermeable as keratinocytes form a scaffold-like

arrangement, held together by keratohyalin and involucrin.

5. Colouring Function: Melanocytes in skin release melanin, which gives it its colour.

Melanin are of three types: eumelanin, which is dark and brown; pheomelanin, found

mainly in females, in lips, nipples, vagina and red hair; and neuromelanin, produced by

catecholaminergic neurons in central nervous system. Eumelanin gives skin and hair their

colour. Pheomelanin causes red hair colour. Lesser level of eumelanin, with absence of

other pigments, gives rise to gray hair colour, while enhanced level of eumelanin, with

absence of other pigments gives rise to yellow hair colour. Skin colour is not produced by

melanin only; two other things are also responsible; yellow pigment (carotene) and blood

flow. In yellow races, there is less amount of melanin, which allows carotene colour to

become prominent. Skin colour is pale if blood flow is reduced, and blue if blood is

deoxygenated.

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6. Biomechanical Function: Skin has elasticity of solids and viscosity of fluids. Variation

in skin elasticity depends upon direction of stress. Skin reacts to stretch in a non-linear

way. If stress is plotted against strain, a non-linear graph is obtained. To start with, a

minor force causes a great increase in length, which is due to rearrangement of elastic

fibres of dermis. Later, a bigger force is needed to distort skin, which requires an

alteration in direction of collagen fibres and displacement of ECM. Once collagen fibres

reach their maximum length, force needed to cause further increase in length rises

sharply.

7. Absorptive Function: Skin is not entirely resistant to absorption. Many liquids can cross

skin barrier, at variable rates. Skin is not permeable to polar molecules like water,

sodium, potassium, but is permeable to alcohols. Further, areas of body are differently

permeable; face, forehead and dorsum of hand are very permeable, and palms of hand less

permeable. Many oil-based medicines are applied on skin, which act locally as well

absorbed systemically.

8. Synthetic Function: Vitamin D is synthesized by skin when ultraviolet radiation falls on

it. Skin also takes part in synthesis of cytokines and growth factors.

9. Waste Disposal Function: Skin helps getting rid of waste substances through sweat

glands, like salts and urea.

10. Psychological Function: Skin plays a significant role in mental health. Skin is very

visible and so has great cosmetic, aesthetic, and cultural importance.

11. Secretory Function: Glands of skin produce sweat and sebum (Barrett et al, 2015).

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Figure 44: Functions of different Constituents of Skin (Venus et al, 2010)

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24. Biochemistry

Skin ie both epidermis and dermis consists of many macro-molecules. Macro- molecules taking part in wound healing are discussed in section of Pathogenesis.

Biochemistry of other molecules ie Keratins, Melanins, and Biological Glues is discussed here. It is followed by discussion on carbohydrate, lipid, and protein metabolism in skin

(Rodwell, 2015 [Bib]).

1. Keratins: Outer two layers of the epidermis are formed from keratin cytoskeleton of dead

keratinocytes. Two primary groups of keratins are α-keratins and β-keratins. The α-

keratins are found in mammals while β-keratins are present in birds and reptiles. Both

forms are right handed helical structure. They are sub-divided into type I ie acidic

keratins, and type II ie basic keratins. Human skin contains about 20 genetically different

keratins. Two most abundant amino acids are glycine and alanine, while third is cysteine.

Long stretches of a-helix are attached to short non-helical segments. Contacts between

two a-helices are produced by amino acid side chain on one edge of each helix. Two

polypeptides form a dimeric coil. Protofilaments are produced from two antiparallel head-

to-tail associated coils. Protofilaments dimerize to form a protofibril, four of which form

a microfibril. Cysteine residues in alpha-keratin affect its macromolecular structure and

function. Intra- and inter-molecular hydrogen bonds and disulphide bridges occur on all

keratins. In cells, keratin type I forms pair with keratin type II. Different keratin types are

expressed in different cell types and different layers of epidermis: cytoskeleton of

epithelial cells contains K14 (type I) & K5 (type II); and K18 (type I) & K8 (type II).

Basal layer contains K13 (type I) & K4 (type II). Spinous and granular layers contain K10

(type I) & K1 (type II). Clear and cornified layers contain K3 (type I) & K12 (type II)

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(Figure 45).

2. Melanins: Melanins are polymorphous polymers of eumelanin, pheomelanin, mixed

melanins and neuromelanin. Melanocytes produce black-brown eumelanin and yellow-

reddish pheomelanin. Eumelanin is a polymer comprising of amino acid units in reduced

or oxidized states. Pheomelanin is mainly benzothiazine derivative. Neuromelanin is

produced in dopaminergic neurons. Melanin absorbs ultraviolet light at a wavelength of

280-320 nm. Both eumelanin and pheomelanin play important protective role in binding

to ions, drugs, and chemicals. Melanin is formed by catalysation of L‐phenylalanine and

L‐tyrosine to form levodopa using phenylalanine hydroxylase, tyrosinase and tyrosinase

hydroxylase 1. These pathways are then sub-divided into eumelanogenesis or

pheomelanogenesis. Melanosomes are elliptic membrane-bound organelles. Synthesis of

matrix proteins and tyrosinase occurs on rough endoplasmic reticulum. Fusion of

premelanosomes with tyrosinase vesicles gives rise to production of melanosome.

Melanosomes migrate into one of dendrites of melanocyte, which transfers it to a

neighbouring keratinocyte. Melanin granules collect above nuclei of keratinocytes and

absorb ultraviolet radiations before reached nucleus. Quick response of melanocyte-

keratinocyte complex occurs to many environmental stimuli (paracrine and/or autocrine),

ultraviolet radiation, melanocyte-stimulating hormone, growth factors, and cytokines.

3. Biological Glues: Barrier function of epidermis is dependent upon transglutaminase-

mediated cross-linking of proteins and lipids. These are called biological glues.

Transglutaminase reaction includes post-translation modification of proteins, creation of

covalent bond amongst a free NH3 group of protein or peptide bound lysine and g-

carboxamide group of protein or peptide-bound glutamine free amine group. Proteins then

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become resistant to mechanical disruption and proteolysis. Quality of stratum corneum

barrier depends on presence of cholesterol, fatty acids, and ceramides. Changes in

concentration of any of these affect barrier quality.

4. Skin Metabolism: Primary source for energy production in epidermis is glucose from

circulation, which diffuses into keratinocytes without effect of insulin. Large proportion

of glucose is catabolized to lactate (even in presence of oxygen). Citric acid cycle does

operate in epidermis, but this cycle is inefficient due to wide fluctuation of temperature

and blood flow in skin. Twenty percent of glucose is metabolized by pentose-phosphate

pathway with production of nicotinamide adenine dinucleotide phosphate (NADPH) and

pentose for both fatty acids synthesis and nucleic acids. Secondary source of energy is

fatty acids derived from both epidermal stores and exogenous sources (when glucose flow

is limited, then fatty acids are metabolized). Glycogens present in small amount under

physiological conditions; however, it is elevated in all types of injury to epidermis to

provide energy which is required for repair of skin wound. Glucose is a substrate also for

synthesis of lipids, polysaccharides, glycoproteins and nucleic acids. Glycosaminoglycans

and proteoglycans are highly charged molecules, and attract water, forming a gel like

substance. Lipid metabolism is also a source of energy for epidermis. Lipids are

components of membranes, are major constituents of permeability barriers, and also

supply energy. Synthesis of lipids takes place from glucose catabolism, amino acids and

circulating fatty acids. Lipogenesis is ongoing in all layers of epidermis. Catabolism is

generally by lipases in outer layers of epidermis. Proteins are only catabolized in extreme

starvation.

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Figure 45: Keratin molecular structure (Stanley, 2015 [Bib]) 160

25. Incidence& Prevalence of Keloids & Hypertrophic Scars

Keloids are found only in human beings (although some cases have been reported in horses and dogs). Keloids are found on average in 6% (range 4.5-16%) of world population.

(Figure 46). Keloids are more common in women than men; this may be an artefact, as women more often get earlobe piercing, the commonest cause and site of Keloid. Sun et al

(2014) reported that Incidence of keloid in ethnic Chinese population was 0.15%, male to female ratio 1:1.33, and women with fibroid of uterus had a 2.25 fold risk of Keloids.

Hypertrophic scars generally form within 1 month after injury. Risk of Hypertrophic

Scars is increased if epithelialization occurs in more than 21 days. Incidence of Hypertrophic

Scar is 40-70% after operations and 30-90% after burns (Niessen, 1999).Alster (2003) reported that Hypertrophic scars are found in 1.5% to 4.5% (average 3%) of the world population. They can occur anywhere in body, more commonly across areas of tension and flexor surfaces. Lesions are at the beginning red and elevated, but change with time into pale and flat scars. Not enough data is available about epidemiology of Hypertrophic Scar, as it is generally thought to be a variation of the normal.

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Figure 46:B; Age of onset of Keloid among 1075 patients (Tirgan, 2015 [Bib])

162

26. Aetiology of Keloids & Hypertrophic Scars

Hypertension, Autoimmunity, and Spontaneous; or extrinsic ie Piercing, Tattooing, Epilation,

Buns, Bites, Vaccination, Infection, Irritation, Inflammation, Sutures, Foreign Body, and

Drug Induced.

Intrinsic Factors:

1. Race: A black person is 15 times as likely to develop a Keloid as a person of white race.

Coloured persons have intermediate predisposition. Tendency may be related to actual

colour of skin rather than race. Fitzpatrick (1988) scale is a classification of human skin

colour, which has six categories (Figure 47). It is thought that individuals at far end of

this scale ie Type V, and VI may be more susceptible to keloids.

2. Genetics: Half of people with Keloids will have other members of the family also

develop Keloids. Keloids have autosomal dominant inheritance with incomplete clinical

penetrance and variable expression. Nine upregulated genes are found in Keloids. These

genes, especially novel nuclear protein 1 (NNP-1) contribute to Keloid formation Na et

al, 2004). Mutations of apoptosis genes, like p53, bcl-2 and Fas, are found in keloid

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fibroblasts, causing decreased rates of apoptosis. MacLeod (1912) published an article in

which tendency of keloid formation was mentioned as ‘The patient had shown no

tendency to keloidal formation previously. ’Bloom (1947) presented a case of multiple

keloids in twin sisters, pointing to genetic predisposition of Keloids. Wallace (1950)

presented a case of acne keloid, in which he used the term keloidal tendency. Bloom

(1956) published a review article on hereditary nature of keloids, and also described a

family with keloids in five generations. Moschella (1962) described tendency of keloid

formation in cousins, suggesting genetic basis. Ramakrishnan et al (1974) reported a set

of twins who formed keloids simultaneously after vaccination. Marneros et al (2001)

described four pairs of twins with keloids. Hosalkar et al (2007) reported increased

incidence of Keloids in patients of multiple hereditary exostoses; an autosomal dominant

condition.

3. Age: Most common age of development of Keloids is 20 years (range 10 to 30 years).

They are unusual in children and elderly

4. Sex: Women are more likely to develop Keloids as compared to men. This may be an

artefact, due to customary earlobe piercing in females.

5. Site: External ear (especially lobule), neck, upper torso, shoulder, sternum, back, and face

are most common areas afflicted by Keloids. Keloids are very rare on palm, sole,

genitalia, and eyelids; however, with current body piercing practices they are reported on

genitalia, eyebrow, eyelid, umbilicus, nose, nipple, and tongue (Neiburger et al, 2006).

6. Tension: Tension or mechanical strain in wound is more likely to give rise to Keloids.

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Scars perpendicular to muscle fibres are more flat and narrow, and have less collagen

than those which are parallel to muscle fibres.

7. Direction: If direction of wound is across normal skin lines, incidence of Keloids is

increased, as compared to those parallel to normal skin lines. Ogawa has stressed

importance of mechanobiology of scarring in many articles (2011).

8. Hormones: Keloids tend to enlarge during pregnancy. This is considered to be due to

hormonal changes in pregnancy.

9. Syndromes: Keloids are a part of Rubinstein-Taybi syndrome. Geominne (1968)

described a syndrome of torticollis, cryptorchidism, and kidney dysplasia, and keloids.

Touraine-Solente-Gole syndrome includes pachydermia, periostosis and keloids.

Spontaneous Keloids were reported in Dubowitz syndrome (Paradisi et al 1994). Gulec

(2001) reported a case of Noonan syndrome with tendency for keloid formation. Yavuzer

et al (2001) reported plantar keloid in a case of Proteus syndrome. Jimbow et al (1988)

reported keloidal lesions with an unusual accumulation of type IV collagen in progeria.

Botella et al (1976) reported development of keloid in two cases of hydroa vacciniforme,

a rare skin disease. Raskin (1975) suggested association of osteopoikilosis with keloid.

10. Hypertension: Snyder et al (1996) and Arima et al (2015) reported association of keloids

with hypertension. Hypertension is thought to be a risk factor for Keloids (Huang &

Ogawa, 2014).

11. Autoimmunity: Sako and Worwick (2015) reported extensive keloids in a patient

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suffering from pemphigus vulgaris, an autoimmune disease of skin. Oittinen (2007)

reported multiple nonsyndromic spontaneous keloids in a patient suffering from allergic

diseases.

12. Spontaneous: Keloids can occur without any known injury, infection, or inflammation.

Labert (1851 AD), Ellerbroek (1951), and Pedoya (1954) described spontaneous keloids.

Extrinsic Factors:

13. Piercing: It is main cause of earlobe keloid formation in genetically prone people.

Keloids have now been reported from other areas of cosmetic piercing ie eyebrow,

umbilicus, and genitalia. MacLeod (1912) published an article about Keloid formation

following ear-piercing.

14. Tattooing: It sometimes causes Keloid formation in genetically prone persons. These

Keloids are more difficult to treat, due to presence of multiple ink colours.

15. Epilation: Vogt (1973) reported formation of keloid after electro-epilation.

16. Buns: Deep partial thickness or full thickness burn can cause large Keloids, especially if

it has taken more than three weeks to heal. These Keloids are very stubborn, due to its

large size and stiff texture. Cubison et al (2006) studied correlation between healing time

and formation of keloids in children due to scalds.

17. Bites: Keloids have been reported after insect bites, especially those of hornets and

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honey-bees. Dore (1918) presented a case of keloid following dog bite on face in a child.

18. Vaccination: BCG (and previously Smallpox) vaccination can cause Keloid at the site.

Kunzfeld (1952) published an article on development of keloids after vaccination.

Plaschkes and Kaplan (1962) described keloid formation after BCG vaccination.

19. Infection: Wounds with infection and foreign body are more likely to end-up in Keloids

than uninfected wounds.

20. Irritation: Prolonged irritation of scar can cause high levels of profibrotic cytokines,

leading to Keloid formation.

21. Inflammation: Chickenpox, Acne, Folliculitis, Herpes Zoster etc can lead to Keloid

production. Thomas (1946) presented a case of multiple keloids following varicella.

22. Sutures: Wounds stitched with absorbable sutures are more likely to develop Keloids

than these stitched with non-absorbable sutures.

23. Foreign Body: Contact of wound with foreign body is also considered a cause of Keloid

formation. This is especially relevant in cases of body piercing, when piercing is

immediately followed by a foreign (eg ear pin) insertion, in direct contact with wound.

24. Drug Induced: Shivaswamy and Thappa in 2007 reported a case of extensive keloids due

to cyclosporin treatment in a pemphigus vulgaris case. Scot et al (1994) reported linear

keloids within striae distensae due to anabolic androgenic steroids abuse in athletes.

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Figure 47: Fitzpatrick Scale of Skin Colour (Fitzpatrick, 1988)

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27. Pathogenesis of Keloids & Hypertrophic Scars

Due to struggle to restore barriers, re-establish normal vascularity, and restore mechanical integrity of injured structure, perfect repair is forgone due to urgency to come back to normal function. Conversely, regeneration is perfect rebuilding of previous structure and absence of scar. Although regeneration of skin is goal of wound healing, it is only found in foetus, or in some other tissues like bone, liver and epithelium. In wound healing in adults, regeneration is sacrificed for early repair.

Wound-Healing Phases: Phases of normal wound healing are inflammation, proliferation, and maturation. These phases are not water-tight compartments; there is a lot of overlap

169 between them. They are described separately for the purpose of clear understanding only

(Bhardwaj & Deb, 2013 [Bib]; Brunicardi, 2015 [Bib]; Thorne, 2014 [Bib]; Townsend et al,

2012 [Bib]) (Figure 48).

Inflammatory Phase: It has following components:

Haemostasis and Inflammation: During an acute injury, blood vessel damage causes initial severe vasoconstriction, followed by vasodilation and increased vascular permeability.

Erythrocytes and platelets plug injured capillaries, leading to cessation of haemorrhage.

Activation of platelets by binding to exposed type IV and V collagen from damaged endothelium results in platelet aggregation. Initial contact amongst platelets and collagen needs factor VIII.

Increased Vascular Permeability: Platelet binding to damaged capillary endothelium causes conformational changes in platelets that trigger intracellular signal transduction pathways which lead to activation of platelets and release of active proteins. Platelet alpha granules are organelles which store PDGF, TGF-β, IGF-I, fibronectin, fibrinogen, thrombospondin, and factor VIII. Dense bodies in platelets comprise vasoactive amines, eg serotonin, which lead to vasodilation and increased vascular permeability. Mast cells adherent to endothelial surface release histamine and serotonin, resulting in increased vascular permeability, which causes extravasation of plasma. Clotting cascade is started using intrinsic and extrinsic pathways. As platelets become activated, membrane phospholipids bind factor V, which allows interaction with factor X. Membrane-bound prothrombinase activity is generated and potentiates thrombin production exponentially. The thrombin itself activates platelets and catalyzes conversion of fibrinogen to fibrin. Fibrin strands trap erythrocytes to form clot and seal

170 wound.

Agents of Inflammatory Phase: Many cells and substances partake in inflammatory phase.

Chemokines: Chemokines stimulate migration of different cell types, particularly inflammatory cells, into wound and are participants in regulation of different phases of wound healing. The CXC, CC, and C ligand families bind to G protein–coupled surface receptors called CXC receptors and CC receptors. Macrophage chemoattractant protein

(MCP-1, or CCL2) is induced in keratinocytes after injury. Expression of this chemokine is sustained in chronic wounds and results in prolonged presence of polymorphonuclear cells

(PMNs) and macrophages, leading to prolonged inflammatory response and pathological scarring. CXCL1 (GRO-α) is a potent PMN chemotactic regulator and is increased in acute wounds. It is also involved in reepithelialization. Interleukin-8 (IL-8, or CXCL8) expression is increased in acute and chronic wounds. It is involved in reepithelialization and induces leukocyte expression of matrix metalloproteinases (MMPs), which stimulates remodelling. It is also a strong chemoattractant for PMNs and participates in inflammation. Relatively low levels of IL-8 are found in fatal wounds and may be why fatal wounds have decreased inflammation and heal without scars. Expression of keratinocyte-produced chemokine interferon inducible protein 10 (IP-10 or CXCL10) is elevated in acute wounds as well as chronic inflammatory conditions. It impairs wound healing by increasing inflammation and recruiting lymphocytes to wound. It also inhibits proliferation by decreasing reepithelialization and angiogenesis and preventing fibroblast migration. Stromal cell-derived factor-1 (SDF-1, or CXCL12) is produced by endothelial cells, myofibroblasts, and keratinocytes and is involved in inflammation by engaging lymphocytes to wound and helping angiogenesis.

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Polymorphonuclear Cells: The release of histamine and serotonin leads to increased vascular permeability of capillary bed. Complement factors such as C5a and leukotriene B4 promote neutrophil adherence and chemoattraction. In the presence of thrombin, endothelial cells exposed to leukotriene C4 and D4 release platelet-aggregating factor, which further enhances neutrophil adhesion. Monocytes produce many inflammatory mediators, which further promote endothelial-neutrophil adherence. Enhanced capillary permeability and many chemotactic factors help extravasation of neutrophils to injured area. As neutrophils migrate, they release lysosomes and enzymes into extracellular matrix, which further facilitates neutrophil migration. Vasodilation combined with enhanced vascular permeability gives rise to features of inflammation; rubor (redness), tumor (swelling), calor (heat), and dolor (pain).

Macrophages: Macrophage is crucial to wound healing because it causes release of cytokines.

Macrophages in wound appear when neutrophils disappear. Monocytes reach there by chemotaxis in 24 to 48 hours. Activated integrin promotes adhesion-mediated gene induction in monocytes which transforms them to macrophages, resulting in enhanced phagocytosis and selective expression of cytokines and signal transduction elements by messenger RNA, including early growth response genes EGR2 and c-Foes. Macrophages contain receptors for immunoglobulin G (Fc receptor), C3b (CR1 & CR3), and fibronectin (integrin receptors) that allow recognition of opsonized pathogens and help phagocytosis.

Lymphocytes: T lymphocytes appear in great number at fifth day, and peak at seventh day. B lymphocytes are only involved in downregulating healing as wound closes. Lymphocytes control fibroblasts by producing stimulatory or inhibitory cytokines. Macrophages clear foreign debris like bacteria or enzymatically degraded proteins, and work as antigen- presenting cells to lymphocytes. This activates lymphocyte growth and release of different

172 cytokines. T cells synthesize IFN-γ, which stimulates macrophage to release cytokines. IFN-γ also causes reduced production of prostaglandins, which increases effect of inflammatory cytokines.

Proliferative Phase: As haemostasis and inflammation resolve, scaffolding is placed for wound repair by angiogenesis, fibroplasia, and epithelialization. Granulation tissue is formed, consisting of capillaries, fibroblasts, macrophages, collagen, and fibronectin. Growth factors change granulation tissue, especially fibroplasia.

Angiogenesis: Angiogenesis is required for a healing wound setting. After injury, basement membrane is destroyed, causing migration of cells. Endothelial cells reform tubules. Later, basement membrane reforms and results in capillary maturation. After injury, endothelium is exposed to numerous soluble factors and comes in contact with adhering blood cells. These interactions result in upregulation of cell surface adhesion molecules, like vascular cell surface adhesion molecule-1 (VCAM-1). Matrix-degrading enzymes, such as plasmin and metalloproteinases, are released and activated, and degrade endothelial basement membrane.

Disruption of basement membrane permits migration of endothelial cells in wound, promoted by fibroblast growth factor (FGF), PDGF, and TGF-β. Injured endothelial cells express adhesion molecules, like integrin αvβ3, that facilitates attachment to fibrin, fibronectin, and fibrinogen and thus facilitates endothelial cell migration along provisional matrix scaffold.

Fibroplasia: Fibroblasts arise from normal mesenchymal cells of connective tissue. After trauma, inactive fibroblasts reach inflammatory site due to chemoattraction. Here they divide and produce different ingredients of extracellular matrix. Due to activation by macrophage- and platelet-derived cytokines and growth factors, fibroblasts, normally arrested inG0 phase,

173 undergo replication. Platelet-derived TGF-β stimulates fibroblast proliferation indirectly by releasing PDGF. The fibroblast can also stimulate replication in an autocrine way due to release of FGF-2. To continue proliferating, fibroblasts require further stimulation by factors such as EGF or IGF-. Although fibroblasts require growth factors for proliferation, they do not need growth factors to survive. Time needed by mesenchymal cells to change into fibroblasts is responsible for interval amid trauma and collagen formation at injury site. This period consists of three to five days, and is known as lag phase.

Epithelialization: Re-epithelialization of wounds starts shortly after trauma. Initially, wound is sealed by a clot and later by epithelial cells migrating across defect. Keratinocytes in basal layer of residual epidermis or appendages migrate to wound surface. Epithelialization is due to a series of changes in keratinocytes. Cells are stimulated to migrate. Attachments to neighbouring and adjoining cells and to dermis are loosened, as demonstrated by intracellular tonofilament retraction, dissolution of intercellular desmosomes and hemidesmosomes linking epidermis to basement membrane, and production of cytoplasmic actin filaments.

Migratory cells dissect wound by separating dead from living tissue. Degradation of ECM is caused by MMP-1 and plasminogen activator. Migratory cells are also phagocytic and remove debris from wound. Cells behind front edge of migratory cells begin to proliferate. If basement membrane is not intact, it is repaired first. Local release of EGF, TGF-α, and KGF and enhanced expression of their receptors may also stimulate these events.

Agents of Proliferative Phase: Many cells and substances partake in proliferative phase of wound healing.

Extracellular Matrix: ECM stabilizes physical structure of dermis. Glycosaminoglycans

174

(GAGs), polysaccharide chains, and fibrous proteins like collagen, elastin, fibronectin, and laminin are produced by cells in ECM. The wound matrix changes in composition as healing progresses. Provisional matrix acts as a scaffold for cellular migration and consists of fibrin, fibrinogen, fibronectin, and vitronectin. Later GAGs and proteoglycans are formed.

Collagens, the predominant scar proteins, form at the end.

Collagen Fibres: They are main constituent of skin, and form 25% of total protein quantity.

Proline- and glycine-rich collagen molecule is a long, triple-stranded helical structure that consists of three collagen polypeptide α chains wound around one another in a rope like super-helix. With its ring-like structure, proline provides stability to helical conformation in each α chain, whereas glycine, due to small size, allows tight packing of three α chains to form superhelix. There are 20 types of collagen. Type I is the most common collagen of body, found mainly in skin and bone. Collagen polypeptide chains are produced on ribosomes and enter endoplasmic reticulum (ER) lumen as pro–α chains. These precursors contain amino-terminal signal peptides to direct them to ER. Within lumen of ER, some of prolines and lysines undergo hydroxylation to make hydroxyproline and hydroxylysine.

Hydroxylation results instable triple-stranded helix through formation of interchain hydrogen bonds. The pro–α chain combines with two others to make procollagen, a hydrogen-bonded, triple-stranded helical molecule. After secretion into ECM, proteases cleave propeptides of procollagen to form collagen monomers. These monomers combine to form collagen fibrils.

Elastic Fibres: Elastic fibres in ECM provide resilience to allow recoil after stretching. Elastic fibres are mainly composed of elastin. Elastin consists of hydrophobic and alanine- and lysine-rich α-helical segments which alternate along polypeptide chain. The alanine- and lysine-rich α-helical segments form cross links amongst neighbouring molecules. Elastic

175 fibres comprise an elastin core enclosed by a layer of microfibrils that consist of many specific glycoproteins like fibrillin. Elastin-binding fibrillin is essential for integrity of elastic fibres. In nonwounded circumstances, there is little elastin degradation, probably because of elastin’s hydrophobic nature, which makes interior of this highly folded protein inaccessible.

Glycosaminoglycans: They are polysaccharides consisting of disaccharide units, a sulphated amino sugar, and uronic acid. Their negative charge attracts osmotically active cations, like

Na+, which causes a lot of water to be stored into matrix. Hyaluronan is the simplest GAG. It is composed of repetitive nonsulfated disaccharide units and is found in adult tissues, but is especially prevalent in fatal tissues. Its abundance in foetal wounds is a factor in scarless wound healing seen in foetus. Unlike other GAGs, hyaluronan is not covalently attached to any protein.

Proteoglycans: They are a group of glycoproteins and GAG chains. The number and types of

GAGs attached to core protein varies much, and GAGs can be modified by sulfonation.

Because of their GAGs, proteoglycans provide hydrated space in between cells.

Basal Lamina: Basal laminae are thin sheets of specialized ECM which segregate cells and epithelia from adjacent connective tissue. Basal lamina is attached to connective tissue by special fastening fibrils, and is collectively called basement membrane.

Maturational Phase: Wound contraction occurs by centripetal movement of full thickness of surrounding skin and reduces size of scar. Fibroblasts in a contracting wound undergo change to stimulated cells, referred to as myofibroblasts. These cells have alpha smooth muscle actin in bundles named as stress fibres. The actin appears at day 6 after injury, continues at higher

176 levels for 15 days, and disappears by 4 weeks, when cell undergoes apoptosis. It appears that a stimulated fibroblast develops contractile ability related to formation of cytoplasmic actin- myosin complexes. When this stimulated cell is placed in fibroblast-populated collagen lattice, contraction occurs even faster. The tension that is exerted by fibroblasts’ attempt at contraction appears to stimulate actin-myosin structures in their cytoplasm. Fibroblasts develop a linear arrangement inline of tension that when removed, causes cells to round up.

The actin microfilaments are linearly arranged in long axis of fibroblast. They are associated with dense bodies that allow attachment to surrounding ECM. Fibronexus is attachment entity that connects cytoskeleton to ECM and spans cell membrane in doing so. MMPs also appear to be important for wound contraction. It has been demonstrated that MMP-3 strongly affects wound contraction. Different populations of fibroblasts respond to contraction stimulus in a heterogeneous fashion. Stromelysin-1, with participation of β1 integrins, allows modification of attachment sites between fibroblasts and collagen fibrils. Similarly, cytokines such as TGF-β1 affect contraction by increasing expression of β1 integrin.

Remodelling: At this stage, fibroblast decrease in number and capillary network decreases.

Wound strength rises sharply in 1 to 6 weeks and then plateaus up to 1 year. When compared to normal skin, scar tensile strength is 30%. An increase in strength occurs after 21 days, as a result of cross linking. Epidermo-dermal interface in a healed wound does not have rete pegs.

Due to this there is increased fragility of skin, and predisposes neo-epidermis is injured even with minor injury. Scar is not as functional as normal skin, sweat glands, sebaceous glands, and hair follicles do not reform in scar.

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Figure 48: Phases of Normal Wound Healing (Townsend et al, 2012 [Bib]) 178

28. Pathogenesis (Abnormal)

Classic theory is that Keloids and Hypertrophic Scars are considered different entities.

