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APPLICATIONS OF HUMAN BONE MATERIALS AND SYNTHESIZED

BIOMATERIALS FOR BONE-RELATED TISSUE ENGINEERING

A Dissertation

Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

Of the requirements for the Degree

Doctor of Philosophy

Qing Yu

December, 2016

APPLICATIONS OF HUMAN BONE MATERIALS AND SYNTHESIZED

BIOMATERIALS FOR BONE-RELATED TISSUE ENGINEERING

Qing Yu

Dissertation

Approved: Accepted:

______Advisor Department Chair Dr. William J. Landis Dr. Coleen Pugh

______Committee Member Dean of the College Dr. Nita Sahai Dr. Eric J. Amis

______Committee Member Dean of the Graduate School Dr. Coleen Pugh Dr. Chand Midha

______Committee Member Date Dr. Marnie Saunders

______Committee Member Dr. Ge Zhang

ABSTRACT

Engineered bone grafting has been considered as one of the alternative methods for bone regeneration in both fundamental research and clinical applications to address bone disorders. Bone graft materials, autologous bone, allogeneic bone and synthetic polymer scaffolds have been commonly utilized surgically as substrates for bone grafting. In this dissertation, periosteum, a thin membrane in which progenitor cells can develop into osteoblasts to regenerate bone tissue, has been applied in three different studies to determine its capability to induce new bone formation.

In the first study, human periosteum-wrapped bone allografts were implanted subcutaneously in athymic mice followed by sample harvest and gene expression analysis and histological assessment. The second study developed a tissue-engineering approach to generate a functional tendon-to-bone enthesis.

In this instance, the constructs were fabricated from human periosteum-wrapped allograft bone and tenocyte- and chondrocyte-seeded biomaterials. The constructs were then implanted with and without mechanical force by either tethering them to the trapezius and gluteus maximus muscles of athymic mice or not tethering them at all. Biomechanical, histological, and histochemical properties of these tendon-to-bone enthesis models were analyzed following their

iii implantation. The third study was designed to determine the possible effects of bromine- or silicon-functionalized poly(lactic acid) (Si-PLA) scaffolds on skeletal development. To examine initially the cytotoxicity of bromine on periosteal cells, a PrestoBlue® assay was performed on human periosteal cell-seeded brominated PLA scaffolds over a 21-day time period.

With application of histological and gene expression analysis, new bone formation and resorption were detected in human-periosteum allografts implanted for different time periods. Correlated histological and gene data showed that periosteum has the capability of inducing bone regeneration in allografts and in tendon-to-bone enthesis models. Tissue-engineered enthesis models fabricated with periosteum-allograft and chondrocyte- and tenocyte- seeded scaffolds provide a novel method for healing enthesis defects in regenerative medicine. In addition, results from bromine cytotoxicity studies of human periosteal cells imply subsequent Si-PLA experiments with minimal numbers of bromine residues on the backbone of PLA. These tissue engineering investigations suggest that both allograft bone and biosynthetic polymers have great potential in regenerative medicine applications for bone.

DEDICATION

To my mother, who has been the most supportive and loveliest person in the world.

ACKNOWLEDGMENTS

Firstly, I would like to express my deep and sincere appreciation to Dr. William

J. Landis, my dearest advisor, for his continuous support of my Ph.D. study and related research, for being a tremendous mentor, for his patience, encouragement, motivation, and omniscience. His guidance in not only my writing and research but also my life is invaluable. Besides work, he and his wife

Jane Landis treat me like their family member. I could not have imagined a better advisor and mentor for my graduate study. A special thanks to Mrs. Robin

Jacquet for her help with the implantation of the constructs into nude mice, the analysis of gene expression, the amount of time and effort she devoted in ensuring my success in laboratory research. She also showed me the path to faith in God with her love.

My sincere thanks also go to my committee members, Dr. Coleen Pugh

(Department of Polymer Science) for her assistance in polymer synthesis, Dr.

Nita Sahai (Department of Polymer Science) for her insights into my research,

Dr. Marnie Saunders (Department of Biomedical Engineering) for providing instrument access to perform tensile testing, and Dr. Ge Zhang (Department of

Biomedical Engineering) for her encouragement and guidance in my career and my life. I thank Dr. John Elias (Department of Research, Akron General Medical

Center) for his help with preliminary tensile testing and Gunze, Ltd. (Kyoto,

Japan) for supplying the polymer materials. I would like to thank Dr. Hitomi

Nakao, Dr. Philip McClellan, Dr. Narihiko Hirano, and Mr. Josh Bundy (the Landis

Laboratory, Department of Polymer Science) for their assistance with sample preparation and implantations and Dr. Colin Wright, Dr. Xiang Yan, and Mr.

Haidong Zhu (Department of Polymer Science) for their assistance in polymer synthesis. I gratefully acknowledge support from the National Science

Foundation (DMR-1006195; Dr. Coleen Pugh, PI, Department of Polymer

Science, University of Akron) for aspects of this dissertation concerning polymer synthesis. I would especially like to thank the Northeast Ohio Medical University

Comparative Medicine Unit for animal housing and care, the Gift of Hope Organ

& Tissue Donor Network (Itasca, IL) and Dr. Susan Chubinskaya (Rush

University, Chicago) for human donor tissue, and the Musculoskeletal Transplant

Foundation (Jessup, PA) for donation of allograft bone. I also would like to express respect and gratitude to the families of donors for access to tissues.

Words cannot express how grateful I am to my mom and dad, my grandparents, and my aunts and uncles -- all my sweetest family members -- for their support and love as always. Xing and my friends, Tianyi, Shuo, Jing, Fan,

Sharon, and Chris have encouraged me to walk through the most difficult time.

The love, prayers, compassion, and generosity of my church people have made me a stronger person.

“The Lord is my shepherd, I lack nothing.” (Psalm 23:1, NIV)

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TABLE OF CONTENTS

Page

LIST OF FIGURES …………………………………………………………………….xii

LIST OF TABLES …………………………………………………………………..…xvii

LIST OF SCHEMES……………………………………………………………..…...xviii

CHAPTER

I. A HUMAN PERIOSTEUM-BONE ALLOGRAFT ...... 19

1.1 Introduction ...... 19

1.1.1 Basic Concepts of Periosteum ...... 19

1.1.1.a Anatomical Aspects ...... 20

1.1.1.b Microscopic Considerations ...... 23

1.1.2 Basic Concepts of Bone ...... 24

1.1.2.a Components of Bone ...... 26

1.1.2.b Functions of Bone ...... 27

1.1.2.c Bone Remodeling ...... 28

1.1.2.d Mechanisms of Bone Remodeling ...... 33

1.1.3 Application of Polymerase Chain Reaction Analysis in Osteogenesis Studies ...... 36

1.1.4 Role of Periosteum in Bone Healing ...... 39

1.1.5 Utilization of Periosteum in Tissue Engineering of Bone Grafting ..... 41

1.2 Materials and Methods ...... 48

1.2.1 Experimental Materials...... 48

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1.2.2 Experimental Methods ...... 49

1.2.2.a Fabrication of Periosteum-bone Allografts ...... 49

1.2.2.b Specimen Implantation, Harvesting, and Processing ...... 50

1.2.2.c Specimen Staining ...... 53

1.2.2.d Quantitative Polymerase Chain Reaction Assessment ...... 54

1.2.2.e Statistical Analysis ...... 60

1.3 Results and Discussion ...... 60

1.3.1 Histological Results ...... 60

1.3.2 RT-qPCR Results ...... 69

1.3.3 Discussion ...... 77

II. A HUMAN TENDON-TO-BONE ENTHESIS MODEL ...... 83

2.1 Introduction ...... 83

2.1.1 Basic Concepts of an Enthesis ...... 83

2.1.1.a The Flexor Tendon and Rotator Cuff ...... 85

2.1.1.b Surgical and Mechanical Challenges in Healing of a Tendon-to- Bone Enthesis ...... 87

2.1.1.c Type II Collagen and Scleraxis ...... 89

2.1.1.d Muscle Loading in Tendon-to-Bone Enthesis Development ...... 90

2.1.2 Tissue-engineering Strategies ...... 91

2.1.2.a Application of Mesenchymal Stem Cells ...... 92

2.1.2.b Utilization of Biological Factors ...... 93

2.1.2.c Usage of Mechanical Loading ...... 94

2.1.2.d A Human Tendon-to-bone Enthesis Model ...... 96

2.2 Materials and Methods ...... 98

2.2.1 Experimental Materials...... 98

2.2.1 Experimental Methods ...... 100

2.2.2.a Periosteum, Tenocyte and Chondrocyte Isolation and Cultivation ...... 100

2.2.2.b Construct Model Fabrication ...... 103

2.2.2.c Specimen Implantation, Harvest, Fixation and Embedding ...... 106

2.2.2.d Biomechanical Testing ...... 109

2.2.2.e Histological Staining ...... 112

2.2.2.f Immunohistochemistry of Type II Collagen and Scx ...... 112

2.3 Results and Discussion ...... 115

2.3.1 Tensile Testing Results ...... 116

2.3.2 Histology Results ...... 127

2.3.3 Discussion ...... 144

III. SILICON-FUNCTIONALIZED POLY(LACTIC ACID) ...... 151

3.1 Introduction ...... 151

3.1.1 Silicon and Bone Health ...... 152

3.1.1.a Chemistry of Silicon ...... 152

3.1.1.b The Function of Silicon in Animal Growth ...... 154

3.1.1.c The Function of Silicon in Bone Formation ...... 158

3.1.2 Bromine Toxicity ...... 162

3.1.3 Scaffolding in Tissue Engineering ...... 164

3.2 Materials and Methods ...... 166

3.2.1 Experimental Materials...... 166

3.2.2 Experimental Methods ...... 167

3.2.2.a PLB Synthesis and Analysis ...... 167

3.2.2.b PLB Scaffold Fabrication ...... 169

3.2.2.b Periosteal Cell Expansion, Freezing, and Thawing ...... 171

3.2.2.c Cell Seeding ...... 173

3.2.2.d Cytotoxicity Assay ...... 175

3.3 Results and Discussion ...... 176

3.3.1 PLB Synthesis Results ...... 177

3.3.2 Cytotoxicity Study Results ...... 179

3.3.3 Discussion ...... 189

BIBLIOGRAPHY ...... 195

APPENDIX ...... 212

LIST OF FIGURES

Figure Page

1.1. A light micrograph of the periosteum covering the human femoral midshaft ...... 20

1.2. A light photomicrograph of bovine periosteal cells isolated and cultured in a petri dish for 10 days ...... 22

1.3. Cell populations present in the outer and inner layers of the periosteum . 24

1.4. Anatomical structure of a human long bone ...... 26

1.5. Diagrammatic representation of the hierarchical structural organization from the gross anatomical level to the sub-microscopic level ...... 30

1.6. Hematoxylin and eosin staining of a human periosteum-allograft bone specimen ...... 31

1.7. Bone remodeling process (from left to right). The signals received by lining cells (LC) on the bone surface initiate the bone remodeling process. Osteoclast (Oc) precursors attach to the surface and fuse to become multinucleated ...... 32

1.8. The relationship of osteoblasts, osteoclasts, and osteocytes during bone resorption ...... 35

1.9. Schematic outline of PCR ...... 38

1.10. A series of panels showing intact and bisected representative middle phalanx specimens harvested after 10 and 20 weeks of implantation in athymic (nude) mice ...... 45

1.11. Panels showing histological staining of paraffin-embedded sections of constructs composed of midshafts of P(LA-CL) ...... 46

1.12. A schematic of fabrication of human periosteum-allograft bone ...... 50

1.13. Methods and materials for creation of human periosteum allograft constructs ...... 52

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1.14. H&E-stained histological sections and enlargements of 10-week human allograft models (with and without periosteum) ...... 63

1.15. H&E-stained histological sections and enlargements of 20-week human allograft models (with and without periosteum) ...... 65

1.16. H&E-stained histological sections and enlargements of 40-week human allograft models (with and without periosteum) ...... 67

1.17. Light micrographs of H&E and TRAP staining results of a 10-week human periosteum-allograft construct ...... 68

1.18. Relative mean fold changes (10-week data utilized as the calibrator) in mRNA expression of murine-specific genes determined by RT-qPCR ..... 71

1.19. Relative mean fold changes compared to 10-week samples in mRNA expression of murine-specific genes determined by RT-qPCR ...... 72

1.20. Relative mean fold changes (10-week data used as calibrators) in mRNA expression of human osteoblast-related genes by RT-qPCR ...... 76

1.21. Relative mean fold changes derived from a comparison of 20- and 40- week to 10-week samples in mRNA expression of human osteoclast- related genes determined by RT-qPCR ...... 76

1.22. Relative mean fold changes of 20- and 40-week specimens compared to 10-week specimens in mRNA expression of human SOST obtained with RT-qPCR ...... 77

2.1. Schematics of the (A) tendon and (B) ligament attachment to bone across a functional gradation region consisting in order of unmineralized fibrocartilage, mineralized fibrocartilage, and bone ...... 84

2.2. A schematic of the cross-section of a rat supraspinatus tendon-to-bone enthesis ...... 88

2.3. A: Intact human knee from a 43-year-old donor ...... 102

2.4. A schematic of a fabricated tendon-to-bone enthesis model ...... 104

2.5. Light photographs of experimental materials ...... 105

2.6. The experimental group design for the enthesis study ...... 107

2.7. Unseeded aspect of a tendon-to-bone enthesis construct ...... 108

2.8. Tensile testing as set up in the Akron General Medical Center ...... 110

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2.9. The seeded aspect of a tendon-to-bone enthesis sample clamped by custom grips mounted to the arm of the traveler by a universal joint in the testing machine ...... 111

2.10. A 40-week tendon-to-bone untethered construct (Group 1) implanted subcutaneously in an athymic mouse ...... 115

2.11. Maximum load of 20- and 40-week seeded and unseeded aspects of untethered (NT) enthesis models from the Elias Laboratory ...... 117

2.12. Maximum load of 20- and 40-week seeded and unseeded aspects of untethered (NT) enthesis models from the Elias Laboratory ...... 118

2.13. Stiffness of 20- and 40-week seeded and unseeded aspects of untethered enthesis models from the Elias Laboratory ...... 119

2.14. Tensile testing curves of the unseeded and seeded aspects of a representative 20-week tendon-to-bone enthesis model examined from the Saunders Laboratory ...... 121

2.15. Maximum loads of 20-week seeded and unseeded aspects of nontethered (NT) and tethered (T) enthesis models ...... 122

2.16. Stiffness of seeded and unseeded aspects of 20-week untethered (NT) and tethered (T) enthesis models from the Saunders Laboratory ...... 124

2.17. Maximum loads of 40-week seeded and unseeded aspects of non-tethered (NT) and tethered (T) enthesis models ...... 124

2.18. Stiffness of 40-week seeded and unseeded aspects of non-tethered (NT) and tethered (T) enthesis models from the Saunders Laboratory ...... 125

2.19. Maximum loads of 20- and 40-week seeded and unseeded aspects of tethered (T) enthesis models from the Saunders Laboratory ...... 126

2.20. An H&E-stained histological section of a representative 20-week untethered tendon-to-bone enthesis specimen (Group 1) after tensile testing ...... 129

2.21. An H&E-stained histological section from a representative 20-week untethered tendon-to-bone enthesis model from Group 1 ...... 130

2.22. An H&E-stained histological section of a representative untethered 40- week tendon-to-bone enthesis model (Group 1) ...... 131

2.23. Light micrographs of a picrosirius red-stained 40-week untethered tendon- to-bone enthesis model specimen from Group 1 ...... 132

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2.24. Light micrographs of an H&E-stained section and enlargements of a representative enthesis specimen after application of tethered mechanical forces for 20 weeks in vivo (Group 2) ...... 134

2.25. A light micrograph of a tensile-tested tethered 40-week tendon-to-bone enthesis model stained with H&E ...... 135

2.26. Light micrographs of an H&E-stained tethered Group 2 40-week tendon-to- bone enthesis model ...... 136

2.27. Light micrographs of an untethered 20-week tendon-to-bone enthesis model wrapped with periosteum (Group 1) and stained with anti-type II collagen ...... 138

2.28. Light micrographs of an untethered 40-week tendon-to-bone enthesis model wrapped with periosteum (Group 1) and IHC-stained for type II collagen presence ...... 139

2.29. IHC (A) and H&E (a) staining results showing positive type II collagen and cartilage staining for a representative tensile-tested tethered 20-week tendon-to-bone enthesis model (Group 2) ...... 140

2.30. Scleraxis immunostaining results for representative untethered 20- (A) and 40-week (B) tendon-to-bone enthesis models implanted in athymic mice (Group 1) ...... 142

2.31. IHC staining results for a representative section of a tethered 20-week tendon-to-bone enthesis model (Group 2) to detect scleraxis ...... 143

3.1. Growth curves of chicks fed with/without silicon in their diet ...... 155

3.2. Macropathologic examination results from 4-week-old chick skulls without and with silicon supplements ...... 156

3.3. Cell growth of HOB cells over a 14-day period of culture in vitro ...... 161

3.4. Cell counts at day 6. Each well was originally seeded at a density of 50,000 cells/well ...... 164

3.5. A schematic for fabrication of a PLB scaffold ...... 170

3.6. Light photomicrographs of cadaveric primary human periosteal cell growth from the periosteum tissue cambium layer and expansion in vitro ...... 172

3.7. PLA and PLB (10% and 5% Br) scaffolds before (A) and after (B) seeding with human periosteal cells ...... 175

3.8. A 300 MHz 1H NMR spectrum of a poly[(lactic acid)-co-(2-bromo-3- hydroxypropionic acid)] (PLB) copolymer, [BrA]:[LA] = 20:80 ...... 178

