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Evaluation of Porcine Acellularized Dermal Matrix As a Biomaterial: in Vitro Fibroplasia

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Evaluation of Porcine Acellularized Dermal Matrix As a Biomaterial: in Vitro Fibroplasia EVALUATION OF PORCINE ACELLULARIZED DERMAL MATRIX AS A BIOMATERIAL: IN VITRO FIBROPLASIA Alexis Devon Amour A thesis submitted in confonnity with the requirements for the degree of Master of Science Department of Surgery, Institute of Medical Science University of Toronto 8 COPYRIGHT BY ALEXIS ARlMOUR 2001 The author has granted a non- L'auteur a accordé une licence non exclusive kence allowing th3 exc1usi've permettant h la Natiod Li* of Canada to BiôliotMque nafiode du Canada de reprodwe, 10- distriie or seii reproduire, prêter, distribuer ou copies of'this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format 6lectronique. The author retains ownefsbip of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thése ni des extraits substantiels may be printed or othefwise de celle-ci ne doivent être imprim6s reproduced without the author's ou autrement reproduits sans son permissionC aumkatim Alexis Devon Amour Department of Surgery, Institute of Medical Science, University of Toronto Abstract Acellularized pig dermis (pig ACM) is a biomaterial under study for use as a xenograft dermal substitute, and for modeling demal maaix biology in vitro. The specific objectives of the pnsent project were threefold (1) To chmcterize the structure and acellularity of pig ACM, in cornparison with pig demis and human ACM. (2) To study the effects of pig ACM on fibroplasia in vitro in compatison with other in vitro models of fibroplasia. (3) To evaluate the effects of hyaluronan and heparin on fibroplasia in vitro, af'ter incorporation into pig ACM. Pig ACM was found to be inferior to human ACM as a scaffold for human fibroblasts. Fibroblasts proliferated mon rapidly on pig ACM and differentiated into myofi broblasts to a greater degree. Hyaluronan-enriched pig ACM was associated with decreased myofibroblast differentiation, whereas a greater percentage of fibroblasts differentiated to myofibroblasts on heparin-emiched ACM. Hyaluronan, unlike heparin, may therefore be considered a useful ACM dermal substitute component. Pig ACM was found to be a promising matenal for in vitro modeling of myofibroblast-mediated contraction of dermis. Structural modification of pig ACM would likely impmve its clinical suitability as a xenograft dennal substitute. 1would like to thank the following contributors to this project: Audny Robinson-Seurig - for her dedication and perseverance in initiating this project. Dr. Eva Turley - for her insights and thoughffil discussions. James Ho - for skilled and prompt execution of endless histology samples. Dr. Ron Levine - for his support and assistance in obtaining surgical specimens. Carrie Purcell - for her help in providing pigs used in other research studies. Dr. Cho Pang - for his interest and readiness to help with this project. Dr. Chris Fomst - for his guidance and rnentorship regaràing a career as a surgeon- scientist. Dr. Peter Neligan - for advice, encouragement and his cornmitment to the Surgeon- Scientist program in Plastic Surgery. Robert Chemecki - for technical assistance with scanning electron microscoyy, and for hours of delighthil conversation. Prof. Maurice Ringuette - for introducing me to the mystenes of matrix biology. David Giewercer - for technical assistance with confocal rnicroscopy. Trudey Nicklee - for her enthusiasm and help with histological analysis. Ryan Lausman, Tracy Yeung, Tamara Allred - for their commitment to parts of this project as undergraduate students. Jennifer Hansen, Michael May, Gary Skarja, Ali Khademhosseini - for their friendship and research discussions over the last two years. Xudong Cao - for knowing how to help with any problem. Robert Shenker, Zvi Margaliot, Peter Bray, Nancy de Kleer, Melinda Musgrave - for their advice, camaraderie and support during my time away from nsidency. Dr. Joel Fish - for introducing me to this project and his practical input throughout its progression. Dr. John Semple - for his fiendly and professional support as a supervisor, despite many other cornmitments. Rof. Kimberiy Woodhouse - for her guidance and suggestions throughout this project and thesis report, and her dedication to teaching. Sandra Elliot, Rebecca Lawson-Smith, Allison Brown, Richard Stahl - for their fiiendship and help in the lab. Joanna Fromstein - for her help with everything from cornputers to poster printing, coursework to baking, laser pointers to brainstorming. Catherine Bellingham - for sharing so many things with me and in doing so, malcing difficult experiences fun and great experiences memorabk. Cheryl, Jessica Trygve, Quintin and