[Bib]; Kumar, 2014 [Bib]; Patterson, 2015 [Bib]; Townsend, 2012 [Bib]).

From Histology point of view, both Keloids & Hypertrophic Scars show increased thickness of dermis, and decrease in rete ridges. They have abundant collagen and glycoprotein content. Normal skin has discrete collagen bundles, usually parallel to skin surface, which have random connections between them by fibrillar collagen, like a basket- weave pattern. In Normal Scars, collagen bundles are placed parallel to each other, in a sheet pattern. In Hypertrophic Scars, collagen bundles are more flat, more stretched, more random, like a wavy pattern, with intervening fibroblasts. In Keloids, collagen bundles are almost absent, and collagen fibres are placed haphazardly in disarrayed pattern. Collagen fibres are much large and thick, and fibroblasts are missing from centre of Keloid.

Keloid and Hypertrophic Scar fibroblasts have normal proliferation rates, but

Hypertrophic Scar fibroblasts make collagen at 3 times, and Keloid fibroblasts 20 times greater rate than normal fibroblasts. Rate of apoptosis of keloid fibroblasts is decreased

.Large quantity of extracellular matrix, like fibronectin and proteoglycans are also present.

Fibronectin synthesis decreases during normal wound healing, but production remains high for a long time in Keloids & Hypertrophic Scars. Tenascin C, biglycan, integrins, and decorin

179 may be increased. Expression of dermatopontin (a multifunctional protein of extracellular matrix that influences collagen assembly) is decreased. Angiotensin-converting enzyme

(ACE) is much increased in pathological scar tissue as compared to normal and wounded skin.

This disturbed synthetic activity is facilitated by changed growth factors and cytokines production. There is an important role TGF-β, which is a potent chemotactic factor for fibroblasts and stimulates them to produce major matrix components including collagen.

Disturbances in Transforming Growth Factor Beta (TGFB) signalling, and keratinocyte- fibroblast interactions may be involved. TGFB is produced by macrophages as well

Extracellular Matrix (ECM). TGFB acts by fibroblasts activation, leading to accelerated collagen production. TGFB production is higher in Hypertrophic Scars, and both Keloid and

Hypertrophic Scar fibroblasts respond to lower levels of TGFB than normal fibroblasts.

Expression of its isoforms TGFB1, and TGFB2 is increased in Keloid fibroblasts, while

Hypertrophic Scar fibroblasts produce more TGFB1. Addition of exogenous TGFB2 activates fibroblasts from both Keloids & Hypertrophic Scars. Keloid fibroblasts also have upregulated antiapoptotic gene expression, which can be differentially expressed within different areas of same Keloid. In contrast to elevated collagen synthesis seen in these scars, collagen degradation is low. Both matrix metallo-proteinases (MMP)-1 and MMP-9 are low in Keloids &Hypertrophic Scars and Keloids.

Genetics of keloidal scarring shows varying inheritance patterns, linkage loci

(involving chromosomes 2q23 and 7p11), HLA alleles (including HLA-DRB1*15, HLA-

DQA1*0104, DQ-B1*0501, and DQB1*0503), and a considerable number of dysregulated genes. The P311 gene is upregulated in fibroblasts; this gene induces a myofibroblastic

180 phenotype and stimulates expression of TGF-β1. Mutations in p53 also occur, giving rise to decreased apoptosis of keloid fibroblasts. Heat shock protein 47 (HSP 47) is overexpressed in keloid fibroblasts and is another mechanism involved in collagen accumulation. The relative mass of type III collagen are enhanced. There is also increased production of types I and III collagen mRNA. Production of gli-1 oncogene is markedly increased in keloid fibroblasts.

VEGF, CTGF, PDGF, and Interleukins 4 and 15 are increased. These mediators start different signalling pathways, like AKT/mTOR and SMAD pathways, which eventually lead to activation and proliferation of fibroblasts (Brunicardi, 2015 [Bib]; Kumar, 2014 [Bib];

Patterson, 2015 [Bib]) (Figure 49).

Dieburg (1852 AD), for the first time, gave a description of histopathology of Keloid.

Hofs (1952), for the first time, published an article on Pathogenesis of Keloids. Robinson

&Hamilton (1953) studied role of heparin in fibroproliferative disorders. They found enhanced heparin tolerance in keloid patients. This was the first time that molecular basis of keloid formation was described. Zimmer (1957) described use of paper chromatography in diagnosis of keloid. Hummel (1958) described histochemistry technique to study pathogenesis of keloid. Amante et al (1963) described histochemistry of keloid collagen using chromatography. Bairti and Depetris (1963) described ultrastructure of keloid collagen using electron microscopy and crystallography. Linares et al (1972) described electron microscopic features of keloids. Kischer (1974) performed scanning and transmission electron microscopy of fibroblast from normal skin and keloids. He, along with Shetlar, in the same year, also studied histochemistry of normal skin and keloids. Bailey et al (1975) studied characteristics of collagen of normal and hypertrophic scars. They maintained that hypertrophic scar collagen was like embryonic collagen. Baur et al (1975) observed presence of myofibroblasts in hypertrophic scars, and called them contractile fibroblasts.

181

Moulton-Levy (1984) reported that keloids had a multicellular origin and that they did not develop clonally as did most neoplasms. Tsuji and Sawabe (1987) reported presence of large number of elastic fibres in hypertrophic scars. Kreig and Heckmann (1989) described, for the first time, role of cytokines, through stimulation of fibroblasts, in formation of keloids.

Arakawa et al (1996) reported decreased collagenase gene production in keloid fibroblasts.

Garner et al (1998) wrote that epidermis regulated dermal fibroblast activity, and lack of epidermis led to excess collagen production. Lee et al (1999) studied level of TGF beta 1, 2, and 3 in keloids. Wu et al (1999) reported that intralesional collagenase caused collagen degradation in explanted hypertrophic scars.

Feng et al (2000) reported that TNF-alpha had different biological effects on fibroblasts derived from hypertrophic scar or normal dermis; it decreased cytoactivity of hypertrophic scar fibroblasts, and increased that of normal skin fibroblasts. Lim et al (2001) investigated effect of keloid keratinocytes on fibroblast proliferation. Leake et al (2003) reported that urokinase-mediated plasminogen activation system was involved in spread of keloids beyond wound margins due to degradation of extracellular matrix. Luo et al (2003) reported apoptosis of keloid-derived fibroblasts induced by Fas gene transfection. They concluded that blockage of upstream apoptosis pathway in keloid-derived fibroblasts was due to nonfunction of Fas protein, and mutation of Fas gene may be the pathogenesis of keloids.

Moulin et al in 2004 compared Fas antibody induced apoptosis of normal skin and hypertrophic scar myofibroblasts. Phan et al (2005) reported that Smad3 signalling played a significant role in keloid formation through epithelial-mesenchymal interactions, and suggested inhibition of Smad signalling for keloid treatment. Wang D. et al (2005) reported making of recombinant human Smad 7 adenoviral vector.

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Figure 49: Abnormal Wound Healing(Townsend et al, 2012 [Bib])

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29. Pathogenesis Theories

1. Genetics Theory: Genetic inheritance of Keloid tendency is shown by the fact that

Blacks are 15 times more prone to form Keloids as compared to Whites, and Keloids are

absent in albinos. Keloid-associated loci have been identified in Japanese, African-

American, and Han Chinese population.

2. Mechanics Theory: This is also called skin injury-wound tension theory. Keloids are

most commonly found on presternal and inter-scapular areas, which have high

mechanical stress; Keloids very rarely occur on scalp and lower legs, which have low

mechanical stress. Ogawa has stressed importance of mechanobiology of scarring (2011).

3. Endocrines Theory: Keloids may be caused by hyperactivity of endocrine glands.

Sebum or sebocyte are most commonly implicated. Keloids are less common on non-

sebaceous areas, like palms and soles. Keloids are more common in young adults, as

sebum production is abundant at this age.

4. Metabolic Theory: Unusual metabolic products, like lactates, have been found in

Keloids. Keloids have higher levels of adenosine triphosphate (ATP). There is possibility

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that abnormal scarring is linked to increased metabolic activity, as Keloids have high

oxygen consumption rate. Wan and Evans (1999) reported increase in free radicals in

hypertrophic scars. Murakami et al (2006) suggested that there was a close relationship

between subcutaneous ischaemia and hypertrophic scar formation.

5. Circulatory Theory: Many patients of keloids present with hypertension. Angiotensin-

converting enzyme (ACE) is significantly higher in keloid. Hypertension may adversely

affect Keloids, or Keloids and hypertension may have a common aetiology.

Antihypertensive drugs are being used in treatment of Keloids, eg verapamil.

6. Immunological Theory: Immunological changes may also be involved in Keloid

formation, because eluates of Keloids contain antinuclear antibodies against fibroblasts.

Further, some immunoglobulins level is increased in Keloids. Laurentaci and Dioguardi

(1977) studied histocompatibility antigens (HLA) in keloids and hypertrophic scars.

Janssen de Limpens and Cormane (1982) reported that keloid patients had antinuclear

antibodies against fibroblasts. Yagi et al (1979) suggested that immune reaction to sebum

could cause keloid. Minet et al (1983) suggested immunological basis of keloid

formation. Smith et al (1987) described role of mast cells in formation of keloids.

7. Nutritional Theory: Keloids may be due to inadequate fatty acids in nutrition. Patients

with Keloids are being studied for treatment with fatty acids nutrition. Fish oil (omega-3

polyunsaturated fatty acid) has been used in traditional remedies for keloids. Louw (2007)

proposed use of fatty acids for prevention and treatment of keloids. Cabbabe and Korock

(1986) studied role of vitamin C deficiency in wound healing in Guinea pigs. They found

that there was relationship between vitamin C deficiency and hypertrophic scar formation.

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Figure 50: Wound healing in pathological scars has prolonged inflammation phase, inappropriate cytokines & delayed healing response (Huang et al 2013) 186

30. Pathology of Keloids & Hypertrophic Scars

Keloids grow beyond wound margins into adjacent normal skin. This outgrowth is in the form of claws or tentacles. However, there is a sharp margin of demarcation between keloid tissue and adjacent normal skin tissue, and there is no invasion in true sense. Keloids normally do not invade hypodermis. However, if hypodermis or other deeper tissues are also injured in the first instance, then hypodermis and even deeper tissue are also involved.

Hypertrophic Scars do not invade surrounding normal skin, and retain the shape of original wound. There is a sharp margin of demarcation between hypertrophic scar tissue and the adjacent normal skin tissue. Hypertrophic scars do not invade hypodermis.

On Gross Examination, Keloids and Hypertrophic Scars are firm, hard, or nodular in consistency. They are brown, pale, or flesh-coloured. They may be irregularly shaped or linear. Occasionally they may be pigmented. Surface is smooth and shiny. They may have claw-like projections. They are devoid of hair.

On Cut Section, surface is heterogeneous, but smooth. Keloid gives a gritty feeling on cutting. Cut surface is pale or pink in colour; outer area is pink due to cells and blood vessels, while core is pale due to increased collagen deposition. It also shows areas of brown and red colour, due to old or fresh haemorrhage. One pale nodule is usually present in the centre, with irregular spikes going in all directions; it is called collagen nodule. Deeper area is black in colour, due to haemorrhage at the time of excision. A few blood vessels are also visible in deeper part. Overlying epidermis is usually normal; sometimes it is thinned out due to pressure of underlying collagen (Figure 51).

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Figure 51: Gross Pathology of Keloid 188

31. Microscopic Pathology

Keloids are benign unencapsulated tumours. Early keloids show abundant fibrillary collagen. Mature keloids have a characteristic appearance with broad, homogeneous, brightly eosinophilic, swirling, irregularly distributed collagen bundles. Fibroblasts are increased and are found along collagen bundles with an orientation similar to accompanying collagen; centre of keloid may be acellular. The Argyrophilic Nucleolar Organizer Region (AgNOR) count of fibroblasts is significantly increased. There is also excessive deposition of fibronectin. There are also abundant mucopolysaccharides, particularly chondroitin-4-sulfate, between bundles. Keloids have reduced vascularity compared with hypertrophic scars and normal healing wounds. Keloids are usually elevated above surrounding skin surface. The overlying epidermis may be thin, and beneath it, there are often some telangiectatic vessels.

A sparse, chronic inflammatory cell infiltrate may surround these peripheral vessels. A study from Taiwan (Lee et al, 2004) reported that in scars with no detectable keloidal collagen, features favouring diagnosis of keloid were nonflattened epidermis, nonfibrotic papillary dermis, a tongue-like advancing edge, a horizontal cellular fibrous band in upper reticular dermis, and a prominent fascia-like band. With intralesional cryo-needle procedures, swirl pattern of collagen bundles is lost, thickness of collagen layer decreases, bundles become more compact, and there is a loss of proliferating fibroblasts and mast cells in treated site(Patterson, 2015) (Figure 52).

Hypertrophic scars are only slightly elevated above surrounding skin. The collagen bundles are characteristically oriented parallel to skin surface, as are accompanying fibroblasts. They are markedly increased in number, although there is some reduction in number with time. Capillaries are generally perpendicular to skin surface, and these may be

189 surrounded by a sparse inflammatory cell infiltrate within scar. There is little mucin except in early lesions. Elastic tissue is sparse or absent. Subepidermal clefting sometimes develops overlying a scar; bullae are rare.

Mast cells are increased in keloids. Dystrophic calcification and bone formation may be seen in keloids, particularly on abdominal wall. Alpha-smooth muscle actin has also been found in both hypertrophic scars (70%) and keloids (45%). CD34, S-100, and factor XIIIa are not expressed in keloids or hypertrophic scars.

Electron Microscopy: On transmission electron microscopy, Keloids exhibit many fibroblasts with conspicuous Golgi apparatus and ample rough endoplasmic reticulum.

Collagen is separated from surface of fibroblasts by a diffuse amorphous substance.

Myofibroblasts have usually not been demonstrated, although it has been speculated that they may be present in early lesions. Very fine elastic fibres can be seen in hypertrophic scars on transmission electron microscopy, although they cannot be demonstrated by light microscopy. On scanning electron microscopy, the overall impression is of much increased density of collagen. The bundle arrangement is less obvious and the confluence of collagen gives a homogeneous appearance. Fibre diameter is about half to three quarters of that in normal skin, and cross stranding is frequent. The fibres are mostly straight. Capillaries are absent. Some whorled nodules are also seen. No significant differences between keloid and hypertrophic scar are observed but in both, fibre parallelism is usually marked. The steroid- treated hypertrophic scar shows a dense homogeneous arrangement of collagen, with small fibre size. It differs from the active hypertrophic scar only in a relative increase in interstitial space containing mucinous material. Hypertrophic scar treated by compression shows some parallelism of small fibre bundles and some return of waviness (Patterson, 2015).

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Figure 52: Microscopic Pathology of Keloid

191

32. Classifications of Keloids & Hypertrophic Scars

International Classifications of Disease (ICD) has produced following classifications of Keloids:

ICD-9-CM:

 Cheloid (see also Keloid) 701.4

 Hawkins' keloid 701.4

 Hypertrophy, hypertrophic

 scar 701.4

 Kelis 701.4

 Keloid, cheloid 701.4

 Addison's (morphea) 701.0

 cornea 371.00

 Hawkins' 701.4

 scar 701.4

 Keloma 701.4

 Scar, scarring (see also Cicatrix) 709.2

 cheloid 701.4

 hypertrophic 701.4

 keloid 701.4 (WHO, 1998 [Bib])

ICD-10:

L91.0 Hypertrophic scar

Keloid scar

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Keloid

Excl.:

acne keloid (L73.0)

scar NOS (L90.5)

L91.8 Other hypertrophic disorders of skin

L91.9 Hypertrophic disorder of skin, unspecified (WHO, 2015 [Bib])

Malbec and Quaife (1956) tried to give a classification of keloids. Onwukwe (1978) presented his classification of keloids. Keloids can also be classified on the basis of

Morphology as well as Topography (Tigran, 2015 [Bib]).

Morphological Classification: Keloids can be classified according to their appearance:

1. Keloidal Papule: It is slightly raised from skin surface, has flat surface, and distinct

margins.

2. Nodular Keloid: It is hemispherical, raised, and has broad base.

3. Keloid Tumour: It is in the form of a large nodule.

4. Linear Keloid: It is usually formed after surgical incision.

5. Flat Keloid/Keloidal Plaque: It is minimally raised from skin surface.

6. Butterfly Keloid: It is in the shape of a butterfly, and is more common on chest wall,

especially in women (Figure 53)

7. Guttate Keloid: It presents as multiple small keloids in one area of body.

8. Hyper-inflammatory Keloid: It is painful, and infected. It is often found on chest wall in

black hairy men.

9. Superficially Spreading Keloid: It grows rapidly to surrounding area.

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10. Pedunculated Keloid: It has got a small stalk and a large head. It is the easiest keloid to

treat.

11. Bulky Keloid: It is due to coalescence of multiple nodules, or overgrowth of a single

nodule.

12. Massive Keloid: It is very large in size, more than 10 cm in diameter, and spreads in all

directions.

Topographical Classification: Keloids can be classified according to their site:

1. Scalp Keloid: It is usually in occipital area of black men.

2. Ear Keloid: It is due to ear piercing.

3. Earlobe keloid: It is the commonest site of keloid, especially in women, due to piercing

practice.

4. Posterior Auricular Keloid: It is due to operation in this area.

5. Facial Keloids: It is linear or nodular, usually in black men.

6. Neck Keloid: It is linear or nodular, usually in black men.

7. Chest Wall Keloid: It is second commonest site of keloid, especially after sternotomy for

open-heart surgery.

8. Upper Arm Keloid: It occurs after vaccination.

9. Umbilical Keloid: It is due to piercing or laparoscopic operation.

10. Pubic Keloid: It is usually in black men.

11. Other Sites: Keloid can form on any part of body.

12. Rare Sites: Palm of hand, sole of foot, penis, and scrotum. Parsons (1966) presented a

case of keloid of penis. LeFlore and Antonie (1991) reported a case of palmer keloid.

Sandler (1999) reported a case of plantar keloid.

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Figure 53: Butterfly Keloid (Tirgan, 2015 [Bib])

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33. Symptoms& Signs of Keloids & Hypertrophic Scars

Keloids and Hypertrophic Scars usually present after 3 to 12 months of original event.

Clinical diagnosis of Keloid is thought to be snap, and easy; very few clinicians go for any laboratory investigation. However, there are dozens of differential diagnoses of Keloid, and the Author recommends histopathology in all cases of Keloid to safeguard against undiagnosed malignancy (Nagi & Babar, 2015 [Bib]).

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Figure 54: Keloid on Sole of Foot (Google, 2015 [Bib]) 197

34. Investigations of Keloids & Hypertrophic Scars

Usually diagnosis is made on basis of history and examination. Investigations are rarely done. However the Author recommends histopathology in all cases of keloid (Nagi &

Babar, 2015 [Bib]).

Histologically both Keloids & Hypertrophic Scars exhibit increased thickness of dermis in which rete ridges are missing. They show a large quantity of collagen and glycoproteins. Normal skin has discrete collagen bundles, parallel to skin surface, with random connections by fine fibrillar collagen strands. In Hypertrophic Scars, collagen bundles are flat, stretched, random, and wavy, with abundant fibroblasts. In Keloids, collagen bundles are absent, and fibres are connected randomly in loose sheets with a haphazard orientation to skin surface. Collagen fibres are large and thick and fibroblasts are absent in centre of Keloid. Immunohistochemistry shows presence of TGF beta1, alpha-SMA, Desmin, and Periostin. There is also increased amount of CD3+, CD4+, CD 45R0, and a marked increased ratio of CD4 (+): CD8 (+) in keloid tissue. Many other antibodies are also being studied. CD34, S-100, and factor XIIIa are not expressed in keloids.

On transmission electron microscopy, Keloids exhibit many fibroblasts with conspicuous Golgi apparatus and ample rough endoplasmic reticulum. Collagen is separated from surface of fibroblasts by a diffuse amorphous substance. Myofibroblasts have usually not been demonstrated. On scanning electron microscopy, the overall impression is of much increased density of collagen. The bundle arrangement is less obvious and the confluence of collagen gives a homogeneous appearance. Fibre diameter is about half to three quarters of that in normal skin, and cross stranding is frequent. The fibres are mostly straight (Figure 55).

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Figure 55:B; SEM of Keloid (Kemble, 1976) 199

35. Diagnosis of Keloids & Hypertrophic Scars

Gross differential diagnosis is clinical diagnosis of skin lesions which mimic Keloid

(Ogawa, 2009).The term Keloid used without any qualification means Keloid of Alibert:

1. Keloidal Basal Cell Carcinoma: It presents as nodule mimicking a keloid and contains

hyaline keloidal collagen. Abundant collagenous stroma of this condition is a histologic

rarity. Requena et al (1996) reported two cases of nodular keloids on face, with no history

of trauma. Histology showed basaloid cells of basal cell carcinoma, interspersed with

keloidal collagen bundles.

2. Keloidal Squamous Cell Carcinoma: It presents in long standing keloid, of more than

20 years duration. It is found at old age. A few cases have been reported. Radiotherapy

may be a predisposing factor.

3. Keloidal Melanoma: Pseudo-melanoma has been described in a Keloid. Clinically it

resembles superficial spreading malignant melanoma. Histology shows picture of Keloid

with increased number of melanocytes in nests and as isolated cells throughout it.

4. Keloidal Kaposi’s Sarcoma: Keloid formation has been reported in Kaposi’s Sarcoma

lesions. Schwartz et al (1994) reported this for the first time. Acquired Immune

Deficiency Syndrome (AIDS) may be a risk factor in such cases.

5. Keloidal Acne: It is also called Acne Keloidalis Nuchae. It is caused by hairs curling

back into skin. Lesions form on hairy areas of skin like nape of neck. The term is a

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misnomer; it is neither keloid nor acne, but it resembles both. Adamson (1913) reported it

for the first time, and called it Dermatitis Papillaris Capillitii (Kaposi). Little (1918) also

published a case report. Levine (1945) presented a case of folliculitis keloidalis.

6. Keloidal Sycosis: It is also called Sycosis Barbae, or Chronic Folliculitis. It generally

involves beard area of men. It is caused by chronic infection of hair follicles due to

shaving injuries. Chronic folliculitis can also occur on chest of hairy men.

7. Keloidal Scleroderma: It is also called Keloid of Addison, Morphea, or Nodular

Scleroderma. It is localized scleroderma. It presents as patch of hardened skin. Fagge

(1868 AD) wrote an article titled: On keloid, sclerosis, morphea and some allied

affections, in which he differentiated between these diseases. This was an effort to clarify

a long lasting confusion, arising out of a mistake of Addison (Figure 60).

8. Keloidal Lupus Erythematosus: Scar tissue may form in cases of systemic lupus

erythematosus with severe inflammatory reaction, which becomes a keloid in prone

patients. James et al (1984) reported Keloids in two cases of systemic sclerosis

9. Keloidal Sarcoidosis: Sarcoidosis is a systemic granulomatous disease that can involve

any organ of body. It is one of the great imitators. Its hallmark is non-caseating

granuloma. It can occur in a scar, and can be mistaken for a keloid.

10. Keloidal Dermatofibroma: In this lesion a typical dermatofibroma exhibits an area

similar to Keloid in its superficial portion.

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11. Keloidal Blastomycosis: It is also called Blastomycosis, or Lobomycosis. It is a fungal

disease of skin produced by Lacazia loboi. Infection usually occurs in minor wounds or

insect bites. It looks like a chronic keloidal nodule on face or limbs.

12. Acne Keloid: It is a Keloid formed in an Acne scar, usually on face of women. This

condition is distinct from Acne Keloidalis Nuchae, which is on nape of neck of black

men. Wallace (1950) reported it for the first time in a 27 years old girl.

13. Syphilitic Keloid: Syphilis is now rare. In the past keloids have been reported in

syphilitic lesions. MacLeod (1911) presented a case of Keloids forming in scars of

ulcerating syphilitic lesions. Lewis (1946) presented a case of Keloid in syphilis lesion.

14. Striae Keloid: These are linear keloids found on abdomen and thighs. Striae formed due

to obesity or pregnancy become elevated, hypertrophied, ultimately forming true keloids.

They are especially found in athletes taking anabolic steroids.

15. Hawkins’ Keloid: It is also called Warty Tumour of Cicatrix. In first stage, it has a wart-

like appearance. In second stage, it becomes solid. In third stage, it ulcerates and sloughs

at base. Ulcer has raised, thickened, and everted edges.

16. Diffuse Cutaneous Leishmaniasis: It is caused by Leishmania aethiopica, or

Leishmania mexicana. Infection consists of a primary lesion, which slowly spreads to

involve multiple areas of the skin. Its lesions resemble nodular leprosy or keloid.

17. Scleroedema of Buschke: It is Type III scleroedema. It presents with thickening and

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hardening of skin of neck and upper back slowly. It persists for months or years. It is

more common in adult male diabetics.Ricciardi (1948) compared it with keloid.

18. Pyogenic Granuloma: It is an overgrowth of granulation tissue through a hole in

epithelium of a healing wound. If it is not treated in time, it becomes epithelialized, and

its appearance resembles keloid.

19. Foreign Body Granuloma: It is a types of Pyogenic Granuloma in which there is foreign

body in wound. It is more stubborn to heal, and has more chances of mimicking a keloid.

20. Xanthoma: It is deposition of cholesterol rich material in macrophages in skin. It is

usually related to lipid metabolism disorders. Tuberous xanthoma presents as firm,

painless, red-yellow nodule, and mimics a keloid.

21. Lichen Sclerosus

22. Tuberous Sclerosis

23. Erythema Elevatum Diutinum

24. Apocrine Cystadenoma

25. Chondroid Syringoma

26. Trichilemmal Carcinoma: It is a malignant tumour of hair follicle. It arises from outer

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root sheath of hair follicles. It is found on hairy areas of body like scalp or beard.

27. Desmoid Tumour: It is also called Desmoid Fibromatosis, or Aggressive Fibromatosis.

It is a fibrous tissue tumour arising from skin and subcutaneous tissue, usually of

abdominal wall. It involves muscles as well. It may recur after excision.

28. Extra-abdominal Desmoid Tumour

29. Desmoplastic Fibroblastoma

30. Desmoplastic Melanoma: It is a fibrosing type of spindle cell melanoma. It usually

appears as indurated plaque in persistently sun-exposed skin. Often lesion is non-

pigmented, and can be confused with a keloid.

31. Persistent Spitz Naevus: It is a benign tumour of melanocytes, but recurs after excision.

It presents as a raised flesh-coloured or pink-red nodule. Occasionally, multiple or

disseminated lesions can occur. It can be misdiagnosed as keloid.

32. Dermatofibroma: It is also called Benign Fibrous Histiocytoma. It is an asymptomatic

nodule, 3-10 mm in diameter, domed, but sometimes depressed below skin. Surface is

dull, shiny or scaly. It is firm, with variable colour. It stains positive for factor XIIIA.

33. Dermatofibrosarcoma Protuberans: It is a fibrohistiocytic type soft tissue sarcoma that

develops in dermis/hypodermis. It is of intermediate to low-grade malignancy. Sclerosing

or atrophic type particularly resembles a keloid. It stains positive for CD34 antigen.

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34. Malignant Fibrous Histiocytoma: It is also called Pleomorphic Undifferentiated

Sarcoma. Cells are of diverse type, and do not fit into a specific type of sarcoma.

35. Plexiform Fibrohistiocytic Tumour

36. Angiofibroma

37. Angiofibroblastoma

38. Collagenoma

39. Storiform Collagenoma

40. Elastoma

41. Elastofibroma

42. Digital Fibromyxoma

43. Fibroxanthoma

44. Fibroepithelial Polyp

45. Hyaline Fibroma

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46. Pleomorphic Fibroma

47. Acral Fibrokeratoma

48. Giant Cell Fibroblastoma

49. Infantile Desmoid Fibromatosis

50. Infantile Digital Fibromatosis

51. Infantile Fibrous Hamartoma

52. Infantile Lipofibromatosis

53. Infantile Myofibromatosis

54. Juvenile Aponeurotic Fibromatosis

55. Juvenile Calcifying Aponeurotic Fibromatosis

56. Juvenile Calcifying Fibrous Pseudotumour

57. Juvenile Collagenoma

58. Juvenile Elastoma

59. Juvenile Hyaline Fibromatosis

60. Juvenile Palmoplantar Fibromatosis

61. Juvenile Xanthogranuloma

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Figure 56: Morphea (Google, 2015 [Bib])

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36. Microscopic Diagnosis

Microscopic differential diagnosis is basically laboratory diagnosis of Fibrous tissue.

It can be due to repair process, called Fibrosis; or it can be due to neoplasm, called Fibroma or Fibromatosis or Fibrosarcoma. In central nervous system, there are no fibroblasts and no collagen, except in meninges or blood vessel; connective tissue consists of glia cells. So end result of repair is Gliosis, consisting of glia cells containing fibrils. In peripheral nervous system, connective tissue cells are called Schwann cells, and repair process Schwannosis.

Skin:

1. to 61 differential diagnoses of skin fibroses are given in previous Chapter (Figure 57).