3.9. A GPC chromatogram of poly[(lactic acid)-co-(2-bromo-3-hydroxypropionic acid)] (PLB) copolymers ...... 179

3.10. A chart of growth curves for PB assay I of human periosteal cells (43-year- old male, P3) on tissue culture polystyrene (TCPS) control, PLA scaffolds, and PLB ([BrA]:[LA] = [20:80], Mn = 20 kDa, Đ = 2.03, 10 mol%, 5 mol%, or 1 mol% Br) scaffolds over a 21-day period ...... 180

3.11. A representative light photograph of a human-periosteal-cell-seeded PLB (10 mol% Br) scaffold in cell culture media on day 21 ...... 181

3.12. A chart of growth curves for PB assay II of human periosteal cells (43- year-old male, P3) on tissue culture polystyrene (TCPS) control, PLA scaffolds, and PLB ([BrA]:[LA] = [20:80], Mn = 25 kDa, Đ = 1.37, 10 mol%, 5 mol%, or 1 mol% Br) scaffolds over a 21-day period ...... 182

3.13. A chart of growth curves for PB assay III of human periosteal cells (61- year-old female, P3) on tissue culture polystyrene (TCPS) control, PLA scaffolds, and PLB ([BrA]:[LA] = [20:80], Mn = 25 kDa, Đ = 1.37, 10 mol%, 5 mol%, or 1 mol% Br) scaffolds over a 21-day period ...... 183

3.14. A chart of growth curves for PB assay IV of human periosteal cells (61- year-old female, P3) on tissue culture polystyrene (TCPS) control, PLA scaffolds, and PLB ([BrA]:[LA] = [20:80], Mn = 17 kDa, Đ = 2.17, 10 mol%, 5 mol%, or 1 mol% Br) scaffolds over a 21-day period ...... 185

3.15. Human periosteal cell growth-fold change from day 1 to day 21 (solid lines) on PLB scaffolds (1 mol% Br) from PB assay II (A), III (B), and IV (C) ... 189

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LIST OF TABLES

Table Page

1.1. A list of murine-specific primer sets...... 58

1.2. A list of human-specific primer sets...... 59

1.3. Designed primer sequences (mouse-specific)...... 59

1.4. Relative murine-specific gene expression fold changes of 20- and 40-week human allograft specimens (Group 1) detected with RT-qPCR ...... 70

1.5. Relative human-specific gene expression fold changes of 20- and 40-week human periosteum-allograft specimens (Group 2) compared to 10-week samples and detected with RT-qPCR ...... 73

3.1. Weight amounts of designated controlled Br content polymer blends. ... 169

3.2. Average molecular weight and PDI (Đ) of PLB samples, donor information and cell passage of human periosteal cells, and initial cell seeding density for all four PB assays conducted in the study...... 177

3.3. Human periosteal cell viability on TCPS, PLA scaffolds, and PLB (1 mol% Br) scaffolds detected with the PB assay ...... 186

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LIST OF SCHEMES

Scheme Page

3.1. Synthesis of PLB...... 168

xviii

CHAPTER

I. A HUMAN PERIOSTEUM-BONE ALLOGRAFT

1.1 Introduction

This content contains introduction to periosteum, bones, applications of periosteum in current clinical settings.

1.1.1 Basic Concepts of Periosteum

It is now well reported that periosteum plays a cardinal role in bone formation and fracture healing, in part as a result of its particular biochemistry and structure.1 Periosteum is an unusual connective tissue, a thin and tough membrane that lines the outer surface of all bones of vertebrates with the joints of long bones as the only exception. Periosteum consists of an outer fibrous layer that contains numerous fibroblasts and an inner cambium layer that contains progenitor cells that can differentiate to bone cells (osteoblasts) and cartilage cells (chondrocytes).

Periosteal surface

Cortical bone

Figure 1.1. A light micrograph of the periosteum covering the human femoral midshaft. There is an abundance of cells (arrowheads) comprising the cambium layer of the periosteum from an 81-year-old female cadaver stained with

Masson’s trichrome. Scale bar = 25 µm. This figure has been reproduced with permission from Elsevier.2

1.1.1.a Anatomical Aspects

Periosteum is a type of dense fibrous connective tissue that can form a thin membrane tightly attached to the bone through Sharpey’s fibers.3 Sharpey’s fibers are bundles of collagen fibers which provide anchorage for periosteum to connect to bone. Sharpey’s fibers pass through the outer fibrous layer of periosteum, connecting the periosteum to the peripheral and interstitial structure of bone tissue. These fibers are direct continuations of periosteal collagen fibers around which the cortical lamellae grow.

Periosteum covers most of the surface of bones except articular surfaces, sesamoid bone surfaces, and tendon to bone entheses.4 It contains both an

20 outer fibrous layer and an inner osteogenic cellular layer.5 The outer layer is mostly composed of collagen along with nerve fibers that can transmit pain when the tissue is damaged by some factors. Also, the outer layer contains blood vessels, branches that can pass through the bone and supply the bone cells with nutrition. Some of the branches penetrate into the bone along channels called the Volkmann canals.6 Fibers from the inner layer can also pass through the bone to anchor it to the periosteum like Sharpey’s fibers.

The inner layer of periosteum is composed of preosteoblasts and osteoblasts.

This cambium layer is the primary part of the periosteum and changes with age.

The bones of human children have thicker, firmer, and more elastic periosteum than the bones of adults. In childhood, the speed of bone growth reaches its peak while in adulthood the composite cells grow much slower. However, even in adulthood, the cells maintain their function to generate new bone under both normal and abnormal conditions (injury from a traffic accident, for example). As long as the inner periosteal layer generates new cells during growth and repair processes, periosteal blood vessels will cover the bone and normal function follows. In injuries or fractures of bone, the vessels will help aggregate the bone fragments together in several days, and the bone defect will regenerate new osteoblasts to heal the trauma. Periosteum of the inner surface of the skull will remodel to some extent and grow with the dura mater, the protective membrane of the brain. Occasionally, fibrous collagen will take the place of periosteum along with the Volkmann canals, as a result of excess pressure, for example.

According to the experimental observations in this thesis, the periosteum is a membrane that does not extend to or cover the ends of a long bone, which are cartilaginous, or those sites for ligament and tendon tissues. Fresh bovine calf periosteum is much easier to remove from its underlying bone using an elevator compared to tissue that has been kept in a freezer for a week or longer. The fresher specimens have looser and more elastic attachments connecting periosteum to the bone. When periosteum is peeled or lifted from the bone, it will shrink or contract into a smaller size. The inner layer of periosteum is the cardinal part for allograft use in orthopedic surgery and especially for orthopedic oncology (Figure 1.2).

Figure 1.2. A light photomicrograph of bovine periosteal cells isolated and cultured in a petri dish for 10 days. Cells were obtained from the fresh articular periosteum of a 6-month-old calf. Cells are spindle-shaped and interconnected

22 through an extensive network of thin processes. Scale bar = 200 µm; image was taken with an Olympus inverted microscope, model IX70.

1.1.1.b Microscopic Considerations

Generally, the periosteum has an inner cellular layer and outer fibrous layer with the capability to produce collagen and other extracellular matrix molecules.

The periosteal ultrastructure and the organizational function of periosteum were unknown until recently. The first organizational analysis of periosteum was made by Squier and his group members in 19907, who divided periosteum from the skulls and palates of seven beagles into three layers using light and electron microscopy. The periosteum was classified into three zones (Figure 1.3) according to the proportion of cells, fibrous layers and matrix.7, 8 Zone I contained osteoblasts adjacent to cortical bone, Zone II was relatively semitransparent with many more capillaries than those in Zone I, and Zone III consisted of large numbers of fibroblasts and cells intermingled with collagen fibrils.

Figure 1.3. Cell populations present in the outer and inner layers of the periosteum. The periosteum is divided into three zones: Zone I (osteoblasts adjacent to cortical bone), Zone II (part of the inner cambium layer that contains progenitor cells, Sharpey’s fibers, and blood vessel), and Zone III (outer fibrous layer consisting principally of fibroblasts within a collagen fiber extracellular matrix). Figure has been reproduced with permission from SAGE Publishing.8

Recently scientists simplified the periosteum into two zones: an outer fibrous firm layer and an inner proliferative cambium layer.5 The cambium layer has three to four layers of cells; it lies on cortical bone (Figure 1.3) and mainly contains osteoblasts arranged in the layer adjacent to the bone surface. The surface of bone is in an epithelium with a supra-osteoblast layer of smaller, compact cells.9, 10 Normal lamellar bone forms from cortical bone to broaden overall bone width. Primary bone may also form after bone fracture.11-13

In order to describe this research project clearly, some basic concepts of bone and its properties will be detailed in the following section of this chapter.

1.1.2 Basic Concepts of Bone

Bone such as femur or tibia is a rigid organ and it is a type of tissue that plays a significant role in survival as it provides mechanical support to the body, protects internal organs, stores ions in the form of mineral and serves as the source of erythrocytes, leukocytes and other hematopoietic elements. Bone and bone tissue are different terms, and bone includes blood vessels, epithelium,

24 nerves, and marrow while bone tissue is considered part of a bone. Bone tissue can be divided into two types: compact (cortical) bone and spongy (cancellous) bone (Figure 1.4, 5). Compact bone tissue usually is located in the diaphysis of a long bone (such as the shaft of a mature femur) that is covered with periosteum and consists of densely packed osteons that include vascular canals with blood vessels. Cancellous bone tissue consists of irregularly shaped microscopic-sized mineralized organic matrix components (honeycomb-like or sponge-shaped internal structure) with dense connective tissue. The mineralized structure forms bone struts or trabeculae that are mechanically hard. Between the trabeculae are blood vessels, fat cells and undifferentiated mesenchymal cells. In a word, compact bone tissue forms the hard exterior while cancellous bone tissue fills the hollow interior of bone. These aspects of bone tissue produce a bone that is light weight yet having rigid external structure that can support the mechanical requirements of the body.

The total number of bones in the body changes from infancy to adulthood as a result of normal bone fusion. At birth for humans, the number of bones is 270 compared to 206 when one reaches maturity.14 Figure 1.4 presents a general overview of long bone structural features.

Figure 1.4. Anatomical structure of a human long bone. Periosteum covers outer bone surfaces. O.c.s. = outer circumferential system; i.c.s. = inner circumferential system. Figure reproduced with permission from the CRC Press.6

1.1.2.a Components of Bone

The basic components of bone lead to the formation of bone tissue. Long bones may appear as long, tubular segments, flat bones may appear as bilaminar plates, and short bones generally have prismatic structures. Typically in long bones, three different regions can be used to identify those parts: the diaphysis, epiphysis and metaphysis (Figure 1.4). The diaphysis is the central, longest part of long bone while the epiphyses are located on its two ends. The metaphysis lies between the diaphysis and epiphysis. Figure 1.4 shows that the epiphysis and metaphysis are divided by growth cartilage, a cartilaginous layer

26 which may disappear as bone matures.6 All skeletal segments consist of an outer layer of compact bone and an inner zone with bone marrow.

1.1.2.b Functions of Bone

The main functions of bone can be classified into three parts: metabolic, synthetic, and mechanical functions. Metabolic functions consist of aspects described as follows. Bone tissue can store heavy metal ions and elements as well as foreign (abnormal) ions and elements and this property provides bone with the capacity of detoxification. Bone can buffer the blood against excessive pH changes by alkaline salt adsorption and it then may restore proper acid-base balance. Bone can provide a reserve of fat from its yellow bone marrow and mineralized bone matrix stores important growth factors such as transforming growth factors, bone morphogenetic proteins and others. Bone is the major reservoir of the body for mineral ions such as phosphate and calcium without which the general function of the human body would deteriorate and fail. Bone can affect endocrine organs through fibroblast growth factor to change the phosphoric metabolism of the body.15 As for its synthetic function, bone produces blood for all the components of the human body. The core part of this function lies with hematopoietic stem cells from the red marrow in the medullary cavity of long bones and the pores of cancellous bone that are responsible for blood cell generation. Mechanical functions of bone include protection of soft organs such as the brain, the lungs and other organs, and the bone acts as a frame work to support the whole body. Mechanical forces and energy are

27 converted into movement in order to manipulate or partially control the whole body when bones, muscles, joints, ligaments, and tendons function together.16

Bone is also capable of sound transduction as in the inner ear of humans.

1.1.2.c Bone Remodeling

Bone remodeling, also called bone metabolism, is a complex lifelong process whereby mature bone tissue is removed from the bone (bone resorption) and new bone tissue is formed (bone ossification). Bone formation, also known as osteogenesis or ossification, is the process of cellular synthesis of an organic matrix (laying down new bone materials) resulting from the normal action of osteoblasts, one of the principal cell types of bone. Calcification occurs when calcium salt incorporation into soft tissue causes it to become hardened during the process of ossification. Specifically, mineralization or calcification of bone substance is the deposition of solid calcium inorganic orthophosphate phase crystals into the organic matrix that has been synthesized by osteoblasts.17

Calcified bone matrix then contains two components: the mineral substance and organic matrix.

Generally, the organic matrix constituent of bone substance is a composite of many different proteins — collagen being the most common and whose molecules are organized or self-assembled in a specific three-dimensional packing arrangement (Figure 1.5).18 The organic matrix of bone primarily contains type I collagen fibrils that occupy over 90% of the entire extracellular matrix with approximately 10% noncollagenous proteins and proteoglycans.19

The mineral substance of bone is composed of calcium phosphate

20 hydroxyapatite [Ca5(PO4)3(OH)]. Calcification is the first step of bone formation and begins after the assembly of collagen fibrils. Calcium and phosphate ions from the extracellular fluid contribute to apatite formation of bone. The cellular activity and function of osteoblasts changes when these cells become progressively surrounded by an organic and inorganic matrix. As osteoblasts are completely encased by an organic matrix, they increase in maturation and are identified as osteocytes (Figure 1.6). Osteocytes maintain functions different from those of osteoblasts and are connected to one another and to osteoblasts by cellular processes that are enclosed in canaliculi or channels through the bone matrix. This means of intercommunication offers a principal pathway that supports the functional and topological connections between osteocytes, osteoblasts and the extracellular fluid of bone.17

Figure 1.5. Diagrammatic representation of the hierarchical structural organization from the gross anatomical level to the sub-microscopic level.

Structures include cortical and cancellous bone, osteon units (Haversian canals), lamellae formed with collagen fibers, collagen fibrils, collagen molecules, and bone crystals. Figure reproduced with permission from Elsevier.18

The manner by which older bone tissue is resorbed is related to another distinct bone cell, the osteoclast. Osteoclasts are multinucleated giant cells responsible for resorbing (or removing) the mineralized bone matrix and solid calcium inorganic orthophosphate.21 Osteoclasts were first described and recognized in 1873 22, and they are formed by the fusion of mononuclear progenitors of the monocyte family.23-26 Bone resorption by osteoclasts takes place through a multi-step complex pathway that includes the generation of immature osteoclast precursors which have close contact with the bone surface.

The three-dimensional disposition of the collagen fibrils and the properties of the solid calcium inorganic orthophosphate phase will change along with their chemical and physical functions during the time between their formation and resorption.

Figure 1.6. Hematoxylin and eosin staining of a human periosteum-allograft bone specimen. The arrows show osteocytes in their lacunae and surrounded by their mineralized bone matrix. Scale bar = 100 µm.

During this process, osteoclasts demineralize and resorb old bone while osteoblasts form new bone to balance the bone mass in order to adjust the stresses and other forces placed on it (Figure 1.7). When there is an increase in osteoclast activity, it will lead to an increase in bone resorption, which will result in reduced bone mineral density.27 Along with rapidly developed techniques, modern molecular biological investigations have provided more information about the mechanisms and proteins related to bone remodeling.28-30

Figure 1.7. Bone remodeling process (from left to right). The signals received by lining cells (LC) on the bone surface initiate the bone remodeling process.

Osteoclast (Oc) precursors attach to the surface and fuse to become multinucleated. Osteoclasts proceed to resorb a specific site of bone for about 3 weeks. Mononuclear cells clean the resorbed surface during the transition from resorption to formation. A histologically recognizable mark (the cement line [CL]) identifies the reversal from resorption to formation. The process of forming bone tissue is activated and accomplished by osteoblasts (Ob) which secrete new matrix called osteoid (OS). The osteoid will become mineralized. The events of bone resorption and bone formation are classified as the bone remodeling unit

(BRU). At the end of the process, the osteoblasts flatten and transition to lining cells on the newly formed bone surface. Figure reproduced with permission from

John Wiley and Sons.31

1.1.2.d Mechanisms of Bone Remodeling

The homeostasis of bone is carried out by a balance between bone resorption by osteoclasts and bone formation by osteoblasts, the two main processes of bone remodeling.31, 32 Bone remodeling is a coordinated process which is controlled by the bone multi-cellular units including osteoblasts, osteocytes, and osteoclasts with bone resorption (2-4 weeks) and formation (4 months) at the unit.31, 33

Derived from mesenchymal stem cells, osteoblasts play a central role in bone generation by forming multiple bone matrix proteins and regulating the maturation of osteoclasts by soluble factors and related interaction, resulting in bone remodeling. Once the osteo/chondroprogenitor cells have matured, transcription factors such as Runt-related transcription factor 2 (Runx2), Distal- less homeobox 5 (Dlx5), and osterix (Osx) are expressed.34 In addition, Runx2 has been reported to upregulate osteoblast-specific genes: COL1A1 (type I collagen), ALPL (alkaline phosphatase), IBSP (bone sialoprotein), and BGLAP

(bone gamma-carboxyglutamate protein, also known as osteocalcin).35 When an osteoblast approaches its late stage of differentiation, higher expression of

COL1A1, IBSP, and BGLAP are detected.34 Osteoblasts will secrete bone matrix until they are encapsulated in osteoid and then will develop into osteocytes. The new osteocytes generate sclerostin (SOST) whose function is to reduce osteoblastic mineralization.34, 36

Binding of osteoblast-secreted receptor-activator of nuclear factor kappa beta ligand (RANKL) to its nuclear factor kappa beta receptor (RANK) located on

33 osteoclast precursors activates osteoclasts.33 Upon activation of osteoclasts, cathepsin K (CTSK) is expressed, a cysteine protease that stimulates tartrate- resistant acid phosphatase (TRAP). TRAP degrades collagen and other bone matrix components in early events of bone resorption.