Jocelyne Armour - for their infinite help, patience and caring, regardless of how busy 1 am. Peter Jamieson - for his commitment, humour, and invaluable support. iii lm primary or passaged human dermal fibroblasts AC acetyl group ACM acellular dennal matrix BSA bovine serum albuniin CEA cultund epidemal autograft CPm counts per minute DMEM Du belcco 's Modi fied Eagle Medium DMSO dimeth ylsulfoxide DNase deoxyribonuclease EBM-2 endothelial ceIl basal medium ECM extracellular matrix EDTA eth ylene diamine tetra-acetate FBS fetd bovine serum FD fkeeze-dried FITC f luorescein FTSG full thickness skin graft GAG glycosaminogiycan G~Y glycine H&E hematoxylin and eosin HA hyaluronan HA-ACM hyaluronan-enrichcd acellular demal matrix fiACM human acellular dermal matrix HBSS Hank' s balanced salt solution HCl hydmchloridic acid HP heparîn HP-ACM heparin-emiched acellular dermal rnauix MCDB-131 Molecular Cellular Developmental Biology medium #13 1 MEC microvascular endothelial cells Mm f(4.5-dimethyl thiazol-2-y1)-2,bdiphenyl tetrazolium bromiâe MW, average molecular weight II total sample size NaOH sodium hydroxide O.C.T. trade name, not an abbrevation 0.d. outer diameter P# passage number PACM pig acellular dennal maeix PBS phosphate buffe~dsaline PG proteoglycan PMSF phen yl-methyl-su1fon yI-fluoride RHPS RTF rat tail fibroblasts 8 sample size per group, per npeat SEM scanning electron microscopy SMA alpha-smooth muscle actin STSG spli t-thickness skin graft TCA trichloroacetic acid TEM transmission electron microscopy TGW transforming growth factor-beta Absrnet ~a~~a~ea~~aaaaaaaaeeaaaomaeeoaaaeeaaaaaaaoaaaaaoaaaaaoaaaeoaaeaoaaoaaaaaaaaaeaaaaaoaaaaaaaaoaaaaoaeaaaaaaomii A~~~~l~g~cnts~aa~oemaaeaeaaeaaeaaaeeaeaaaaaaaoaeaaeoaaaaaaaaaaaaaaaoeaaaaaooamaaoaeaaaaoaeaoaaommaaaaaoaaaaaaaaaiii List OIAbb~~villfl~~aooeeaaaaoeaaoaoaaaaaeaeaaeaaaaaaeaoaaaaaaaeaaoaaeaeoaaoaoaooaaaaaaaeaaaaaaaaaaaaaeaoaaaaaaeaaaaaaa IV Lht of Tables ea~~oae~oaaaaemoeaaaaaoaaaaaoaaaaaaoeaaaeaeoeeamaaaoaaaaaaoooaaaaaaaaaaaaaaoaameaaaaaaaaaaaaaaemaaaaoaaaoaaaaamx List of Fiwro~~~~~aa~~e~o~o~oa~aoaeoaee~aaaoaeaaaaaa~aeaaaommaaaaaaaoaaaaaamaaaaaeaaaaooaeaoaeaaaaamaaaaaaaaaa Xi Chapter One: INTRODUCTION ..................................................................1 1.1 CLIN~CALPROBLEM.......................................................................................................... 1 1.1.1 Burns ................................................................................................................ 1 1.1.2 Chronic Wounds ............................................................................................... 2 1.2 DERMALSmsm ENGINEERING ................................................................................ 2 1.3 HYPOTHESESAND OBJECTIVES........................................................................................ 4 Chapter TWO:BACKGRO~oooo~o~~~ooooooooooooooooooooo~oooooooo 6 2.1 ANAT~MYOF SKIN........................................................................................................... 6 2.1.1 Dermal extracellular matrix ........................................................................... 6 2.1.1.1 Collagen ........................................................................................... 7 2.1.1.2 Hyalumnan ........................................................................................ 8 2.1.1.3Heparan sulfates ................................................................................ 9 2.1.2 Comparison of pig and human skin .................................................................. 10 2.2 CEU-hhmLNTEaAcrro~s m WOUNDHEALING ......................................................... 11 2.2.1 Stages of wound healing ................................................................................... Il 2.2.2 Inflammation ....................................................................................................12 2.2.3 Angiogenesis ....................................................................................................12 2.2.4 Epithelialization ............................................................................................... 13 2.2.5 Fibroplasia ........................................................................................................ 13 2.2.6 Wound contraction ........................................................................................... 16 2.2.7 Sumrnary .......................................................................................................... 18 2.3 HOSTRES~NSE TO BIOMA~................................................................................. 19 2.3.1 Foreign body nsponse ...................................................................................... 19 2.3.2 Biomaterial integration
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