Musculoskeletal System:

62. Palmer Fibromatosis (Dupuytren)

63. Plantar Fibromatosis

64. Volkmann’s Contracture

65. Myositis Proliferans

66. Nodular Fasciitis

67. Ischaemic Fasciitis

68. Eosinophilic Fasciitis

69. Fibrosarcoma of Soft Tissues

70. Myofibroblastic Sarcoma

71. Tumefactive Fibro-inflammatory Lesions

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72. Tenosynovial Fibroma

73. Fibromatosis Colli

74. Fibrous Dysplasia

75. Osteofibrous Dysplasia

76. Fibrosarcoma of Bones

77. Bone Callus

78. Arthrofibrosis

79. Adhesive Capsulitis

80. Burn Contracture

81. Nodular Synovitis

82. Hypertrophic Osteoarthropathy

Gastrointestinal System:

83. Hypertrophic Gastropathy

84. Hypertrophic Pyloric Stenosis

85. Cystic Fibrosis Intestine

86. Crohn's Disease

87. Intestinal Stricture/Stenosis

88. Peritoneal Adhesions

89. Intestinal Keloid: (Mahboubi et al, 1981)

90. Umbilical Keloid: (Ikard and Wahl, 1990)

91. Cystic Fibrosis of Liver

92. Echinococcosis Fibrosis of Liver

93. Cirrhosis

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94. Sclerosing Cholangitis

95. Common Bile Duct Keloid: (Pruemers, 1962)

96. Cystic Fibrosis of Pancreas

97. Idiopathic Retroperitoneal Fibrosis: (Hughes and Buckley, 1993)

Respiratory System:

98. Pulmonary Fibrosis

99. Idiopathic Pulmonary Fibrosis

100. Cystic Fibrosis of Lung

101. Progressive Massive Fibrosis of Lung

102. Echinococcosis Fibrosis of Lung

103. Inflammatory Pseudotumour of Lung

104. Organizes Pneumonia

105. Solitary Fibrous Tumour of Pleura

106. Mediastinal Fibrosis

Nervous System:

107. Gliosis

108. Meningeal Keloid: (Martinez et al, 1973)

109. Neurofibroma

110. Neurofibrolipoma

111. Neurofibrosarcoma

112. Schwannoma

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113. Schwannosis

Urogenital System:

114. Cystic Fibrosis Kidney

115. Penile Fibromatosis (Peyronie)

116. Bladder Neck Stenosis

117. Uterine Keloid: (Noble, 1959)

118. Fibroid Uterus: (Leppert et al, 2006)

119. Vaginal Keloid: (Ntiri, 1993)

120. Myometrial Hypertrophy.

Cardiovascular System:

121. Coronary Artery Stenosis

122. Atrial Fibrosis

123. Endomyocardial Fibrosis

124. Old Myocardial Infarction

125. Hypertrophic Cardiomyopathy

126. Atherosclerosis

Breast& Endocrine System:

127. Fibroadenoma

128. Fibroadenosis

129. Gigantomastia

130. Scirrhous Carcinoma

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131. Sclerosing Lymphocytic Lobulitis

132. Pseudoangiomatous Hyperplasia

133. Hashimoto’s Thyroiditis

134. Riedel Thyroiditis

Eyes& ENT:

135. Corneal Keloid

136. Conjunctival Keloid: (Lewis, 1951)

137. Proliferative Vitreo-retinopathy

138. Nasopharyngeal Angiofibroma

139. Mucosal Keloid: (Ow, 1989)

140. Gingival Keloid

141. Vocal Cord Keloid: (Sherik, 1961)

Multi-Organ Systems:

142. Myelofibrosis

143. Polyfibromatosis (Gardner)

144. Nephrogenic Systemic Fibrosis

145. Systemic Sclerosis/Scleroderma

146. Calcified Keloid: (Redmond and Baker, 1983)

147. Congenital Keloid (Matthews, 1940)

148. Mienicki's Keloid

149. Foliaceus Keloid (Gougerot, 1950)

150. Suppurative Keloid

151. Ulcerative Keloid (MacCormac, 1934)

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Figure 57: Acne Keloidosis Nuchae

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37. Differentiation of Keloids & Hypertrophic Scars

Keloids are usually differentiated from Hypertrophic Scars by spread beyond primary injury site. Keloids are also thought to keep growing indefinitely against Hypertrophic Scars which stop growing after sometime. Keloids are considered to have symptoms of pruritus, burning sensation, and pain, which are said to be absent in Hypertrophic Scar. But there is no objective, definite, agreed cut-off point between keloid and Hypertrophic Scar. The author considers them as variation on the same theme, with milder form being called Hypertrophic

Scar, and severer form Keloid (Nagi & Babar, 2015 [Bib]) (Figure 58).

In a case presented by MacLeod (1911) differentiation was made between Keloids and Hypertrophic Scars: ‘The President (Dr. T. Colcott Fox) said it was a nice point in the case as to whether the condition was true keloid or hypertrophic scar-tissue. In his own experience the multiple hypertrophic scars of a generalized syphilide tended to resolve spontaneously, and were not true keloids. Dr. Pernet said he regarded the case as one of hypertrophic scarring, not as true keloid.’ Dore (1918) tried to differentiate between keloid and hypertrophic scar: ‘My own feeling was that radium should be applied at once, but Dr.

Whitfield is of the opinion that cases of this type, which he regards as one of hypertrophic scar tissue, and not true keloid, improve spontaneously, and should not be treated for several months.’ Kischer (1984) compared ultrastructure of keloids and hypertrophic scars. He found quiescent fibroblasts in keloids, and active in hypertrophic scars.

McGrouther (1994) suggested not to differentiate between hypertrophic scars and

214 keloids, and to invest our time and resources in research on their treatment. He wrote:

‘Further attempts to achieve a clinical distinction between hypertrophic and keloid scars seem pointless. Research in recent years has shifted from the extracellular components towards the cells themselves. Much more work needs to be done to characterize the activities of the various cell lines and the mechanisms of their control. A key question is whether the cells are due to a different subpopulation of fibroblasts or whether they are normal wound-healing cells acting under some chemical or physical influence.’ Ehlrich et al (1994) described structural and immunochemical dissimilarities of keloids and hypertrophic scars. They reported: ‘Keloids contain large, thick collagen fibers composed of numerous fibrils closely packed together. In contrast hypertrophic scars exhibit nodular structures in which fibroblastic cells, small vessels, and fine, randomly organized collagen fibers are present.’

Zhang et al (2000) reported that hypertrophic scar and keloid could be differentiated by intracellular actin, F actin and F/G ratio; they were increased in hypertrophic scars than in keloids. Roseborough et al (2004) complained about confusing terminology of scars. They wrote; ‘advances in scar management have been hampered by the confusing or ambiguous terminology. There is no consensus on what amount of post-traumatic skin scar formation is normal and what should be considered hypertrophic. In the World Health Organization's

ICD-9, there is no diagnostic code for hypertrophic scar--only keloid is listed. Yet, the medical and scientific literature distinguishes them as different conditions.’

Lee et al (2004) gave histological differentiation of keloid and hypertrophic scar: ‘The features more commonly seen in keloids were: (a) no flattening of the overlying epidermis,

(b) no scarring of the papillary dermis, (c) presence of keloidal collagen, (d) absence of

215 prominent vertically oriented blood vessels, (e) presence of prominent disarray of fibrous fascicles/nodules, (f) presence of a tongue-like advancing edge underneath normal-appearing epidermis and papillary dermis, (g) horizontal cellular fibrous band in the upper reticular dermis, and (h) prominent fascia-like fibrous band.’

Huang et al in 2014 asked a question: Are Keloid and Hypertrophic Scar different forms of the same disorder? They themselves answered: ‘Hypertrophic Scars and Keloids are commonly seen as two different diseases by both clinicians and pathologists. However, as supported by histological evidence showing they share increased numbers of fibroblasts and accumulate collagen products, Hypertrophic Scar and Keloid might be different forms of the same pathological entity, rather than separate conditions. To test this hypothesis, Keloids from patients who underwent scar excisions were examined histologically. In keloid samples, coexistence of hyalinised collagen, which is the most important pathognomonic characteristic of a Keloid, and dermal nodules that are considered to be characteristic of Hypertrophic Scar, was found. Moreover, hyalinised fibres appeared to initiate from the corner of the dermal nodules. Key features of inflammation such as microvessels, fibroblasts, and inflammatory cells, all decreased gradually from the periphery to the centre of keloids, indicative of reduced inflammation in the centre. Thus, we hypothesise that Hypertrophic Scars and Keloid can be considered as successive stages of the same fibroproliferative skin disorder, with differing degrees of inflammation that might be affected by genetic predisposition.’ Burd

(2008) in an editorial titled: So what is a keloid scar? questioned attitude toward keloids: ‘We have individual concepts about keloid, but it is not considered a well-defined category.’

Kose and Waseem (2008) observed that keloids and hypertrophic scars were two

216 stages of same process. They made following Table of Differentiation:

Keloid Hypertrophic Scar

Collagen bundles Large, thick, closely Fine, well-organized, wavy,

packed, random to parallel to epidermis

epidermis

Myofibroblast Absent Present

(α-SMA expressing) Around blood vessel wall Nodular formation

(PCNA expressing) High expression Low expression

Mucin deposition Focal expression in dermis Negative

ATP level High expression Low expression

Hyaluronic acid Thickened, granular/ Major component papillary localization spinous layer dermis

Amorphous substance Diffuse pattern Absent on electron microscopy

Apoptosis Increased/decreased Decreased

p53 level High Low (Kose and Waseem, 2008)

217

(Brunicardi et al, 2015 [Bib]) made following Table of Differentiation:

Keloid Hypertrophic Scar

Incidence Rare Frequent

Ethnicity African, Asian, Hispanic No predilection

Prior injury Yes Yes

Site Neck, chest, ear lobes, Anywhere

shoulders, back

Genetics Autosomal dominant, No

with incomplete

penetration

Timing Symptom-free interval; 4–6 weeks post-injury

may appear years

after injury

Symptoms Pain, pruritus, hyper- Raised, some pruritus,

aesthesia, growth beyond respects wound confines

wound margins

Regression No Frequent spontaneous

Contracture Rare Frequent

Histology Hypocellular, thick, Parallel orientation

wavy collagen fibres in of collagen fibres

random orientation

218

Figure 58: Hypertrophic Scar

219

38. Prophylaxis of Keloids & Hypertrophic Scars

Keloids and Hypertrophic Scars are relatively easy to prevent, very difficult to treat, and near impossible to cure. The best treatment is prevention, especially in patients with a known predisposition to Keloid formation. Patients prone to keloid formation should avoid any other cosmetic surgery. They should also inform the surgeon when undergoing any other type of surgery, so that surgeon takes necessary precautions. Prevention also includes better wound care (Williams et al, 2013 [Bib]). Following treatment methods are also used for prevention of Keloids and Hypertrophic Scars in patients of burns, injury, skin infections, or keloid prone patients undergoing surgery, especially at high risk sites. Whitehill (1954) proposed prophylactic measures in keloid prone patients undergoing surgery. Meyer (1956) warned cosmetic surgeons to be aware of tendency to keloid formation in certain patients, which could ruin outcome of cosmetic operations (Figure 59).

1. Surgical Techniques

2. Steroids, Intralesional

3. Silicone Sheet

4. Pressure Garments

5. Laser

6. Cytotoxics, Intralesional

7. Immunomodulators, Topical

8. Miscellaneous Treatments

9. Symptomatic Treatments

As these methods are also employed for management of Keloids and Hypertrophic

Scars, their details are given in Treatment section

220

Figure 59: Keloid formation after Facelift Operation 221

39. Treatment of Keloids & Hypertrophic Scars

Keloids & Hypertrophic Scars.

Because exact cause of Keloids & Hypertrophic Scars is still not known, many modalities of treatment are being used without much success. Transforming Growth Factor

Beta causes formation of exuberant fibrous tissue. Cytokines which inhibit pro-fibrotic growth factors may be future treatment of Keloids & Hypertrophic Scars. At present there is no randomized controlled trial available evaluating the efficacy of cytokine Interferon

Gamma in the treatment of Keloids & Hypertrophic Scars. As the treatment of Keloids &

Hypertrophic Scars is a challenging problem, development of new and better therapies is need of the hour.

Treatments of keloids and hypertrophic scars are classified into first line, second line

222 etc, based on their popularity with treating physicians, and not on strict scientific evidence.

First Line Treatments: No of Types

1. Surgery 15

2. Steroids, Intralesional 6

3. Silicone Sheet 4

4. Pressure Garments 11

Second Line Treatments:

5. Laser Surgery 7+1

6. Cryosurgery 3+1

7. Radiotherapy 6+5

8. Cytotoxics 6

Third Line Treatments:

9. Immunomodulators, Topical 2

10. Calcineurin Inhibitors, Topical 3

11. Verapamil, Intralesional 2

12. Botulinum Toxins, Intralesional 1

Future Treatments:

13. Cytokines/Anti-Cytokines 3+3

14. Growth Factors/Anti-Growth Factors 3+3

15. Gene Therapy 6

16. Stem Cell Therapy 6

223

17. Nano-Medicine

Miscellaneous Treatments:

18. Acupuncture

19. Antihistamines, Systemic

20. Asiaticoside, Topical

21. Banding

22. Beta AminoPropioNtile fumarate (BAPN), Oral

23. Camouflage/Make-up

24. Captopril/Enalapril, Topical

25. Chemical Peel

26. Citalopram

27. Colchicine, Oral

28. Cyanoacrylates

29. Fatty Acids, Topical

30. Hyaluronidase, Intralesional

31. Hydrogel Dressing

32. Micro-Needling

33. Moist Exposed Burn Ointment (MEBO)

34. Mucopolysaccharide Polysulfuric Acid Ester (Hirudoid)

35. Mugwort Lotion

36. Onion Extract, Topical

37. Penicillamine, Oral

38. Pentoxifylline, Oral

39. Perfinodone

224

40. Phenol, Topical

41. Photodynamic Therapy

42. Phototherapy

43. Prostaglandin E2, Topical

44. Putrescine (tetramethylenediamine)

45. Radiofrequency Waves

46. Retinoids (Retinoic Acid, Tretinoin), Topical

47. Steroids, Topical

48. Surgical Paper Tape

49. Tamoxifen, Topical

50. Tattooing

51. Tranilast

52. Vitamins E, Topical

53. Zinc, Topical

Symptomatic Treatments:

54. Allantoin, Topical

55. Anaesthetics, Topical

56. Analgesics, Systemic and Topical

57. Emollients, Topical

58. Heparin, Topical

59. Massage

60. NSAIDs, Systemic and Topical

61. Physiotherapy

62. Prickly Heat Powder

225

63. Prostaglandin Inhibitors

64. Psychological Counselling

65. Splints, Static and Dynamic

66. Water Bath

Historical Treatments:

67. Aminocaproates

68. Anaesthetics, Infiltration

69. Bee Venom

70. Caustic Potash

71. Charpy’s Method

72. Chloroquine, Intralesional

73. Chymotrypsin

74. Collagen Cream

75. Collagenase, Intralesional

76. Dermabrasion

77. Dibunol (Butylated Hydroxytoluene)

78. Dimethyl Sulfoxide

79. Fibrolysin, Intralesional

80. Gate’s Method

81. Glycerol

82. Haprinoids

83. Histaminase

84. Honey

85. Hyaluronoglucosaminidase

86. Iodine

226

87. Iron

88. Lactic Acid

89. Lemon Juice

90. Lipotalon

91. Magnesium Lithospermate

92. Magnetics

93. Microwaves

94. Minoxidil

95. Mucopolysaccharide Cream:

96. Oestrogen

97. Orgotein(superoxide dismutase)

98. Oxygen

99. Parathyroidectomy

100. Pyrogens

101. Quinones, Oral

102. Scarification

103. Sulphur Douches

104. Thiosinamine, Intralesional

105. Ultrasound

106. Urea

107. Vitamin B

108. Vitamin C

109. Vitamin K

110. Volon A Tincture

111. Xymedone

227

Figure 60:B; Thomas Addison 228

40. Surgery

Surgery is a first-line treatment for Keloids. Different types of surgical operations are used for Keloids. They are customized for individual patient and Keloid. Dore (1918) wrote:

‘With regard to treatment, --- If it is a keloid ---I doubt the advisability of excision, although I have heard it stated that if a keloid is sufficiently widely excised it does not recur.’ Even after about 100 years, the best surgical treatment of Keloid is still not clear, and further research is needed in this regard. Following are different surgical management methods are there for

Keloids and Hypertrophic Scars. Surgery should be customized according to individual patient, individual keloid, and individual site.

1. Surgical Excision and Primary Suturing: This is done for small/linear/pedunculated

keloids. Stitching is done with non-absorbable sutures like polypropylene (Figure 61).

This can be combined with intralesional injections, silicone sheet, compression garments,

and symptomatic treatment. First injection should be intraoperative, in wound margins.

2. Surgical Excision and Split Thickness Skin Grafting: This operation is used for large

or irregular keloids. Special measures are taken to prevent donor site keloid. Porter (1909)

first mentioned skin graft in the treatment of keloids. Pierre (1950) described treatment of

keloids by surgical excision, split-thickness skin grafting, and adjuvant radiotherapy

3. Surgical Excision and Full Thickness Skin Grafting: This is used for small keloids, as

large full thickness graft is not feasible due to donor site problems. It is applied at

cosmetically or functionally important areas eg palm of hand. Opolski (1967) presented a

case of keloid of nose treated with excision and full thickness skin grafting.

229

4. Surgical Excision and Flap Reconstruction: This technique is most often used on

cosmetically or functionally important areas like face and neck. Flaps of large size can be

applied, and donor area closed by primary suturing.

5. Surgical Excision and Acellular Human Dermal (Cadaveric) Allograft (AlloDerm®):

Acellular human (cadaveric) dermal graft is used as a skin substitute. It is available as

commercial preparation, and there is no immunological reaction, as it is not antigenic.

6. Surgical Excision and Tissue Engineered Dermal (Fibroblast &Collagen) Allograft:

Integra® is a bilayered membrane dermal substitute comprising neonatal dermal

fibroblasts applied on collagen matrix. Apligraph® is a bilayered membrane comprising

neonatal dermal fibroblasts applied on collagen matrix.

7. Surgical Excision and Tissue Engineered Epidermal (Keratinocyte) Allograft

(Epicel®): It is cultured keratinocyte autograft. Keratinocytes are taken from the patient

and cultured, and applied to wound as epidermal graft. Teepe et al (1990) described it for

the first time.

8. Shave Excision: This technique has been used for superficial keloids. Humbey knife is

used for this purpose. Mostafa and Abdel-Fattah (1976) recommended shaving and skin

grafting for hypertrophic scars after burns.

9. Shave Excision and Split Thickness Skin Grafting: Sometimes split thickness skin

graft is applied after shave excision. It has better results than shave excision alone.

230

10. Contracture Release with Small Wave Incision: This is used if there is a contracture

associated with keloid. Hyakusoku and Ogawa (2003) described this incision, W-plasty or

Z-plasty can also be used in such cases.

11. Intralesional/Intramarginal/Intrakeloidal Excision: It has relatively better results and

less recurrence rate than extralesional/extramarginal/extrakeloidal excision. Sugawara &

Hashimoto (1967) described hollowing-out method for treatment of keloids.

12. Subcutaneous/Fascial Tensile Reduction Sutures: They minimize tension in dermis,

and decrease recurrence rate after surgery.

13. Debulking Surgery: It is done for large keloids, especially if there is infection or

ulceration. Adjuvant treatment becomes easier after debulking.

14. Liposuction: In this method, fat is removed from abdomen and thighs, and then Keloid is

excised, thereby reducing tension during suturing (Ma et al, 2010).

15. Tissue Expansion: Hagerty and Zubowitz (1986) reported use of tissue expander in

treatment of hypertrophic scars.

Surgical excision has a response rate of 100%, and recurrence rate of 50 to 100%. If surgical excision is combined with adjuvants, recurrence rate is decreased. Berman and

Bieley (1996) in a review article reported a recurrence rate, with surgery alone for keloids from 45% to 100%. If surgery was accompanied by intralesional steroids, relapse rate was

50%. Surgery along with compression treatment on earlobes had zero recurrences. Adjuvant radiotherapy after surgical excision had 10% recurrence rate. Lasers had similar recurrence rate as surgery; relapse decreased if it was accompanied by adjuvant therapy. Interferon-alpha

2b given after surgical excision had 8% recurrence rate.

231

Figure 61: Surgical Excision of a Pedunculated Keloid (Google, 2015) 232

41. Steroids, Intralesional

Steroids are first-line therapy for Keloids. There are two groups of steroid hormones; mineralocorticoids and glucocorticoids. The term steroid used without qualification generally means glucocorticoids. Steroids are normally produced in adrenal cortex from cholesterol.

There are also many synthetic steroids used for treatment of different conditions. Steroid suppresses every component of inflammatory process. It inhibits phospholipase A2, thereby decreasing synthesis of arachidonic acid which is predecessor of prostaglandins and leukotrienes. It increases production of anti-inflammatory cytokines like bFGF, decreases synthesis of proinflammatory cytokines like TGFB 1, VEGF, IGF-1 and interleukins, suppresses cell-mediated immunity, reduces complement synthesis, and decrease activity of leukocytes (Katzung, 2015 [Bib]). In Keloids, it causes inhibition of fibroblast activity and proliferation, decrease in collagen and glycoprotein synthesis, and ultimately fibroblast degeneration. Asboe-Hansen (1955) described treatment of keloids with intralesional injections of a steroid (hydrocortisone acetate) for the first time.

Complications of intralesional corticosteroid treatment are skin atrophy, hypopigmentation or hyperpigmentation, and telangiectasia. Intralesional steroid injections is not feasible for large or multiple keloids, as of large doses of corticosteroids required. Jemec

(1988) reported subcutaneous fat atrophy with intralesional steroid injection.

Response to steroid therapy varies from 50% to 100%, and it has 10% to 50% relapse rate. Their efficacy is dependent on dosage. Steroids may be used as single agent, but if used as adjuvant to other therapies, it gives greater results and lesser side effects. Injection should be given inside keloid, which usually causes much pain. Volume of injection is dependent

233 upon size of Keloid, site of body, and age of patient. Intralesional Steroids used to treat

Keloid and Hypertrophic Scars are given below. One ml injection is sufficient for four cm2

Keloid (Figure 62).

1. Triamcinolone Acetonide: It is the most popular steroid used for intralesional treatment

of Keloids and Hypertrophic Scars. It has intermediate duration of action. It is given in

dose of 40 mg ml-1. Triamcinolone Acetonide is discussed in detail in a separate Chapter.

2. Methylprednisolone Acetate: It is an intermediate acting steroid. It has a half-life of 12

to 36 hours. It is given as intralesional injection of 40 mg ml-1. Injections are given at

intervals of two weeks.

3. DexamethasoneSodium Phosphate: It has long duration of action. Its half-life is 36 to

54 hours. It is given as intralesional injection of 4 mg ml-1. Injections are given at

intervals of three weeks. Velazco and Rosenthal (1959) described use of intralesional

dexamethasone for treatment of keloids. Wilson et al (1965) injected dexamethasone in

post-operative wounds as prophylaxis against keloid formation.

4. Betamethasone Sodium Phosphate: It has long duration of action. Its half-life is 36 to

54 hours. It is given as intralesional injection of 4 mg ml-1. Injections are given at

intervals of three weeks. Viglioglia (1963) described keloid treatment with betamethasone

injection. He used the term intralesional for the first time. Golladay (1988) reported use of

betamethasone sodium phosphate for keloids.

5. Hydrocortisone Acetate: It is a short acting steroid. Its half-life is 8 to 12 hours. It is

used as intralesional injection in a dose of 50 mg ml-1. Injections are given weekly. It is

not popular now due to its short duration of action.

6. Cortisone: It is a short acting steroid. Its half-life is 8 to 12 hours. Historically, it has

been used as intralesional injection of Keloid, in a dose of 50 mg ml-1. Injections are

given weekly. It is no longer used now.

234

Figure 62: Intralesional Steroid Injection

235

42. Silicone Sheet

Silicone is a first-line preventive and curative therapy for keloids and hypertrophic scars. Silicone may be thought as gold standard in non-invasive treatment of Keloids. Mode of action of silicone is not known. It was considered that silicone penetrated skin, but most reports showed no silicone in scar or skin. It was also proved that there was no marked dissimilarity in pressures produced at scar surface, in scar surface temperature and oxygen tension, or water vapour transmissivity of silicon. Quinn et al (1985) for the first time reported use of silicone gel for keloid treatment. Kuhn et al (2001) proved that silicone sheet acted by decreasing fibrogenic cytokines like TGF beta 2.

However, Silicone is thought to prevent and treat Keloids by first occlusion and later hydration of keloid. Effect of enhanced occlusion and hydration on new scar is to give newly formed stratum corneum help in holding optimum amount of water. If the stratum corneum is dry, it signals keratinocytes in epidermis to secrete cytokines, which signal fibroblasts to form large volume of collagen to help water retention in stratum corneum. Silicone Sheet provides amount of occlusion to scar, which is same as in normal skin; so cytokine and fibroblast action and collagen synthesis are markedly reduced, and stratum corneum hydration is normalized. Silicon sheet can be removed very gently, compared with non- silicone sheets; this minimizes injury to freshly made stratum corneum (Figure 63).

Another mechanism of action of silicone sheet is to transfer tension from edges of scar to silicone sheet. This decrease in tension provides good milieu for normal scar formation and significantly reduces rate of keloid formation. Silicon sheet also inhibits the body's natural reaction to inflammation to cause increased blood supply. This decreases blood

236 supply to scar and exuberance of healing, thereby reducing chances of abnormal scar formation. Another mode of action is that silicone sheet generates a negative electric field by producing friction between itself and skin. This static electricity aids in alignment of collagen fibres, thus causing reduction of hypertrophic scars.

Silicone (polydimethylsiloxane) is available in the form of sheet as well as gel, cream, and oil. Silicone Sheet is more popular than Ge, Cream or Oil, as it is economical, and has theoretical advantage of applying some pressure on Keloid. It is a soft, flexible, self-adhesive, semi-occlusive gel sheet, armoured with a silicone membrane support to give it enhanced durability and easy usability. It is applied on intact skin. Silicone sheet application is painless and is maintained for 24 hours a day for 3-6 months. It can be applied under a pressure garment. It has a few side effects like pruritus, contact dermatitis and dry skin. It has a remission rate of 50-100%, and relapse rate of 25-35%.

Non-sheet silicone gel is a viscous silicone preparation that forms a thin membrane on skin. Gel is an invisible, air permeable, water-repellent, and odourless. It has been produced to circumvent some problems faced with silicone gel sheet, like difficulties in securing it, as well as using it on big areas, near joints, or on exposed areas like face and hands. It is applied twice daily as a very thin layer over scar. The treated area becomes non-sticky within 10 to

15 minutes. It dries and makes a flexible, transparent, gas-permeable, water-impermeable sheet. It is not necessary to cover treated area with a bandage. Silicone Sheet or Gel can be applied to wounds immediately after they have become epithelialized.

237

Figure 63: Silicone Sheet

238

43. Pressure Garments

Pressure garments are a first-line treatment for Keloids. Mostly they are made-up of elastic fabric. Other material used to make pressure garments include Dacron, and

Polyurethane. They are either knitted or woven. These are available in the form of jackets, knickers, sleeves, leggings, gloves, stockings, caps, or masks (Figure 64). They can also be made into elastocrepe bandages, tubigrip bandages, and compression earrings.

Mechanism of action of pressure devices is still not clearly understood. Suggested mechanisms of action include hypoxia, biochemical, cellular and collagenous changes.

Pressure may influence collagen production by reducing blood, oxygen, and nutrients supply to the keloid. Pressure may encourage realignment of collagen bundles, restore ECM organization to that of normal healing, and cause loss of fibrogenic α-SMA-expressing myofibroblasts apoptosis. Cellular mechanoreceptors may have an important role in compression therapy, by altering synthesis and action of matrix metalloproteinase (MMP)-28.

Mechanoreceptors induce apoptosis and are involved in integrity of extracellular matrix.

Costa et al (1999) reported that pressure accelerated remodelling phase of repair. Renò et al

(2001) said compression caused prostaglandin E2 release. Renò et al (2003) said compression induced apoptosis in keloid.

There are two ways of making pressure garments. Reduction factor technique is commonly used; it means reducing person’s circumferential size by a fixed percentage without considering cloth tension when sizing the patient. Other technique uses Laplace’s law, based on circumferential size of person and tension profile of cloth. It is impossible to apply pressure on concave areas of body with pressure garments alone. For these areas,

239 custom-built pressure devices are used, which are worn under pressure garments, to increase local pressure. These inserts are made up of foam or rubber, and should be worn continuously.

Pressure garments flatten scars, and improve pliability. A pressure of between 20 and

30 mm Hg is required. Garments are worn for 23 hours a day for up to 1 or more years. For better results, garments should be initiated as early as two weeks after injury; these can be applied even on unepithelized wounds. Scars of more than 6 to 12 months do not remit well.

There can be gradual loss of pressure from cloth. There are also difficulties regarding compliance of patients, especially in summer season. They can lead to overheating, itching, blistering, wound disruption, and stunted bone growth in children. Further, pressure garments are much discomforting in tropical countries. To increase compliance, pressure garments are being made of different colours/designs. Pressure garments have a response rate of 90-100%.

Pressure garments should be washed with hand using detergent, and should be drip dried. They should not be machine-washed, machine-dried, wrung, stretched, or ironed. Two sets of garments are recommended for every patient. In spite of best care, these garments gradually reduce their pressure. So they should be changed after every three months.