RANK, RANKL, and the decoy receptor, osteoprotegerin (OPG), are three key proteins of a system which control bone resorption processes.27 Following the discovery of RANK, RANKL and OPG in the late 1990s, their importance in the maintenance of skeletal structure and their dramatic roles in bone disease were largely unexpected.37 In recent years the understanding of these proteins, in particular their regulation, has greatly increased. RANK was discovered by

Anderson et al.38 by directly sequencing cDNA from a human bone marrow- derived myeloid dendritic cell. RANK is a member of the tumor necrosis factor receptor (TNFR) family.39 Gene expression of RANK has been found on the surface of a wide variety of cells such as osteoclast precursors (circulating monocytes),40 mature osteoclasts,41 dendritic cells,38, 39 mammary gland epithelial cells,42 breast cancer cells43, and prostate cancer cells.44

RANKL is a tumor necrosis factor (TNF)-related cytokine expressed by various bone cells including mature osteoblasts and their immature precursors,45 osteocytes,46 T lymphocytes, B lymphocytes,47 and megakaryocytes.48 The human and murine RANKL proteins share 83-87% homology.49 Human and murine osteoblasts express RANKL once the cells are activated by different cytokines and hormones.38, 50 An increase in the production of RANKL will

34 stimulate bone resorption processes by increasing the differentiation, activation, and survival of osteoclasts.

It has been widely agreed that the expression of RANKL by osteoblasts mainly regulates bone remodeling, but several recent studies provide evidence also implying the essential nature of osteocyte-secreted RANKL in bone remodeling.28, 29, 51 Figure 1.8 demonstrates a tri-partite relationship between osteoblasts, osteoclasts, and osteocytes in bone resorption postulated by

Matsuo.51 Multinucleated osteoclasts erode the bone surface with CTSK, whereas osteoblasts secrete new bone matrix. The osteocytes and its dendrites are embedded in their canaliculi and lacunae expressing RANKL to induce osteoclast activity as well as to suppress osteoblast differentiation with sclerostin secretion.51 In this manner, the osteocytes regulate bone homeostasis through

RANKL and SOST expression.29, 51

Figure 1.8. The relationship of osteoblasts, osteoclasts, and osteocytes during bone resorption. With osteoclasts resorbing old bone matrix and osteoblasts

35 forming new bone matrix, respectively, osteocytes (embedded in their lacunae) regulate the bone remodeling process by secretion of RANKL and SOST. This figure has been reproduced with permission from International Bone and Mineral

Society.51

1.1.3 Application of Polymerase Chain Reaction Analysis in Osteogenesis

Studies

Known as the single most important tool in biological sciences that has been developed in the last 30 years, polymerase chain reaction (PCR) analysis has been widely applied in the research and diagnosis of fields such as forensic biochemistry, pathology, molecular biology and food science.52, 53 This technique was first introduced in 1985 following the identification of a specific DNA polymerase that can withstand repeated heating to high temperatures.54 It is a test-tube method to copy specific target DNA sequences selectively within a given source of DNA, and this technique has been widely applied in general laboratory practice after creation of efficient thermal cycling machines. Although the applications of the PCR are versatile, the technique is a particularly useful tool in DNA probe assays.55, 56

There are five individual components of a basic PCR. These are the source of template DNA containing the target gene sequence to be amplified (Figure

1.9); two short single-stranded sequences of DNA termed primers (amplimers), designed on the basis of previous knowledge of the DNA target sequence; a

36 suitable heat-stable DNA polymerase; DNA precursors; and suitable buffers containing optimum concentrations of salts such as magnesium and potassium.54

Figure 1.9. Schematic outline of PCR. (a) An outline of the temperature cycling in PCR. Step 1: when heated to 94°C, the double-stranded DNA template containing the target sequence of nucleotides for amplification is denatured.

Step 2: when cooled to 40-60°C, the primers anneal to specific sequences and they will flank the target sequence to be amplified. Step 3: on heating the solution to 72°C, the nucleotides are added to the 3’ end of the primers, leading to an efficient DNA synthesis. Temperature cycling steps 1-3 are repeated approximately 30-40 times. Single filled arrows indicate the oligonucleotide primers while newly formed DNA strands are denoted by the arrowheads. (b) An

38 outline of how the products are obtained with the application of PCR. After one cycle, two new single stranded DNA strands are synthesized. Diagonal stripes indicate the strands extending beyond the target DNA sequence for amplification.

After two cycles, the number of new strands will be six, four of which are extending beyond the ends of the target sequence and the rest of which are the target sequence that is to be amplified. After 30 cycles, for example, the desired target sequence has been exponentially amplified more than 105 times. Figure reproduced with permission from BMJ Publishing Group Ltd.52

Thus, by employing different primers, target gene sequences will be amplified through heat cycling. For osteogenic applications, utilizing related primers in reverse transcription-quantitative polymerase chain reaction (RT-qPCR) studies provides a great deal of information about the mechanisms of bone formation or resorption at a genetic level. In this regard, RNA is first extracted from a bone sample followed by reverse transcription into complementary DNA (cDNA). The cDNA is utilized as the template for the RT-qPCR reaction.

1.1.4 Role of Periosteum in Bone Healing

Periosteum lies on bone and its cambium layer is the direct connective tissue layer between bone and periosteum (Figure 1.4). Periosteum can be the reservoir of undifferentiated multi-potential mesenchymal stem cells which are able to differentiate into chondrocyte precursors and osteoblast precursors (as has been mentioned above) and are a source of growth factors that play a key

39 role in bone healing and remodeling processes, especially at the outer surface of a cortical bone.57-61

Scientists have proposed that periosteum regenerates both cartilage and bone from its progenitor cells.61-66 During the process of hematoma formation, platelet degranulation occurs and numbers of factors are released at the fracture site to serve important functions in healing.67 Those factors are called chemoattractants (molecules possessing chemotaxis-inducer effects in motile cells) for the mesenchymal cells originating within the external soft tissue and the bone cavity marrow.68, 69 The numerous growth factors derived from platelets can activate the generation and differentiation of the mesenchymal stem cells of periosteum.67 A fracture callus will form in the middle of a fracture to bridge the fracture fragments with hyaline cartilage.70, 71 After a bone fracture, there will be a vascular disruption that forms a blood clot (a hematoma) as an inflammatory response.67

Meanwhile, these chemoattractants can interact with endothelial cells, attract granulocytes (a category of white blood cells characterized by the existence of granules in their cytoplasm; granulocytes play an important role in inflammatory and repair processes of bone fracture72) and macrophages (cells produced by the differentiation of monocytes in tissues and function in nonspecific defense as well as in helping to initiate specific defense mechanisms of vertebrate animals).

Chemoattractants can also form a major component of the cellular infiltrate and interact with modified lipoproteins, other plasma components, platelets, and

40 additional constituents of the hematoma associated with a bone fracture.73

These various interactions play crucial steps in the bone healing process.68, 74

1.1.5 Utilization of Periosteum in Tissue Engineering of Bone Grafting

Grafting has been applied widely in surgery in order to heal bone defects. An autograft is bone tissue obtained from and used for the same individual while an allograft is taken from another donor of the same species. Typically, bone autografts and allografts are sterilized and devoid of living cells and organic constituents. Annually, more than 500,000 bone grafting procedures occur in the

United States and over 2.2 million segmental bone defects are healed by bone autografts or allografts around the world.75, 76

Segmental bone defects arise from the removal of a piece of bone from an individual as a result of bone resection or bone trauma. Defects may result in a persistent gap between the replacement bone graft and the host bone causing a failed surgery which is painful to the patient. A consequence of a failed surgery may be limb amputation. Under this circumstance, alternative approaches to traditional bone grafts are being investigated. A periosteum-bone allograft is one potentially suitable choice to solve the problem. Numerous studies have suggested that the presence of periosteum would induce the growth, development, and regeneration of bone.77 As introduced in the previous parts of this dissertation, periosteum is known to be pluripotential, containing both chondrogenic and osteoblastic cells, and it could be used to engineer new bone formation in vivo.78 Applied as a graft material, a periosteum-bone allograft may

41 generate new bone tissue that could be characterized through specific techniques to confirm the proper function and expression of genes and the synthesis and secretion of cellular and extracellular proteins of chondrogenic and osteogenic cells.1, 79

Known as a well vascularized ‘osteogenic organ,’ periosteum is a functional fibrous tissue comprising a thin but tough fibrous membrane firmly anchored to bone.80 As noted earlier, the cambium layer of periosteum contains progenitor cells that maintain an ability to differentiate into osteoprogenitor or chondroprogenitor cells that can form bone and cartilage, respectively, under the control of different growth factors. In addition with vascularity provided through the periosteum, nutrition is present for cell viability. Thus, the periosteum plays an important role in bone formation.

A number of studies have assessed the significance of wrapping periosteum around segmental allografts that may be applied to repair bone defects.

Previous results from the Landis Laboratory have demonstrated that osteoblasts and chondrocytes retrieved from the periosteum maintain their viability during implantation and infiltrate into experimental tissue-engineered bone models.77

Animal studies have indicated increased bone formation and improved union when allografts are wrapped with periosteum.81 Additionally, the clinical use of periosteum to induce healing of bony non-unions has been supported by numerous case studies.82, 83 From a recently published paper, laser capture microdissection and quantitative RT-PCR analysis were used to demonstrate gene expression of bovine type II collagen, aggrecan, osteopontin, osteocalcin

42 and bone sialoprotein in new cartilaginous growth plates of tissue-engineered model digit constructs, and data obtained were consistent with results from normal growth plates from bovine bone.84-87 Furthermore, other recent research has shown that periosteal cells enhanced new bone formation in tissue- engineered bone better than bone marrow or mesenchymal stem cells.82

In another published paper, human-shaped middle phalanges were fabricated with midshaft scaffolds of poly(L-lactide-co-ε-caprolactone) [P(LA-CL)], hydroxyapatite-P(LA-CL), and β-tricalcium phosphate-P(LA-CL) wrapped with or without bovine periosteum (Figures 1.10-11).88 Results revealed the capabilities of periosteum of inducing vascular development, the gene expression of bovine- specific type II collagen, aggrecan, and bone sialoprotein, and the maintenance of a human phalanx shape.88 Figure 1.10 illustrates that midshafts of the constructs without periosteum wrapping were observed with relatively higher vascularization compared to those covered by the fibrous periosteal tissue, without consideration of the copolymer used for fabrication. Further, non- wrapped midshafts were thinner than the qualitatively firmer periosteum-wrapped midshafts, regardless of the type of the polymers.88

A comparison obtained between constructs with and without periosteum wrapping implied that periosteum generated additional cells that secreted extracellular matrix (ECM) and induced mineralization in midshaft models.

Histological comparisons among constructs with or without periosteum were derived through implantation in athymic mice and application of various procedures including toluidine blue, Safranin-O red, Alizarin red, and von Kossa

43 staining (Figure 1.11). For 10-week implanted constructs with and without periosteum, toluidine blue staining clearly marked the midshaft construct regions and showed that the presence of periosteum led to greater cell proliferation.

Safranin-O red staining demonstrated that periosteum wrapped about the polymeric scaffolds produced more proteoglycans than those same molecules detected in unwrapped scaffolds. The presence of calcium revealed by Alizarin red and phosphate by von Kossa staining indicated a higher level of mineralization for periosteum-wrapped scaffolds than that in unwrapped scaffolds. Such tissue-engineering results have documented that periosteum plays a crucial role in new bone formation on biodegradable polymeric materials.88

Figure 1.10. A series of panels showing intact and bisected representative middle phalanx specimens harvested after 10 and 20 weeks of implantation in athymic (nude) mice. Construct midshafts were composed of copoly(L-lactide/ε- caprolactone) [P(LA-CL)] and were either without (top four panels) or wrapped with calf periosteum (bottom four panels). After implantation, there were noticeable differences between constructs with and without periosteum in terms of midshaft vascularization and thickness. Scale bar = 1 cm. Figure reproduced with permission from Mary Ann Liebert, Inc., publishers.88

Figure 1.11. Panels showing histological staining of paraffin-embedded sections of constructs composed of midshafts of P(LA-CL). Constructs were prepared with or without periosteum and retrieved from nude mice after 10 weeks of implantation. Toluidine blue (A), Safranin-O (B), Alizarin red (C), and von Kossa

(D) staining of the sections indicate basic tissue morphology, proteoglycan

(cartilage) development, calcium deposition, and presence of phosphate.

Staining of tissue sections in the bottom panel (A-D) is as noted for tissue sections in the top panel. Scale bar = 1 cm. Figure reproduced with permission from Mary Ann Liebert, Inc., publishers.88

The studies described above all support the concept that periosteum may promote union at an allograft-host bone junction that is otherwise limited in scope and flawed in design with a bone graft alone. By developing periosteum-allograft constructs in vitro and in vivo before implantation into a segmental bone defect, their molecular, biochemical, structural, and biomechanical nature could be investigated with the prospect of improving such bone defect healing. Based on the concepts defined above, the hypothesis to be tested in this study is that a construct tissue-engineered with human periosteum, wrapped about a human bone allograft will be suitable for healing segmental bone defects. The overall aim of this thesis study is to determine if human periosteum, wrapped around a human bone allograft and supported with an appropriate bio-environment, will promote new bone formation into the allograft and, if so, new bone formation in a model of a segmental defect.

One goal for this study is to assess the molecular and histological events that occur in allograft bone wrapped with periosteum in minimally load-bearing sites in vivo.89 To approach this goal, allograft bone constructs will be wrapped with fresh, human periosteum harvested from cadaveric human limbs of donors.

Constructs will be cultured for approximately one week and then implanted for

10, 20, and 40 weeks in athymic mice. After harvesting the constructs at all three designated times, assessment will be made by histological staining to determine general cell morphology and to quantitate the area of new osteoid formation in the periosteum-allograft constructs and the penetration depth of the periosteal cells infiltrated into the allografts. RT-qPCR analysis will be applied to evaluate

47 the levels of human bone-specific and murine-specific gene expression in the construct tissues.

1.2 Materials and Methods

This section includes experimental materials and methods that applied in the thesis study.

1.2.1 Experimental Materials

Fresh periosteal strips (approximately 1 cm × 5 cm) were harvested within 24 hours of donor death from knees from 52- to 71-year-old male human cadaver limbs (obtained from the Gift of Hope Tissue & Organ Donor Network, Itasca, IL, through Rush University Medical Center, Rush University, Chicago, IL). Human femoral cortical strut bone allografts (donor age 16 to 53 years) were obtained from the Musculoskeletal Transplant Foundation (MTF, Jessup, PA) and were sectioned under sterile conditions into 43 cubes, 1 cm3 each. Athymic mice purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN) were housed at the Northeast Ohio Medical University Comparative Medicine Unit (NEOMED,

Rootstown, OH) and maintained in compliance with the regulations of the

Institutional Animal Care and Use Committee of NEOMED. Medium 199 (M199,

Mediatech, Inc., Manassas, VA) supplemented with 10% Fetal Bovine Serum

(purchased from HyClone/Thermo Scientific Co. Waltham, MA), 1% antibiotic/antimycotic (Mediatech, Inc.), and 0.2% primocin (InvivoGen, San

Diego, CA) constituted the complete feeding or medium used to culture periosteum allografts. M199 referred to below is the complete feeding medium.

1.2.2 Experimental Methods

This section includes fabrication of specimens, animal surgery, histology and gene expression methods applied in the thesis study.

1.2.2.a Fabrication of Periosteum-bone Allografts

Human femoral cortical strut bone allografts (from 16- to 53-year-old donors,

MTF Non-Transplantable Tissue Program) were sectioned with a Dremel saw under sterile conditions into 20 blocks of 1 cm3 each (Figure 1.13-A) and incubated (37°C, 5% CO2, M199 medium) for 24 hours.

Muscle, adipose tissue, and fascia were removed with surgical instruments from donor knees to expose periosteum. Human periosteum strips, each of a size about 1 cm wide by 3.5 cm in length, were harvested with an elevator and a pair of forceps from the distal femur of 54- to 71-year-old cadaver donors (Gift of

Hope Tissue & Organ Donor Network) and were placed in M199 medium

(cambium layer facing down) for 1 week. Bone allografts without periosteum served as experimental controls (Group 1). After one week of culture, the periosteum strips were wrapped about twenty-two human allograft blocks

(cambium layer down and facing the allograft, Group 2) and secured with 5-0

Nylon sutures. Periosteum-bone allograft constructs and allograft blocks alone were cultured for one week at 37°C and 5% CO2 in an incubator and the culture medium was changed every two days. The experimental design is illustrated in

Figure 1.12.

49 Figure 1.12. A schematic of fabrication of human periosteum-allograft bone.

Group 1: human allograft bone specimen (N = 7). Group 2: human periosteum- allograft bone construct (N = 22). The cambium layer of periosteum strips faced the bone graft so that periosteal progenitor cells might migrate and infiltrate the human allograft. The complete constructs were secured with Nylon 5-0 sutures.