Lawrence (1898 AD) from Australia mentioned pressure therapy of keloids for the first time. Lintilhac et al (1966) described role of prolonged elastic compression in prevention of hypertrophic scars. Kloti and Pochon (1979) reported usefulness of Jobst compression suits in prevention and treatment of post-burn keloids in children. Le Coultre and Graber

(1985) used plastic face mask, and silicone gloves & stockings for treatment of keloids.

240

Figure 64: Pressure Garments 241

44. Laser Surgery

Laser (Light Amplification by Stimulated Emission of Radiation) is a high intensity, monochromatic, coherent light. Laser is named after the medium that produces laser beam eg

CO2. Medium can be gas, plasma, liquid, or solid. Sometimes an organic dye mixed in a solvent is used as lasing medium; a high energy source like flashlamp is used to pump lasing liquid. Laser Power is measured in Watts, Fluence (energy absorbed by tissues) in J cm-2,

Wavelength in nm, Spot in mm, and pulse in msec. Surrounding tissues are cooled to prevent damage. Multiple treatment sessions are required at intervals of one to three months. One treatment session takes one hour on average. Local anaesthesia may be required (Figure 65).

Laser energy is absorbed by haemoglobin, producing heat and causing coagulation of tissues. This further causes hypoperfusion and hypoxia, which ultimately changes collagen production by fibroblasts and collagen destruction due to metalloproteinase production. There is also heating of collagen fibres with disruption of disulphide bonds. Later new collagen forms, with realignment of collagen fibres. There is also decreased fibroblast growth and collagen type III production. There is also downregulation of TGF-β1 and rise in MMP-13

(collagenase-3) action. Laser also causes production of bFGF. Laser also causes fall mast cells number, and histamine production.

Depending upon their mechanism of action, lasers are of three types: Ablative Laser, which destroys mainly epidermis, eg CO2, Nd:YAG; Fractional Laser (multiple dots), which destroys both dermis and epidermis equally, eg CO2, Nd:YAG; and Non-ablative Laser, which destroys dermis only, and leaves epidermis intact, eg PDL, IPL. Depending upon their duration, lasers are of two types: Continuous Laser, which is applied as continuous wave eg

242

CO2, Nd:YAG; and Pulsed Laser, Q switched Laser, Pulsed Pump Laser, which is applied in pulses eg PDL, IPL. Depending upon their distance, lasers are of two types: Contact Laser, which acts in direct contact with lesion; and Non-contact Laser, which acts at a distance.

For Keloid treatment with pulsed laser, fluence ranges from 4.5-5.5 J cm-2 with spot size of 10 mm. Single pulses are given in an adjacent, non-overlapping manner. For ablative laser fluence of 14 J cm-2 is used. Side effects include temporary swelling, redness, and pigmentation. Wavelengths from 488 nm to 1064 nm are used for management of hypertrophic scars; 585 is most effective, as oxyhaemoglobin absorption peak is 542 nm.

Landthaler et al (1981) reported, for the first time, use of lasers in management of keloids. Richard and Boulnois (1983) reported Helio-Neon laser for keloids. Lenz (1985) reported punctiform argon ion laser for treatment of keloids. Kantor et al (1985) described management of ear keloids using CO2 laser. Hulsbergen Henning et al (1986) described management of keloids with Argon laser. Sherman and Rosenfeld (1988) reported use of

Nd:YAG laser in management of keloids. Astler (1994) reported treatment of keloids by pulsed dye laser.

Intense Pulse Light (IPL) is produced by polychromatic, noncoherent, broad-spectrum pulsed light source (xenon lamp). It emits 390 nm to 1200 nm wavelength light. It is based on principle of absorption of photons by exogenous or endogenous chromophores in skin; main chromophores of skin are haemoglobin, melanin, and water. Transmission of energy to target tissues produces heat and later necrosis due to selective photothermolysis. Wavelength is chosen according to absorption peak of the target chromophore. Pulse duration ought to be smaller as compared to thermal relaxation time. For Keloids treatment, wavelength of 500 nm and 550 nm is used.Laser-Assisted Skin Healing (LASH) is another method of treatment of keloids. Capon et al (2009).

243

Figure 65: Laser Treatment of Keloid

244

45. Cryosurgery

Cryosurgery means destruction of a lesion with localized freezing temperature; it is basically a cold burn. It is mainly used for skin lesions. Cryosurgery involves rapid freezing and slow thawing, with multiple freeze-thaw cycles, and multiple sessions. Cryosurgery uses solid carbon dioxide, liquid nitrogen, or gaseous argon; liquid nitrogen is the most popular.

Application is in the form of direct contact of stick/probe, or spray of liquid; spray transfers heat faster and is more damaging to lesions, but firm pressure and use of lubricating jelly speed freezing time when using a stick/probe. Compressed gases like nitrous oxide (N2O) or carbon dioxide (CO2) can also be employed to get low temperatures required for cryosurgery; gas is allowed to expand using a valve in metal tip of cryosurgical probe, thus making tip to very cold. Mechanism of action is freezing of tissue; when tissue is frozen, it is injured by ice crystal formation within cells, vascular thrombosis and stasis, anoxia, and release of free radicals and toxins. Tissue destroyed is in the form of an ice ball. Frozen tissue ends up with peripheral erythema, followed by oedema, bulla formation, exudation and mummification.

Lesions generally heal leaving a fine scar four weeks after treatment. Local anaesthesia may be needed (Figure 66).

Cryotherapy is relatively safe, with rare side effects and contraindications. Pain, bulla formation and oedema are main temporary side effects; hypopigmentation and/or peripheral hyperpigmentation is the most common long-term adverse effect. Contraindications to cryotherapy include cryofibrinogenaemia, cryoglobulinaemia, Raynaud’s disease, agammaglobulinaemia, and multiple myeloma.

For treatment of Keloids, generally liquid nitrogen spray is used. Tissue temperature

245 of -30° C to -60° C is required. Freeze time is upto 30 seconds, and thaw is allowed its own time. Ice ball should have a margin of 2 mm normal tissue. One to three freeze-thaw cycles are required, and management is done in three sessions, at intervals of two months. Treatment is more successful in Keloids that are recent and smaller in size.

Intralesional cryotherapy is emerging as an alternative to external cryotherapy. It causes destruction of Keloid with minimal damage to epidermis. A lumbar puncture needle is passed through long axis of keloid, from one side to other, passing liquid nitrogen through it with an intravenous drip set for two freeze-thaw cycles of 20-30 seconds each for 5-10 sessions. It freezes keloids from inside out. This new procedure is safe, well tolerated, and more efficacious than topical cryotherapy (Abdel-Meguid, 2015).In case of bigger keloids, multiple needle insertions are required to freeze it completely. Simultaneous use of multiple needles can also be done. This procedure is done under local anaesthesia.

Response rate with cryosurgery is 50 to 75%. van Leeuwen et al in 2015 did a meta- analysis of intralesional cryosurgery for Keloids. Eight trials fulfilling inclusion criteria were included. Mean scar volume reduction was 51% to 63%, but no complete scar elimination was produced. Keloid relapse rate was 0% to 24%. Hypopigmentation was found in

Fitzpatrick 4-6 patients. Symptoms of pain and itching reduced markedly in most trials.

Pierce (1974) described treatment of keloids with cryosurgery, for the first time.

Babin and Ceilley (1979) combined cryotherapy with intralesional triamcinolone. Muti and

Ponzio (1983) emphasized the use of nitrogen protoxide cryotherapy for keloids.

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Figure 66: Cryosurgery for Keloid

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46. Radiotherapy

Radiotherapy is second-line therapy for Keloids. As monotherapy, it is used mainly for large Keloids that are refractory to other treatments, and also cannot be excised due to anatomical or other constraints. It is usually used after surgical excision of Keloids; sometimes pre-operative radiotherapy is also used. Radiotherapy is contraindicated in children, as it causes growth retardation, and increased incidence of malignancies.

Radiotherapy uses ionizing radiation, which acts by inhibition of proliferation of fibroblasts by causing damage to their DNA. Its response rate is60-90%and recurrence rate 10-100%.As adjuvant to surgery, its response rate is 80%.Side effects (45%) are paraesthesia, hyperpigmentation, pruritus, erythema, pain, and secondary malignancies (Figure 67).

Different types of radiotherapy sources are used for treatment of keloids, including kilovoltage (superficial), orthovoltage (deep), supervoltage, and megavoltage X-rays

(photons), gamma rays (photons), and megavoltage electrons (beta rays). Superficial X-rays machine can deliver homogeneous doses to skin surface, but have a high absorption rate to any bone below irradiated area. Electrons are delivered using high-energy lineal accelerators, with the lowest energy levels (4–6 MeV) being most appropriate for keloid treatment. With linear accelerator, dose homogeneity is easily achieved, and treatment can be applied for five minutes per day of treatment. However, entry-surface doses are too low with electrons and require a bolus dose in contact with skin. Radiotherapy dose for Keloids ranges from 15 to 30

Gray (Gy), usually given in fractions, with 3 to 5 Gy in each fraction (day). Dose may vary according to size and location of Keloid. Radiotherapy is started within 24 hours of excision, with treatment time less than seven days. High dose single fraction radiotherapy is also used.

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Morris & Dore (1909) mentioned X-rays radiotherapy for treatment of Keloids for the first time. Vasileva (1954) wrote an article on the treatment of keloids with superficial X- rays. Arnold and Grauer (1959) described treatment of keloid by surgical excision and prophylactic full-dose radiotherapy. Kik et al (1974) reported side effects of radiotherapy of keloids in children. Levy et al in 1976 reported post-operative low dose superficial x- irradiation after keloid excision. Ogawa et al (2003) studied recurrence rates in keloids treated with surgery and postoperative electron-beam irradiation.

Brachytherapy is also used in the treatment of Keloids. It is also used immediately after surgical excision. It is considered that brachytherapy treats keloids more efficiently than teletherapy. Dose of brachytherapy is higher than teletherapy ie 30-40 Gray. Brachytherapy can be of low-dose-rate (<2 Gy h-1) given over period of days, medium-dose-rate (2-12 Gy h-

1) given over period of hours, high-dose-rate (>12 Gy h-1) given over period of minutes, or pulsed-dose-rate given in short pulses of once per hour. It can be given both as contact

(surface applicator) as well as interstitial (implantation of wire/seeds) mode. Placement of radiation source is usually temporary and is removed after treatment, but sometimes it is permanent eg radioactive seeds, which gradually decay and need not be removed.

Radioactive wire passes through a catheter placed in wound during surgical excision of

Keloid, while radioactive seeds are themselves implanted into wound during surgery.

Brachytherapy gives beta radiation using Cobalt60, Strontium90, or Yttrium90.Gamma radiation is given usingIridium192, and electrons are given by I125.

Dore (1918) mentioned radium as treatment of keloid. Hudson (1934) presented a case of radium needle therapy for keloid. Jakob and Balz (1959) described treatment of keloids with radioactive strontium and yttrium. Nicoletis et al (1967) described interstitial radiotherapy with iridium. Malaker et al (1976) reported keloid treatment with excision and irradiation delivered by an iridium wire.

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Figure 67:B; Radiotherapy Machine 250

47. Cytotoxics

Cytotoxics are second-line therapy for Keloids. They are usually used as adjuvant to surgical excision. They can be combined with other intralesional agents. Some of cytotoxics are given as topical on wound just after keloid excision, and some are given systemically.

Cytotoxic drugs have many side effects, some of which are serious (Katzung, 2015 [Bib]).

Onwukwe (1980) reported, for the first time, treatment of keloids with antineoplastics ie methotrexate. Datubo-Brown (1990) reported use of systemic chemotherapy in treatment of keloids. Important cytotoxics used for treatment of Keloids are described below.

1. 5 Fluorouracil, Intralesional; Topical on Wound: It is a fluorinated pyrimidine

analogue. It is an antimetabolite that prevents conversion of uridine into thymidine by

blocking thymidylate synthase. Thymidine is necessary for DNA replication; lack of

thymidine results in death of rapidly dividing cells. Therefore, 5-FU inhibits proliferation

of fibroblasts, and limits uncontrolled production of collagen fibres by fibroblasts. It also

inhibits proliferation and differentiation of myofibroblasts. This is considered to be due to

inhibition of proinflammatory cytokines and growth factors eg TGF Beta 1. It is used in

keloids both in isolation and in combination with other substances. It is used as

intralesional injection in a dose of 50 mg/ml per session at weekly intervals, with a total

dose of 500 mg. Response rate is 50%, and recurrence rate 20%.Thrice weekly injections

have also been used, with a response rate of 90%. Best response is found with new,

symptomatic and inflamed Keloids, whereas older, non-inflamed, asymptomatic Keloids

respond less. Side effects include erythema, pain, burning sensation, hyperpigmentation,

and ulceration. 5 FU can be combined with other intralesional injections eg

Triamcinolone acetonide. Uppal et al (2001) reported use of 5-fluorouracil for wound

irrigation after extralesional excision of keloids (Figure 68).

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2. Bleomycin, Intralesional: Bleomycin is a glycopeptide antitumour antibiotic derived

from Streptomyces verticellus that is widely used as an antineoplastic, antibacterial and

antiviral agent. Bleomycin decreases collagen synthesis, and increases apoptosis.

Bleomycin is used as intralesional injection or tattoo in a dose of 1.5 IU/ml to treat

keloids; 3-5 treatments are given at monthly intervals. Response rate of 90% is reported.

Side effects include pain, ulceration, hyperpigmentation, and pulmonary fibrosis. Espana

et al (2001) described use of intralesional Bleomycin for management of hypertrophic

scars. Naeini et al (2006) reported Bleomycin tattooing as treatment for large keloids.

3. Mitomycin C, Topical on Wound: Itis an antitumour antibiotic, which is produced by

Streptomyces cespitosus. It has both antineoplastic as well as antiproliferative properties.

It acts by alkylating and cross-linking DNA at adenosine and guanine nucleotides,

thereby blocking DNA, RNA and protein formation. It prevents fibroblast growth and

reduces scar production (Simmen et al, 2003). It is applied topically to wound bed after

excision of keloid for five minutes in a preparation of 0.4 mg/ml.

4. Methotrexate, Oral: Methotrexate is a folic acid derivative as well as antagonist. It is an

antimetabolite, competitive inhibitor of dihydrofolate reductase. Blocking of reduction of

dihydrofolate to tetrahydrofolate results in stoppage of DNA production due to inhibition

of another enzyme. It is given by oral route in a single dose of 20 mg every four days for

three months (Viera et al, 2010).

5. Doxorubicin (Adriamycin), Intralesional: It is an anthracycline. It irreversibly

inactivates prolyl 4-hydroxylase and inhibits collagen alpha-chain assembly. Another

mechanism of inhibition of collagen synthesis is inhibition of enzyme prolidase, which is

important enzyme for collagen resynthesis. It is given as intralesional injection.

6. Thiotepa, Intralesional: It is an alkylating agent, which is used for treatment of keloids.

It is given as intralesional injection.

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Figure 68:B; 5- Fluorouracil

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48. Immunomodulators, Topical

Immunomodulators are drugs used to regulate or modify immune system of body.

They either augment (immunostimulants) or diminish (immunosuppressants) immune response. They are also called biological response modifier. Immunomodulators include cytokines, growth factors, corticosteroids, cytotoxic agents, enzyme inhibitors, and immunoglobulins. Some of them are normally present in body. They are used as third-line drugs for treatment of Keloids (Katzung, 2015 [Bib]).

1. Imiquimod: It belongs to imidazoquinolone group, which is a class of immunoenhancing

drugs. It acts by stimulating macrophages by attaching to cell surface receptors, like Toll-

like receptors (TLR) 7/8, thus causing production of proinflammatory cytokines, mainly

interferon alpha, IFN gamma, TNF alpha and interleukin. Cytokine activation is due to a

highly complex process concerning innate and acquired immunity through receptors on

macrophages, Langerhans cells, and dendritic cells. Tyring (2001) suggested using topical

Imiquimod for prevention and management of keloids. Jacob et al in 2003 reported that

imiquimod acted by altering apoptosis genes. It is applied topically as a 5% cream

(Aldara®) daily for eight weeks. It is applied after surgical wound is fully healed. Side

effects of imiquimod include erythema, pain, ulceration, hyperpigmentation and

hypopigmentation (Figure 69).

2. Resimquimod: It is another drug of imidazoquinolone group. It is 100 times more potent

than imiquimod. It is also used as a topical agent for treatment of Keloids. It is available

as 0.01% gel. It is applied twice weekly. Side effects include erythema, pruritus,

ulceration, body aches, fever, and tiredness.

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Figure 69:B; Imiquimod 255

49. Calcineurin Inhibitors, Topical

Calcineurin Inhibitors are immunosuppressants used for different indications. They are used as third-line treatment of Keloids. Kim et al (2001) recommended topical use of rapamycin analogues, including tacrolimus, for keloids.

1. Tacrolimus: It is a macrolide antibiotic derived from an actinomycete Streptomyces

tsukubaensis. It is an immunosuppressant agent, which acts by many different

mechanisms, including inhibition of calcineurin, a protein phosphatase involved in

activating T-cells. It also suppresses production of interleukin 2. Oral form is generally

used to prevent rejection of transplanted organs. A patient of atopic dermatitis who

applied topical tacrolimus to his lesion found that his concomitant keloid resolved

completely; that led to use of Tacrolimus for psoriasis and keloid treatment (Katzung,

2015 [Bib}). Tacrolimus is used as 0.1% ointment (Protopic®). It is applied two times a

day for 12 weeks. Side effects of Tacrolimus ointment are stinging, burning sensation,

itching, tingling, headache, stuffy nose, skin infections, abdominal pain, muscle pain, and

enlarged lymph nodes. Rare side effects include nephrotoxicity, and risk of malignancies.

A few patients who used Tacrolimus ointment developed skin malignancy (Viera, 2010)

(Figure 70).

2. Sirolimus (Rapamycin): It is a macrolide antibiotic derived from Streptomyces

hygroscopicus. It inhibits the proper functioning of a serine/threonine protein kinase. It

inhibits mammalian target of Rapamycin (mTOR), inhibits response to interleukin 2, and

reduces extracellular matrix deposition. The mTOR is a regulator of collagen expression.

It prevents gli-1 signal transduction, and resultantly restores normal apoptosis, alongwith

reduced production of the ECM. Side effects include cough, skin rash, and face swelling.

It is used for skin diseases and Keloids as 0.1% ointment (Katzung, 2015 [Bib])

3. Cyclosporine: It is a peptide antibiotic; its mechanism of action is similar to Tacrolimus.

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Figure 70:B; Tacrolimus 257

50. Verapamil, Intralesional

Verapamil is a phenylalkylamine, a papaverine derivative. It is a calcium channel blocker, which inhibits entry of calcium ions from extracellular matrix to cytoplasm. It inhibits L-type and T-type channels, and has more affinity for depolarized channels than for non-depolarized channels. It stimulates procollagenase synthesis, and enhances collagenase action of ECM. It decreases synthesis and secretion of collagen, glycosaminoglycans, and fibronectin because the process by which these components are released from fibroblasts requires calcium. It also inhibits interleukin 6, VEGF, TGF beta 1, and proliferation of fibroblasts, causing depolymerization of actin filaments, and change in cell form from bipolar to spherical, apoptosis and ultimately decrease in fibrosis. It is given as intralesional injection in wound margins after excision of Keloid. It is given in a dosage of 2.5 mg/ml daily for 15 days. Topical verapamil is also used as a scar modulator. It is available as 15% gel.

Verapamil has also been used to treat other fibrotic conditions like Dupuytren’s contracture.

Lee and Ping (1990) studied use of calcium antagonists to retard extracellular matrix production. Lee R. C. et al (1994) used intralesional verapamil in keloid patients (Figure 71).

Side effects of verapamil include sinus bradycardia, hypotension, pulmonary oedema, atrioventricular block, constipation, heartburn, vertigo, light-headedness, and headache

(Katzung, 2015 [Bib]).

Verapamil is a third-line drug in treatment of keloids. It is only used as an adjuvant treatment along with surgery or other intralesional treatments. Topical treatment is rarely used. Liu et al (1998) reported inhibitory action of tetrandrine, another calcium channel blocker, on growth of human skin fibroblast.

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Figure 71:B; Verapamil 259

51. Botulinum Toxins, Intralesional

It is an exotoxin of neurotoxin type produced by Clostridium botulinum, an anaerobic gram-positive bacterium. Botulinum toxin is the most potent poison occurring naturally; 100 kg of it can kill the whole population of the world. It is 150-kDa protein comprising of a light chain (50 kDa), attached by single disulphide bond to a heavy chain (100 kDa). This molecule has three lobes, representing light chain, amino-terminal part of heavy chain, and carboxyl-terminal part of heavy chain. It prevents neuromuscular transmission by blocking receptors at presynaptic terminals of motor nerves, thus preventing release of acetylcholine.

Botulinum Toxin is of eight different types; A, B, C1, C2, D, E, F, and G. Type A is generally as therapeutic agent. It is being used for different types of dystonias, spasmodic dysphonia, intractable cervical pain, cricopharyngeal dysphagia, gustatory sweating, hyperhiderosis, blepharospasm, achalasia cardia, and wrinkles of face. It has been used for treatment of anal fissure (Figure 72).

It is being used as intralesional injection in management of Keloids. In order to decrease antibody resistance, least effective dosage is used. A preparation of 35 units ml-1, and total dosage of 70 to 140 units per sitting are being used, at three months interval for nine months. Another dosage recommendation is 2.5 units cm-2, at monthly sessions for three months (Viera et al, 2010). It is a third-line drug for management of keloids. Zhibo and

Miaobo (2008) suggested use of botulinum toxin A for management of keloid.

Complications of Botulinum Toxin are ptosis, ectropion, diplopia, bruising, haematoma, dryness of mouth, and headache.

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Figure 72: Botulinum Toxin 261

52. Cytokines & Anti-Cytokines

Cytokines are polypeptide molecules. The transmit signals amongst immune system cells, and also amongst other cells and tissues. Cytokines comprise a heterogeneous group and consist of interferons, TNF, chemokines, and growth factors. They perform main role in maintaining body milieu as well as in body defence. Modulation of production and activity of cytokines, and their use as drugs is under intensive as well as extensive research. Different methods are being used to further develop cytokines as therapeutic agents (Katzung, 2015

[Bib]).

Interferon alfa is a cytokine produced by a recombinant DNA technology using

Escherichia coli. Recombinant interferon alpha is produced in a number of forms, and is most effective against leukaemia, lymphoma, multiple myeloma, and chronic type C hepatitis.

Interferon alpha 2b is being used in management of Keloids. It is given in dose of 1.5 million

IU/ml two times a day for four days. Remission rate of 50% and relapse rate of 20% is reported. It may cause altered pigmentation. Berman and Duncan (1989), for the first time, used an Interferon, Alfa-2b, for treatment of keloids. Ghahary et al (1993) reported that keloid formation due to burns was due to increased expression of fibronectin mRNA, and interferon alfa-2b which downregulates this expression might be used in treatment of keloids.

Sahara et al (1993) reported decrease of in vitro keloid fibroblast contraction in the presence of interferon alfa-2b. Ghahary et al (1995) reported that interferon alpha-2b more useful than interferon-gamma in management of fibroproliferative diseases, like keloid. Berman and

Flores (1997) reported therapeutic advantage of postoperative interferon alpha 2b over triamcinolone acetonide.

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Interferon beta is a cytokine produced by modified Escherichia coli, and appears in a number of forms. It is used to treat multiple sclerosis. It is administered by sub-cutaneous injection and slows advance of affliction as well as reduces frequency of attacks. It is not used for treatment of keloids. Interferon gamma is discussed in a separate Chapter.

Interleukin 10 is another antifibrotic cytokine. IL-10 is the main suppressor of inflammation. It regulates fibroblast differentiation and proliferation. It downregulates IL-6 and IL-8, the profibrotic cytokines. In a study in animal were treated with IL-10, two days prior to production of wounds. Three days after injury, level of inflammatory mediators was reduced and collagen formation was absent. There was rebuilding of normal skin structure in contrast to wounds given placebo. Recombinant human Interlukin-10 (Ilodecakin) has been used in clinical trials. Intralesional injection of Ilodecakin to wound margins decreases incidence of Keloid formation (Figure 73).

Anti-Cytokine therapies include tumour necrosis factor alpha inhibitors (Etanercept,

Enbrel®), and mannose-6-phosphate inhibitors (Juvidex), which act by decreasing collagen synthesis. Castgnoli et al (1993) reported that TNF alpha is essential for wound repair and keloid might be a result of a low level of TNF alpha. Soluble Cytokine Receptors control cytokine activity, and are a future treatment of Keloids (Katzung, 2015 [Bib]).

Cytokines and related products under research in preclinical phase include Nexagon, an anti-connexin oligonucleotide; EGS-001, a bone morphogenetic protein-7 polypeptide; stabilized 20-mer oligo-deoxynucleotide, anti-sense to CTGF; and Inhibitors of procollagen-

C-Proteinase. Those in Phase I trial include IgG1 antibody binding to domain 2 of CTGF; and

24 amino acid peptide analogue of HSP 20 (Occleston et al, 2008).

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Figure 73: Effects of Cytokines on Fibroblasts to promote or prevent fibrosis (Backovic et al, 2010) 264

53. Growth Factors& Anti-Growth Factors

Growth Factors are protein molecules, which regulates cell division & cell survival.

They are secreted by cells of body, and function in an autocrine or paracrine manner. Growth factors attach themselves to cell surface receptors, causing cell growth and differentiation.

Some growth factors are multipotent, producing cell growth in various types of cells, whereas other growth factors are specific for a special type of cell. Growth factors may work as stimulators or inhibitors. They also increase or decrease cell migration, or work as chemotactic agents. Some growth factors stimulate apoptosis or angiogenesis. Growth factors are also responsible for wound repair. In this setting, they can be profibrotic or antifibrotic.

For therapeutic purposes, growth factors are produced by genetic engineering using E coli.

These are called human recombinant growth factors.

TGF-beta 3 can be used to decrease scar production and to manage. Exogenous addition of TGF-β3 is found to reduce fibronectin and collagen deposition in wound healing and scarring. Recombinant TGF beta 3 is available with generic name of Avotermin

(Juvista®).Topical Avotermin has shown good results in Keloid treatment. Tian et al (2000) reported that TGF beta 3 had action opposite to that of TGF beta 1 and 2 ie it helps maturation of scars. Miller and Nanchahal (2005) suggested that increasing the relative ratio of TGFbeta-3 to TGFbeta-1 and TGFbeta-2 will minimize scarring. Occleston et al (2008) reported prevention of keloid by TGF beta 3.Ferguson et al (2009) used TGFB3 for the treatment of scars, and found it significantly better. Mroweitz and Seifert (2009) reported use of recombinant TGF-B3, interleukin 10, and imatinib mesylate for treatment of keloids. Han et al (2012) described anti-motility signalling mechanism of TGFβ3.

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Basic fibroblast growth factor (bFGF) activates growth of various types of cells. It possesses angiogenic as well as mitotic functions, thereby manipulating remodelling, healing, neovascularization, and also enhancing tumour growth. It also markedly inhibits change of mesodermal progenitor cells to myofibroblasts. It also stimulates wound repair and enhances quality of scar by controlling ECM formation. Tan et al (1993) described fibroblast growth factor (FGF) and its action in regulation of various ECM proteins (Figure 74).

Hepatocyte growth factor (HGF) has angiogenic, antiapoptotic, antifibrotic, and regenerative, functions. It changes levels of various cytokines, like VEGF and TGF-β1, and may reduce formation of keloid. Ono et al injected HGF gene in edges of incised wounds on dorsum of rats and concluded that it decreased apoptosis as well as growth of fibroblasts and caused accelerated healing and improved scarring.

Anti-Growth Factor therapies include VEGF inhibitor Bevacizumab (Avastin®), Anti

TGF beta-1, and anti TGF beta-2. These agents can be used to treat keloids. Antisense TGF-

β1 oligonucleotides cause decrease in expression of TGF-β1 gene, and give rise to long term reduction of TGF-β-mediated keloid formation. Soluble growth factors receptors are also a future treatment for keloids, as they control activities of growth factors.

MicroRNAs (miRNAs) may be profibrotic and their upregulation causes fibrosis, while anti-fibrotic miRNAs inhibit give rise to reduction in fibrosis. Antifibrotic miRNAs may be upregulated with the use of viral vectors, and so may be used to treat keloids.

Opposite to this, profibrotic miRNAs can be downregulated by using anti-miRNAs, sponges, erasers and masks. MicroRNA treatment may be useful in management of keloids (Babalola et al, 2013).

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Figure 74: TGFB3 versus Placebo for the Treatment of Scars (Ferguson et al, 2009) 267

54. Gene Therapy

Many different studies have been done to find out role of gene therapy in management of Keloids. Some important studies are reproduced below (Townsend et al, 2012 [Bib]).

Ha et al (2003) reported gene therapy for keloids. A cDNA encoding human HGF was inserted into a replication-defective type 5 adenovirus vector by homologous recombination of intracellular plasmid DNA; so a recombinant vector having HGF (Ad-HGF) was produced.

Ad-HGF and Ad-GFP were expanded, purified, and transfected to cultured rabbit ear scar fibroblast to see transmission efficacy and level of HGF. To study action of Ad-HGF on pathological scar, Ad-HGF was injected in a hypertrophic scar, which is akin to human hypertrophic scars. After 32 days of a single intradermal injection of Ad-HGF most of scars markedly flattened, and some even became similar to normal skin. Microscopy showed that in most healed wounds of Ad-HGF group, dermis was thin, and fibrous tissue, hair follicles and sebaceous glands were present, and immune response against HGF was absent.

Li [Lu] and Gao, (2005) did a study to substitute dysfunctional Fas gene and rebuild blocked Fas signal employing two types of recombinant Adenovirus having human Fas gene.

Then keloids fibroblasts were transfected by Adenovirus, and levels of pre and post exposure

Fas protein were checked. After that characteristics of new Fas protein was studied.

Expression of Fas protein in transfected keloid fibroblasts was much improved. Apoptosis was also seen in these fibroblasts. Recombinant Adenovirus with Fas gene can transfect Fas gene in keloid- fibroblasts and produce much better expression of Fas protein. New Fas gene can rebuild blocked Fas signal. Luo et al (2003) reported apoptosis of keloid-derived fibroblasts induced by Fas gene transfection.

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Xu et al (2008) used recombinant adenovirus-mediated double suicide gene CDglyTK for keloid treatment. Biological behaviour of keloids is same as that of malignancy, as these spread outside the original wound, and do not regress. A study was done to find out lethal and non-lethal action of double suicide gene on hypertrophic scar fibroblasts. Bcl-2 and BAX perform a significant action in apoptosis produced by double suicide gene. Recombinant adenovirus CDglyTK suicide genes were reconstructed by use of modified AdEasy system.

Lethal and non-lethal actions were detected two days later by using microculture tetrazolium assay. Alterations in fibroblasts were checked by using H & E stain, and apoptosis was measured using terminal deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assay. Bcl-2 and BAX were measured by IHC and quantitative real-time PCR. Lethal and non-lethal actions of CDglyTK were increased in keloid fibroblasts. Apoptosis is a major mechanism causing fibroblast death in keloids, and Bcl-2 and BAX perform a significant part in apoptosis. They concluded that CDglyTK double suicide gene treatment was useful in killing keloid fibroblasts, thereby reducing fibrosis.

Lee et al (2011) studied relaxin inhibition of collagen formation in stimulated fibroblasts. In a study, an adenovirus producing relaxin (dE1-RGD/lacZ/RLX) was formed and role of relaxin expressing adenovirus on expression of different ECM constituents in keloid spheroids was investigated. Levels of type I and III collagen, fibronectin and elastin were studied by IHC in keloid spheroids transduced with relaxin producing adenovirus. It revealed that level of main ECM constituents (e.g. type I and III collagen, elastin and fibronectin) was much decreased in keloid spheroids transduced with dE1-RGD/lacZ/RLX

(Figure 75).

Wang et al (2013) studied TRAP-1-like protein (TLP), which efficiently regulates

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Smad 2- and Smad 3-dependent signal expression in TGF-β pathway. Relation between TLP and Type I/III collagen formation was a good finding for wound repair and gene treatment of keloids. To study actions of TLP on collagen formation in human skin fibroblasts, lentiviral vectors encoding TLP was built to transfect uninjured human skin fibroblasts. Levels of collagen I/III and phosphorylation of Smad 2 and Smad 3 in fibroblasts were studied after

TLP treatment. Comparison of TLP expression in uninjured skin and in keloid was also done, and action of TLP on cell viability was studied using MTT assay. TLP level in Keloid was significantly increased as compared to normal skin.

Wang D. et al (2005) reported formation of recombinant human Smad 7 adenoviral vector. Recombinant human Smad 7 adenoviral vector was built by direct DNA cloning and then transfected into 293 cells for virus packaging. After amplification and purification, the recombinant adenovirus was used to transfect keloid fibroblasts. Smad 7 mRNA transcription of transfected cells was measured using RT-PCR. It revealed increased level of adenovirus mediated Smad 7 mRNA in keloid cells. It was concluded that recombinant Smad7 adenoviral vector may be expressed in cultured cells.

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Figure 75: Gene Therapy (Google, 2015 [Bib])

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55. Stem Cell Therapy

Lau et al in 2009, for the first time, explored role of stem cells in wound repair and keloid prevention. Skin is an ideal model for studying wound healing, involving interaction amongst different types of cells. Disruption of such processes causes defects in healing of wounds, causing keloid formation. Role of stem cells in tissue regeneration and wound healing is being studied extensively. So a large number of stem cell treatments for skin wounds and keloids are being developed (Townsend et al, 2012 [Bib]) (Figure 76).

Stem cells, which develop into progenitor cells and subsequently differentiated cells, perform a specific role in process of wound repair. Among the most important cells which take part in wound healing, adipose-derived stem cells (ADSC), bone marrow stem cells

(BMSC), epidermal stem cells (ESC), human Wharton's jelly stem cells (hWJSC), induced pluripotent stem cells (iPSC), and mesenchymal stem cells (MSC) are most important.

Activity of these cells is strictly regulated by various growth factors, like EGF, FGF, PDGF,

TGF, and VEGF. Any disorders in functioning of stem cells and biological activity of growth factors can lead to defects in healing, eg delayed wound healing or creation of hypertrophic scars (Pikula et al 2015).

Adipose-derived stem cells (ADSCs) have use in many fibroproliferative disorders.

They act by decreasing collagen formation. A trial was done to study role of intralesional

ADSCs injection on hypertrophic scar production using rabbit ear model. Intralesional injections of ADSCs gave rise to scars with almost normal appearance and markedly reduced elevation in comparison to control. Furthermore, collagen was laid uniformly and there was lower level of alpha-smooth muscle actin (Zhang et al, 2015).

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Bone marrow stem cells (BMSC) are present in inflammatory phase of wound healing. They consist of fibrocytes and keratinocyte-like cells (KLC). These cells help in regeneration and repair of wound, but they can also have adverse effects. BMSC have potential avenues for control and treatment of keloids (Curran and Ghahary, 2013).

Epidermal stem cells (ESCs) from hypertrophic scar and normal skin were compared.

Epidermal stem cells were separated, enriched and adhered from hypertrophic scars (scar group) and normal skins (control group). Morphology and growth characteristics of primary epidermal stem cells were observed. ESCs in hypertrophic scar had same characteristics with

ESCs in normal skin. However, ESCs from hypertrophic scar were lower in number than that from normal skin (Zhou et al, 2014).

Human Wharton's jelly stem cells (hWJSC) help in wound healing. Wharton's jelly stem cells increase fibroblast growth and migration, enhance re-epithelialization and help whole process of wound healing by paracrine signalling. Wharton's jelly is a good mesenchymal stem cell store because procuring these ells is not painful or invasive. Apart from their role in wound healing, they have an important effect in Keloid treatment (Kamolz et al, 2014)

Induced pluripotent stem cells (iPSCs) are cloned embryonic-like stem cells, produced from adult mouse fibroblasts. A study found that iPSC conditioned medium (iPSC-

CM) markedly decreases keloid fibroblast activation. It was seen that iPSC-CM significantly decreased collagen I level, and alpha-smooth muscle actin showed a markedly low level in human dermal fibroblasts stimulated with TGF beta 1. This study suggests that iPSC-CM can prevent keloid formation by decreasing fibroblast activation, reducing inflammatory process,

273 and lowering contractility function of fibroblasts (Ren et al, 2015).

Mesenchymal stem cells (MSCs) also reduce tissue fibrosis. A study was done to investigate action of MSCs on hypertrophic scar fibroblasts and ECM production using paracrine method, and if antifibrotic functions of MSCs used TGF beta based activation. In vivo trials revealed that MSC conditioned media (CM) infusion in a mouse skin fibrosis model produced marked reduction in dermal fibrosis. Microscopy and IHC analysis for alpha-smooth muscle actin showed that TGF beta 3 of CM facilitated treatment effects could decrease matrix production and myofibroblast proliferation & differentiation. It was concluded that TGF beta 3 mediated the diminishing action of MSCs on both proliferation of human keloid fibroblasts and production of ECM. It also reduced skin fibrosis in mouse model, so giving new knowledge and leading to MSC-based treatment for keloids (Wu et al,

2015).

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Figure 76: Stem Cell Therapy (Google, 2015 [Bib] 275

56. Nano Medicine

Nanotechnology is the basis of Nano-Medicine. Nanotechnology is defined as usage of matter with sizes in nanometre range. Uses of this very small size technology is there for decades. They cover different fields and industries like electronics, energy, cosmetics, and food.

Nanomedicine is use of nanotechnology in health facilities. Main utility of nanomedicine is usage of nanoparticles to change pharmacokinetics of drugs. Nanomedicines are utilized in management of patients afflicted with a large number diseases like malignancies. Nanomedicines which are in use today are resolving problems encountered in gaining full advantage of drug molecules employed. Some medicines are mildly soluble in water and human body has difficulty in absorbing them to cure the disease. In some instances, medicine is absorbed normally but body eliminates it prior to treatment of disease.

Many drugs have adverse actions due to less delivery at the site of pathology. Nanomedicines perform a significant role in ensuring that enough drug enters the body, that drug which enters the body stays in body for significant time and has reached especially to parts of body which require it. Another filed of nanotechnology and nanomedicine is diagnosis of disease.

By tracing individual molecules, it is possible to diagnose disease quickly, so that treatment can be started in time (Figure 77).

Clinically approved nanomedicines carriers include polymers, polymer-protein conjugates, micelles, liposomes, nanoparticles, nanocrystals, and nanoemulsions. These formulations carry antineoplastics, antifungals, hormones, analgesics, vaccines, iron, interferon alfa and radiodiagnostics.

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Mao et al (2010) investigated scar penetration efficacy of various sizes of ethosomes encapsulating 5-Fluorouracil. They prepared three kinds of ethosomes with different sizes by extruding the vesicles through polycarbonate membrane filters. Their encapsulation efficiency of Fluorouracil (5-FU), and scar-penetration efficiencies were checked. The prepared ethosomes were 216, 107, and 65 nm in diameter respectively, and exhibited good dispensability. Their encapsulation efficiency of 5-FU were 12%, 34%, and 41%, respectively. Ethosomes of 65 nm diameter showed maximal fluorescence penetration efficacy that could reach deep layer of hypertrophic scar.

Zhang et al (2012) investigated delivery of drugs by ethosomes in human hypertrophic scar. Percutaneous ethosome penetration was studied in vitro in keloid as well as normal skin. The 5-FU that penetrated hypertrophic scar and normal skin in 24 hours was most profuse in ethosomes through scar, trailed with hydroethanolic solution through scar, ethosomes through skin, and hydroethanolic solution through skin.

Mohammadzadeh (2013) hypothesized that Nanosilver exhibited significant physical, chemical and biological characteristics, and might interact with cells and modify proteins and enzymes which neutralize free radical injury to cells. Polymeric micelles were a good material to fulfil this objective, due to their capability to cross multiple biological barriers. As they are of small size (20–150 nm), these micelles are capable to block glomerular clearance, and metabolism by liver. So exterior and interior of micelles can be changed with specific drugs for diseased cells and pH-sensitive silver binding linkers for sustained release. Micelle- coated nanosilver may decrease the antioxidant defence mechanism of dermal cells, due to which apoptogenic factors such as cytochrome C are produced, which lead to programmed keloid cell death.

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Figure 77: Different structures of nanomedicines and their sizes (BSNM, 2015)

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57. Miscellaneous Treatments

These are given in alphabetical order.

1. Acupuncture: Hunter (2011) reported treatment of keloid with acupuncture.

2. Antihistamines, Systemic: Histamine H1 blockers also have antiproliferative properties

which inhibit synthesis and deposition of collagen by keloid fibroblasts via decrease of

release of TGF β 1. Topol et al (1981) recommended antihistamines to inhibit

proliferation of fibroblasts of skin, scar, and keloid. Venugopal et al (1999) reported that

chlorphenaramine maleate decreased proliferation rate and DNA synthesis in fibroblasts.

3. Asiaticoside, Topical: It is a derivative of plant Centella asiatica (Indian pennywort). It

is used for treatment of Keloids as an ingredient of Gotu Kola, a herbal medicine. It

enhances expression of Smad 7, which has an inhibitory effect on scar fibroblasts. Bosse

et al (1979) reported use of oral Madecassol (Asiaticoside) for treatment of keloid. Qi et

al (2000) reported that Asiaticoside acted by inhibition of fibroblast proliferation.

4. Banding: Parikh et al in 2008 reported treatment of keloids with suture banding.

5. Beta AminoPropioNitrile fumarate (BAPN), Oral: It is a lysyl oxidase inhibitor. It

interferes with collagen cross-linking, thereby making it more susceptible to degradation.

Peacock (1978) proposed beta-aminopropionitrile for control of scar tissue.

6. Camouflage/Make-up: It is a simple treatment, which reduces stigma of disfiguring

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keloids. It is effective in covering or concealing these lesions. Gunter (1978) proposed

camouflage of keloids and hypertrophic scars in exposed areas. Camouflage may also

promote physiological changes in Keloids (Kazuki, 2001) (Figure 78).

7. Captopril/Enalapril Topical: Local renin-angiotensin system in dermis has a part to

play in regulation of collagen synthesis. Its inhibition by captopril in skin results in

blockage of angiotensin II, suppression of TGF beta 1, and release of interleukin-6,

thereby blocking fibroblast proliferation and collagen production. It is used topically as

5% ointment (Ardekani). Iannello et al (2006) reported use of enalapril for treatment of

keloid.

8. Chemical Peel: It is done using trichloroacetic acid. It removes superficial surface of

Keloid, and regenerated surface is better than original.

9. Citalopram: Bláha et al (1999) reported use of citalopram for prophylaxis of

hypertrophic scars in burn patients.

10. Colchicine, Oral: It is an antimicrotubular agent. It has been used as 1 mg orally for one

year, as adjuvant to surgical excision. It especially inhibits biosynthesis of collagen and

growth of fibroblasts. Colchicine also enhances action of collagenase, increases

destruction of collagen, and reduces collagen production. Peacock (1978) proposed

colchicine for control scar tissue.

11. Cyanoacrylates: Scuderi et al (2010) studied efficacy of topical cyanoacrylates in

treatment of hypertrophic scars. He reported them effective.

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12. Fatty Acids, Topical: Fish oil (omega-3 polyunsaturated fatty acid) is used in traditional

remedies for keloids. Louw in 2007 proposed use of fatty acids for treatment of keloids.

13. Hyaluronidase, Intralesional: It is an enzyme that digests hyaluronic acid. It also digests

connective tissue, including collagen. It is available as recumbent human enzyme. It is

given intralesionally as 150 mg/ml injection. Braun-Falco and Weber (1951) described

treatment of keloids with local injection of hyaluronidase.

14. Hydrogel Dressing: It is an occlusive dressing, with effects similar to silicone sheet.

Ricketts et al (1996) reported use of non-silicone hydrogel dressing for keloids.

15. Micro Needling: It creates controlled scar injury. Skin is punctured with a microneedle,

which produces injury. New collagen and elastin, and new capillaries are formed in these

wounds. This neovascularization and neocollagenesis causes decrease in size of Keloids.

16. Moist Exposed Burn Ointment (MEBO): Atiyeh et al in 2003 reported significantly

superior scar quality in wound treated with MEBO.

17. Mucopolysaccharide Polysulfuric Acid Ester (Hirudoid): Stuttgen et al (1990)

recommended its use for keloids and hypertrophic scars.

18. Mugwort Lotion: It is derived from Artemisia yomogi, a Chinese herb. It is said to have

antihistaminic properties.

19. Onion Extract, Topical (Mederma): Onion extract (Allium cepa) belongs to Liliaceae

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family. It contains quercetin, fructose, xylose, galactose, glucose, mannose, allylsulfides,

flavonoids, cycloalliin, selenium, thiosulfinates, and sulphur and seleno compounds. It is

available as 12% Allium cepa gel. It is applied three times a day for three to six months.

Chadzynska (1987) reported treatment of keloid with allantoin, heparin and extractum

cepae ointment (Contractubex).

20. Penicillamine, Oral: It is a prototype thiol, which inhibits synthesis cross-links. Uncross-

linked collagen is more susceptible to collagenase degradation. Moyanhan (1974)

reported use of penicillamine in treatment of keloids in children.

21. Pentoxifylline, Oral: It is an analogue of methyl xanthine theobromine. It causes

decrease in collagen, fibronectin, and glycosaminoglycan formation by fibroblasts.

Berman and Duncan (1990) reported that it inhibited growth of keloid fibroblasts.

22. Pirfenidone: It reduces fibrosis through downregulation of production of growth factors

and procollagens I & III. Shetlar et al (1998) reported treatment of keloid implants with it.

23. Phenol, Topical: Topical Phenol (40%) is used for treatment of Keloids. It acts as

chemical cautery (Mseddi et al, 2014).

24. Photodynamic Therapy: It acts by formation of free radicals. It is done with topical

application of 20% Aminolevulinic acid (ALA), followed by blue light photodynamic

illumination for 14-18 hours. Castro et al (1989) studied photodynamic therapy with

Nd:YAG laser on fibroblasts sensitized to Q-switch II dye. Campbell (2010) used ALA.

25. Phototherapy: Phototherapy with ultraviolet light A1 and B is being used in treatment of

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Keloids. Vilcek and Babor (1961) reported on use of phototherapy with ultraviolet rays

for treatment of keloid.

26. Prostaglandin E2 Topical: It is a powerful blocker of fibroblast migration, growth, and

collagen production, and a promoter of collagen destruction.

27. Putrescine (tetramethylenediamine): Polyamines, of which putrescine is simplest, are

growth factors necessary for cell division. Dolynchuk (1996) proposed use of Putrescine

for treatment of keloids.

28. Radiofrequency Waves: Keloids are treated with radiofrequency waves, followed by

intralesional steroid injection, in three to four sessions. There are both objective and

subjective benefits: Lambranzi (1955) published an article on use of radio waves for

keloids. Radiofrequency waves alone are not effective (Meshkinpor et al, 2005)

29. Retinoids (Retinoic Acid, Tretinoin), Topical: They act by inhibiting DNA production.

They also have a blocking action on TGF β 1 mediated collagen type I gene expression.

They have response rate of 50-100%. They are applied three times a day for three to six

months, as 0.05 or 0.1% cream. Hansen (1979) reported their use for treatment of

hypertrophic scars. Janssen de Limpens (1980) reported their use for keloids.

30. Steroids, Topical: Steroids are used topically as ointment. They are of limited value.

Another method is to use steroid-impregnated tape, which is applied to keloid for 12

hours a day. Cordran tape contains a steroid called flurandrenolone. Maggio (1953)

described for the first time treatment of keloids with local steroids. Mancini (1954)

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reported prevention of keloid by local topical hydrocortisone. Shell and Inmon (1959)

described use of cortisone ointment to prevent keloid formation. Ratzer (1970) described

flurandrenolone tape as occlusive dressing for keloids.

31. Surgical Paper Tape: It has mechanism of action and efficacy similar to hydrocolloid

dressing. Reiffel (1995) reported prevention of keloids by paper tape application.

32. Tamoxifen, Topical: It is a competitive inhibitor of oestradiol at receptor level. It

inhibits fibroblast formation by inhibiting TGF-beta activity, and reduces rate of collagen

biosynthesis via RNA transcription modification. It is used as topical ointment.Hu et al

(1998) suggested topical tamoxifen for treatment of excessive dermal scarring.

33. Tattooing: It is another procedure with which keloid is masked with ornamental tattoos.

34. Tranilast: It is a special type of antihistamine. Waseda (1989) reported use of Tranilast

in keloids. Suzawa et al (1992) studied mechanism of action of Tranilast in keloids.

35. Vitamin E, Topical: It is also called Tocopherol. It is a known antioxidant that promotes

healing by limiting effects of free radicals, as well as inhibiting inflammation and excess

collagen production. It is applied thrice daily for three to six months. Edgerton et al

(1951) described treatment of keloids with vitamin E for the first time.

36. Zinc, Topical: It is used in the form of zinc oxide. It reduces keloid elevation index.

Sodenberg et al (1982) reported keloids and hypertrophic scar treatment with zinc tape.

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Figure 78: Camouflage of Keloid: above; before, below; after (Tirgan, 2015 [Bib])

285

58. Symptomatic Treatments

These treatments give symptomatic relief in patients of Keloids and Hypertrophic

Scars. These are given in alphabetical order.

1. Allantoin, Topical: It is a keratolytic agent, which softens skin, and controls itching and

burning sensation. Schoppa (1969) described use of allantoin and heparin for prevention

of keloids in childhood burns. Chadzynska (1987) reported treatment of keloid with

allantoin, heparin and extractum cepae ointment (Contractubex).

2. Anaesthetics, Topical: Pramoxine is a topical anaesthetic available as 1% cream, which

is applied thrice daily for relief from pruritus.

3. Analgesics, Systemic: They give relief from pain and swelling. Systemic paracetamol is

used.

4. Emollients, Topical: They include white petroleum jelly, creams, lotions, oils, and

ointments. They soften skin and give soothing effect. Many commercial oils have been

marketed for keloids (Figure 79).

5. Heparin, Topical: It removes local swelling, and keeps the keloid moist. It also reduces

pain and discomfort. Keller (1955) published a report on treatment of keloid with heparin.

Schoppa (1969) described use of allantoin and heparin for prevention of keloids in

childhood burns. Chadzynska (1987) reported treatment of keloid with allantoin, heparin

and extractum cepae ointment (Contractubex).

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6. Massage: It controls swelling. It should from distal to proximal direction. Pressure should

be enough to blanch thumb nail of massager. It provides local soothing, comfort, and

reduces pain and discomfort.

7. NSAIDs, Systemic and Topical: Topical Bufexamac and Salicylic Acid are popular.

Diclofenac is also used. Achten et al (1973) described use of bufexamac for treatment of

keloids.

8. Physiotherapy: It prevents stiffness and deformities. It should be continued for many

months. Meyer and Lepinay (1962) described its role in treatment of keloids.

9. Prickly Heat Powder: It contains menthol and camphor. It has a cooling effect and

controls burning sensation. It is especially in hot weather and hot climate. It should

preferably be applied after a bath or shower.

10. Prostaglandin Inhibitors: Wester et al (1989) mentioned use of prostaglandin inhibitors

in treatment of keloids.

11. Psychological Counselling: It prevent mental stigma attached with disfigurement,

especially for keloids on exposed areas. Women are especially prone to psychological

problems. Kelly (1978) noted: ‘Keloids are medically benign, but often psychologically

and cosmetically malignant lesions.’

12. Splints, Static and Dynamic: They provide local rest, and prevent deformities. They

should be continued for many months. Hurtado and Crowther (1985) reported methyl

methacrylate stent for treatment of ear keloids.

13. Water Bath: It keeps the keloid soft and moist. It has local soothing effect. Whirlpool

bath should be used if facilities are available. It is especially helpful in summer season.

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Figure 79: White Petroleum Jelly 288

59. Historical Treatments

There are many treatments which have historically been used for Keloids. They are no longer used now. These are given in alphabetical order.

1. Aminocaproates: Nicoletis (1966) described their use in treatment of keloids.

2. Anaesthetics, Infiltration: Kolokolova et al (1971) used procaine block for pruritus of

keloids.

3. Bee Venom: Kapulan (1973) described use of bee venom for treatment of keloids.

4. Caustic Potash: Labert (1851) described its use for treatment of keloids.

5. Charpy’s Method: Piera (1950) reported a case of keloids treated by Charpy’s method.

6. Chloroquine, Intralesional: Sharapova and Khamaganova (1973) reported use of

intralesional injection of chloroquine in treatment of keloid (Figure 80).

7. Chymotrypsin: Bureau et al (1967) described treatment of keloids with chymotrypsin.

8. Collagen Cream:

9. Collagenase, Intralesional: Mandl (1982) described use microbial collagenase from

Clostridium histolyticum for intralesional injection for keloid treatment.

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10. Dermabrasion: Luikart et al (1956) reported use of dermabrasion for treatment of keloids.

Roenigk (1977) recommended dermabrasion for keloids.

11. Dibunol (Butylated Hydroxytoluene): Kolchina and Shakhnes (1985) reported use of

dibunol in treatment of keloids.

12. Dimethyl Sulfoxide: Engel (1966) described its use in treatment of keloids.

13. Fibrolysin, Intralesional: Porter (1909) described its use for treatment of keloids.

14. Gate’s Method: Lisbonne (1960) described it for management and prevention of keloid.

15. Glycerol: Pfeiffer (1968) described usage of glycerol for treatment of keloids.

16. Haprinoids: Saipt (1964) described treatment of keloids with haparinoids.

17. Histaminase: Schirren et al (1963) described treatment with histaminase as prophylaxis

against keloid formation in burn patients.

18. Honey:

19. Hyaluronoglucosaminidase: Wernsdoefer (1962) described its use in treatment of keloids.

20. Iodine:

21. Iron: Michel et al (1961) reported use of Vitamin B, Vitamin C, and Iron for keloids.

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22. Lactic Acid: Kerl et al (1981) reported treatment of keloids with calmurid, a combination

of lactic acid and urea.

23. Lemon Juice: Reuter (1973) reported treatment of keloids with lemon juice.

24. Lipotalon: Stark et al (1994) reported treatment of keloids with intralesional Lipotalon, a

microsomal corticoid.

25. Magnesium Lithospermate: Shigematsu (1994) reported that magnesium lithospermate

extracted from Chinese medicinal herb Salviae Miltorrhizae Radix inhibited collagen

synthesis by skin fibroblasts without affecting DNA or noncollagen protein synthesis.

26. Magnetics: Muhlbauer (1974) reported use of magnetics to prevent keloid formation.

27. Microwaves: Shafranov et al (1985) reported use of microwaves for treatment of keloids.

28. Minoxidil: Pinol et al (1990) used minoxidil in keloids, as it inhibited fibroblasts.

29. Mucopolysaccharide Cream:

30. Oestrogen: Kuhn (1963) used steroid-vitamin A-oestrogen lotion for keloids in women.

31. Orgotein (superoxide dismutase): Grosser et al (1984) used it for treatment of keloids.

32. Oxygen: Pankova (1952) used subcutaneous injection of oxygen for treatment of keloids.

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33. Parathyroidectomy: Olivier and Barasch (1946)and Minissale (1951)recommended

parathyroidectomy for treatment of Keloids

34. Pyrogens: Pavlova & Bolkhovitinova (1970) described treatment of keloid with pyrogens.

35. Quinones, Oral: Kelly and Pinkus (1958) used oral quinones for keloid treatment.

36. Scarification: Lawrence (1898 AD) from Australia treated a Keloid by scarification.

37. Sulphur Douches: Biett (1833 AD) reported its use for keloid treatment.

38. Thiosinamine, Intralesional: Porter (1909), Kollner and Stein (1957) reported treatment of

keloids with thiosinamine.

39. Ultrasound: Kuiter et al (1955) reported treatment of keloids with ultrasonic energy.

40. Urea: It is hygroscopic and keratolytic. Pfeiffer (1968) described its use for keloids.

41. Vitamin B: Michel et al (1961) reported use of Vitamin B for management of keloids.

42. Vitamin C: Michel et al (1961) and Szabo et al (1971) reported usage of Vitamin C for

keloid management.

43. Vitamin K: Mienicki and Kossakowska (1963) described use of vitamin K for keloids.

44. Volon A Tincture (salicylic acid & triamcinolone acetonide): Schmid (1967) described

treatment of keloids with Volon A tincture.

45. Xymedone: Shafikov et al (1993) reported use of Xymedone, a pyrimidine derivative for

prevention of keloids in burn patients.

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Figure 80:B; Chloroquine 293

60. Multi-Modal Therapy

No single therapy for Keloids and Hypertrophic Scars is effective. Therefore, management of these lesions is largely multimodal. Two, three, and even four modes of treatment can be combined to give acceptable results. Miscellaneous and Symptomatic

Treatments are used with all these regimens.

1. Surgery Steroids

2. Surgery Steroids Silicone 90%

3. Surgery Steroids Silicone Pressure

4. Steroids Silicone

5. Steroids Silicone Pressure

6. Silicon Pressure

7. Surgery Radiotherapy (Figure 81)

8. Surgery Radiotherapy Silicone

9. Surgery Radiotherapy Silicone Pressure

10. Surgery Cytotoxics

11. Surgery Cytotoxics Silicone

12. Surgery Cytotoxics Silicone Pressure

13. Cryosurgery Pressure

14. Laser Pressure

15. Surgery Immunomodulators

16. Surgery Calcineurin Inhibitors

17. Surgery Verapamil

18. Surgery Botulinum Toxins

19. Steroid Cryosurgery 85%

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Figure 81: Multimodal Therapy (Surgery+Radiotherapy) (Jones et al, 2015) 295

61. Complications of Keloids & Hypertrophic Scars

Following complications may occur in Keloids and Hypertrophic Scars:

1. Infection& Ulceration: Long-standing Keloids with a dry rough surface can develop

cracks on its surface. This is especially so if there is some element of accompanying

contracture. These cracks can become secondarily infected. If not treated in time, these

cracks coalesce to form ulcers.

2. Suppurative Keloids: The development of suppuration and sinus formation in a keloid is

rare. This complication occurs mainly in beard, neck, and presternal areas of black hairy

men. This occurs due to entrapment of hair and sebaceous glands in a growing keloid.

3. Malignant Transformation: Untreated Keloids having prolonged irritation and rubbing

can develop squamous cell carcinoma after 2-3 decades. It is slow growing, painless,

without lymphatic spread, as local lymphatics have already been destroyed. Horton et al

(1953) reported a case of malignant change in keloids. Tumour is usually well-

differentiated squamous cell carcinoma (Onuigbo, 2006) (Figure 82).