1.2.2.b Specimen Implantation, Harvesting, and Processing

After one week of incubation, the constructs were implanted subcutaneously in six-week-old athymic (nude) mice (Harlan Sprauge Dawley, Inc.). To implant the constructs, an incision (~1.5 cm) was made with a sterile scalpel on the dorsum of the animal. The constructs were placed in the subcutaneous dorsum of the athymic mice with application of a pair of sterile scissors to open a pocket.

Each athymic mouse was implanted with two constructs on either side of its dorsum (Figure 1.12-E). The incision was closed with PGA sutures.

50 Mice were harvested after 10-, 20-, and 40-weeks of implantation. Constructs were retrieved with sterile surgical scissors and scalpels. For the two constructs on the dorsum of the athymic mice, the left sample was taken for histology and the right one was reserved for gene expression. For histological analysis, specimens were fixed in 20 mL 10% neutral buffered formalin (NBF, Electron

Microscopy Sciences, Hatfield, PA) for 1 week and then were decalcified in

Immunocal solution (Decal Chemical Corp., Tallman, NY) for up to 4 weeks until completely decalcified. Insertion of a 22 gauge needle was used to verify the completion of demineralization. The demineralized specimens were embedded in paraffin for histological assessment. For gene expression analysis, the harvested samples were immersed immediately in RNAlaterTM (Ambion, Austin,

TX) to preserve RNA. Undecalcified, RNAlater-treated constructs were first cooled at -4°C for 24 hours and then transferred to a -80°C freezer for storage until RNA isolation.

Figure 1.13. Methods and materials for creation of human periosteum allograft constructs. A: Human femoral cortical strut bone allografts were sectioned under sterile conditions into 43 blocks of 1 cm3 each and incubated (37°C, 5%

CO2, 24 hours, M199). B: A photograph of the human proximal tibia procured within 24 hours of death of a 52-year-old male was dissected to provide 31 strips of periosteum. C: Human periosteum strips harvested from the proximal tibia.

D: Periosteum strips wrapped about the allograft bone segments, sutured and placed in M199. E: An athymic mouse implanted with two human periosteum- bone allografts on each side of its back (arrows). F: A human periosteum-bone allograft construct harvested from an athymic mouse after 20 weeks of implantation. The arrow points out the periosteum on the construct. The figure shows the periosteum closely associating with the bone allograft. Scale bars = 2 cm (A, D, E), 3 cm (B, C, F).

1.2.2.c Specimen Staining

Followed fixation and decalcification, the specimens were rinsed in deionized water three times followed by three changes of 70% ethanol, 15 minutes each.

The samples were then placed in plastic cassettes and processed through a series of ethanol and xylene rinses in a processor (Model ASP300S tissue processor, Leica Biosystems, Inc., Buffalo Grove, IL) prior to embedding the processed samples in paraffin wax blocks. After sectioning, 5-7 µm (in thickness) sections were treated variously with hematoxylin and eosin (H&E), picrosirius red (PSR), and tartrate-resistant acid phosphatase (TRAP), respectively. H&E was applied to determine general cell morphology, the quantity and location of new bone formation in the constructs, and the depth of penetration of periosteal-derived cells into the allograft segment of constructs.

Picrosirius red staining was utilized to assess collagen content.90 TRAP staining was used to identify possible osteoclasts. Stained sections were photographed

53 and image montages were created with Microsoft Image Composite Editor version 2.0.3.0 (Microsoft, corp., Seattle, CA).

1.2.2.d Quantitative Polymerase Chain Reaction Assessment

PCR results were obtained from RNA isolated from all samples in both Group

1 (human allograft bone only) and Group 2 (human periosteum-allograft bone).

The quantitative levels of mouse and human bone-specific and cartilage-specific genes expressed over the time of construct implantation were evaluated through real time RT-qPCR. Protocols followed the routinely performed procedures in the

Landis Laboratory and are outlined below.84

RNA was isolated from the implanted construct samples (Group 1 and Group

2) which were placed in RNAlaterTM after harvest from athymic mice (10, 20, and

40 weeks). The constructs were first ground to powders with a cryogenic mill

(SPEX SamplePrep 6870 Freezer/Mill, Thomas Scientific, Waltham, MA) under liquid nitrogen. The powders of each specimen were transferred to 50 mL conical tubes with addition of 3-5 mL TRI Reagent® (Molecular Research Center,

Inc., Cincinnati, OH) following the manufacturer’s manual. A commercial

E.Z.N.A. Total RNA Kit I (Omega Bio-Tek, Inc., Norcross, GA) was utilized for column purification of RNA. DNase digestion was performed with an optional

RNase Free DNase set (E1091) from Omega Bio-Tek, Inc. An Eppendorf

BioPhotometer (Eppendorf, Hauppauge, NY) was used to obtain 260/280 ratios as an indication of the quality and the quantity of RNA samples (values ~2.0 are considered as high quality of purified RNA).

Purified RNA samples from experimental groups and discarded surgical tissues (human) were reverse-transcribed to corresponding cDNA

(complementary DNA) based on a previously reported protocol.91 Concisely, 1

µg of purified RNA was mixed with AmbionTM 10X first strand buffer, oligo (dT) primers, dNTPs (2'-deoxynucleoside 5'-triphosphates), random hexamers,

RNase inhibitor, and M-MLV reverse transcriptase (Life Technologies, Corp.,

Carlsbad, CA) in a volume of 20 µL. Negative controls (minus RT, -RT) were selected RNA specimens (from both Group 1 and Group 2) reverse-transcribed without M-MLV reverse transcriptase reactions. All reactions were left at room temperature for 10 min following incubation in a 37 °C heating block for 1 hr. The reaction was inactivated by 5 minutes of incubation at 95 °C before transferring cDNA samples to ice for cooling.

With each reaction presumably 80% efficiency, the final cDNA concentration was 40 ng/µL from 1 µg of purified RNA. Stock samples for generation of standard curves in PCR were mixtures of cDNA from human-periosteum allograft samples and discarded surgical human tissue RNA samples. A standard curve was composed of a serial dilution of 20 ng/µL, 10 ng/µL, 5 ng/µL, 1 ng/µL, 0.5 ng/µL, and 0.1 ng/µL from the stock solution.

Gene expression levels of mouse-specific decorin (DCN), tumor necrosis factor superfamily, member 11 (TNFSF11, or RANKL), type I collagen (COL1A1), cathepsin K (CTSK), tumor necrosis factor receptor superfamily, member 11a

(TNFRSF11A, or RANK), alkaline phosphatase (ALPL), sclerostin (SOST), actin

(beta, Actb), and beta-2-microglobulin (B2m) were normalized to the mouse-

55 specific housekeeping gene, cyclophilin D (PPID). In addition, human-specific

DCN, RANKL, COL1A1, ALPL, bone integrin-binding sialoprotein (IBSP), bone gamma-carboxyglutamate protein (BGLAP, also known as osteocalcin, OC), osterix (OSX), CTSK, RANK, and SOST were normalized to the housekeeping gene, human large ribosomal protein (RPLP0, P0).

Purchased human-specific and murine-specific primer sets (Applied

Biosystems, Foster City, CA) and previously designed murine-specific primers were used as listed in Tables 1.1 and 1.2, respectively. Designed mouse- specific primers were synthesized by Sigma-Genosys (The Woodlands, TX).

Primer sequences were determined with application of the BLAST program

(National Center for Biotechnology Information; www.ncbi.nlm.nih.gov, Table

1.3).

Gene expression was analyzed following the relative standard quantification methodology and the 7500 Real time PCR System (Applied Biosystems, Foster

City, CA).91 Each 20 µL reaction well (96-well plates) for qPCR analysis contained 10 µL of SYBR Green® Master Mix (designed primer sets) or 10 µL of

TaqMan® Master Mix (purchased primer sets, Applied Biosystem), 7 or 8 µL of sterile water, 1 µL of forward primer and 1 µL of reverse primer (SYBR Green®

Master mix), 1 µL (20x) of purchased primers (TaqMan® Master Mix), and 1 µL of cDNA. In order to confirm that no DNA contamination or primer-dimer formation had occurred in the analysis samples, minus RTs, no template controls

(NTC, primer and master mix only), and buffer blanks were used as negative

56 specimens. The standard solutions were applied in triplicate to generate a standard curve while each sample was applied in duplicate.

Designed mouse-specific primers (PPID, COL1A1, and SOST) containing

SYBR Green® were first activated at 95 °C for 10 min, followed by amplification with 40 cycles of 95 °C for 15 s, 60 °C for 1 min, and 72 °C for 30 s. The last cycle heated the plate incrementally by degree to 95 °C to generate a melting curve. Dissociation curves of designed primer sets were extracted utilizing the

7500 software v2.0.4 (Applied Biosystems) to obtain the melting temperatures of sample amplification products. The qPCR for primers purchased from Applied

Biosystems utilizing TaqMan® Master Mix was performed by heating the 96-well plate at 50 °C for 2 min and activating the polymerase at 95 °C for 10 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s (annealing and extension). Once the baseline value was defined, an increase of PCR product

(amplicon) at a specific cycle (threshold cycle, CT) was detected by fluorescence measurements. After amplification was complete, generation of relative standard curves was performed (7500 software v2.0.4, Applied Biosystems) and the relative quantity of each sample was calculated.

Because the amounts of input RNA varied between samples, the relative quantities of each amplified product for all samples were normalized to their respective housekeeping gene (human or murine). The quality of initial isolated

RNA was determined by the ratio of relative quantity of P0 to the relative quantity of beta-actin, both used as stable reference genes. Samples which had a ratio less than 0.5 or greater than 2.0 were considered poor quality or containing

57 degraded RNA and they were removed from analysis. The normalized value of each 20- and 40-week sample was compared to that of its respective 10-week normalized value and plotted as fold change of gene expression over time.

Errors in fold changes were expressed as standard errors of mean values.

Table1.1. A list of murine-specific primer sets.

Gene Name Gene Symbol Catalog Number Cyclophilin D PPID F1577; R1694 Decorin DCN Mm00514535_m1 Receptor activator of nuclear factor RANKL Mm00441906_m1 kappa-B ligand Collagen, type I, alpha 1 COL1A1 F66; R194 Alkaline phosphatase ALPL Mm00475834_m1 Cathepsin K CTSK Mm00484039_m1 Receptor Activator of nuclear factor RANK Mm00437132_m1 kappa-B Actin, beta Actb Mm00607939_s1 Sclerostin SOST F1038;R1105 Beta-2-Microglobulin B2m Mm00437762_m1

Table1.2. A list of human-specific primer sets.

Gene Name Gene Symbol Catalog Number Large ribosomal protein RPLP0 4326314E Decorin DCN Hs00370384_m1 Receptor activator of nuclear factor RANKL Hs00243522_m1 kappa-B ligand Collagen, type I, alpha 1 COL1A1 Hs00164004_m1 Osteocalcin OC Hs00609452_g1 Cathepsin K CTSK Hs00166156_m1 Receptor Activator of nuclear factor RANK Hs00187192_m1 kappa-B Integrin-binding sialoprotein IBSP Hs00173720_m1 Osterix OSX Hs01866874_s1 Sclerostin SOST Hs00228830_m1 Alkaline phosphatase ALPL Hs00758162_m1

Table1.3. Designed primer sequences (mouse-specific).

GenBank Product Product Gene Accession Primer Sequence (5' to 3') Size Melting Number (bp) Point (°C) GGGTCACCACATGAA F GAACTGTACTA Cyclophilin D BC019778 118 77 CATGCCCGATGGTTT R ATACACA CCAAGAAGACATCCC F Collagen, type TGAAGTCA U08020 129 81 I, alpha 1 TGCACGTCATCGCAC R ACA AGCCTGCGTGGGCT F AGATAG Sclerostin NM024449 66 80 ACGTAGCCCAACATC R ACACTCA

1.2.2.e Statistical Analysis

Statistical analysis was performed by applying a one-way analysis of variance

(ANOVA) test with IBM SPSS Statistics Version 22.0. (IBM Corp., Armonk, NY).

A student’s t-test (parametric, unpaired, two-tailed test) was also utilized to identify statistically significant differences (p ≤ 0.05) between normalized expression levels of human-specific genes by comparison of 20-week results to

10-week counterparts and then 40-week results to 10-week counterparts. The student’s t-test was repeated with the murine-specific genes and the same time point comparisons.

1.3 Results and Discussion

This section includes results and discussion collected from histology and gene expression study.

1.3.1 Histological Results

Periosteum remained viable after 10, 20, and 40 weeks of implantation as it was observed intact around the peripheral of the allograft bone regardless of the age and gender of patients. Histological assessment was performed on control specimens (allograft bone alone) implanted for 10, 20, and 40 weeks and specimens wrapped with periosteum after 10, 20, and 40 weeks of implantation.

Apparent cell infiltration from the host-mouse in allograft bone pores was observed from 10-, 20-, and 40-week allograft bone alone samples (Figure 1.14-

A, 15-A, and 16-A). No regenerated bone tissue was detected in the control specimens. Observations from specimens with different implantation times in

Group 1 expressed thin layers of mouse encapsulation tissue around the periphery of the allograft bone. Vascular elements were found within the cavities that compromised the allograft bone (Figure 1.14-A, 15-A, and 16-A).

Specimens from Group 1 are compared after 10, 20, and 40 weeks of implantation and H&E staining (Figure 14-A, 15-A, and 16-A). With increasing implantation time, specimens from Group 1 show a slight qualitative increase in the depths of mouse tissue infiltration and the presence of vascular elements.

Micrographs taken from all Group 2 samples indicate viable periosteum after

10, 20, and 40 weeks of implantation. Composite light micrographs illustrating periosteum-allograft specimens implanted for 10, 20, and 40 weeks are shown in

Figure 1.14-B, 15-B, and 16-B, respectively. Bone resorption by possible osteoclasts was present at the interface of periosteum and allograft bone (Figure

1.14-B and 15-B). New bone tissue formed along the periosteum-allograft bone interfaces is marked in 10-, 20-, and 40-week human allograft-bone sections

(Figure 1.14B, 15-B, and 16-B). Vascular elements were observed in periosteum and bone cavities that compromised allograft bone construct (Figure 14-B, 15-B, and 16-B).

Figure 1.14. H&E-stained histological sections and enlargements of 10-week human allograft models (with and without periosteum). A: a 10-week allograft bone (AB) control sample with partial mouse fibrous tissue infiltration (arrow) in bone cavities formed by vascularization. Such bone cavities are apparent in other specimens, and they are shown in respective figures. A region of the tissue (a) is enclosed and shown enlarged in the inset. Enlargement a: apparent cell infiltration from the host-mouse in one of many allograft bone (AB) pores. B: visible periosteum (P) wrapped around the periphery of a 10-week human allograft bone construct (AB). This specimen contains numerous cavities or pores as in A resulting from vascularization of the allograft. Three regions (a, b, and c) are shown enlarged in the insets. Enlargement a: human periosteum (P) infiltration present in the allograft bone (AB, arrow); b: a site of possible resorption by putative osteoclasts (arrow) at the interface between periosteum

(P) and allograft bone (AB); it appears that allograft bone tissue has been eroded by the osteoclasts in this image; c: newly forming bone osteoid containing several osteocytes along the original periosteum (P)-allograft bone (AB) interface

(arrows). Scale bars = 1000 µm (panels A, B), 200 µm (panels A-a, B-a), 100 µm

(panels B-b, c).

Figure 1.15. H&E-stained histological sections and enlargements of 20-week human allograft models (with and without periosteum). The bone cavities are shown in different shapes from Figure 1.14 as the specimens were sectioned in a transverse fashion. A: a 20-week allograft bone (AB) control sample with murine encapsulated fibrous tissue (arrow) infiltration of the allograft. A region of the tissue (a) is enlarged and shown in the inset. Enlargement a: mouse tissue infiltration observed in many pores of the bone graft; vascular elements (arrow) found in one pore of the human allograft bone (AB). B: viable periosteum (P) present around an allograft bone (AB) construct after 20 weeks of implantation in an athymic mouse. Three regions are shown enlarged in the insets.

Enlargement a: bone lining cells (arrows) observed at the interface between allograft bone (AB) and periosteum (P); b: blood vessels (arrows) detected in pores of allograft bone (AB); c: allograft bone being resorbed by presumptive osteoclasts (arrow) in the area where periosteum (P) is attached to the allograft bone (AB). Scale bars = 1000 µm (panels A, B), 200 µm (panels A-a, b), 100 µm

(panels B-a, b, c).

Figure 1.16. H&E-stained histological sections and enlargements of 40-week human allograft models (with and without periosteum). A: mouse fibrous tissue

(arrows) infiltration observed in a 40-week allograft bone (AB) specimen. Three regions (a, b, and c) are shown enlarged in the insets. Enlargement a, b, and c all show mouse tissue infiltration and vascular elements (arrows) in cavities in various areas of the allograft bone (AB). B: Periosteum (P) wrapped around the periphery of a 40-week human allograft bone (AB) sample. The empty space between allograft bone and periosteum is an artifact attributable to sectioning the construct. Three sites (a, b, and c) are enlarged in the insets to show construct detail. Enlargement a: vascular elements (arrow) present in an interior allograft bone pore; b: bone formation (arrow) observed at the interface between periosteum (P) and allograft bone (AB); c: new bone matrix formation (arrow) observed at the interface of periosteum (P) and allograft bone (AB). Scale bars =

1000 µm (panels A, B), 200 µm (panels A-a, b, B-b, c), 100 µm (panels A-c, B-a).

To identify possible osteoclasts in human periosteum-allograft samples, TRAP staining was applied. Micrographs from both H&E- and TRAP-stained sections were obtained. In Figure 1.17-A, H&E staining indicates newly formed bone beneath reversal lines in the construct and the presence of a resorption cavity in the tissue. Figure 1.17-B shows a TRAP-stained presumptive osteoclast within its resorption cavity. This image result demonstrated that bone graft remodeling had occurred in the construct during its implantation. No notable osteoclasts were found in control samples (10, 20, or 40 weeks of implantation).