4. Psychological Effects: Patients with disfiguring Keloids and Hypertrophic Scars usually

experience many psychological effects. These psychological effects can affect patients’

family and work life. Rei Ogawa (2008) described Keloids a serious disease like cancer.

He wrote keloids produced severe mental pain and anguish, comparable to that suffered

by persons suffering from malignancies. Only people actually suffering from keloids were

capable of understanding this thing in full sense. He concluded that unless behaviour of

doctors toward keloids and keloid patients was modified, a large number of keloid

patients would continuously suffer needless mental trauma. He further said that this

attitude was not pardonable considering the level of medical science at present time.

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Figure 82: Malignant Transformation in a Keloid

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62. Prognosis& Follow-Up of Keloids & Hypertrophic Scars

Sometimes a keloid may regress spontaneously and also become free of symptoms.

When a Keloid stops growing, it becomes stable in character, and is very unlikely to start growing again. Response/Regression/Remission rates are different with different treatments, varying from 00 to 100%. Recurrence/Relapse rates are also different with different treatments, varying from 50 to 100%. With surgical treatment alone, and in patients of keloid tendency, keloid size may double after treatment; so response rate becomes -100%, and recurrence rate +200%. Multimodal therapy has relatively better prognosis than single mode therapy. Two, three, and even four modes of treatment are combined for better prognosis.

Follow-up is done for at least 1 year, preferably 18 months, after successful treatment.

Patient is called every three months, and progression of keloid noted. If patient is advised prolonged ambulatory treatment, like silicon sheet or pressure dressing, he is called for more frequent visits. Photography is much helpful to monitor size and morphology of keloid.

Different types of keloidometer are also available, but they are generally not used by the clinicians. Different types of Keloid Index have also been prepared, like Vancouver Scar

Scale, but again they are not used clinically. So by and large monitoring of keloids is subjective. Hambleton et al (1992) reported monitoring of progress of hypertrophic scar by ultrasound measurement of thickness (Figure 83).

Treatment of recurrences should be started as soon as possible. Treatment of recurrence is same as that of a fresh keloid. However as keloids becomes old, they become harder and more difficult to treat

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Figure 83:B; Vancouver Scar Scale (VSS) 299

63. Animal Model of Keloids & Hypertrophic Scars

Keloids are only found in human beings. This is the main hindrance for scientific research on keloids, as direct research on human bring is not feasible, and is also unethical.

Therefore research on keloids has mostly been clinical, with limited evidence base. So far no successful animal model of Keloids has been produced. Therefore efforts are going on to establish an animal model of Keloids. The other mode of research is tissue culture model.

Fibroblasts are isolated form human keloid tissue, and cultured in artificial media. Different experiments are done on these fibroblasts to study aetiology, pathogenesis, and treatment of keloids. But it is found out that fibroblasts are not the only culprits in keloidogenesis; other cells and biochemicals are as important in keloid formation (Figure 84).

Robinson et al (1951) published a report on use of tissue culture technique to study keloid pathology. Garner et al (1995) studied scar contraction by using keloid and skin fibroblast-populated collagen lattices. Koch et al (1997) reported use of serum-free keloid fibroblast culture in research on keloids. Kuhn et al (2001) also used fibroblast populated collagen lattice as a model. For this they prepared fibroblast cultures and collagen lattices, and placed them on each other. They used it to study wound contraction.

Szava et al (1961) published an article in which they reported animal studies of burns leading to pathological changes in connective tissue. Kelly (1964) implanted polyvinyl sponge in albino rat. Ketchum et al (1967) performed animal studies to see effect of steroids on mature collagen. Szabo et al (1971) reported explanation of human keloid tissue in chick embryo. Bazin et al (1975) produced an animal model in rats for this purpose; it was the first animal model of wound healing and hypertrophic scars. Shetlar et al in 1985 reported, for the

300 first time, animal model for study of keloids. Robb et al (1987) developed similar animal model for study of keloids. However Shetlar, in his letter to the editor, asserted prior claim on this model (Shetlar 1987).Morris et al (1997), for the first time, developed rabbit ear hypertrophic scar model.

Li et al (2001) reported establishment of experimental animal model for hypertrophic scar on rabbit ears by creating round and rectangular wounds. Aksoy et al (2002) produced hypertrophic scar animal model in guinea pigs by skin excision and coal tar application.

Matthews et al (2004) reported that osteoarthritis induced by cruciate ligament transection in animal models produced a scar-like mass on medial side of knee joint, which might be used as model of hypertrophic scars. Hochman et al (2005) from Brazil reported Keloid heterograft in hamster cheek pouch as a new animal model. Yang et al in 2007 reported development of a new hypertrophic scar model by transplanting full thickness human skin on backs of nude mice. Ninety percent of mice undergoing full-thickness human skin grafting formed durable hypertrophic scars. These scars were red, hard, and raised from skin surface even six months later. Microscopic examination revealed showed collagen production and inflammatory cells.

Ramos et al (2008) in an article titled: Is there an ideal animal model to study hypertrophic scarring? described five different types of animal models: heterologous keloid implant in immunodeficient animals; heterologous keloid implant in immune privileged site; hypertrophic scar production through chemical trauma; hypertrophic scar production in anatomically specific site; and porcine model. They noted that ideal animal model should help study of pathophysiology, histopathology, and molecular reactions, and to test preventive and therapeutic regimens. These animal models should also be helpful to study mechanism of scar formation and pathological wound healing. They wrote that female red

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Duroc pig was a novel model, showing its likeness to humans. Full-thickness human skin grafts in nude mice also represented improvement in hypertrophic scarring animal model.

Zhu et al (2008) suggested need of research for correlation between rabbit ear anatomy and hypertrophic scar production.

Supp et al (2012) described hypertrophic scar or keloid production by use of tissue engineering technique in immunodeficient mouse.Wang H. et al (2005), Wang and Luo

(2013), established human keloid animal model by use of tissue engineering technique.

Human keloid fibroblasts were moved to poly(lactic-co-glycolic acid) (PLGA) copolymer and cultured for seven days. Then composite of fibroblasts and PLGA (experimental group), and PLGA only (control group) were implanted into subcutaneous pouches in nude mice.

Implanted specimen were harvested on 30, 60, 120, and 180 days for microscopic examination. All mice stayed alive after experiment. Implants of experimental group went on growing in size from days 30 to 180, while implants of control group reduced in size.

Employing different histology stains, fibroblasts and collagen in implants were kept under observation throughout. Excessive quantities of fibroblasts and collagen were present in implants of 180 days, and they showed microscopic characteristics as found in human keloids. Under transmission electron microscopy, implant fibroblasts also had copious amount of rough endoplasmic reticulum in their cytoplasm.

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Figure 84: Animal Model of Keloid 303

64. Role of TGFB 1 in Keloids & Hypertrophic Scars

There is an important role of TGF-β in wound repair; it is a potent chemotactic factor for fibroblasts and stimulates them to produce major extracellular matrix components including collagen. Disturbances in TGF Beta signalling, and keratinocyte-fibroblast interactions may be involved. TGFB is produced by macrophages as well as Extracellular

Matrix. TGFB acts by fibroblasts activation, leading to accelerated collagen production.

TGFB production is higher in Hypertrophic Scars, and both Keloid and Hypertrophic Scar fibroblasts respond to lower levels of TGFB than normal fibroblasts. Expression of its isoforms TGFB1, and TGFB2 is increased in Keloid fibroblasts, while Hypertrophic Scar fibroblasts produce more TGFB1. Addition of exogenous TGFB2 activates fibroblasts from both Keloids & Hypertrophic Scars. Keloid fibroblasts also have upregulated antiapoptotic gene expression, which can be differentially expressed within different areas of Keloid.

Studies involving antibodies to TGFB show that its activity can be blocked and fibrosis decreased (Brunicardi, 2015 [Bib]; Kumar, 2014 [Bib]) (Figure 85).

Peltonen et al (1991) studied co-localization of type I & VI collagen and TGF beta 1 mRNA. They noted, for the first time, that TGF beta 1 was responsible for fibrosis. Babu et al

(1992) reported modified reaction to TGF beta 1 by keloid fibroblasts. Bleacher et al (1993) compared foetal tissue repair with adult wound healing, and effect of TGF beta. In a review they wrote: ‘Studies of fetal repair have already suggested mechanisms that might favorably alter adult healing. It has been recognized that the addition of TGF-beta to fetal wounds causes an adult-like healing response with fibrosis and inflammation. A subsequent study using neutralizing antibody to TGF-beta in adult wounds showed enhanced healing with a more normal dermal architecture with fewer macrophages, fewer blood vessels, and less

304 collagen.’ Ghahary et al (1993) reported increased expression of mRNA of TGF beta I & III procollagen in keloids. Messadi et al (1994) examined TGF beta 1 synthesis by fibroblasts of normal and fibrotic tissues. Younani et al (1994) studied changes in collagen synthesis by

TGF beta in keloid fibroblasts. Ghahary et al (1995) studied immunolocalization of TGF-beta

1 in keloids and uninjured skin. Schmid et al (1998) reported increased level of TGF beta I &

II receptors in granulation tissue and keloid. Lee et al (1999) studied TGF beta 1, 2, & 3 levels in keloids. Wang et al (1999) wrote that addition of TGF beta 2 caused enhanced collagen production in keloids, and anti-TGF beta2 antibody reduced collagen production.

Tredget et al (2000), using three sets of keloid and normal fibroblasts from same patients, treated nonconfluent and near confluent fibroblasts with TGF beta, and cell growth

18 and collagen synthesis were measured using cell counting and O2 isotopic uptake into hydroxyproline before analysis by gas chromatography-mass spectrometry. Keloid and normal fibroblasts were examined for formation of TGF beta protein using ELISA for TGF beta 1, TGF beta 2, and TGF beta 3 after acidification of medium samples from 96 hour cultures. Keloid and normal fibroblasts were treated with IFN alpha 2b or IFN gamma or both for 96 hours. Quantitative RT-PCR and Northern blot analysis were done using internal standards for human TGF beta 1. They concluded that TGF-beta stimulated both keloid and normal fibroblast growth. Collagen production was higher in keloid than in normal fibroblasts and was maximally increased at 75 pM TGF-beta. TGF beta stimulated collagen production was blocked by IFN alpha or IFN gamma or both in an additive fashion. They supported use of IFN alpha and IFN gamma for treatment of keloids. Wu et al (2000) studied level of mRNA of TGF-beta and TIMP-1 in keloid. They concluded: ‘The average integral optical density of mRNA for TGF-beta and TIMP-1 increased by 8 and 7 folds (P < 0.001) related to normal skin and the expression of them showed highly positive correlation.’ Lu et al (2004) described expression of TGF beta and tits receptors in keloids.

305

Figure 85: TGF Beta effects on wound healing (Werner & Grose, 2003) 306

65. Role of DMSO in Keloids & Hypertrophic Scars

Dimethyl Sulfoxide (DMSO) is a colourless odourless fluid, formed as a wood and paper industry by-product. It is an organosulfur compound having formula of (CH3)2SO. It is a universal polaraprotic solvent that dissolves both polar and nonpolar chemicals, and is miscible in organic solvents and water. FDA permits it for management of interstitial cystitis and as preservative of organs for transplant. Further, skin manifestations of scleroderma resolve following topical applications of DMSO. Topical application of DMSO provides pain relief and control of inflammation in persons having arthritis and those suffering from trauma. DMSO also increases transmission of many chemicals via skin; therefore, solution of idoxuridine and DMSO is used for herpes zoster treatment. Engel (1966) described use of dimethyl sulfoxide in treatment of keloids (Figure 86).

Side effects of DMSO are redness and itching upon topical use, intravascular haemolysis when given in intravenous drip, and abdominal pain when given orally. DMSO has an unusual property that many patients perceive a garlic like taste in mouth after DMSO application on skin

DMSO was used to make transparent solution of Triamcinolone Acetonide in this study. Commercially available Triamcinolone Acetonide injection is in the form of a milky suspension, and it is not possible to do a blinded trial when comparing it with a transparent solution like Interferon Gamma. DMSO’s role in treatment of Keloid has not been studied, but depending upon its properties, it can be strongly guessed that its effect on Keloid would be positive rather than negative. Therefore it may have improved the efficacy of

Triamcinolone Acetonide, and narrowed difference of efficacy between Triamcinolone

Acetonide and Interferon Gamma. In this way it would not adversely affect outcome of study.

307

Figure 86:B; DMSO 308

66. Role of Triamcinolone Acetonide in Keloids & Hypertrophic Scars

Montgomery & Portnoy; Murray; and Thivolet & Pellerat, (1963), for the first time, simultaneously described use of Triamcinolone Acetonide injection for management of keloids. Vallis (1967) used Dermojet mechanical injection device for intralesional

Triamcinolone Acetonide injection in Keloids. Wrong (1969) proposed that all keloids should be treated and preferred treatment was intralesional triamcinolone acetonide. It is the most common steroid used for intralesional injection of Keloids and Hypertrophic Scars. It is a synthetic glucocorticoid. It has intermediate duration of action, extended over a period of weeks. It suppresses every component of inflammatory process. It inhibits phospholipase A2, thereby decreasing synthesis of arachidonic acid which is forerunner of prostaglandins and leukotrienes. It increases production of anti-inflammatory cytokines like bFGF, decreases synthesis of proinflammatory cytokines like TGFB 1, VEGF, IGF-1 and interleukins, suppresses cell-mediated immunity, reduces complement synthesis, and decrease activity of leukocytes. In Keloids, it causes inhibition of fibroblast activity and proliferation, decrease in collagen and glycoprotein synthesis, and ultimately fibroblast degeneration. It softens and flattens Keloids, and also gives symptomatic relief from pain and itching. It is most useful in new keloids, and less in old keloids. It also gives symptomatic respite from pruritus, pain, and burning sensation (Figure 87).

Triamcinolone Acetonide (Kenacort-A®) is given in a standard dose of 40 mg ml-1for four cm2 Keloid. First injection should be intraoperative if keloid is large and is excised. It is given at 2-6 weeks intervals. Usually three injections are given, but treatment may be continued for as long as lesions are active. In larger lesions, upto two ml injection can be given in one sitting; repeat injections are given weekly, at different sites. There is no upper

309 limit of size of Keloid for treatment by intralesional injections, as the drug is not absorbed systemically. Generally speaking, injection should not be injected too often, in too high dose, or sub-dermally. It can be given in full strength or diluted with aqua pro injection, normal saline or local anaesthetic (lignocaine). Typical regimens for triamcinolone acetonide intralesional injections include: 40 mg/ml for a thick keloid, and 20 mg/ml for a moderate keloid, and 10 mg/ml for a thin keloid (Doherty, 2015 [Bib]).

It has a response/regression/remission rate of 50-100%, and recurrence/relapse rate of

30-100%.Response rate is increased when triamcinolone acetonide is combined with surgical excision. Other adjuvant therapies include laser, irradiation, 5-FU, and cryosurgery. In a study surgery alongwith intraoperative triamcinolone acetonide injection, and then injection every week for two to five weeks, and then every month for 4 to 6 months gave satisfactory results. Total symptomatic respite was attained with this treatment in all cases in five weeks after operation. Objective response was found in 91.9% patients (Tang, 1992).

Side effects of intralesional Triamcinolone Acetonide may be early or delayed. Early side effects are non-specific, localized to injection site. Delayed side effect may be local or systemic. Local side effects include lipoatrophy of skin and subcutaneous tissue, hypopigmentation (leukoderma), hyperpigmentation (brown marks), telangiectasis, and steroid acne. Systemic side effects are very rare when proper dose and technique is used.

These include congestive heart failure, diabetes mellitus, Cushing’s syndrome, hirsutism, hypertrichosis, fractures, depression, and glaucoma. Hypersensitivity reactions are very infrequent; they are urticaria and anaphylaxis. Humbert et al (2001) presented a case of

Hoigne's syndrome (pseudo-anaphylactic reaction) with intralesional triamcinolone acetonide.

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Figure 87:B; Triamcinolone Acetonide 311

67. Role of Interferon Gamma in Keloids & Hypertrophic Scars

Interferon gamma is a cytokine produced by genetically modified Escherichia coli containing DNA which encodes human proteins. It comprises a core of six α-helices and an extended sequence at C-terminal portion. The α-helices in the core are coded as 1 to 6.

Biologically active dimer is produced by anti-parallel non-covalent inter-locking of two

16,465 Dalton monomers. End-result is a single-chain polypeptide having 140 amino acids. It has a specific activity of 20 million IU mg-1. It is of two types; 1a and 1b. Interferon gamma

1a is used to treat T cell lymphoma. Interferon gamma 1b is used as an adjuvant treatment for decrease of frequency and severity of serious infections in patients with Chronic

Granulomatous Disease. It is also used to slow progress of Severe Malignant Osteopetrosis. It reduces collagen I, II, III by decreasing mRNA and reducing TGFB 1 level (Figure 88).

Interferon Gamma 1b is available in brand names of Actimmune® (InterMune, USA), and Imukin® (Boehringer Ingelheim, Germany). Each vial contains 2 million IU (100 µg) recombinant human interferon gamma in 0.5 ml. It is given with the frequency of thrice weekly for three weeks, or once weekly for 10 weeks. It is used as intralesional injection in a dose of 10 µg, 50 µg or 100 µg per 0.5 ml. It is stored in a refrigerator (2-8 C), but should not be frozen. Response rate is reported in only one study, and that is 30%.

The most common side effects of IFNG 1b injection are influenza type symptoms like fever, headache, chills, myalgia, and fatigue. Other adverse reactions are nausea, vomiting, diarrhoea, anorexia, weight loss, skin rash, local pain, depression, abdominal pain, arthralgia, and backache. These side effects are mild, transient, and relieved by symptomatic treatment.

These effects are with systemic administration. With intralesional injection, there is very little

312 systemic absorption of drug and only reported side effects is mild headache.

Granstein et al (1990), for the first time reported effect of Interferons on collagen production. They reported that IFN gamma inhibited collagen production by myofibroblasts and synovial fibroblast-like cells. Ghahary et al (1995) reported that IFN gamma and alpha-

2b regulated levels of collagenase and tissue inhibitor of metalloproteinase-1 mRNA in keloid & skin fibroblasts differently. Harrop et al in 1995 investigated effects of IFN gamma on cell proliferation, collagen production, and levels of types I & III procollagen mRNA in human postburn keloid fibroblasts. They reported that treating proliferating fibroblast cultures for 5 days with IFN gamma (1000 U/ml) resulted in 51% reduction in hypertrophic scar cell proliferation. Kikuchi et al (1995) studied actions of many growth factors on type I collagen metabolism, in Keloid and normal fibroblasts. Six fibroblast cell strains, which were taken from Keloid or normal skin, showed same growth responses to PDGF, TGF Beta 1,

IFN Gamma, and histamine. In contrast, Keloid fibroblasts revealed markedly increased growth response to EGF than normal fibroblasts. Treatment with IFNG (100 U/ml) decreased collagen I production in both groups; effect was significantly greater in Keloid fibroblasts.

TGFB1 treatment upregulated collagen I production in both groups.

Ladin et al (1998) studied p53 and apoptosis changes in keloids and keloid fibroblasts.

Keloid lesions and Keloid fibroblasts showed less apoptosis as compared to normal controls.

Keloid fibroblasts showed increased apoptosis when treated with hydrocortisone, IFN

Gamma, and hypoxia, in comparison to normal adult fibroblasts. Cen and Yan (1999) reported that IFN Gamma downregulated proliferation of fibroblasts, decreased differentiation of myofibroblasts, and induced apoptosis, and might be used in management of keloids. Tredget et al (2000) reported that TGF-beta stimulated both keloid and normal

313 fibroblast growth. Collagen production was higher in keloid than in normal fibroblasts and was maximally increased at 75 pM TGF-beta. TGF beta stimulated collagen production was blocked by IFN alpha or IFN gamma or both in an additive fashion. They supported use of

IFN alpha and IFN gamma for treatment of keloids. Liu et al (2009) reported that Interferon

Gamma could down-regulate level of Smad 3, and up-regulate level of Smad 7 in a time- and dose-dependent manner, and reduce level of CTGF and alpha SMA in basic state or stimulated by TGF Beta. It reveals a marked inhibitory effect on TGF Beta/Smad signal pathway. They concluded that it was a significant finding in management of keloid and hypertrophic scar by Interferon Gamma. Hasegawa et al (2003) reported that Interferon

Gamma failed to antagonize Transforming Growth Factor Beta mediated fibrotic response in

Keloid fibroblasts; so it might not be useful for Keloid & Hypertrophic treatment.

Granstein et al (1990) treated eight patients of keloids with interferon gamma. Six out of eight patients who completed treatment course showed decrease in height at treated site, with mean decrease of 30.4% in treated lesion versus 1.1% in control lesion. Larrabee Jr et al

(1990) treated 10 cases of keloids with interferon gamma. All lesions reduced in width and height. Five of 10 keloids reduced by 50% in width. Pittet et al (1994) treated 4 cases of keloids with IFN Gamma. IFN gamma decreased symptoms and size of lesions. Broker et al

(1996) treated keloids in nine patients with excision followed by Interferon Gamma injections. Three out of four patients who completed the trial felt that they had significant recurrence.

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Figure 88: Interferon Gamma commercial preparation

315

68. Technique of Intralesional Injection in Keloids & Hypertrophic Scars

Porter (1909) mentioned about intralesional treatment of Keloid. He, however, did not use this term. Braun-Falco and Weber (1951) described intralesional treatment of keloids.

Lesion and surrounding area is cleaned with alcoholic prep pad. Intralesional injection is given directly into Keloid, using a 23 gauge needle. Injection is intradermal, not subcutaneous, or subepidermal. It is given in middle depth of keloid, in reticular dermis.

Needle should be at 30° angle to skin or less. Most commonly used gadget is insulin syringe.

It has a short needle, which prevents its inadvertent entry into hypodermis. Further, syringe and needle are manufactured as one piece; so there is no risk of separating needle from syringe, and showering drug in the eyes of surgeon, due to high pressure needed for an intrakeloidal injection. The initial dose per injection site varies depending on the lesion being treated. Generally 0.25 ml is injected per square centimetre of Keloid. Total dose should not normally exceed 2 mL per session. Injection is repeated every 2 to 6 weeks. Treatment can be continued till the keloid has flattened to surrounding skin surface.

A better alternative is dental syringe, which is made-up of metal, and has thumb ring, and finger bars, for giving high pressure, without deforming piston head, and injuring thumb of doctor, as is common with plastic syringes. In this syringe a carpule is used, which is basically a glass cartridge, with a rubber stopper at back end, and aluminium cap with rubber diaphragm on front end. Drug is filled in this carpule with an ordinary syringe through its diaphragm. Then this carpule is loaded in syringe barrel. Harpoon of piston rod penetrates and engages stopper of carpule. A special double-sided dental needle is applied to it. Syringe tip and needle are threaded for a secure fix. Pressure dental syringe is also available, which can be used for hard, thick Keloids. Sagher (1989) used it for first time.

Mechanical injection devices are a still better option for intralesional injection of

316 keloid. Most popular such gadget is Dermojet®. It is a needleless syringe, also called needle- free injector, or jet-injector. It injects drug in skin or keloid by intradermal injection, without the use of an overt needle. It is fitted with a head-tip delivering single dose of 0.1 ml with one push, and can deliver 40 doses ie four ml with one filling. Drug is loaded by unscrewing injector head-tip, and putting drug in it with an ordinary needle-syringe. Then head-screw is tightened again. Device is cocked by holding it vertically, injection head-tip up, and folding the loading leader towards main body of device. Tip of the head is placed perpendicular to keloid, in contact with it, and firing button on the top of device is pressed. Depth of penetration of injector is four to six mm; therefore, for thin keloids, injector head is placed a few mm superficial to keloid, or at acute angle to it. For a thick Keloid, injections are given at three mm interval; in this way a four cm2 keloid needs 36 injections or 3.6 ml of drug,.

Dermojet is also available with interchangeable head-tip of 0.1ml and 0.05.ml dose; the 0.05 ml tip-head gives 1.8 ml drug in 36 injections, and is suitable for moderate or thin keloids.

Interchangeable multi-jet heads are also available, which can give three to five injections with one push, with total dose of 0.01 ml. A characteristic papule with a small hole on tip provides immediate clue that drug has been injected. Syrijet is another needle-less syringe, which can deliver drug to skin or mucosa in the form of high pressure jets. It holds 1.8 ml carpule, and is calibrated to deliver 0.05-0.2 ml drug in one go. Vallis (1967) used it for first time. Ono

(1999) used electric syringe pump (Figure 89).

Drug can be given in full strength, or diluted with aqua pro injection, normal saline or lignocaine. Typical regimen for intralesional injections include full dose per ml for thick keloid, half dose per ml for moderate keloid, and quarter dose per ml for thin keloids.

Intralesional injection should not be given if there is active infection at the site or in surrounding areas. Local side effects of intralesional injection include pain, bleeding, bruising, infection, dermatitis, impaired wound healing, abscess, necrosis, and ulceration.

317

Figure 89: Dermojet 318

69. OBJECTIVE

The objective of this study is: To compare the efficacy of intralesional Interferon

Gamma and Triamcinolone Acetonide for the treatment of Keloids & Hypertrophic Scars.

70. HYPOTHESIS

Null Hypothesis (H0): Intralesional Interferon Gamma and Triamcinolone Acetonide have equal efficacy for the treatment of Keloids & Hypertrophic Scars.

Alternate Hypothesis (HA): Intralesional Interferon Gamma is more efficacious than

Triamcinolone Acetonide for the treatment of Keloids & Hypertrophic Scars.

71. OPERATIONAL DEFINITION

Operational Definition is: Efficacy; Decrease in volume of Keloids & Hypertrophic

Scars

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72. PILOT STUDIES

Before the start of main project, the Author had to do a few pilot studies, as exact guidelines about this project were not available in literature. Keloids are only found in human beings. This is the main hindrance for scientific research on keloids, as direct research on human bring is not feasible, and is also unethical. Therefore research on keloids has mostly been clinical, with limited evidence base. So far no successful animal model of Keloids has been produced. Therefore efforts are going on to establish an animal model of Keloids. Four of these pilot studies were about Keloid Animal Model.

Commercially available Triamcinolone Acetonide injection is in the form of a milky suspension, and it is not possible to do a blinded trial when comparing it with a transparent solution like Interferon Gamma. So a study was done to make a transparent solution of

Triamcinolone Acetonide. The author wants to treat Keloids with Interferon Gamma, but information about its dose was not available. A pilot study was done regarding that. Finally, a

Pilot study was done to find out population variance and hypothesized difference. These studies are listed below. Details are described in Chapters 89-94.

1. Production of Keloid Animal Model (Jan 11)

2. Development of New Techniques for Production of Rabbit Ear Keloid Model(Feb 11)

3. Dosing of Transforming Growth Factor Beta 1 for Production of Rabbit Ear Keloid

Model (Mar 11)

4. Mannan & Hannan Technique for Production of Rabbit Ear Keloid Model (Apr 11)

5. Production of Multiple Keloids in one Animal (May 11)

6. Estimation of Hypothesized Difference and Population variance for Keloids (Jun 11)

7. Dosing of Interferon Gamma for Treatment of Keloids in Rabbits (Jul 11)

8. Production of Transparent Solution of Triamcinolone Acetonide (Aug 11)

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73. MATERIALS & METHODS

Main Outcome Measure: Decrease in volume of Keloid & Hypertrophic Scar.

Study Design: Prospective Randomized Triple Blind Active Controlled Trial.

Study Setting: Department of Pathology and Experimental Research Laboratory, University of Health Sciences, Lahore, Pakistan (Figures 90-93).

Study Duration: Two Years.

Sample Size: Gross = 240; Net = 187 subjects (Keloids & Hypertrophic Scars) divided into control and interventional groups. Sample size (n) was calculated by the formula of

2 휎2 Comparing Two Means as follows: 푛 = (푍 + 푍 ) × 2 × , where Zα/2 is the critical 훼/2 훽 푑2 value of the normal distribution at α/2, Zβ is the critical value of the normal distribution at β,

σ2 is the population variance, and d is the hypothesized difference. Using this formula sample size came out to be 80 in each group ie total 160 subjects. Fifty percent extra subjects were included to cater for failure to develop Keloid, through-and-through perforation of rabbit ear, infection, mortality, or they were reserved for follow-up of Keloid behaviour, or were punch biopsied for histopathology. Manual Calculation of Sample Size is shown in Appendix II.

Automatic Calculation of Sample Size using ‘Select Statistics’ software is shown in Figure

94.

Sampling Technique: Simple Random Selection (three stages), and Allocation (three stages).

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Sample Selection: Inclusion Criteria: Subjects with Keloids & Hypertrophic Scars.

Exclusion Criteria: Subjects with infected Keloids & Hypertrophic Scars.

Data Collection Procedure: Universal Protection was used ie Surgical Gown, Surgical Cap,

Surgical Mask, Surgical Goggles, Examination Gloves, and Surgical Shoe Covers (Figure

95). Six Rabbit Cages were taken, and marked as No 1 to No 6 with Permanent Marker

(Figure 96). Rabbit Cages were placed in a ventilated room fitted with Air Conditioner and

Fan Heater (according to season), and Exhaust Fan (Figure 97). Thirty New Zealand White

Rabbits were taken from UHS breeding facility (Figure 98), and five were put in each Cage

(Figure 99, 100).