Figure 1.17. Light micrographs of H&E and TRAP staining results of a 10-week human periosteum-allograft construct. A: a scalloped resorption cavity (arrows) created by an osteoclast (s) beneath newly formed bone reversal lines in an H&E stained 10-week allograft bone (AB) wrapped with periosteum (P), B: osteoclast(s) stained as red/pink (arrows) by TRAP within its resorption cavity in an allograft bone (AB) construct wrapped with periosteum (P). Scale bars = 100

µm (panel A), 50 µm (panel B).

1.3.2 RT-qPCR Results

For human allograft bones (Group 1) and human periosteum wrapped- allograft (human) constructs (Group 2), the relative mean of fold changes of murine- and human-specific gene expression was calculated for specimens implanted for 10, 20, and 40 weeks with application of RT-qPCR. Relative fold changes of gene expression were obtained by comparing data from 20- and 40- week specimens to 10-week samples, respectively. Linear trend lines indicating overall traces of related fold changes of mRNA expression levels of each gene

(murine- and human-specific) were automatically generated with Microsoft Excel

2013 (Microsoft Corp., Redmond, WA; Figures 2.18-22, respectively). The p values were identified with a student’s t-test (parametric, unpaired, two-tailed test) and one-way ANOVA. Statistical significance was determined as p ≤ 0.05.

For human allograft only bone specimens (Group 1) implanted for 20 weeks, the relative fold-change values when compared to 10-week samples (calibrators) of murine-specific DCN, RANKL, COL1A1, ALPL, RANK, and CTSK were 0.96 ±

0.55, 1.61 ± 0.39, 0.57 ± 0.14, 1.61 ± 0.55, 0.81 ± 0.18, and 0.27, respectively

(Table 1.4, error not applicable for CTSK as only one specimen was available for examination). The 40-week specimens had respective values of 0.53 ± 0.14,

0.70 ± 0.12, 0.58 ± 0.22, 0.73 ± 0.22, 1.04 ± 0.26, and 0.53 ± 0.20 when also compared to 10-week samples (Table 1.4). Murine-specific SOST was not detected in 20- or 40-week samples (data not shown). Moreover, human-specific gene expression was not detected in specimens in Group 1 regardless of implantation time (data not shown). After 20 and 40 weeks of implantation, the

69 levels of mouse-specific DCN, RANKL, COL1A1, ALPL, and RANK resulted in no statistically significant differences when compared to 10-week specimens (Table

1.4).

Table 1.4. Relative murine-specific gene expression fold changes of 20- and 40- week human allograft specimens (Group 1) detected with RT-qPCR. The data are presented as the fold change ± standard error of the mean at 20 and 40 weeks compared to 10 weeks of implantation and after standardization to a cyclophilin D housekeeping gene reference. Error was not applicable for CTSK as only one specimen was available for examination. There were no statistically significant differences in the levels of any of the murine-specific genes analyzed from 20- and 40-week samples when compared to 10-week specimens. A student’s t-test (parametric, unpaired, two-tailed test) identified p values between

10- and 20-week samples and between 10- and 40-week samples. The gene expression by osteocytes was not detected (N.D.) in any of the specimens. N =

2 (10 weeks), 1 (20 weeks, CTSK), 2 (20 weeks, other genes), and 3 (40 weeks).

Murine 20-week vs. 10-week 40-week vs. 10-week Gene Fold Change p Value Fold Change p Value Symbol DCN 0.96 ± 0.55 0.95 0.53 ± 0.14 0.38 Osteoblast RANKL 1.61 ± 0.39 0.26 0.70 ± 0.12 0.14 Activity COL1A1 0.57 ± 0.14 0.62 0.58 ± 0.22 0.53 ALPL 1.61 ± 0.55 0.48 0.73 ± 0.22 0.58 Osteoclast RANK 0.81 ± 0.18 0.43 1.04 ± 0.26 0.90 Activity CTSK 0.27 N/A 0.53 ± 0.20 N/A Osteocyte SOST N.D. N/A N.D. N/A Activity

Figures 1.18 and 1.19 show relative fold changes of the levels of mRNA expression of murine-specific DCN, RANKL, COL1A1, ALPL, RANK, and CTSK from specimens (Group 1) following 10, 20, and 40 weeks of implantation. Trend lines were plotted for each individual gene to reveal any change in the level of gene expression over 40 weeks of time. Trend lines for DCN, RANKL, CTSK, and COL1A1 all displayed reduced gene expression levels over the 40-week time frame.

Figure 1.18. Relative mean fold changes (10-week data utilized as the calibrator) in mRNA expression of murine-specific genes determined by RT-qPCR. Data represent mean fold changes in mRNA expression for four human-specific preosteoblast and osteoblast activity-related genes: DCN (A), RANKL (B),

COL1A1 (C), and ALPL (D). N = 2 (10 weeks), 2 (20 weeks), and 3 (40 weeks).

A, B, C, and D all show a decrease in trend lines over 40 weeks of specimen implantation. Bars represent standard error of mean values of fold changes given for each gene.

Figure 1.19. Relative mean fold changes compared to 10-week samples (Figure

1.18) in mRNA expression of murine-specific genes determined by RT-qPCR.

Data represent mean fold changes in mRNA expression for two human-specific osteoclast activity-related genes: RANK (A) and CTSK (B). N = 2 (10 weeks), 2

(20-week RANK), 1 (20-week CTSK, error bar not applicable), and 3 (40 weeks).

Bars represent standard error of mean values of fold changes given for each gene.

Relative gene expression fold changes of human-specific DCN, RANKL,

COL1A1, OC, IBSP, OSX, ALPL, RANK, CTSK, and SOST from 20-week specimens (human periosteum-allograft bone, Group 2) compared to 10-week samples (Group 2) gave respective values of 1.05 ± 0.28, 1.87 ± 0.29, 0.79 ±

0.25, 2.19 ± 0.65, 1.11 ± 0.39, 1.01 ± 0.15, 0.89 ± 0.37, 2.44 ± 0.99, 1.07 ± 0.22, and 15.18 ± 5.77 (Table 1.5). The fold changes of the 40-week samples (Group 72

2) also compared to 10-week specimens (Group 2) had in sequence 0.97 ± 0.10,

5.04 ± 2.00, 0.54 ± 0.03, 1.62 ± 0.46, 1.04 ± 0.24, 1.79 ± 0.99, 0.53 ± 0.12, 1.21

± 0.54, 1.20 ± 0.12, and 32.98 ± 12.44, respectively (Table 1.5). For samples implanted for 20 and 40 weeks, only the levels of RANKL and SOST gene expression increased statistically significantly at 20 and 40 weeks when compared to 10-week samples, with p values less than 0.05 (highlighted in Table

1.5).

Table 1.5. Relative human-specific gene expression fold changes of 20- and 40- week human periosteum-allograft specimens (Group 2) compared to 10-week samples and detected with RT-qPCR. The data are presented as the fold change ± standard error of the mean at 20 and 40 weeks compared to 10 weeks of implantation and after normalization to the large ribosomal protein (P0) housekeeping reference gene. A student’s t-test (parametric, unpaired, two- tailed test) identified p values of samples at different time points. The data are considered statistically significant for p ≤ 0.05. The fold changes of RANKL and

SOST both showed statistically significant increases (p values highlighted) at 20 weeks and 40 weeks when compared to 10-week samples. N = 6 (10 weeks), 8

(20 weeks), 3 (40 weeks, RANK, CTSK, and SOST), and 5 (40 weeks, other genes).

Human 20-week vs. 10-week 40-week vs. 10-week Gene Fold Change p Value Fold Change p Value Symbol DCN 1.05 ± 0.28 0.90 0.97 ± 0.10 0.92 RANKL 1.87 ± 0.29 0.03 5.04 ± 2.00 0.03 COL1A1 0.79 ± 0.25 0.51 0.54 ± 0.03 0.06 Osteoblast OC 2.19 ± 0.65 0.14 1.62 ± 0.46 0.23 Activity IBSP 1.11 ± 0.39 0.82 1.04 ± 0.24 0.92 OSX 1.01 ± 0.15 0.96 1.79 ± 0.99 0.39 ALPL 0.89 ± 0.37 0.82 0.53 ± 0.12 0.19 Osteoclast RANK 2.44 ± 0.99 0.27 1.21 ± 0.54 0.80 Activity CTSK 1.07 ± 0.22 0.78 1.20 ± 0.12 0.56 Osteocyte SOST 15.18 ± 5.77 0.04 32.98 ± 12.44 0.01 Activity

Figure 1.20 shows fold changes in levels of mRNA expressed by human osteoblast-related genes, DCN, RANKL, COL1A1, OC, IBSP, OSX, and ALPL at different harvest time points for specimens (Group 2). The presence of osteoblasts in specimens was verified by the detection of increasing expression levels of the osteoblast-related genes RANKL, OC, and OSX with longer implantation times (Figure 1.20-B, D, and F). Human-specific RANK and CTSK were detected in samples implanted for up to 40 weeks, results which imply the activation of osteoclasts and remodeling of allograft bones (Figure 1.21-A, B).

Figure 1.21 suggests increasing activity of osteoclasts (expression of RANK and

CTSK) in human-periosteum allograft bone for all three implantation time points.

Figure 1.22 illustrates the levels of mRNA expression of human-specific SOST at

20 and 40 weeks. SOST levels were statistically significantly different from sampling at 10 weeks where a 15-fold increase was observed at 20 weeks and a

30-fold increase at 40 weeks, respectively. In this Group, the mRNA expression levels of only human-specific RANKL and SOST at 10, 20, and 40 weeks were 74 detected as statistically significantly different by one-way ANOVA (p = 0.014 and

0.016, respectively).

Figure 1.20. Relative mean fold changes (10-week data used as calibrators) in mRNA expression of human osteoblast-related genes determined by RT-qPCR.

Data represent mean fold changes in mRNA expression for seven human- specific genes: DCN (A), RANKL (B), COL1A1 (C), OC (D), IBSP (E), OSX (F), and ALPL (G). Trend lines were plotted for each individual gene to demonstrate expression levels over 40-week time frames. N = 6 (10 weeks), 8 (20 weeks), and 5 (40 weeks). Bars represent standard error of mean values of fold changes given for each gene. A student’s t-test (parametric, unpaired, two-tailed test) was applied to calculate p values. *p < 0.05.

Figure 1.21. Relative mean fold changes derived from a comparison of 20- and

40-week to 10-week samples in mRNA expression of human osteoclast-related

76 genes determined by RT-qPCR. Data represent mean fold changes in mRNA expression for two human-specific genes, RANK (A), CTSK (B). N = 6 (10 weeks), 8 (20 weeks), and 3 (40 weeks). Bars represent standard error of mean values of fold changes given for each gene.

Figure 1.22. Relative mean fold changes of 20- and 40-week specimens compared to 10-week specimens in mRNA expression of human SOST obtained with RT-qPCR. Data represent mean fold changes in mRNA expression for human-specific SOST over 10, 20, and 40 weeks of specimen implantation. N =

6 (10 weeks), 8 (20 weeks), and 3 (40 weeks). Bars represent standard error of mean values of fold changes given for each gene. A student’s t-test (parametric, unpaired, two-tailed test) was applied to calculate p values. *p < 0.05, **p ≤ 0.01.

1.3.3 Discussion

Delayed-union or non-union between the host bone and the graft are problematic in orthopedic cases of current clinical treatments of segmental bone defects. The aims of this experiment were to explore alternative approaches for improvement of healing in such surgical situations with utilization of human

77 periosteum to prevent further orthopedic interventions. A pilot study of human- periosteum allografts from the Landis Laboratory showed in earlier histological studies that human cadaveric periosteum-allograft constructs were remodeled after 20 weeks of implantation in athymic mice.89 The experiments of this dissertation now expand the implantation time from 20 weeks to 40 weeks and with periosteum tissue and bone allografts from various patients. In this novel tissue-engineering model, human periosteum was wrapped around human allograft bone and implanted subcutaneously into immune-deficient mice for 10,

20, and 40 weeks. New data demonstrated that periosteum-wrapped allografts were observed viable up to 40-week implantation time points. Further, histological and gene expression results provide evidence for the presence of both bone formation and resorption in human-periosteum constructs only.

Specimens without periosteum yielded no similar data (Group 1). After 10, 20, and 40 weeks of implantation in the mice, osteoblasts migrated from the human periosteum into the allograft bone, and these cells formed new osteoid. Data indicate active remodeling of allograft bone by periosteum in samples at all three implantation time points. Preliminary histological observations of these specimens demonstrate osteoclasts or their precursors and suggest new bone osteoid is greater in areas in 20- and 40- compared to 10-week implants.

Interestingly, bone formation was also observed to be increased in allograft implants with more trabecular structure than cortical, presumably because of ease of cell infiltration into the more porous trabecular framework (data not shown). Histological micrographs demonstrate that both new bone formation and

78 old bone resorption in human periosteum allografts were induced by human periosteum as new bone tissue was not found in any of the specimens in Group

1 regardless of implantation time. Notably, the initial bone remodeling was present at the interface of the cambium layer of periosteum and allograft bone.

RT-qPCR results comparing the levels of mRNA expression between 20-week or 40-week to 10-week counterpart samples generated relative fold-change values. Murine-specific genes (DCN, RANKL, COL1A1, and ALPL) were detected at a minimal level in human allografts (Group 1) after implantation for

10, 20, and 40 weeks. Such gene expression might be derived from cells in the murine-encapsulation tissue around or within the construct or possibly from an immune response from the host mice.92, 93 Bone formation was not detected in any of the allograft-only bone histological sections. The mRNA levels of each murine-specific gene analyzed in the human allografts (Group 1) harvested at different time points resulted in no statistically significant differences comparing

20- and 40-week specimens to 10-week counterparts. The detection of murine- specific RANK and CTSK from human allograft bone-only constructs implanted for up to 40 weeks indicates the possible presence of osteoclasts in these specimens; such cells could originate from the murine hematopoetic system and they would presumably resorb the implanted allograft bone over time.

Histologically, however, there was no apparent visible resorption or degradation of allograft-alone specimens.

For human-periosteum allografts (Group 2), gene expression is markedly higher for human-specific OC, RANKL, RANK, and SOST for 20-week

79 periosteum samples compared to 10-week samples. The relatively higher fold- change values of RANKL, OC, OSX and SOST all indicate the activity of osteoblasts and presence of osteocytes (a marker of the formation of established bone matrix) during implantation. The gene expression of type I collagen

(encoded as COL1A1), alkaline phosphatase (ALPL), osteocalcin (OC), and bone sialoprotein (IBSP) demonstrating osteoblast activity necessary to build and mineralize new bone matrix shows the wrapped periosteum was osteoinductive during the implantation period of specimens. It is of note that the gene expression levels of RANKL and SOST at 20 and 40 weeks both increased in a statistically significant manner when compared to 10 weeks, a result indicating increased bone formation activity and the presence of increasing numbers of osteocytes in newly formed bone matrix and mature bone. The increase in osteocytes would presumably lead to increasing expression of RANKL and

SOST as signals for the induction of osteoclasts and osteoblasts. This result would be consistent with the major function of osteocytes in regulating bone homeostasis.28, 29, 51 As additional support to this concept, the increased RANKL level and the presence of RANK and CTSK after longer implantation periods also illustrate the activation of osteoclasts in various aspects of bone resorption.

Further, the gene expression results here have demonstrated that cellular activity and bone remodeling (bone formation and resorption) occurred in these human- periosteum allograft models, implanted for up to 40 weeks, and these data correlate with the histological results. All these results confirm conclusions from

80 pilot experiments that the periosteum is viable, healthy and productive after 40 weeks of implantation.89

Human periosteum from different aged donors showed regenerative capabilities when wrapped around human allografts and implanted in athymic mice for up to 40 weeks. This and previous experiments demonstrate the positive influence of periosteum on inducing bone remodeling (new bone formation and bone resorption) when applied to a human allograft. When specimens were implanted for longer time periods, remodeling of the allografts appeared to increase because osteoblast- and osteoclast-related genes were expressed at higher levels when compared to 10-week samples. Some comparisons were statistically significantly different.

The hypothesis of this thesis study is that a construct tissue-engineered with human periosteum, wrapped about a human bone allograft will be suitable for healing segmental bone defects. From the summary of information here, the experiments detailed above now indicate possible application of tissue- engineered periosteum-allografts in healing of segmental bone defects.

Periosteum is viable and periosteum-allograft constructs develop with new bone formation after implantation in vivo. The tissue-engineering strategy described in this chapter provides a potential means of improving delayed-union or non-union at the healing sites of segmental bone defects or bone fractures. The potential of periosteum and its resident cells could be utilized in current novel tissue- engineering methods and innovative tissue regenerative medicine.

Future work relative to this project includes numerous approaches. These might involve additional implants, microcomputed tomographic (microCT) analysis of 3D-fabricated or printed human periosteum allograft models to examine bone mineral density in specimens implanted for 10, 20, and 40 weeks, immunohistochemical staining (to identify the presence of several other human- specific proteins), and biomechanical testing of the constructs.

CHAPTER

II. A HUMAN TENDON-TO-BONE ENTHESIS MODEL

2.1 Introduction

This content contains introduction to enthesis and tissue-engineered strategies utilized in current clinical settings.

2.1.1 Basic Concepts of an Enthesis

The loss of a tendon-to-bone insertion site and overuse injuries of that site are common and difficult problems in the field of orthopedic injuries of the shoulder, hand, elbow, knee, heel and foot. Necessary surgical interventions such as reconstruction or grafting solutions are currently applied to treat the injuries.

Tears of flexor tendons of the hand, for instance, require surgical attachment of a tendon to its bony insertions. In other words, the surgeons anticipate natural attachments to form at the interface of tendon-to-bone entheses. The developments may be beneficial to the patient but uncertain since possible complications may occur, including recurrent pain, peri-tendinous adhesion, persistent tendon lacerations and malfunction of fingers because of limited mobility.94 In addition, removal of injured body parts may be necessary as a result of traumatic injuries and the procedure may likely cause emotional upset of the patient, dysfunction of the flexor tendon, or joint contracture.94, 95 In these

83 cases, the outcomes of such surgical procedures do not meet the patient expectations. The problematic results have led to research studies of the insertion site where tendon meets bone.

Figure 2.1. Schematics of the (A) tendon and (B) ligament attachment to bone across a functional gradation region consisting in order of unmineralized fibrocartilage, mineralized fibrocartilage, and bone. Images show tendon or ligament tissues stained with toluidine blue (A) and hematoxylin and eosin (B), respectively. The figure has been reproduced with permission from Annual

Reviews (Annual Review of Biomedical Engineering).96

The musculoskeletal system connects the body through multiple types of connective tissues, including tendon, bone and ligament. Tendon and ligament are both fibrous connective tissues. The former attaches muscle to bone as it serves to enable the movement of bones, and the latter connects bone to bone as it stabilizes the bone structures by holding them together. The attachment of tendon or ligament to bone is called a tendon-to-bone insertion site or “enthesis.”

A tendon-to-bone insertion site has four-zones that gradually transit from tendon 84 to unmineralized fibrocartilage, mineralized fibrocartilage, and to mineralized bone tissue (Figure 2.1).96-98 Ligament or tendon attaches to bone either by fibrous tissue or by fibrocartilage.99, 100 As a matter of fact, tendon dissipates only 7% of work to heat applied to stretch it, and the entheses act to decrease stress concentration between tendon and skeletal tissue in order to avoid the intensified local tension at the interface of bony and tendon tissue.101, 102

As a complex interface, the enthesis plays as a critical bridge between soft tissue and bone, displays a transitional trend of cell phenotypes, exhibits gradations of biomechanical properties, reduces stress concentrations, and assists in process of mobility.103 The gradations also possess an important role in mechanically regulating functions of the tendon-to-bone insertion sites as the enthesis would prevent possible forceful stress from damaging the interface.97, 104

With surgical or natural repair processes, the healing of such regions will result in fibrovascular tissue formation at the repair site.105-108 The following pages will discuss several developing solutions for fabricating constructs with specific biomechanical properties and healing of a tendon-to-bone enthesis.

2.1.1.a The Flexor Tendon and Rotator Cuff

As has been described in the previous paragraph, the tendon connects muscle to bone in the way of enabling movement of bone when muscle contraction occurs. The flexor tendons slide through tight tunnels (“tendon sheaths”), which stabilize tendons and help them function effectively. The ability to flex the finger, for example, consists of a series of flexor muscles in the

85 forearm and their tendons are inserted into the bones of finger. The injury of a flexor tendon might cause the loss of bending of the fingers or thumb. In this case, the flexor digitorum profundus tendon (FDP) attaching to the distal phalanx and the flexor digitorum superficialis tendon binding to the middle phalanx demonstrate the specific type of tendon-to-bone insertion site characterized by the four-zone enthesis.94 The retinacula (sheath) structures serve as strong fibrous bands by wrapping around the flexor tendons in order to maintain the flexor tendons in position during flexion.

Based on the way flexor tendons are attached to bone, a minor injury to the flexor tendon might be more complex and result in the loss of bending of the fingers or thumb. Such injuries to flexor tendons remain the most difficult problem among all hand surgeries as surgeons expect optimal outcomes for their patients.109 Currently, surgeons repair these types of insults by approximating the ends of the cut tendon with special designated stiches. However, the surgery might lead to hand/finger malfunction with restricted motion since the tendon-to- bone insertion site does not regenerate at all well.

The rotator cuff is a group of tendons and muscles in the shoulder which connects the upper arm (humerus) to the shoulder blade (scapula).110 The rotator cuff tendons help stabilize the shoulder, allow lifting and rotation of the arms by supporting the muscles.110 A rotator cuff tear refers to an injury to the tendon that is generally caused by aging or repeated usage. The tear of a rotator cuff may result in damage or loss of the tendon-to-bone enthesis, and the insult may lead further to malfunction and instability of the rotator cuff.

Approximately 30% of people over 60 years of age suffer rotator cuff tears and accompanying pain.111, 112 Although the surgery for repairing rotator cuff tears is common for orthopedic surgeons, it may require replacement of tendons or joints and the patients experience less than optimal results since the quality of the resulting tissue differs greatly from the normal tissue.113

2.1.1.b Surgical and Mechanical Challenges in Healing of a Tendon-to-Bone

Enthesis

The tendon-to-bone enthesis possesses a graded connective tissue transition that includes four distinct zones categorized by their structure. Microscopically, each distinct zone transitions from tendon to bone in composition and structure.

To begin with, type I collagen and tenocytes are highly aligned in tendon.

Secondly, uncalcified fibrocartilage has type II collagen of marked content along with type III collagen and a small amount of type X collagen, decorin, and aggrecan. Similarly, with a large amount of type II collagen, the mineralized cartilage consists of significant type X collagen but scarce levels of aggrecan.

The type I collagen fibers are highly aligned in the direction of tensile force in tendon but type II fibers are less oriented in the insertion site (Figure 2.2).97, 114

Additionally, the insertion site possesses a transitional decrease in tissue organization with an increase in mineral content.97 The complexity of collagen and mineralization in this region makes the augmentation and repair of the tendon-to-bone insertion site more difficult.

The functional insertion site gradations, known to be very poorly regenerating during healing of the four-zone enthesis, have been a challenge to orthopedic surgeons in the restoration of tendon following flexor tendon injuries.109 This repair includes the achievement of mechanical stabilization of tendon-to-bone during healing, the accomplishment of a natural gradation structure from soft to hard tissue while healing, and the realization of a developed transitional area with a population of graded multi-cell phenotypes (tenocytes, chondrocytes, and bone-related cells) maintained along with the transitional mechanical structure.115

Figure 2.2. A schematic of the cross-section of a rat supraspinatus tendon-to- bone enthesis. Blue shading indicates the concentration of mineralization in the gradation of tissue. As is shown in the enlargements, the collagen fibers

88 possess greater orientation in tendon than in bone. This figure has been reproduced with permission from Informa Healthcare.115

2.1.1.c Type II Collagen and Scleraxis

Fibrocartilage differentiation from tenocytes at the enthesis includes changes in cell phenotypes. Immunohistochemistry is a process utilized to identify cell types in tissue by targeting antigens with the application of antibodies. To detect growth and development of specific tissue in the zones of tendon-to-bone enthesis models, type II collagen and transcription factor scleraxis (Scx) were selected targets for immunohistochemical (IHC) staining.

Type II collagen usually can be observed in articular and hyaline cartilage. In adult articular cartilage, type II collagen represents more than 90% of the collagen.116 In a tendon-to-bone enthesis, both uncalcified and calcified fibrocartilage zones express type II collagen.103, 117 The scleraxis protein is a member of the basic helix-loop-helix superfamily of transcription factors, and it is a transcription factor necessary for tenogenesis. Scleraxis can be detected in progenitor cells and cells from tendinous connective tissue and is responsible for coordination of the differentiation of such cells.118-120 The early expression of scleraxis from the progenitor cells contributes to possible tendon development and connective tissue attachment (such as muscle-tendon attachment).118

Scleraxis is a requirement for developing a functional tendon-to-bone enthesis.121

Kilian et al. have conducted experiments to determine how scleraxis affects the maturation of the tendon-to-bone insertion (an enthesis) in mice.121 The studies

89 involved conditionally deleted Scx in some mice compared to normal mouse controls.121 Results obtained over postnatal days 7-56 of the animal models showed that deletion of Scx directly caused defective entheses and led to impaired and poorly functioned tissue mechanical properties.121

2.1.1.d Muscle Loading in Tendon-to-Bone Enthesis Development

Physical conditions affect the growth and homeostasis of the musculoskeletal system. Mechanical loading/force, one of many physical factors, is essential for skeletal growth and maturation.122, 123 To generate a proper insertion site, mechanical loading in the development of a tendon-to-bone enthesis contributes to fibrocartilage generation, mineral aggregation, and the arrangement of collagen fibers.124 Elimination of muscle forces will result in bone and joint malformation and structural and functional impairment of the enthesis as well.

Experiments have been conducted to estimate the role of mechanical loading, to be more specific, muscle loading, in the healing of a tendon-to-bone enthesis in a canine model.123, 125 Damaged loaded and unloaded flexor tendon-to-bone entheses models in the healing process were tested in various aspects to determine bone mineral density and functional enthesis mechanical properties.125

Removal of muscle loading in the repair process led to an enthesis with a narrower range of motion as well as decreased biomechanical properties (higher stiffness in the loaded canine group).125 The authors also reported a possible balance of the mechanical loading in the skeletal system to provide proper load to avoid overload tensile force.125

Schwartz et al. have investigated the influence of muscle loading on the development in mice of a functional tendon-to-bone enthesis at the microscale.123

The collagen fiber alignment and mineral characteristics of both paralyzed rotator cuffs and functionally normal supraspinatus tendon entheses were examined.123

A significant reduction of collagen fiber alignment was detected in the toxin- induced paralyzed mouse enthesis model examined and compared to its counterpart functional enthesis model, a result indicating a decreasing quality of the tissue in the affected mice.123 A related experiment conducted by

Thomopoulos et al. revealed that reduction of muscle loading in mice postponed development of tendon-to-bone insertion sites postnatally.126 Examination of the supraspinatus enthesis during postnatal development of animals with paralyzed and functional entheses showed decreased muscle volume, delayed fibrocartilage formation, and less mineralization in the affected tissues.126 These results directly implied that muscle loading plays a crucial role in early development of the enthesis.126

2.1.2 Tissue-engineering Strategies

The quality of a tissue-engineered tendon-to-bone strategy is focused on four aspects of reconstruction, the ability to include a gradation in cell phenotypes, regeneration of natural-like tissue biomechanical properties, the capability of reducing stress, and facilitating movement. Numerous of experiments and tests from many laboratories have been performed in order to approach this strategy to regenerate the four-zone enthesis region. Several current tissue-engineering

91 experiments demonstrating models that possess the insertion site gradations of an enthesis are described in the following paragraphs from three aspects, applications of mesenchymal stem cells, biological factors and mechanical loading.

2.1.2.a Application of Mesenchymal Stem Cells

A common sense approach to enthesis reconstruction is based on the fact that seeding multiple cell types onto specific tissue composites may be useful although challenging for clinical application. Mesenchymal stem cells (MSCs) have been investigated in this regard since they can be derived from several types of tissues and then induced to differentiate into the target cell phenotypes.

Nourissat et al. conducted a study of MSC treatment and chondrocyte injection in a damaged enthesis of a rat model.127 The Achilles’ tendon along with the enthesis of the rat model was destroyed and chondrocytes and MSCs were then injected into separate individual animals. After 15, 30 and 45 days, the formation of a new enthesis at the healing region was determined macroscopically through IHC staining of type II collagen, glycosaminoglycan

(GAG) development and growth of columnar chondrocytes. Compared to the control group that had no injections, only the chondrocyte and MSC groups had produced a new entheses with stronger biomechanical properties. What was most interesting was that an aligned enthesis with columnar chondrocytes was only observed after treatment with MSCs. Gulotta et al. hypothesized that the utilization of bone marrow-derived MSCs would improve the biological

92 environment around the repair to promote regeneration of the natural insertion site and to prevent the formation of scar tissue.128 In this experiment, MSCs were used at the healing rotator cuff insertion site of Lewis rats. However, compared to the animal models that were not treated with MSCs, no difference was detected in the improvement of the structure or the strength of the healing tendon attachment site.128 The authors then followed with experiments with scleraxis (Scx)-modified bone marrow-derived cells.129 As noted above, Scx is a fundamental transcription factor for guiding tendon development in embryogenesis,129 so these studies were designed to demonstrate that the application of Scx-transduced MSCs would improve healing of a tendon-to-bone enthesis accompanied by stronger attachment compared to MSCs alone. The work showed that genetically modified MSCs can regenerate a tendon-to-bone enthesis with improved biomechanical results.129

2.1.2.b Utilization of Biological Factors

Another potential enthesis repair pathway follows from the fact that changes in cell phenotype result in changes in tissue composition and structure through the secretion of biological factors. In this case, growth factors might play a crucial role in regulating cell phenotype and gradation of soft-to-hard tissue as the growth factor concentration gradients in the polymeric scaffold may help differentiation of cells along the transitional region.130 Wang et al. presented a possible solution to the difficulties of culturing several types of enthesis cells by applying controlled delivery of growth factors, bone morphogenetic protein 2

(BMP-2) and insulin-like growth factor I (IGF-I), over modified gradients combined with specific scaffolds.130 On the surface of the silk constructs investigated and incorporating BMP-2 and IGF-I, human MSCs were cultured in osteogenic and chondrogenic media. A trend of a graded increase in calcium, glycosaminoglycan deposition, and types I, II, and X collagen gene transcription was obtained with augmenting BMP-2 content and reduced IGF-I content. The resulting gradients had similar trends to the unmineralized-to-mineralized fibrocartilage in normal enthesis insertion sites, a result indicating the application of biodegradable materials such as PLGA and silk had potential to control cell differentiation with additional effects of different biological factors, in this case, in healing of tendon-to-bone insertion sites.130

2.1.2.c Usage of Mechanical Loading

Besides cell phenotypes and biological factors, which play important roles in the development of an enthesis transitional area, biomechanical driving forces are also a critical consideration in cell differentiation. Biomechanical forces, electromagnetic fields, and ultrasound have all been shown to influence the regulation and the differentiation of MSCs.131-133

As noted above, mechanical loading is of great importance in regulating the graded transition in cell morphology of an enthesis, its mineral content, and its tissue biomechanical properties.134-136 Chen et al., for example, demonstrated the effects of repeated mechanical stretching on the mRNA expression of typical osteoblast marker genes and tendon-related genes in human MSCs seeded onto

6-well plates pre-coated with type I collagen.137 Here, the mRNA expression of types I and III collagen and tenascin-C significantly increased in MSCs when the cells underwent 10% stretching for 48 hours.137 The effects from stretching still existed after these MSCs had rested for 48 hours.137 This study is one of several supporting the application of mechanical factors to induce the formation of a potential graded tendon-to-bone attachment site with addition of MSCs. Other methodology such as that utilizing electrospun PLGA nanofibers as a substrate and applying simulated body fluid to generate a mineralization gradient is also a possible alternative solution to tissue engineering strategy for reconstructing an enthesis.138

Tatara et al. hypothesized that unloading muscle would restrain bone development and improve bone resorption at an enthesis and that the removal of loads leading to malformed bones could be changed by modulating osteoclast activity.139 With comparison of muscle-loaded and unloaded mice, the latter resulted in a delay of endochondral ossification at entheses as well as the presence of several defects in bone volume and cancellous bone structure.139

This localized paralysis model resulted in joint level deformities and mineralization defects in the animal. In addition, significant decreases in tissue biomechanical properties occurred with changes in composition and structure of the affected entheses. The study demonstrated that the bone remodeling at developing entheses requires muscle forces.139 In a related experiment,

Schwarz et al. performed a study to monitor the growth of the murine supraspinatus tendon insertion site starting from postnatal day 1 to day 28. The

95 distribution of mineral of the murine supraspinatus enthesis development at the micrometer-scale was obtained using X-ray micro-computed tomography (µCT) and Raman microprobe spectroscopy in the absence of muscle forces.140, 141 On postnatal day 7, early mineral gradients were detected at the tendon-to-bone interface. Further investigation of mineral morphology at the nanoscale with transmission electron microscopy (TEM) showed that the shape of mineral particles at the enthesis interface varied throughout enthesis development.

Additional results revealed that, with an increasing bone mineral density

(evaluated by µCT), the mineral-to-collagen ratio (evaluated by Raman spectroscopy) remained stable over time. The authors implied that the features of entheses discussed above have a meaningful mechanical significance directly relevant to the development of a tendon-to-bone attachment.140, 141

2.1.2.d A Human Tendon-to-bone Enthesis Model

Recent innovations in tissue engineering are approaching the realization of creating live tissue for clinical applications. For example, previous experiments from the Landis Laboratory have shown that models of human phalanges and interphalangeal joints can be generated with cells implanted on biodegradable polymer scaffolds and developed for more than one year in the subcutaneous tissue spaces of athymic mice.88, 142-144 These models provide firm and reproducible evidence that implanted osteoblasts, chondrocytes and tenocytes can produce respective bone, cartilage and tendon in the engineered constructs.

As such, the phalanx models are considered to be clinically useful as a guide or

96 template applicable to fabrication of a tissue-engineered enthesis. In this regard, it must be demonstrated that tendons can be grown and secured to the phalanx bone in a fashion that mirrors the biologically superior four-zone enthesis existing in the normal tendon-to-bone interface (Figure 2.1-a). Experiments reported above have shown their potential applications in regenerating the tendon-to-bone insertion site but are still limited in the achievement of natural four-zone enthesis gradients. Based on the need of a tissue-engineered enthesis model consisting of transitional cell phenotypes and a structure that can sustain appropriate forces of mechanical loading, a human periosteum-allograft bone tendon-to-bone enthesis model has been designed in this laboratory. The model is shown schematically and described in the following paragraphs in this Chapter.