Rabbits (5) in each cage were marked as Code A to Code E with Eosin (Figure 101) at following sites: RF (Right Fore-limb)=A; RH (Right Hind-limb)=B; LH (Left Hind-limb)=C;

LF (Left Fore-limb)=D; FH (Fore-Head)=E. Keloids to be produced were given Nos as follows: Right Ear Ventral Surface; Caudo-Proximal=, Caudo-Distal=II, Cranio-Distal=III,

Cranio-Proximal=IV; Left Ear Ventral Surface; Caudo-Proximal=V, Caudo-Distal=VI,

Cranio-Distal=VII, Cranio-Proximal=VIII. In this way a total of 240 (8x30) Keloids were to be produced.

TGFB1 Injection 50 µg (stored at -40°C in Ultra Low Temperature Freezer) was taken. It was prepared in 10 ml Water for Injection in 10 ml Syringe. A 0.2 ml (1 µg) of it was taken in Insulin Syringe. It was put it in Micro Burette Infusion Set, diluting with Water for Injection 99.8 ml, making it 100 ml (Figure102). A 0.1 ml (1 ng) of it was taken in in each of 200 Insulin Syringes. These syringes were stored at -20°C in freezer compartment of

322

Refrigerator.

Surgical Instruments ie Iris scissors straight, Iris scissors curved, Spencer stitch scissors, Lister bandage scissors, Mosquito artery forceps straight, Mosquito artery forceps curved, Adson forceps plain, Adson forceps curved, Castroviejo caliper, Mayo-Hegar needle holder, Scalpel handle No. 3, Sponge bowl, Kidney tray (Figure 103) were taken from

Instrument Cupboard (Figure 104) and sterilized in Hot Air Sterilizer at 160°C for 90 minutes. Surgeon washed-up with Alcoholic Surgical Hand Disinfectant, and wore Surgical

Gloves (Figure 105).

Rabbits were anaesthetized with Ketamine Injection (75 mg/kg intra-peritoneal), and

Xylazine Injection (15 mg/kg intra-peritoneal) in 5 ml Syringe (Figure 106). Rabbit Ears were disinfected with Sterilized Gauze soaked with Normal Saline with a Drip Set hung on a

Drip Stand (Figure 107), and Alcohol Prep Pad. Proposed points of Keloids were marked 20 mm apart on Rabbit Ears (Figure 108) with Gentian Violet and Castroviejo Caliper, saving major vessels (Figure 109). Thickness of Ear at each marked point was measured with Digital

Micrometre Gauge (Figure 110).

TGFB1 0.1 ml (1 ng), prepared in Insulin Syringes was injected at each point marked, and waited for 1 minute (Figure111, 112). Skin Biopsy Punch 8 mm (Figure 113) was taken and a disc of ventral skin, ventral perichondrium, cartilage, and dorsal perichondrium was excised, leaving behind only dorsal skin, at each point injected (Figure 114). Iris scissors,

Scalpel handle, Scalpel Blade No. 15, Adson forceps, Sponge bowl, Kidney tray, and

Magnifying Lamp (Figure 115, 116). If bleeding occurred, it was controlled with Mosquito artery forceps (Figure 117). If inadvertently dorsal skin was injured, it was stitched with 4/0

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Surgical Silk and Mayo-Hegar Needle Holder. Thickness of dorsal skin was measured with

Digital Micrometre Gauge. Wounds were dried with Sterilized Gauze and Hot Air Dryer

(Figure 118). Wounds were dressed with Hydrocolloid Dressing, using Lister bandage scissors (Figure 119). Routine post-operative care was taken. No analgesics or sedatives were required. No antibiotics were given. Routine wound care was taken. If stitches had been applied, they were removed with Spencer stitch scissors. Dressing was allowed to fall off spontaneously (Figure 120, 121). If stitches were applied, they were removed with Spencer stitch scissors.

At 4 Weeks, Out of 240 subjects, 53 dropped out due to failure to develop Keloid, through-and-through perforation of rabbit ear, infection, mortality, or they were reserved for follow-up of Keloid behaviour (four), or were punch biopsied for histopathology (four).

Therefore net subjects (Keloids) were 187.

Animal models of Keloids & Hypertrophic Scars were produced by injecting

Transforming Growth Factor Beta 1 on ventral surface of ear of randomly selected New

Zealand White Rabbits, and excising a punch of skin and cartilage. Subjects (Keloids &

Hypertrophic Scars) were allocated randomly to control (Triamcinolone Acetonide), and interventional (Interferon Gamma) groups.

Net Subjects (Keloids) were randomized by using Random Numbers generated on

Computer and Internet, using C++ Programme (Figure 122), and a Random Numbers Table was printed on Multi-Function Colour Printer (Figure 123).

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IFNG Injection 100 µg (Figure 124) was taken (stored at -40°C in Ultra Low

Temperature Freezer). It was prepared it in Water for Injection 10 ml in 15 ml Falcon Tube.

A 0.1 ml (1 µg) was taken in in each of 100 Insulin Syringes. The syringes were stored at -

20°C in freezer compartment of Refrigerator.

Triamcinolone Acetonide Powder 100 mg (Figure 125) was measured using Micro

Balance. It was dissolved in DiMethyl SulfOxide (DMSO) 10 ml in 15 ml Falcon Tube. It was sterilized by Syringe Filter (Figure 126). A 0.1 ml (1 mg) was taken in each of 100

Insulin Syringes. These syringes were stored at -20°C in freezer compartment of Refrigerator.

A Research Assistant was appointed. He was asked to put each group of Insulin

Syringes in a separate Syringe Container, label one group as Group 1, and other as Group 2, without informing the researcher (Figure 127). He was asked to record it on a Letterhead

Paper, seal it in an Envelope, store it in a Safe Deposit Locker, and keep it there till completion of data analysis (Triple Blinding) (Figure 128).

Height and Diameter of each Keloid was measured with Digital Micrometre Gauge

(after deducting volume of dorsal skin) (Figure 129-132). Volume of each Keloid was

휋ℎ calculated using Formula of Volume of Spherical Cap: 푉 = (3푎2 + ℎ2), where a is radius 6 of the base of the cap, and h is height of the cap (Figure 133).

Contents of Insulin Syringes were injected in each Keloid according to Random

Numbers Table (Figure 134).

Keloids & Hypertrophic Scars were treated with intralesional injection of

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Triamcinolone Acetonide or Interferon Gamma, and decrease in volume was noted.

At 8 Weeks (4+4), Height and Diameter of each Keloid was measured with Digital

Micrometre Gauge (after deducting volume of dorsal skin).

Four IFNG-treated and four TAC-treated Keloids were punch biopsied for histopathology. These as well as four Keloids biopsied at four weeks were processed, blocked, microtomed, stained with haematoxylin and Eosin (H & E), and examined under microscope.

Volume of each Keloid was calculated using Formula of Volume of Spherical Cap:

휋ℎ 푉 = (3푎2 + ℎ2), where a is radius of the base of the cap, and h is height of the cap. All 6 data was recorded in Performa (Figure 135, 136) and Data Sheet (Figure137, 138) as well as in software. Photographs were taken with Digital Still Camera. Catalogue of Products used in this study is shown in Figures 139-144.

Data Analysis Procedure: Data was analyzed manually and also using Microsoft Excel,

International Business Machines Statistical Package for Social Sciences and other softwares.

Z-Test was applied to measure statistical significance. A p-value of ≤ 0.05 was considered statistically significant.

Reduction in Volume of each Keloid was calculated. Arithmetic Means of Reductions in Volume of Keloid in each Group was calculated.

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1 Standard Deviation of each Mean was calculated using Formula:푆 = √ ∑푁 ( 푥 − 푁 푁 𝑖=1 𝑖

2 푥̅) , where x1, x2,---,xN are the observed values of the sample items and x̅ is the mean value of these observations, and 푁 is size of each sample.

푥̅1−푥̅2−∆ Z-Test was applied using Formula: 푧 = , where x1 and x2 are the means of the 휎 2 휎 2 ̅ ̅ √ 1 + 2 푛1 푛2 two samples, Δ is the hypothesized difference between the population means (0), σ1 and σ2 are the standard deviations of the two populations, and n1 and n2 are the sizes of the two samples. Manual Calculation of Z-Test is shown in Appendix II. Automatic Calculation of Z-

Test is shown in Table 34.

The p-value was calculated on the basis of Z-Test using Z-Distribution Table. Manual

Calculation of p-value using Z-Distribution Table is shown in Table 35-36. Automatic

Calculation of p-value is shown in Table 37.

All these calculations were repeated using Microsoft Excel 2013, and International

Business Machines Statistical Package for Social Sciences version 23.0 software. In-silico

Project Support for Life Sciences web page was used for Z-Test calculation. Social Science

Statistics Home Page was used to calculate p-value.

The Research Assistant was asked to open the code (De-Blinding done). Drug 1 was found to be Triamcinolone Acetonide (Control Group), and Drug 2 Interferon Gamma

(Interventional Group).

327

Figure 90: Experimental Research Laboratory UHS, Outer View 328

Figure 91: Experimental Research Laboratory UHS, Inner View

329

Figure 92: Animal Operation Theatre UHS 330

Figure 93: Pathology Department UHS

331

Figure 94: Sample Size Calculation (Select-Statistics, 2015 [Bib])

332

Figure 95: The Author with Universal Protection

333

Figure 96: Rabbit Cage Numbering

334

Figure 97: Rabbits in Cages with Air-Conditioner and Exhaust Fan

335

Figure 98: Animal Breeding Facility UHS

336

Figure 99: Rabbits in a Cage

337

Figure 100: Animal Keeping

338

Figure 101: Rabbit Coding with Eosin

339

Figure 102: TGFB1 Injection in Micro Burette in Freezer Compartment

340

Figure 103: Surgical Instruments

341

Figure 104: Surgical Instruments Cupboard

342

Figure 105: Preparations for Operation

343

Figure 106: Anaesthetizing Rabbit

344

Figure 107: Disinfecting Rabbit Ear

345

Figure 108: Keloid Sites

346

Figure 109: Marking Keloid Sites with Gentian Violet & Graduated Caliper

347

Figure 110: Measuring Thickness of ear with Digital Micrometre Gauge

348

Figure 111: Injecting TGF Beta 1 at Keloid Points with Insulin Syringe

349

Figure 112: Dorsal Surface of Ear showing swellings at Sites of TGFB 1 Injections

350

Figure 113: Skin Punch

351

Figure 114: Punch of ear Skin and Cartilage has been cut

352

Figure 115: Punch area seen through Magnifying Lamp

353

Figure 116: Four Punches of Skin and Cartilage excised

354

Figure 117: Bleeding being controlled with Artery Forceps

355

Figure 118: Wounds being dried with Hot Air Dryer

356

Figure 119: Wounds dressed with Hydrocolloid Dressing

357

Figure 120: Post-operative Case

358

Figure 121: Keloids developed on Rabbit Ear

359

Figure 122: Random Numbers Generation (Code::Blocks, 2015 [Bib]) 360

Figure 123:B; Random Number Table

361

Figure 124: Interferon Gamma

362

Figure 125: Triamcinolone Acetonide

363

Figure 126: Syringe Filter for Sterilization of Triamcinolone Acetonide

364

Figure 127: Blinded Drugs in Insulin Syringes

365

Figure 128: Triple Blinding Documents

366

Figure 129: Keloids on Rabbit Ear

367

Figure 130: Keloids Marked for Measurement

368

Figure 131: Keloid Thickness Measurement

369

Figure 132: Keloid Diameter Measurement

370

Figure 133: Spherical Cap Volume Calculation (Wolfram-MathWorld, 2015 [Bib])

371

Figure 134: Drug 1 or 2 being injected in Keloids

372

Figure 135:B; Blank Performa

373

Figure 136:B; Filled Performa

374

Figure 137:B; Data Sheet

375

Figure 138:B; Filled Data Sheet

376

Figure 139:B; Products and Manufacturers’ Directory 1

377

Figure 140:B; Products and Manufacturers’ Directory 2

378

Figure 141:B; Products and Manufacturers’ Directory 3

379

Figure 142:B; Products and Manufacturers’ Directory 4

380

Figure 143:B; Products and Manufacturers’ Directory 5

381

Figure 144:B; Products and Manufacturers’ Directory 6

382

74. RESULTS

A total of 240 subjects (Keloids) were included in the study. Out of 240 subjects

(Keloids), 53 dropped out due to mortality, infection, or through-and-through perforation of rabbit ear. Therefore net subjects were 187. Out of net 187 subjects, 93 were in control

(Triamcinolone Acetonide), and 94 in interventional (Interferon Gamma) group

Mean reduction in volume of Keloids & Hypertrophic Scars in Triamcinolone

Acetonide group subjects was 6.56661 mm3 (range 43.763 to -22.959), and in Interferon

Gamma group subjects 13.49791 mm3 (range 44.504 to -16.519). Standard Deviation of

Mean for Triamcinolone Acetonide group was 12.579920, and for Interferon Gamma group

10.064669; combined Standard Deviation was 11.874364.

Z-Test was applied to check differences between 2 groups. Z-Test value came out to be -4.1574. The p-value was 0.000016, which is highly significant. Results are given in

Tabulated and Graphical form in Tables No. 1 to 37 on pages that follow.

In addition, a new technique was invented for production of Keloid Animal Model.

This model was produced on Rabbit Ears. This is the first successful Keloid Animal Model ever produced. The technique has been named and patented as Mannan & Hannan Technique for Production of Rabbit Ear Keloid Model. Details of this technique are given in a separate

Chapter. Production of Keloid was confirmed on clinical, gross, µscopic examinations.

Clinically, significant Keloids were produced. They were hard in consistency, dome- shaped, red in colour, with smooth surface. They were devoid of hairs. These Keloids were

383 followed-up for one year, and many of them were present even at that time. Of course, it was not possible to ask about symptoms of Keloids from Rabbits.

On gross examination, excised Keloids were hard in consistency, and could be palpated as definite nodule between thumb and fore-finger. They had red and smooth surface.

On cutting the specimen, there was gritty feeling. Cut surface was pale in colour.

Histopathology of four Punch Biopsied Keloids showed homogeneous, brightly eosinophilic, irregularly distributed, glassy, hyalinized collagen. Fibroblasts were scant, and oriented in direction of collagen (Tables 38, 39). Histopathology of four Punch Biopsied

Keloids treated with IFNG showed moderately decreased amount of hyalinized collagen and fibroblasts. Histopathology of four Punch Biopsied Keloids treated with TAC showed mildly decreased amount of hyalinized collagen and fibroblasts, and relative increase in interstitial space containing mucinous material.Changes due to these treatments were quantitative (on gross examination) rather than qualitative (on microscopic examination). Other stains, including Immunohistochemistry stains, were also used. Gomori’s Trichrome stains Keloids showing wavy collagen bundles as blue or green. Mallory’s Trichrome and Masson’s

Trichrome are also used for this purpose, which also stain collagen bundles as blue or green.

Movat's Pentachrome stains collagen fibres as yellow. Osmium Tetroxide stains collagen as brown. Periodic Acid-Schiff (PAS) stains collagen as pink. Phosphotungstic Acid-

Hematoxylin (PTAH) stains collagen fibres as red. Van Gieson stains collagen fibres as red.

Verhoeff stains collagen fibers as red.PicroSirius Red uses polarized light to visualize collagen, which appear bright yellow or orange, and thin fibers look green. Periostin is a secreted extracellular matrix protein, which is upregulated in Keloids. Immunohistochemistry staining was also done using anti-Periostin antibodies, which stained collagen as brown

(Table 40).

384

Table 1:B; Group 1 (TAC) Raw Data (1-1)

385

Table 2:B; Group 1 (TAC) Raw Data (1-2)

386

Table 3:B; Group 1 (TAC) Raw Data (1-3)

387

Table 4:B; Group 1 (TAC) Raw Data (1-4) 388

Table 5:B; Group 2 (IFNG) Raw Data (2-1)

389

Table 6:B; Group 2 (IFNG) Raw Data (2-2)

390

Table 7:B; Group 2 (IFNG) Raw Data (2-3)

391

Table 8:B; Group 2 (IFNG) Raw Data (2-4)

392

Table 9:B; Comparative Statistics of all Groups

393

Table 10:B; Group 1 Detailed Statistics

394

Table 11:B; Group 1 Frequencies & Percentages

395

Table 12:B; Group 1 P-P Plot 396

Table 13:B; Group 1 Q-Q Plot

397

Table 14: Group 1 Histogram with Normal Curve

398

Table 15: Group 1 Bar Chart

399

Table 16: Group 1 Boxplot

400

Table 17 Group 1 Pie Chart

401

Table 18:B; Group 2 Detailed Statistics

402

Table 19:B; Group 2 Frequencies & Percentages

403

Table 20:B; Group 2 P-P Plot

404

Table 21:B; Group 2 Q-Q Plot

405

Table 22: Group 2 Histogram with Normal Curve

406

Table 23: Group 2 Bar Chart

407

Table 24: Group 2 Boxplot

408

Table 25: Group2 Pie Chart

409

Table 26:B; Group 1+2 Detailed Statistics

410

Table 27:B; Group 1+2 Frequencies & Percentages

411

Table 28:B; Group 1+2 P-P Plot

412

Table 29:B; Group 1+2 Q-Q Plot

413

Table 30: Group 1+2 Histogram with Normal Curve

414

Table 31: Group 1+2 Bar Chart

415

Table 32: Group 1+2 Boxplot

416

Table 33: Group 1+2 Pie Chart

417

Table 34: Z-Test Calculation (In-Silico, 2015 [Bib])

418

Table 35:B; Z Distribution Table (1) (University-of Wisconsin, 2015 [Bib])

419

Table 36:B; Z Distribution Table (2) (University-of-Wisconsin, 2015 [Bib])

420

Table 37: P-Value Calculation (Social-Science-Statistics, 2015 [Bib])

421

Table 38: Normal Rabbit Skin, H & E Stain

422

Table 39: Keloid in Rabbit, H & E Stain 423

Table 40: Keloid in Rabbit, Periostin Stain 424

75. DISCUSSION

This prospective randomized triple blind active controlled trial comparing the efficacy of intralesional Interferon Gamma and Triamcinolone Acetonide for the treatment of Keloids

& Hypertrophic Scars was carried out at Department of Pathology and Experimental

Research Laboratory, University of Health Sciences, Lahore. It consisted of gross 240 (net

187) subjects (Keloids).

In this study mean reduction in volume of Keloid with Triamcinolone Acetonide was

6.56661 mm3 (range 43.763 to -22.959), and with Interferon Gamma 13.49791 mm3 (range

44.504 to -16.519). Interferon Gamma group had more central tendency, while

Triamcinolone Acetonide group had more dispersion. The p-value was 0.000016, which is highly significant.

Literature search showed no Randomized Controlled Trial comparing efficacy of

Interferon Gamma with Steroids or Placebo in the treatment of Keloids & Hypertrophic

Scars. However some studies were available which investigated role of Interferon Gamma in the treatment of Keloids & Hypertrophic Scars.

Granstein et al (1990) used recombinant human IFNG to manage Keloids in eight patients. Patients were treated with 0.01 or 0.1 mg of recombinant human IFNG injection in one lesion and placebo in other lesion, thrice weekly for 3 weeks. Three days after last injection, biopsies were done on experimental and control lesions. Six out of eight patients who completed treatment course showed decrease in height at treated site, with mean decrease of 30.4% in treated lesion versus 1.1% in control lesion. Keloids treated with

425

Interferon Gamma showed changes in both epidermis and dermis. Epidermis developed thinning of suprapapillary plates, compact hyperkeratosis, focal or diffuse parakeratosis, exocytosis of lymphocytes, and enhanced amount of mucin. Dermis had a decreased amount of thicken collagen bundles and active fibroblasts and enhanced number of inflammatory cells and amount of mucin. They recommended that Interferon Gamma was feasible in treatment of Keloids.

Larrabee Jr et al (1990) studied efficacy and toxicity of intralesional Interferon

Gamma injection in management of Keloids & Hypertrophic Scars in 10 patients. All lesions reduced in width and height. Five of 10 keloids reduced by 50% in width. They recommended that IFNG should be given intralesionally once weekly, upto 0.05 mg per injection, for 10 weeks. There were no serious side effects. Most common side effect was minor headache.

Pittet et al (1994) investigated therapeutic effects of intralesional IFN gamma on human hypertrophic scars in four patients. IFN gamma decreased symptoms and size of lesions; immunofluorescence study revealed that alpha-SMA level was reduced in myofibroblasts. Further, fibroblast culture showed that IFN gamma reduced replication and expression of alpha-SMA. They suggested that IFN gamma could be a useful treatment of hypertrophic scars.

Broker et al (1996) treated keloids in nine patients with excision followed by

Interferon Gamma or placebo injections. Three weeks after excision, each patient received 10 weekly injections of Interferon Gamma in one wound, and normal saline placebo in other.

Out of nine, patients, only three completed complete course of 10 injections. Seven patients

426 were available evaluation at 12 weeks, and only four at one year. Statistical test was not done due to low number, but three out of four patients felt that they had significant recurrence.

Granstein et al (1990), for the first time reported effect of Interferons on collagen production. They wrote that Interferons alpha, beta, and gamma reduced collagen production by skin fibroblasts. Further, IFN gamma also inhibited enhanced collagen production by fibroblasts from scleroderma patients. IFN gamma also inhibited collagen production by myofibroblasts and synovial fibroblast-like cells.

Ghahary et al (1995) made twelve distinct fibroblast cell strains, six from hypertrophic scar and six from normal dermis of same persons. He found rise in collagenase mRNA with up to 4000 IU/ml of IFN alpha 2b, but maximum rise in tissue inhibitor of metalloproteinase mRNA expression and maximum reduction in mRNA for type I procollagen at 2000 IU/ml. After this cells were treated with either IFN alpha 2b (2000

IU/ml) or IFN gamma (1000 IU/ml) for 96 hours. It produced rise in tissue inhibitor of metalloproteinase-1 and collagenase mRNA (81% and 54%, respectively) in IFN alpha 2b treated hypertrophic scar fibroblasts. IFN alpha 2b had same effects on normal skin fibroblasts. But cells treated with IFN gamma revealed rise in tissue inhibitor of metalloproteinase-1 mRNA (78% in hypertrophic scar and 56% in normal dermal fibroblasts) but fall (59% and 42%, respectively) in collagenase mRNA. Significantly increase collagenase activity was present in conditioned medium from IFN alpha-2b treated fibroblasts in comparison with that from IFN gamma treated fibroblasts.

Harrop et al in 1995 investigated effects of IFN gamma on cell proliferation, collagen production, and levels of types I & III procollagen mRNA in human postburn keloid

427 fibroblasts. They reported that treating proliferating fibroblast cultures for 5 days with IFN gamma (1000 u/ml) resulted in 51% reduction in hypertrophic scar cell proliferation. A 34% decrease in collagen production was seen in hypertrophic scar fibroblasts after treatment with

IFN gamma (1000 u/ml) for 48 hr. Northern blot analysis demonstrated 55 and 36% decrease in type I and type III procollagen mRNA levels, respectively, after treatment for 12 hours with IFN-gamma (1000 u/ml).

Kikuchi et al (1995) studied actions of many growth factors on [3H]thymidine incorporation and procollagen type I carboxyterminal propeptide (P1CP) formation, which reflected type I collagen metabolism, in Keloid and normal fibroblasts. Six fibroblast cell strains, which were taken from Keloid or normal skin, showed same growth responses to

PDGF, TGF Beta 1, IFN Gamma, and histamine. In contrast, Keloid fibroblasts revealed markedly increased growth response to EGF than normal fibroblasts. P1CP production was

4.4 times higher in six strains of Keloid fibroblasts than in six controls. Treatment with IFNG

(100 U/ml) decreased P1CP production in both groups; effect was significantly greater in

Keloid fibroblasts. TGFB1 treatment upregulated P1CP production in both groups. Histamine treatment increased P1CP production in Keloid fibroblasts, but not in uninjured skin fibroblasts. They concluded that Interferon Gamma decreased production of type 1 collagen, more so in Keloids.

Ladin et al (1998) studied p53 and apoptosis changes in keloids and keloid fibroblasts.

Twenty keloid paraffin blocks were randomly selected for an immunoperoxidase assay with antibodies against Fas, p53, bcl-2, and bcl-x proteins using target antigen-retrieval method.

Apoptosis was detected in keloids and normal skin and in keloid and normal fibroblasts by the TdT-mediated dUTP nick-end labelling (TUNEL) assay on tissue sections, fibroblast

428 cultures, and by flow cytometry for cell suspensions. Keloid lesions and Keloid fibroblasts showed less apoptosis as compared to normal controls. Keloid fibroblasts showed increased apoptosis when treated with hydrocortisone, IFN Gamma, and hypoxia, in comparison to normal adult fibroblasts.

Cen and Yan (1999) isolated cultured fibroblasts from hypertrophic scars of 10 patients. Fibroblasts were allocated to two groups; one group was treated with IFN gamma

(100 IU/ml, 5 days) and other was control. Proliferative activity in both groups was investigated and compared; it was downregulated in IFN gamma group. In IFN gamma group, differentiation of myofibroblasts were reduced and decreasing ratio was 3.2% at 2nd day and up to 10.5% at 8th day. Apoptotic ratio was 17.7% in IFN gamma group, and 10.9% in control group. The difference was statistically significant. They concluded that IFN

Gamma downregulated proliferation of fibroblasts, decreased differentiation of myofibroblasts, and induced apoptosis, and might be used in management of keloids.

Tredget et al (2000), using three sets of keloid and normal fibroblasts from same patients, treated nonconfluent and near confluent fibroblasts with TGF beta, and cell growth

429 fibroblasts and was maximally increased at 75 pM TGF-beta. TGF beta stimulated collagen production was blocked by IFN alpha or IFN gamma or both in an additive fashion. They supported use of IFN alpha and IFN gamma for treatment of keloids.

Liu et al (2009) studied effects of IFN Gamma on TGF Beta/Smad pathway in Keloid fibroblasts, and its mechanism of action in treatment of pathological scar. Fibroblasts from

Keloid tissue of 3 patients were separated and cultured in vitro. They were divided into control group, TGF Beta group, Interferon IFN group, and TGF Beta+IFN Gamma group.

Expression of mRNA and protein of CTGF, and alpha SMA protein were detected by different techniques. They reported that Interferon Gamma could down-regulate level of

Smad 3, and up-regulate level of Smad 7 in a time- and dose-dependent manner, and reduce level of CTGF and alpha SMA in basic state or stimulated by TGF Beta. It reveals a marked inhibitory effect on TGF Beta/Smad signal pathway. They concluded that it was a significant finding in management of keloid and hypertrophic scar by Interferon Gamma.

Hasegawa et al (2003) studied if Interferon Gamma antagonized Transforming

Growth Factor Beta stimulated fibrotic reaction in Keloid fibroblasts. They concluded that

Interferon Gamma failed to antagonize Transforming Growth Factor Beta mediated fibrotic response in Keloid fibroblasts; so it might not be useful for Keloid & Hypertrophic treatment.

This is the only study in which Interferon Gamma has failed to antagonize TGF Beta activity in Keloid fibroblasts.

For this study a successful keloid animal model was produced. This was done on rabbit ear using a new technique, which has been named as Mannan & Hannan technique.

Previously most of the studies have been done on keloid fibroblasts since 1951 (Robinson,

430

1951; Garner, 1955; Koch, 1997; Kuhn, 2001). These fibroblasts were isolated from human keloid tissue, and cultured in artificial media. Efforts have been going on for development of keloid animal model since 1961 (Szava, 1961; Mancini & Quaife, 1962; Kelly, 1964;

Ketchum, 1967; Szabo, 1971). Bazin et al (1975) produced first animal (rat) model of wound healing and hypertrophic scar.

Shetlar et al (1985) reported, for the first time, animal model for study of keloids.

Robb et al (1987) developed similar animal model for study of keloids. Morris et al (1997), for the first time, developed rabbit ear hypertrophic scar model. Li et al (2001) reported establishment of experimental animal model for hypertrophic scar on rabbit ears by creating round and rectangular wounds. Aksoy et al (2002) produced hypertrophic scar animal model in Guinea pigs by skin excision and coal tar application. Hochman et al (2005) reported

Keloid heterograft in hamster cheek pouch as a new animal model. Yang et al in 2007 reported development of a new hypertrophic scar model by transplanting full thickness human skin on backs of nude mice.

Ramos et al (2008) in an article titled: Is there an ideal animal model to study hypertrophic scarring? described five different types of animal models: heterologous keloid implant in immunodeficient animals; heterologous keloid implant in immune privileged site; hypertrophic scar production through chemical trauma; hypertrophic scar production in anatomically specific site; and porcine model. Philandrianos et al (2014) developed animal model using human keloid containing epidermal and dermal tissue implanted on dorsum of athymic mice. Zhu et al (2008) suggested need of research for correlation between anatomy of rabbit ear and creation of hypertrophic scar.

431

Wang H. et al (2005), Wang and Luo (2013), established human keloid animal model by use of tissue engineering technique. Human keloid fibroblasts were moved to poly(lactic- co-glycolic acid) (PLGA) copolymer and cultured for seven days. Then composite of fibroblasts and PLGA (experimental group), and PLGA only (control group) were implanted into subcutaneous pouches in nude mice. Implanted specimen were harvested on 30, 60, 120, and 180 days for microscopic examination. All mice stayed alive after experiment. Implants of experimental group went on growing in size from days 30 to 180, while implants of control group reduced in size.

76. CONCLUSION

Treatment of Keloids & Hypertrophic Scars with Interferon Gamma is significantly more efficacious than with Triamcinolone Acetonide.