From the background noted above, the overall objective of this study is to develop a tissue-engineering approach by using human periosteum, allograft bone, and biodegradable polymer sheets seeded with human tenocytes and chondrocytes for generation of a functional four-zone tendon-to-bone insertion site, an “enthesis.” The concept for this work is the phalanx model mentioned above that has been well characterized and developed previously.89, 143, 144 In addition, in unpublished results from the Landis Laboratory, the phalanx model has been used to investigate the facts of mechanical forces on the growth and development of both bone and cartilage in this construct. These experiments involved in part the suturing (tethering) of the phalanx model to opposing anatomical features of athymic mice into which the constructs were implanted.

Results obtained demonstrated increased growth of tethered models compared

97 to growth of the same models implanted in other mice but left without suturing.

The clear differences in the two experimental groups serve as the basis for the tethered vs. untethered constructs of a four-zone enthesis in this Chapter.

The tendon-to-bone enthesis model was designed to mimic a normal human four-zone enthesis. By fabricating a tendon-to-bone enthesis model, a gradient region originates from soft tissue (tenocytes seeded onto a bio-polymer), unmineralized fibrocartilage (at the interface between the tenocytes seeded onto a bio-polymer and chondrocytes seeded onto the same bio-polymer), mineralized fibrocartilage (at the interface between chondrocytes seeded onto a bio-polymer and human allograft bone), and mineralized tissue (bone) is expected after an initial culture period and then longer-term implantation in athymic (nude, nu/nu) mice. The complete tissue-engineering procedure included fabrication of tendon- to-bone constructs, cultivation in vitro of the constructs for 1 to 2 weeks, implantation of the constructs in the athymic mice serving as bioreactors in vivo, harvest of constructs from the mice, immunohistological assessment of retrieved specimens, and biomechanical tensile testing of such specimens.

2.2 Materials and Methods

This section includes experimental materials and methods that applied in the thesis study.

2.2.1 Experimental Materials

Human cadaveric knees were obtained from the Gift of Hope Organ & Tissue

Donor Network (Itasca, IL, through Rush University Medical Center, Rush

University, Chicago, IL), placed into sterile containers with sterile saline within 24 hours of donor death, and shipped on ice (4 °C) to the Landis Laboratory at the

University of Akron. Five knees from 43- to 77-year-old donors otherwise free of pathology contributed to this study. Human femoral cortical strut bone allografts were obtained from the Musculoskeletal Transplant Foundation (MTF, Jessup,

PA). Three donors ranging in age from 19- to 54-year-old contributed allograft bone.

Phosphate buffered saline (PBS) was obtained from Sigma-Aldrich (St. Louis,

MO). Medium 199 (M199), Dulbecco’s minimum essential medium (DMEM),

Ham’s F12 medium (F12), and antibiotic/antimycotic (penicillin/streptomycin) were purchased from Mediatech, Inc. (Manassas, VA). Fetal bovine serum

(FBS) was purchased from HyClone/Thermo Scientific Co. (Waltham, MA).

Primocin was produced by InvivoGen (San Diego, CA). Basic fibroblast growth factor (FGF-2, 1 µg/µL) was ordered from Kaken Pharmaceutical Co., Ltd.

(Tokyo, Japan). Osteogenic protein-1 (OP-1/bone morphogenetic protein-7) was obtained from Rush University Medical Center (Chicago, IL). Type 1 and type 2 collagenase were ordered from Worthington Biochemical Co. (Lakewood, NJ).

Pronase was purchased from EMD Chemicals, Inc. (San Diego, CA). For identification of type II collagen and scleraxis by immunohistochemistry, anti-type

II collagen (II-II6B3, mouse anti-chicken MIgG1; Developmental Studies

Hybridoma Bank [DSHB], Iowa City, IA) and anti-Scx (bs-12364R, rabbit anti- mouse IgG; Bioss, Inc., Woburn, MA) were utilized.

Sterile poly(caprolactone-co-L-lactic acid) (PCL/PLLA) sheets (10 cm x 10 cm x 2 mm), nano poly(glycolic acid) (nPGA) sheets (10 cm x 10 cm x 2 mm) and

PGA sheets (10 cm x 10 cm x 0.5 mm) were obtained from the GUNZE Ltd.

(Osaka, Japan). Athymic mice were purchased from Harlan Sprague Dawley,

Inc. (Indianapolis, IN) were housed at the Northeast Ohio Medical University

(NEOMED, Rootstown, OH) and maintained in compliance with the regulations of the Institutional Animal Care and Use Committee of NEOMED.

2.2.1 Experimental Methods

This section includes experimental methods that applied in the dissertation study.

2.2.2.a Periosteum, Tenocyte and Chondrocyte Isolation and Cultivation

M199 supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.2% primocin constituted the complete feeding or medium used to culture periosteum.

DMEM/F12 (1:1) supplemented with 10% FBS, 1% penicillin/streptomycin, 0.2% primocin, and 1% FGF-2 was utilized for the separate culture of either chondrocytes or tenocytes. M199 and DMEM/F12 referred to below are the complete feeding media.

Muscle, adipose tissue, and fascia were removed utilizing surgical scissors from donor knees (Figure 2.3-A) to expose tendon and cartilage. Tendon strips of varying size, articular cartilage pieces (approximately 5 x 5 x 2 mm in length, width, and thickness, respectively) and fresh periosteal strips (approximately 1 ×

5 cm) were harvested with the use of sterile surgical scissors and scalpels.

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Femoral cortical bones were sectioned into 1 cm3 cubes with a Dremel saw under sterile conditions and stored in sterile 24-well plates for later usage (Figure

2.3-B). Periosteum was dissected into rectangular strips (approximately 1 × 5 cm, Figure 2.3-C) and placed in petri dishes containing M199.

Tendon strips were minced in PBS in a petri dish using sterile surgical scissors and sterile razor blades and tissues were then transferred to 50 mL

Falcon tubes containing 40 mL serum-free DMEM/F12 media. Minced specimens were left on ice until digestion in 0.4% type 2 collagenase and 0.1% protease in serum-free DMEM/F12 media. Falcon tubes with tissue in digestion media were placed on a 37 °C shaker (Forma Scientific, Marietta, OH) for 14 hours.

Cartilage was dissected from cadaveric knee joints into pieces (approximately

5 x 5 x 2 mm in dimensions) with a disposable No. 10 surgical scalpel. Cartilage pieces were stored in 50 mL Falcon tubes with 40 mL serum-free DMEM/F12 media. As with tendon, the cartilage samples in Falcon tubes were placed on a

37 °C shaker (Forma Scientific, Marietta, OH) for 14 hours and digested with 40 mL serum-free DMEM/F12 media containing 0.5% type 2 collagenase.

FBS (5 mL) was added to Falcon tubes in order to stop the digestion of either tendon or cartilage. Supernatants of digestion solutions were removed with a pipette, filtered, and placed in new Falcon tubes with 20 mL fresh complete media. Filtered supernatants were then centrifuged and re-diluted with 2-5 mL complete DMEM/F12 media for cell counting with a hemocytometer. After

101 counting, tenocytes and chondrocytes were individually seeded at a density of 5 x 105 cells/mL into T175 culture flasks (Greiner bio-one, Monroe, NC).

Human periosteum, tenocytes, and chondrocytes were cultured at 37 °C and 5%

CO2 in an incubator. Media were changed every other day. The tenocytes and chondrocytes were cultured for one to two weeks.

Figure 2.3. A: Intact human knee from a 43-year-old donor. B: Segmental human allograft bone dissected into approximately 1 cm3 cubes with a Dremel saw and placed in separate wells of a 24-well culture plate. C: Human periosteum strips (approximately 1 x 5 cm) collected from the donor knee and placed in petri dishes. D (from left): A PGA sheet (1 x 4 cm), a PCL/PLLA sheet

(1 x 2 cm), and a PGA sheet wrapped with a PCL/PLLA sheet secured with two sutures placed in a petri dish. Scale bars = 2 cm.

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2.2.2.b Construct Model Fabrication

Models consisted of four separate aspects that are sutured together (Figure

2.4). These are an unseeded PCL/PLLA sheet, a human allograft bone segment wrapped with human periosteum, sheets of nPGA and PGA seeded with human chondrocytes, and a PCL/PLLA sheet seeded with human tenocytes. The model was intended to mimic the bone, fibrocartilage, and tendon aspects of an intact enthesis. The unseeded PCL/PLLA sheet was used for imparting a mechanical force to the model as each construct was implanted, sutured and tethered to the trapezius and gluteus muscle in a nude mouse (See below).

Human allograft bone segments were initially immersed in M199 for 24 hours prior to wrapping each of them with periosteum. Human periosteum strips were wrapped around the allograft bone with the cambium layer of the periosteum facing the allograft bone (Figure 2.4-II). At the overlap of the strips, they were sutured with Nylon 5-0 thread (Figure 2.5-A). Following the suturing, the human periosteum-allograft bone constructs were cultured in M199 in 12-well plates for one week.

PCL/PLLA sheets were cut into 1 x 2 cm rectangular sheets (Figure 2.4-I) with a sterile surgical scalpel and nPGA and PGA sheets were cut into 1 x 1 cm squares (Figure 2.4-III). Rectangular-shaped PGA sheets (1 x 4 cm) were cut to cover the PCL/PLLA sheets (Figure 2.4-IV). After one-two weeks of culture, tenocytes and chondrocytes were removed from culture flasks and 50 µL aliquots of chondrocytes at a concentration of 100 x 106 cells/mL were directly seeded onto each nPGA and PGA double sheet (Figure 2.5-B). Similarly, 150 µL

103 aliquots of tenocytes were seeded to the PGA-covered PCL/PLLA sheets at a concentration of 50 x 106 cells/mL. Subsequently, each individual PCL/PLLA sheet (Figure 2.4-I), a human periosteum-wrapped allograft bone (the bone tissue, Figure 2.4-II), a chondrocyte-seeded nPGA and PGA double layer (the cartilage tissue, Figure 2.4-III), and the tenocyte-seeded sandwich (the tendon attached to the bone, Figure 2.4-IV) were sutured together to form a tendon-to- bone enthesis model (Figure 2.4, Figure 2.5-C & D). Each aspect of the model was secured with PGA 5-0 suture (nPGA/PGA to periosteum) and Nylon 5-0 thread (the remaining sutures).

Figure 2.4. A schematic of a fabricated tendon-to-bone enthesis model. The model consists of an unseeded PCL/PLLA sheet (I), human allograft bone wrapped with human periosteum (II), nPGA and PGA sheets seeded with human chondrocytes (III), and a PCL/PLLA seeded with human tenocytes (IV). Sutures, indicated by the crossed threads, connect the various aspects of the model.

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Figure 2.5. Light photographs of experimental materials. A: Human periosteum strips wrapped about allograft bones and secured with sutures. B: Human chondrocyte-seeded PGA and nPGA double sheets immersed in complete

DMEM/F12 media in a 6-well plate. C: Intact constructs consisting of four aspects individually labeled and corresponding to the enthesis model in Figure

2.4: an unseeded PCL/PLLA sheet (I), human allograft bone wrapped with human periosteum (II), nPGA and PGA sheets seeded with human chondrocytes

(III), and a PCL/PLLA sheet seeded with human tenocytes (IV). D: An enlargement of a complete construct consisting of four aspects labeled respectively as above. Each aspect corresponds to the enthesis model shown in

Figure 2.4: an unseeded PCL/PLLA sheet (I), human allograft bone wrapped with human periosteum (II), nPGA and PGA sheets seeded with human chondrocytes (III), and a PCL/PLLA sheet seeded with human tenocytes (IV).

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Arrows in C and D indicate chondrocyte-seeded nPGA/PGA sheets. Scale bars

= 2 cm.

2.2.2.c Specimen Implantation, Harvest, Fixation and Embedding

Experiments were continued by subcutaneous implantation of the tendon-to- bone enthesis constructs into athymic (nude) mice. OP-1 (0.7 µg/µL) was pipetted directly onto the nPGA sheets prior to implantation. The experimental design is illustrated in Figure 2.6. Two incisions, each of length ~1.5 cm, were made with a sterile scalpel on the animal dorsum. The complete constructs were implanted in the subcutaneous dorsum of athymic mice with reverse action of a pair of sterile scissors opening a pocket, one construct per mouse, with both seeded (PCL/PLLA/tenocytes) and unseeded (PCL/PLLA) construct ends untethered to any muscle in animals (Group 1, Figure 2.6) or tethered proximally to the trapezius (the PCL/PLLA/tenocyte-seeded aspect) and distally to the gluteus maximus (the PCL/PLLA-unseeded aspect) muscle of the mice (Group 2,

Figure 2.6 and 2.7). 5-0 Nylon sutures were used for tethering scaffolds to the animal muscles. The incisions were closed with PGA sutures.

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Figure 2.6. The experimental group design for the enthesis study. The tendon- to-bone enthesis model has four aspects as indicated: an unseeded PCL/PLLA sheet (I), human allograft bone wrapped with human periosteum (II), nPGA and

PGA sheets seeded with human chondrocytes (III), and a PCL/PLLA sheet seeded with human tenocytes (IV). Group 1 constructs were implanted subcutaneously and without tethering (suturing) in the dorsum of nude mice.

Group 2 constructs were implanted in the same way as that of Group 1 but with both PCL/PLLA/tenocyte-seeded aspects and PCL/PLLA-unseeded aspects sutured (as indicated by black Nylon thread in the diagram) respectively to the trapezius and gluteus muscle of the nude mice. The arrows indicate implied mechanical forces applied to the tethered constructs as a mouse walks or runs about its cage.

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Figure 2.7. Unseeded aspect of a tendon-to-bone enthesis construct, indicated by an arrow, sutured to the gluteus maximus muscle of an athymic mouse. Scale bar = 2 cm.

Mice were sacrificed after 20- and 40-weeks of implantation and constructs were retrieved with use of sterile scissors and scalpels. The specimens for tensile testing were immediately transferred to phosphate buffered saline (PBS) and left on ice until further testing. The samples for histological assessment were placed in 20 mL 10% neutral buffered formalin (NBF, Electron Microscopy

Sciences, Hatfield, PA) for fixation for 1 week. After fixation, the samples were immersed in Immunocal solution (Decal Chemical Corp., Tallman, NY) for 4 weeks or until they were completely decalcified. After decalcification, the specimens were rinsed in deionized water three times followed by three changes of 70% ethanol, 15 minutes each. The samples were then placed in plastic cassettes and processed through a series of ethanol and xylene rinses in a

108 processor (Model ASP300S tissue processor, Leica Biosystems, Inc., Buffalo

Grove, IL) prior to embedding the processed samples in paraffin wax blocks.

2.2.2.d Biomechanical Testing

Uniaxial biomechanical testing was performed to evaluate biomechanical properties of tendon-to-bone enthesis models. The specimens were tested within 2 hours of sample harvest. Intact tendon-to-bone enthesis specimens were transferred from the athymic mice to 20 mL vials containing PBS (4 °C) to maintain specimen viability. Both the seeded and unseeded aspects of 20- and

40- week specimens were separately clamped to custom-made polycarbonate friction grips (mounted to a loading traveler arm by universal joints; Figure 2.8 and 2.9) and were strained to failure while the periosteum-bone allograft aspect was fixed to a platform in the testing device. Biomechanical tensile tests were performed in the research laboratory of Dr. John Elias, at the Akron General

Medical Center, and of Dr. Marnie Saunders, at the University of Akron. Their individual custom-designed testing machines were both set to loading rates of

0.5 mm/sec with 10-pound load cells. Biomechanical properties of tendon-to- bone enthesis models were evaluated in terms of load-displacement curves of each individual load-to-failure test.

The testing machine in the Elias Laboratory was facilitated with an MTS

FlexTest 40 controller along with MaxTRAQ (Innovision Systems, Inc., MI), a specimen marker system. Each sample was labeled by black Indian ink. In order to hold each specimen firmly, custom grips for small-sized samples were

109 utilized (Figure 2.8-A) and an adjustable wrench was applied to fix the periosteum-bone aspect to a platform. A 5-mm-gauge length (Figure 2.8-B) was prepared with markers labelled on the enthesis models for cameras to track the motion of the markers as each specimen was strained during testing.

Figure 2.8. Tensile testing as set up in the Akron General Medical Center. A:

The unseeded aspect of a marker-labelled tendon-to-bone construct was secured to custom grips to hold the specimen. An arrow indicates one of the markers labelled on the construct. B: The grips were fixed on the traveler of the testing machine. An arrow indicates a 5-mm-gauge length. Scale bars = 1 cm.

Tensile testing in the Saunders Laboratory utilized a small-scale loading machine designed for small-sized samples145. Both seeded and unseeded aspects of the enthesis constructs were tested separately by connecting them to

110 custom-made polycarbonate friction clamp grips. The periosteum-bone allograft of a tendon-to-bone specimen was clamped and fixed to adjustable rough surface brass blocks (Figure 2.9) mounted to a platform of the testing device.

Tests were performed at the interface of the enthesis model with gauge length less than 1 mm to measure the biomechanical properties of the specimens.

Figure 2.9. The seeded aspect of a tendon-to-bone enthesis sample clamped by custom grips mounted to the arm of the traveler by a universal joint in the testing machine. The periosteum-bone allograft of the sample was secured by clamping it to two brass blocks with rough surfaces to hold the bone aspect rigidly. Scale bar = 2 cm.

After tensile testing, specimens including their ruptured tendon aspects were immediately transferred to 10% NBF followed by decalcification, processing, and embedding described previously (2.2.2.c).

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2.2.2.e Histological Staining

Immunohistochemical and histological analyses were also performed on sections of tendon-to-bone enthesis constructs retrieved after implantation.

Typically, paraffin-embedded sample blocks were sectioned with a C-profile stainless steel knife using a microtome (model RM2255, Leica Biosystems, Inc.).