77. RECOMMENDATION

This study should be replicated in human beings.

432

78. PATENT

Mannan & Hannan Techniquefor Production of Rabbit Ear Keloid Model

Rabbits were anaesthetized with Ketamine Injection (75 mg/kg intra-peritoneal), and

Xylazine Injection (15 mg/kg intra-peritoneal) in 5 ml Syringe. Rabbit Ears were disinfected with Sterilized Gauze soaked with Normal Saline with a Drip Set hung on a Drip Stand, and

Alcohol Prep Pad. Proposed points of Keloids were marked 20 mm apart on Rabbit Ears with

Gentian Violet and Castroviejo Caliper, saving major vessels. Thickness of Ear at each marked point was measured with Digital Micrometre Gauge. TGFB1 0.1 ml (1 ng), prepared in Insulin Syringes was injected at each point marked, and waited for 1 minute. Skin Biopsy

Punch 8 mm was taken and a disc of ventral skin, ventral perichondrium, cartilage, and dorsal perichondrium was excised, leaving behind only dorsal skin, at each point injected. Iris scissors, Scalpel handle, Scalpel Blade No. 15, Adson forceps, Sponge bowl, Kidney tray, and Magnifying Lamp. If bleeding occurred, it was controlled with Mosquito artery forceps.

If inadvertently dorsal skin was injured, it was stitched with 4/0 Surgical Silk and Mayo-

Hegar Needle Holder. Thickness of dorsal skin was measured with Digital Micrometre

Gauge. Wounds were dried with Sterilized Gauze and Hot Air Dryer. Wounds were dressed with Hydrocolloid Dressing, using Lister bandage scissors. Routine post-operative care was taken. No analgesics were required. No antibiotics were given. Routine wound care was taken. If stitches had been applied, they were removed with Spencer stitch scissors. Dressing was allowed to fall off spontaneously. Keloids formed in four weeks in majority of the lesions produced.

433

79. GLOSSARY

1. Active Controlled: A randomized controlled trial in which interventional agent is

compared with another standard agent (usually the present gold standard) rather than a

placebo.

2. Adjuvant: In addition to. This term is usually used in relation to more than one treatment

given one after the other.

3. Apoptosis: Self destruction of cell. It is needed to get rid of unwanted and abnormal

cells.

4. Aqua Pro Injection: Distilled water used as diluent for injections. It is considered a

medicine.

5. Bar Chart/Graph: A chart or graph which uses thin bars to represent data. Height of bar

represents quantity of each datum.

6. Blinding: A research method in which researcher does not know the group of material he

is using ie control or interventional.

7. Boxplot: It is a type of statistical graph, in which a rectangle represents second and third

quartiles. First and fourth quartiles are shown as lines at both ends of rectangle.

8. C++: A computer programming language. It is used for making diverse types of

programmes.

9. Caudal: Related to or towards feet. This term is used mostly in relation to animals

(Quadrupeds).

10. cDNA Microarray: An orderly arrangement of DNA sequences on a glass slide, to study

simultaneous expression of multiple genes.

11. Chemokines: Low molecular weight cytokines which are able to induce chemotaxis in

leukocytes.

434

12. Chemotaxis: Attraction of leukocytes towards injured tissue in response to chemicals

(chemokines) produced there.

13. Cicatrix: Mesh term for Scar eg cicatrix, hypertrophic. This term is rarely used by

authors.

14. Collagen: A type of protein, which is main constituent of dermis, and gives it mechanical

strength.

15. Collagenase: An enzyme which digests collagen. There are different types of

collagenases present.

16. Confidence Level: Statistical probability that value of a parameter will lie in a stated

range of values.

17. Cranial: Related to or towards head. This term is used mostly in relation to animals

(Quadrupeds).

18. Critical Value: A point in test statistic beyond which null hypothesis is rejected.

19. Cytokine: An immune-regulatory protein (as interleukin or interferon) that is secreted by

cells especially of immune system.

20. Cytoskeleton: A collection of filaments and tubules in cytoplasm which maintains cell

shape. They are mainly proteins.

21. D-Day: Decisive Day. Day to start an adventure etc. This term was first used during

Second World War.

22. De-Blinding: In research, it means removal of blinding to find out nature of material

used.

23. De-Bulking: Partial excision of a tumour to decrease its size. This term is usually used in

relation to malignancies.

24. Digital Micrometre Gauge: A digital-mechanical gadget to measure size in

micrometres.

435

25. Distal: Away from centre of the body. This term is usually used in relation to extremities.

It is also used in relation to rabbit ears.

26. Down-Regulation: Process by which a cell decreases production of a sub-cellular

component in response to a stimulus.

27. Efficacy: The power to produce a desired result or effect. This term is usually used for

drugs or other modes of treatment.

28. Elastin: A protein found in dermis and elastic tissues. It gives elasticity to skin. It is

metabolically inactive.

29. Electrophoresis: The movement of charged particles (ions) in a fluid or gel medium

when a current is passed through it.

30. Exocytosis: Release of cell secretions to outside by fusion of vesicular membrane with

cell membrane.

31. Experimental Research: Use of laboratory animals in medical research. This term was

coined to avoid agitation of animal right activists.

32. Extracellular Matrix: The substance in which tissue cells (as of connective tissue) are

embedded.

33. Fas Gene: A gene which encodes one of the many proteins that are responsible for

apoptosis.

34. Fibroblast: Cell producing fibres, especially collagen. They are the main cell of tissue

repair. Their origin is controversial. Later they change into myofibroblasts.

35. Fibrocytes: They are one of the circulating cells of blood, and are precursors of

fibroblasts.

36. Fibrosis: Formation of fibrous tissue as a result of repair after injury, inflammation or

other noxious stimulus.

37. Fore-Limb: Front limbs. This term is usually used for animal waking on four feet

436

(Quadrupeds).

38. Frontispiece: Front illustration and title in normal font, placed facing the main title page

of a book etc. This thesis also contains a frontispiece.

39. Gene Therapy: Replacement of abnormal genes in cells with normal genes to correct

genetic disorders.

40. Gentian Violet: A synthetic violet dye of aniline, used as an antiseptic, and also as a vital

stain.

41. Gray (in radiotherapy): The absorption of one joule of radiation energy per one

kilogram of matter.

42. Grey Literature: It is a type of literature produced by non-publishing organizations, eg

Theses.

43. Growth Factor: A protein capable of stimulating or inhibiting cellular growth.

44. Hereditary: A disease capable of being transferred from parents to offspring in genes

and DNA.

45. Hind-Limb: Back limbs. This term is usually used for animal waking on four feet

(Quadrupeds).

46. Histogram: A statistical graph containing rectangles with area proportional to a variable

and width equal to class interval.

47. Homo sapiens: Species to which human beings belong. It is the highest form of life.

48. Hyperkeratosis: It is the thickening of stratum corneum of epidermis of the skin.

49. Hypertrophic Scar: Excessive overgrowth of dense collagen tissue, often red, pink, or

purple in appearance, at site of a healed skin defect.

50. Hypothesis: An assumption made on the basis of limited evidence. It is tested by

experimentation.

51. Hypothesis, Alternate: This is the hypothesis to be accepted if the null hypothesis is

437

rejected.

52. Hypothesis, Null: The hypothesis that there is no significant difference between specified

populations.

53. Immunofluorescence: A method of finding site of an antigen (or antibody) in tissues by

reacting it with a fluorescent labelled antibody (or antigen).

54. Immunohistochemistry: Determination of specific antigens on tissue sections by use of

immunological markers, which are enzymes.

55. In-situ Hybridization: It is use of DNA or RNA probe to identify complementary DNA

or RNA in cells.

56. Interferon Gamma: It is a cytokine secreted by T-lymphocytes. It is used for

management of keloids.

57. Intralesional: In the lesion eg Keloid. This term is usually used for giving injections in

the lesion

58. Keloid: An exuberant scar which grows beyond margins of original wound, continues to

grow, and has other symptoms like pruritus.

59. Keratinocyte: An epidermal cell which produces keratin. They are continuously

regenerating cells

60. Ketamine: A synthetic non-barbiturate general anaesthetic. It is given intravenously,

intramuscularly, or (in animals) intraperitoneally.

61. LASER: An intense beam of light which maintains its intensity for long distances. Lasers

are used in cutting, alignment, and in surgery.

62. Mannan & Hannan Technique: A new technique to produce rabbit ear Keloid model.

63. Matrix metalloproteinases: A group of proteolytic enzymes in extracellular space. They

require a metal (zinc or calcium) for their functioning.

64. Model, animal: A living organism used for experimentation for benefit of human beings.

438

65. Myofibroblast: A cell that is amid a fibroblast and a smooth muscle cell in phenotype. It

is involved in wound contraction.

66. New Zealand White Rabbit: A breed of domestic rabbits which, despite its name, was

developed in USA. These rabbits are most commonly used in research.

67. Papyrus: A paper-like material made from stem of a water plant. It was used in ancient

Egypt for writing.

68. Parakeratosis: presence of live nucleated keratinocytes in the stratum corneum of skin. It

may be due to accelerated keratinocyte regeneration.

69. Perichondrium: A fibrous connective tissue layer covering the non-articular cartilage.

70. Pie Chart/Graph: A statistical graph, of the shape of an apple-pie. It is divided in

sectors. Size of each sector is proportional to quantity of each.

71. Pilot Study: A small preliminary study to check feasibility of main study. Sample size is

determined on basis of this study.

72. Plagiarism: Copying language or thoughts of another person, without giving credit to

original author.

73. Polymerase Chain Reaction: A molecular pathology test technique in which multiple

copies of DNA are made.

74. Population: The whole pool of subjects under study out of which a representative sample

is taken.

75. Power of Test: Probability of making correct diagnosis if alternate hypothesis is true.

76. P-P Plot (Probability-Probability Plot: It is a statistical graph showing how close two

statistic sets match

77. Procollagen: Soluble precursor of collagen formed by fibroblasts and other cells in

process of collagen synthesis.

78. Prospective: A study to happen at a future date using future data. It is opposite of

439

Retrospective.

79. Protein p53: A tumour suppressor gene which binds with DNA, and down-regulates cell

division.

80. Proximal: Nearer to centre of body. This term is usually used in relation to extremities. It

is also used in relation to rabbit ears.

81. Punch: A tool for circular piercing or cutting. It is used for core biopsy of skin, Keloid

tissue etc.

82. P-Value (Probability Value): A statistic showing how significant findings of a study are.

Lesser p-value means more significance.

83. Q-Q Plot (Quantile-Quantile Plot): A statistical graph comparing two probability

distributions by drawing their quantiles against each other.

84. Randomized: A sampling method in which each member of population has an equal

chance of inclusion in sample.

85. Recombinant: A biological agent produced by manipulating genetic material of a host

organism eg bacteria.

86. Recurrence: To happen or occur again after treatment or cure. This term is mainly used

for tumours.

87. Sample: A set of data randomly selected from the population. Sample is considered of

population.

88. Scar: A mark left on skin after a wound, burn, or ulcer. It can be normal or pathological.

89. Southern Blot: A molecular pathology technique for DNA sequences, in which DNA

fragments are placed on a gel, and then transferred to another material, like a blotting

paper. Southern was name of the scientist who developed this technique. Modified forms

of this test are called Northern Blot and Western Blot (these are not names of scientists).

90. Spherical Cap: The region of a sphere which lies above a given plane. This term is used

440

in mathematics.

91. Standard Deviation: A statistic describing how much values of a group deviate from

mean value of group.

92. Statistical Significance: A statistic showing that results of a particular study are not due

to chance.

93. Suprapapillary Plates: The small area of epidermis between rete pegs.

94. Telangiectasis: Dilatation of superficial capillaries of skin, appearing in clusters of red or

pink colour, especially in white skin people.

95. Transfect: Introduce genetic material by infecting a cell with free nucleic acid. This

method is used in gene therapy.

96. Transforming Growth Factor Beta: A regulatory cytokine which has multiple

functions. It can increase or decrease many cellular functions. It has three isoforms.

97. Triamcinolone Acetonide: A synthetic cortico-steroid that is most commonly used as

intralesional injection for keloid.

98. Triple Blind: A study in which the identities of control group and interventional group

are withheld from the statistician as well. Other variants are Single Blind and Double

Blind.

99. Up-Regulation: Process by which a cell increases production of a sub-cellular

component in response to a stimulus.

100. Variance: It is mean of squared differences from actual mean. It is square of standard

deviation.

101. Xylazine: It is an alpha 2 adrenergic agonist. It is used in animal for sedation during

anaesthesia.

102. Z-Test: It is a statistical test to find out if two population means are different. It is

used if number of subjects is 30 or more.

441

80. BIOGRAPHIES

1. Addison, Thomas: (1793 AD-1860 AD). A British doctor. He belonged to Guy’s

Hospital, and is included in Great Men of Guys. Many diseases and syndrome bear his

name. He, however, made a mistake on Keloid.

2. Alibert, Jean Louis Marc: (1768 AD-1837 AD). He was a French Dermatologist. He

was a pioneer of Dermatology. He coined the term Keloid. He wrote many books on

diseases of skin.

3. Ambroise Pare: (1510 AD-1590 AD). A French barber-surgeon. He was an expert of

treatment of wounds, especially gunshot wounds. He wrote the first book on wounds in

1560. He accidently discovered that hot oil was bad for wounds, and started using water

for wound dressings.

4. Az-Zahrawi, Abu al-Qasim Khalaf ibn al-Abbas: (936-1013). Latinized as Abulcasis

or Albucasis, Az-Zahrawi was born in Qurtaba (Cordoba) in Andalusia (Spain) in the

reign of Khalifa (Caliph) al-Hakam II. He was a court physician and surgeon for more

than 50 years. He wrote a book titled al-Tasrif which is considered the first textbook of

surgery. He invented many surgical instruments

5. Bell, Benjamin: (1749-1806). He belonged to Edinburgh, Scotland. He wrote a book

titled: A system of surgery in six volumes (1783). Chapter XXXVI of this book, in

Volume V, was on Wounds.

6. Breasted, James H.: (1868-1935). He translated Edwin Smith Surgical Papyrus in 1930.

It was published by University of Chicago Oriental Institute, of whom Breasted was

Director.

7. Djoser: (c.2670 BC. One of the most famous contemporaries of king Djoser was his

vizier, Imhotep.

442

8. Gordon (slave): (1863).He, also called Whipped Peter, was first person photographed

with Keloids (from being whipped).

9. Hammurabi: (1792-1750 BC). He was sixth Amorite king of Babylon. He is famous for

his code of law, which is the most ancient written record of law ever found.

10. Horemheb: (1323-1295 BC). Last pharaoh of 18th dynasty of kings of ancient Egypt. He

had scars and keloids on face.

11. Imhotep: (2650-2600 BC). He was vizier of king Djoser of Egypt. It is speculated that he

wrote or re-wrote Edwin Smith Surgical Papyrus.

12. Rayer, Pierre Francois Olive: (1793-1867). He was a French Physician and

Dermatologist. He wrote a book on diseases of the skin (1826 AD). Later he shifted to

Nephrology

13. Retz, Noel: (1758-1810). He was son of a military doctor. He became navy doctor, and

then Royal doctor. Apart from medicine, he also wrote on many other subjects. He was

the first doctor to describe a keloid, though he considered it herniation of fat.

14. Roger of Salerno: (1140-1195).He was a Salernitan (Italian) surgeon who wrote a work

of medicine titled Practica Chirurgiae (The Practice of Surgery) around 1180). It is also

called Chirurgiae Magistri Rogerii (The Surgery of Master Rogerius).

15. Scultetus, Joannis: (1595-1645). First academically trained surgeon (compare with

barber).He wrote a book titled Armamentarium chirurgicum (Surgical arsenal) in 1655. It

contained a complete catalogue of all known surgical instruments, methods of bandaging

wounds and splinting fractures.

16. Smith, Edwin: (1822-1906).He was an American Egyptologist. He bought the Papyrus in

Luxor, Egypt in 1862, in parts, put it together, and tried to translate it, but failed. His

daughter gave this Papyrus to New York Historical Society after his death. This Papyrus

is named after him as Edwin Smith Surgical Papyrus.

443

15. BIBLIOGRAPHY

This Bibliography includes books and other materials relevant to Keloids.

1. Anatomy, Histology, and Embryology Books (2+1+1)

2. Physiology, Biochemistry, and Pharmacology Books (2+1+1)

3. Pathology Books (10)

4. Surgery Books (7)

5. Plastic Surgery Books (3)

6. Dermatology Books (5)

7. Experimental Animal Research, and Zoology Books (1+1)

8. Biostatistics, and Mathematics Books (1+1)

9. Ancient Texts (3)

10. Artwork (4)

11. Blogs (2)

12. Classical Works (17)

13. Computer Programmes (8)

14. Conference Proceedings (1)

15. Electronic Books (2)

16. Figures (2)

17. Generics (5)

18. Magazines Articles(1)

19. Online Databases (13)

20. Theses (6)

21. Report (1)

22. Web Pages (9)

444

1. ADOBE 2015. Acrobat Reader DC (software). San Jose, CA: Adobe.

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3. ALIBERT, J. L. M. 1806. Description des maladies de la peau; observées a l'hôpital saint-

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of the best methods used in their treatment]. 1 ed. Paris: Chez Barrois.

4. ALIBERT, J. L. M. 1825. Description des maladies de la peau, observées à l'Hôpital

Saint-Louis, et exposition des meilleures méthodes suivies pour leur traitement {French}

[Description of diseases of the skin, observed at the Saint-Louis Hospital, and exhibition

of the best methods used in their treatment]. 2 ed. Paris: Chez Barrois.

5. ALIBERT, J. L. M. 1833. Clinique de l'Hopital Saint-Louis, ou traite complet des

maladies de la peau, contenany la description de ces maladies et leurs meilleurs de

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10. ANONUMOUS n.d. Ornamental Scarification Keloid in an African boy. Anonymous.

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17. BARRETT, K. E., BARMAN, S. M., BOITANO, S. & BROOKS, H. 2015. Ganong's

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504

83. PUBLICATIONS

The Author produced following Publications on the basis of this research project.

Abstracts of these Publications are given on pages that follow:

1. Surgical Adjuvant Intralesional Cytokines versus Steroids for Hypertrophic Scars &

Keloids (Research Report)

2. Keloids & Hypertrophic Scars: Treatment with Intralesional Interferon Gamma versus

Triamcinolone Acetonide (Thesis)

3. Keloids & Hypertrophic Scars: (Review Article)

4. Keloids & Hypertrophic Scars: Epidemiology and Management (Book)

5. Keloids & Hypertrophic Scars: Comparison of Treatment with Intralesional Interferon

Gamma versus Triamcinolone Acetonide (Original Article)

6. History of Keloids & Hypertrophic Scars: From 3500 BC To 1915 AD (Review

Article)

7. Production of Keloid Animal Model (Original Article)

8. Development of New Techniques for Production of Rabbit Ear Keloid Model Original

Article)

9. Mannan & Hannan Technique for Production of Rabbit Ear Keloid Model (Original

Article)

10. Production of Multiple Keloids in one Animal (Original Article)

11. Dosing of Transforming Growth Factor Beta for Production of Keloid Animal Model

(Original Article)

12. Dosing of Interferon Gamma for Treatment of Keloids in Rabbit Ear Model (OA)

13. Production of Transparent Solution of Triamcinolone Acetonide (Original Article)

14. Estimation of Hypothesized Difference between effects of Interferon Gamma and

Triamcinolone Acetonide on Keloids, and Population Variance of Keloids.(OA)

505

84. Surgical Adjuvant Cytokines versus Steroids for Keloids (Report)

Abstract

Introduction: Keloids and hypertrophic scars are an exuberance of fibroplasias in cutaneous repair process. Presently there is no single reliable and effective treatment protocol for keloids and hypertrophic scars. But surgical excision followed by intralesional steroids is the most popular. However, with current treatment modalities, average recurrence rate is 75%.

Therapies aimed at molecular targets like growth factors are considered the future of the treatment of keloids and hypertrophic scars. At present there is no randomized controlled trial available evaluating the efficacy of cytokines in the treatment of keloids and hypertrophic scars. As the treatment of keloids and hypertrophic scars is a challenging problem, development of new and better therapies is need of the hour.

Objective: To compare the efficacy of intralesional Interferon Gamma and Triamcinolone

Acetonide for the treatment of keloids and hypertrophic scars.

Hypothesis: Intralesional Interferon Gamma is more efficacious than Triamcinolone

Acetonide for the treatment of keloids and hypertrophic scars.

Operational Definition: Efficacy- Decrease in volume of keloids and hypertrophic scars.

Material and Methods: It was a prospective randomized triple blind active controlled trial of two years duration and gross 240 (net 187) subjects (keloids), carried out at Department of

506

Pathology and Experimental Research Laboratory, University of Health Sciences, Lahore.

Animal models of keloids and hypertrophic scars were produced by injecting Transforming

Growth Factor Beta 1 on ventral surface of rabbit ear, and excising a punch of skin and cartilage. Subjects (keloids) were allocated randomly to interventional (Interferon Gamma), and control (Triamcinolone Acetonide) groups. Keloids and hypertrophic scars were treated with intralesional injection of Interferon Gamma or Triamcinolone Acetonide, and decrease in volume was noted. The data was entered and analyzed using Microsoft Excel 2010 and

International Business Machines-Statistical Package for Social Sciences version 22.0 software. Z-Test of significance was applied. A p-value of ≤ 0.05 was considered statistically significant.

Results: Out of net 187 subjects, 93 were in Group 1 (Control), and 94 in Group 2

(Interventional). Mean reduction in volume of keloid in control (Triamcinolone Acetonide) group subjects was 6.56661 mm3 (range 43.763 to -22.959), and in interventional (Interferon

Gamma) group subjects 13.49791 mm3 (range 44.504 to -16.519). The p-value was 0.000016, which is highly significant.

Conclusion: Treatment of keloids and hypertrophic scars with Interferon Gamma is highly efficacious.

Recommendation: Human Phase of this Project should be done as soon as possible.

507

85. Keloids: Treatment with Interferon Gamma versus Triamcinolone (Thesis)

Abstract

Introduction: Keloids & Hypertrophic Scars are basically an overabundance of fibrosis in cutaneous healing process. Presently there is not a single curative treatment available for

Keloids & Hypertrophic Scars. But surgical excision (for large Keloids only) followed by intralesional steroid (Triamcinolone Acetonide) injection (first injection should be intraoperative, in wound margins) is considered as gold standard treatment for Keloids &

Hypertrophic Scars. Profibrotic growth factors like Transforming Growth Factor Beta cause formation of exuberant fibrous tissue during wound healing process. Cytokines like

Interferon Gamma which inhibit profibrotic growth factors are considered as future treatment of Keloids & Hypertrophic Scars. At present no randomized controlled trial is available evaluating efficacy of Interferon Gamma for the treatment of Keloids & Hypertrophic Scars.

Objective: To compare the efficacy of intralesional Interferon Gamma and Triamcinolone

Acetonide for the treatment of Keloids & Hypertrophic Scars.

Hypothesis (HA): Intralesional Interferon Gamma is more efficacious than Triamcinolone

Acetonide for the treatment of Keloids & Hypertrophic Scars.

Operational Definition: Efficacy; Decrease in volume of Keloids & Hypertrophic Scars.

Main Outcome Measure: Decrease in volume of Keloid & Hypertrophic Scar.

Design: Prospective Randomized Triple Blind Active Controlled Trial.

Setting: Department of Pathology and Experimental Research Laboratory, University of

Health Sciences, Lahore, Pakistan.

Duration: Two years.

Sample Size: Gross = 240; Net = 187 subjects (Keloids & Hypertrophic Scars) divided into

508 control and interventional groups.

Sampling Technique: Simple Random Selection (three stages), and Allocation (three stages).

Subjects (Keloids & Hypertrophic Scars) were allocated randomly to control (Triamcinolone

Acetonide), and interventional (Interferon Gamma) groups. Keloids & Hypertrophic Scars were treated with intralesional injection of Triamcinolone Acetonide or Interferon Gamma, and decrease in volume was noted.

Data Analysis Procedure: Data was analyzed manually and also using Microsoft Excel,

International Business Machines Statistical Package for Social Sciences and other softwares.

Z-Test was applied to measure statistical significance. A p-value of ≤ 0.05 was considered statistically significant.

Results: Out of net 187 subjects, 93 were in control (Triamcinolone Acetonide), and 94 in interventional (Interferon Gamma) group. Mean reduction in volume of Keloids &

Hypertrophic Scars in Triamcinolone Acetonide group was 6.56661 mm3 (range 43.763 to -

22.959), and in Interferon Gamma group 13.49791 mm3 (range 44.504 to -16.519). The p- value was 0.000016 which is highly significant.

Conclusion: Treatment of Keloids & Hypertrophic Scars with Interferon Gamma is significantly more efficacious than with Triamcinolone Acetonide.

Recommendation: This study should be replicated in human beings.

Key Words: Cicatrix, Hypertrophic; Cytokines; Injections, Intralesional; Interferon Gamma;

Keloid; Model, Animal; Steroids; Transforming Growth Factor Beta; Triamcinolone

Acetonide.

509

86. Keloids & Hypertrophic Scars (Review Article)

Abstract

Wounds are as ancient as history of life on this earth. Every Homo sapiens, and also other species suffer from injuries, which give rise to wounds, which ultimately give rise to scars, some of which become abnormal. Hypertrophic scar is the most significant abnormal scar, which sometimes grows further to become Keloid.

Abnormal scarring, which is now called Keloid, is said to be first described in Edwin

Smith Surgical Papyrus. The oldest known graphic representation of Keloids is ornamental, on face of Aztec god Xipe Totec (2600 BC). Bronze statue of King of Ife Empire of Yoruba in Nigeria (1300 AD) was discovered with Ornamental Keloids on face. The first scientific description of Keloid came in 1785 AD from Noel Retz. The term Keloide was first used by

Alibert (1816 AD) in his article titled Note sur la keloide (Note on the Keloide) published in

Journal Universel des Sciences Medicales (International Journal of Medical Sciences).He used many synonyms for Keloids: Keloide, Kelodes, Kelos, Cheloide, Cancroide, Tubercules

Durs, Cancelli, Cancroma, Cancre Blanc, and Le Crebe.

Keloids are found only in human beings (although some cases have been reported in horses and dogs). Keloids are found on average in 6% (range 4.5-16%) of world population.

Prevalence of Keloids is higher in non-White population, being on average 10% (range 15-

20%). Keloids are 15 times more common in Blacks than in Whites. Incidence of Keloids is

10-15% in all types of wound taken together, irrespective of any other factor. Even in coloured races, people with dark complexion are more likely to develop Keloids than those

510 with fair complexion. Albinos are least affected by this disease, suggesting some role of melanocytes or melanin in production of this disease. Most common age of development of

Keloids is 20 years (range 10 to 30 years.

Hypertension, Autoimmunity, and Spontaneous; or extrinsic ie Piercing, Tattooing, Epilation,

Buns, Bites, Vaccination, Infection, Irritation, Inflammation, Sutures, Foreign Body, and

Drug Induced.

Thomas Addison wrote in his article on Keloids (1854 AD): ‘In regard to treatment little can be said’. Unfortunately, this is still true even in 2015 AD. Presently there is no single effective treatment protocol for Keloids & Hypertrophic Scars. Treatment at present is aimed at restoration of normal function, relief of symptoms, cosmesis, and prevention of recurrence. The list of therapies includes surgical excision, intralesional steroids, radiotherapy, silicone sheets, pressure garments, laser, cryosurgery, intralesional chemotherapeutic drugs, and topical retinoids etc. Often two, three, four, or even more modalities of treatment are combined to achieve acceptable results. Unfortunately a universal approach in Keloids & Hypertrophic Scars treatment is yet to be identified. But surgical excision followed by intralesional steroids is the most popular. However, with current treatment modalities, average recurrence rate is 75%. Treatment is more effective on younger

Keloids & Hypertrophic Scars.

511

87. Keloids & Hypertrophic Scars: Epidemiology and Management (Book)

Abstract

Abnormal scarring, which is now called Keloid, is said to be first described in Edwin

Alibert (1816 AD) in his article titled Note sur la keloide (Note on the Keloide) published in

Journal Universel des Sciences Medicales (International Journal of Medical Sciences).He used many synonyms for Keloids: Keloide, Kelodes, Kelos, Cheloide, Cancroide, Tubercules

Durs, Cancelli, Cancroma, Cancre Blanc, and Le Crebe.

Keloids are found only in human beings (although some cases have been reported in horses and dogs). Keloids are found on average in 6% (range 4.5-16%) of world population.

Prevalence of Keloids is higher in non-White population, being on average 10% (range 15-

20%). Keloids are 15 times more common in Blacks than in Whites. Incidence of Keloids is

10-15% in all types of wound taken together, irrespective of any other factor. Even in coloured races, people with dark complexion are more likely to develop Keloids than those

512 with fair complexion. Albinos are least affected by this disease, suggesting some role of melanocytes or melanin in production of this disease. Most common age of development of

Keloids is 20 years (range 10 to 30 years.

Hypertension, Autoimmunity, and Spontaneous; or extrinsic ie Piercing, Tattooing, Epilation,

Buns, Bites, Vaccination, Infection, Irritation, Inflammation, Sutures, Foreign Body, and

Drug Induced

Keloids & Hypertrophic Scars.

513

88. Keloids: Comparison of Treatment with Interferon µ vs Triamcinolone (OA)