Sections 5-7 µm thick were floated in a 42 °C water bath to remove wrinkles before they were mounted on glass slides (Superfrost Excell, Thermo Scientific,

Waltham, MA). The slides were left on a 42 °C hot plate for a minimum of 2 hours and were then stored in slides boxes prior to staining. For histological examination, slides were stained with hematoxylin and eosin (H&E) for observation of basic tissue and cellular morphology and picrosirus red for formation of collagen fibers.84, 146, 147

2.2.2.f Immunohistochemistry of Type II Collagen and Scx

For immunohistochemistry, the slides from the two experimental Groups 1-2 were stained with the following antibodies: anti-type II collagen (DSHB) and anti-

Scx (Bioss, Inc.). These two antibodies were chosen to identify and detect fibrocartilage formation and tendon formation, respectively. Prior to usage, these antibodies were diluted with antibody diluent buffer (BondTM Primary Antibody

Diluent, Leica Biosystems, Inc.) to working concentrations. The validity of interpretations of the staining was based on the comparison between positive

112 and negative controls. Negative controls were tendon-to-bone sections that had no primary antibody applied.

For immunohistochemical (IHC) staining, tendon-to-bone sections on slides were placed flat and facing up on a slide tray in a 60 °C oven to melt paraffin.

After a minimum of 4 hours in the oven, the slides were removed to a glass slide holder for an additional hour in the oven. The slides then went through three fresh changes of xylene (10 minutes each, E K Industries, King of Prussia, PA) to deparaffinize wax. The slides were rehydrated with two changes of 100% ethanol (2 x 10 minutes each) and two changes of 95% ethanol (2 x 5 minutes each). Each slide was placed on a benchtop to dry. A heat gun (5 amp,

93 °C/149 °C, Master Appliance Corp, Racine, WI) was applied to heat the back of the slides to ensure complete removal of water. The specimens were placed in a humidified plastic chamber to avoid dehydration followed by two changes of deionized water. Antigen retrieval was conducted by incubating the slides in a

37 °C oven for 10 minutes with 0.1 % pronase (Calbiochem, San Diego, CA), diluted in PBS. Samples were then washed with two changes of PBS (2 minutes each) to rinse off residual pronase. Background Buster (INNOVEX, Richmond,

CA) was added to each section as the protein blocking buffer (30 minutes).

Slides were incubated for 1 hour at room temperature with primary antibody against type II collagen (dilution 1:10, DSHB). Negative controls were covered with Tween 20/tris-buffered saline (TTBS) to keep the sections hydrated.

Following three changes of TTBS washes (5 minutes each), secondary antibody

(ImmPRESS, Vector Laboratories, Burlingame, CA) was added to the slides for

113

30 minutes. Endogenous peroxidases were blocked with 0.3% hydrogen peroxide in methanol for 5 minutes. The slides were then rinsed with three changes of TTBS (5 minutes each) to remove peroxidase blocking buffer and secondary antibody residue. To visualize type II collagen presence, 3, 3’- diaminobenzidine (DAB, 1 tablet + 7 mL PBS + 3-4 drops of 0.3% H2O2, MaxTag

DAB tablets, Rockland, Limerick, PA) solution was applied to the sections.

Following three changes of TTBS washes (5 minutes each), the slides were counterstained in Mayer’s hematoxylin (Electron Microscopy Sciences, Hatfield,

PA) for 1 minute, dehydrated through 70%, 95%, 100% ethanol and three changes of xylene, mounted with DPX (Electron Microscopy Sciences), and coverslipped for observation under an optical (bright-field) microscope.

IHC staining for scleraxis followed the exact same deparaffinization and rehydration procedure of IHC staining for type II collagen. After rehydration in deionized water (2 x 5 minutes), the slides were moved to a Coplin jar and fixed in 10% NBF (VWR, Radnor, PA) overnight at 4 °C. The following day, the NBF was changed to Immunocal (2 x 1 hour; Decal, Tallman, NY). The slides were rinsed with two changes of deionized water (5 minutes each) followed by addition of peroxide blocking in 0.3% H2O2 in methanol (30 minutes). Antigen retrieval was accomplished by incubating samples in sodium citrate/EDTA/Tween 20 (pH

6.0) at 60 °C (72 hours).

Following antigen retrieval, slides were moved to a humidified plastic chamber and washed with two changes of PBS (5 minutes each). Non-specific proteins on the sections were blocked with addition of Background Buster (30 minutes;

114

INNOVEX, Richmond, CA). Anti-Scx (dilution 1:100, Bioss Inc.) was applied to target samples overnight at 4 °C. Negative controls were covered with TTBS to keep the sections hydrated. The rest of the procedure (visualization of the staining, counterstaining with hematoxylin, and dehydration) followed precisely the protocol as described above for type II collagen IHC staining.

2.3 Results and Discussion

The constructs were carefully harvested from the dorsum of athymic mice with surgical instruments. Any tissue encapsulating the constructs was removed as completely as possible utilizing a pair of surgical scissors (Figure 2.10).

Figure 2.10. A 40-week tendon-to-bone untethered construct (Group 1) implanted subcutaneously in an athymic mouse. The insert shows the construct immediately following harvest from the mouse and prior to fixation in 10% NBF.

Scale bars = 1 cm.

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2.3.1 Tensile Testing Results

Biomechanical properties of the tendon-to-bone enthesis models were analyzed by load-displacement curves obtained from tensile testing using the customized instruments from both the Elias and Saunders Laboratories. As was noted in Materials and Methods, the tensile testing instruments from both laboratories were designed with different parameters but both obtained load-to- failure measurements for tested specimens. The unseeded and seeded aspects of an individual tendon-to-bone enthesis model were tested for comparison within the same Group or between Groups after specimen implantation for different time periods.

Tensile testing data from the Elias Laboratory were collected from 20- and 40- week untethered seeded and unseeded aspects of enthesis models. It is notable that the tests were performed with a 5 mm gauge length at the enthesis interface to allow the labeling of a marker for a camera to track the specimen motion during loading. The average maximum loads of 20-week samples (N = 2 for each untethered unseeded and seeded aspects) were 4.89 ± 0.06 N and 8.00 ±

1.05 N, respectively (Figure 2.11), while the average maximum loads of 40-week specimens (N = 3 for each unseeded and seeded aspects) were 2.35 ± 0.52 N and 2.76 ± 1.12 N, respectively (Figure 2.11). The data were translated differently by comparing 20-week seeded with unseeded aspects and 40-week seeded with unseeded aspects (Figure 2.11), and by comparing 20-week seeded aspects to 40-week seeded aspects and 20-week unseeded aspects to 40-week unseeded aspects (Figure 2.12).

116

An unpaired student’s t-test indicated that there was no statistical significant difference in maximum load between untethered 40-week unseeded and seeded aspects of models (p = 0.70, Figure 2.11). Statistically significant differences were found in maximum load values between untethered 20- and 40-week unseeded aspects (p = 0.02, Figure 2.11), maximum load values between untethered 20- and 40-week seeded aspects (p = 0.03, Figure 2.11), and maximum load values between untethered 20-week unseeded and seeded samples (p = 0.05, Figure 2.12). The statistical results implied possible polymer degradation with longer implantation time as the values of maximum load for untethered 40-week specimens (both unseeded and seeded aspects) were significantly less than those for the 20-week specimens (Figure 2.11).

Maximum Load of 20- and 40-week Unseeded and Seeded Aspects of Untethered Enthesis Models

Unseeded Seeded N = 2, 20 wks, NT N = 3, 40 wks, NT 10.00 9.00 * 8.00 7.00 6.00 5.00 *

4.00 Load Load (N) 3.00 2.00 1.00 0.00 20 week NT 40 week NT

Figure 2.11. Maximum load of 20- and 40-week seeded and unseeded aspects of untethered (NT) enthesis models from the Elias Laboratory. Values are

117 means ± standard error of the mean. The asterisk denotes a statistically significant difference between the two untethered 20-week unseeded sides when compared to their seeded counterpart halves. A student’s t-test (parametric, unpaired, two-tailed test) was applied to identify p values. *p < 0.05.

Maximum Load of 20- and 40-week Unseeded and Seeded Aspects of Untethered Enthesis Models N = 2, 20 wks, NT 20 week NT 40 week NT N = 3, 40 wks, NT 9.00 8.00 7.00 6.00 * 5.00 4.00 Load Load (N) 3.00 2.00 1.00 0.00 Unseeded Seeded

Figure 2.12. Maximum load of 20- and 40-week seeded and unseeded aspects of untethered (NT) enthesis models from the Elias Laboratory. Values are means ± standard error of the mean. Asterisks denote a statistically significant difference between untethered 20- and 40-week seeded aspects and 20- and 40- week unseeded counterparts. A student’s t-test (parametric, unpaired, two-tailed test) was applied to identify p values. * p ≤ 0.05.

118

The stiffness of the samples was derived from the slope of the load displacement curve of each examined specimen. The average stiffness of the

20-week untethered unseeded (N = 2) and seeded (N = 2) aspects was 1.39 ±

0.05 N/mm and 3.81 ± 2.52 N/mm, respectively, while the average stiffness of

40-week specimens (N = 3, both unseeded and seeded aspects) was 0.76 ± 0.37

N/mm and 1.38 ± 0.19 N/mm, respectively (Figure 2.13).

An unpaired student’s t-test indicated that there was no statistically significant difference in stiffness between untethered 20-week unseeded and seeded aspects of models (p = 0.31, Figure 2.13) nor between untethered 40-week unseeded and seeded aspects of models (p = 0.14, Figure 2.13).

Stiffness of 20- and 40-week Unseeded and Seeded Aspects of Untethered Enthesis Models Unseeded Seeded N = 2, 20 wks N = 3, 40 wks 7.00 6.00 5.00 4.00 3.00

2.00 Stiffnes (N/mm) Stiffnes 1.00 0.00 20 week NT 40 week NT

Figure 2.13. Stiffness of 20- and 40-week seeded and unseeded aspects of untethered enthesis models from the Elias Laboratory. The unseeded and seeded aspects from specimens implanted for 20 weeks displayed relative

119 greater stiffness compared to 40-week samples. Values are means ± standard error of the mean.

Tensile testing data from the Saunders Laboratory were collected from 20- and 40-week untethered and tethered seeded and unseeded aspects of enthesis models. Samples were tested at the interface where tendon meets bone to obtain the biomechanical properties of the possible enthesis formation. The stiffness of the samples was obtained by calculating the slope of the load- displacement curves of all samples. A typical displacement-over-load plot of a

20-week tethered tendon-to-bone enthesis model (Figure 2.14) showed that the cell-seeded portion of one representative enthesis model had a greater value of maximum load as well as stiffness (steeper slope) compared to the unseeded aspect of the model.

Load-Displacement Graph for a 20-week Tethered Tendon- to-bone Enthesis Model (Unseeded and Seeded Aspects) 16 Seeded 14 Unseeded 12 10 8

Load Load (N) 6 4 2 0 0 0.5 1 1.5 2 2.5 3 3.5 Displacement (mm)

120

Figure 2.14. Tensile testing curves of the unseeded and seeded aspects of a representative 20-week tendon-to-bone enthesis model examined from the

Saunders Laboratory. The cell-seeded portion showed a greater value of maximum load as well as stiffness (slope) compared to measurements of the unseeded aspect.

The average maximum loads of the 20-week untethered unseeded (N = 2) and seeded (N = 2) aspects were 8.91 ± 7.00 N and 27.85 ± 7.69 N, respectively

(Figure 2.15). The average maximum loads of the 20-week tethered samples were 5.69 ± 1.47 N (N = 3, unseeded) and 12.05 ± 1.24 N (N = 3, seeded)

(Figure 2.15). The maximum loads of the unseeded and seeded aspects of 20- week tethered enthesis model were statistically significantly different (p = 0.02).

Moreover, the average maximum load of the seeded areas of the 20-week untethered enthesis specimens was statistically significantly different from that of the tethered samples (p = 0.03, Figure 2.15). Figure 2.16 shows the corresponding stiffness of 20-week untethered samples was 4.52 ± 0.97 N/mm

(N = 2, unseeded) and 10.12 ± 0.16 N/mm (N = 2, seeded) and the tethered samples had stiffness of 5.19 ± 2.01 N/mm (N= 3, unseeded) and 7.72 ± 1.18

N/mm (N =3, seeded), respectively. With the application of mechanical force

(tethering unseeded and seeded aspects to the dorsum of the mice), seeded aspects of untethered tendon-to-bone enthesis models after 20 weeks of implantation exhibited relatively greater stiffness than that of the tethered specimens.

121

Maximum Load of 20-week Unseeded and Seeded Aspects of Untethered and Tethered Enthesis Models N = 2, 20 wks, NT Unseeded Seeded N = 3, 20 wks, T 40.00 * 35.00 30.00 25.00 20.00

Load (N) 15.00 10.00 5.00 0.00 20 week NT 20 week T

Figure 2.15. Maximum loads of 20-week seeded and unseeded aspects of nontethered (NT) and tethered (T) enthesis models from the Saunders

Laboratory. Values are means ± standard error of the mean. The unseeded and seeded aspects of untethered specimens had relative greater values of maximum loads compared to the loads from the tethered specimens.

As for the 40-week samples, the average maximum loads of the untethered unseeded (N = 2) and seeded (N = 2) aspects were 2.20 ± 0.57 N and 12.95 ±

11.38 N, respectively (Figure 2.17), while the average maximum loads of 40- week tethered specimens (N = 3, both unseeded and seeded aspects) were 3.89

± 1.79 N and 6.83 ± 0.50 N, respectively (Figure 2.17). No statistically significant

122 difference was found between unseeded and seeded aspects of the untethered or tethered enthesis models after 40 weeks of implantation.

The average stiffness of the 40-week untethered unseeded (N = 2) and seeded (N = 2) aspects of the enthesis models was 1.03 ± 0.09 N/mm and 10.56

± 0.20 N/mm, respectively (Figure 2.18). The average stiffness of the 40-week tethered unseeded (N = 3) and seeded (N = 3) aspects was 2.25 ± 1.09 N/mm and 5.41 ± 4.42 N/mm, respectively (Figure 2.18). An unpaired t-test indicated that there was a statistically significant difference in stiffness between only untethered 40-week unseeded and seeded aspects of the enthesis models (p =

0.0003, Figure 2.18).

Stiffness of 20-week Unseeded and Seeded Aspects of Untethered and Tethered Enthesis Models N = 2, 20 wks, NT Unseeded Seeded N = 3, 20 wks, T 12.00

10.00

8.00

6.00

4.00 Stiffness (N/mm) Stiffness

2.00

0.00 20 week NT 20 week T

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Figure 2.16. Stiffness of seeded and unseeded aspects of 20-week untethered

(NT) and tethered (T) enthesis models from the Saunders Laboratory. Values are means ± standard error of the mean. The values of the stiffness of unseeded aspects were relatively less than those from the seeded portions of a 20-week tendon-to-bone enthesis model, regardless of application of mechanical forces.

Maximum Load of 40-week Unseeded and Seeded Aspects of Untethered and Tethered Enthesis Models Unseeded Seeded N = 2, 40 wks, NT N = 3, 40 wks, T 14.00

12.00

10.00

8.00

Load Load (N) 6.00

4.00

2.00

0.00 40 week NT 40 week T

Figure 2.17. Maximum loads of 40-week seeded and unseeded aspects of non- tethered (NT) and tethered (T) enthesis models from the Saunders Laboratory.

Values are means ± standard error of the mean.

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Stiffness of 40-week Unseeded and Seeded Aspects of Tethered and Untethered Enthesis Models Unseeded Seeded N = 2, 40 wks, NT N = 3, 40 wks, T 12.00 * 10.00

8.00

6.00

4.00 Stiffness (N/mm) Stiffness

2.00

0.00 40 week NT 40 week T

Figure 2.18. Stiffness of 40-week seeded and unseeded aspects of non-tethered

(NT) and tethered (T) enthesis models from the Saunders Laboratory. Values are means ± standard error of the mean. The asterisk denotes a statistically significant difference between untethered 40-week unseeded and seeded sides.

A student’s t-test (parametric, unpaired, two-tailed test) was applied to identify p values. * p < 0.05.

A student’s t-test indicated that the maximum loads of the seeded aspects of

20-week tethered enthesis specimens yielded statistically significantly greater values than the loads from 40-week tethered tendon-to-bone enthesis models

(Figure 2.19). In addition, the maximum loads (Figure 2.19) and stiffness (data not shown) of both unseeded and seeded aspects of 20-week tethered

125 specimens showed relatively higher average values compared to those of 40- week tethered specimens, respectively.

Maximum Load of 20- and 40-week Seeded and Unseeded Aspects of Tethered Enthesis Models N = 3, 20 wks, T Unseeded Seeded N = 3, 40 wks, T 14.00 * 12.00

10.00

8.00

6.00 Load Load (N)

4.00

2.00

0.00 20 week T 40 week T

Figure 2.19. Maximum loads of 20- and 40-week seeded and unseeded aspects of tethered (T) enthesis models from the Saunders Laboratory. Data shown here are from Figure 2.15 (20-week tethered) and Figure 2.17 (40-week tethered).

Values are means ± standard error of the mean. Maximum loads of seeded aspects of 20- week tethered tendon-to-bone enthesis samples were statistically significantly higher than those of the 40-week tethered samples. *p < 0.05.

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2.3.2 Histology Results

Tendon-to-bone insertion models retrieved from athymic mice were stained with hematoxylin and eosin (H&E) to evaluate the structural morphology of the tissue-engineered enthesis and picrosirius red to visualize collagen within the sections. Immunostaining of anti-type II collagen and anti-Scx was utilized to access the presence of chondrocytes and tenocytes in the enthesis constructs.

The composite light micrograph in Figure 2.20 shows a representative section of a 20-week tensile-tested tendon-to-bone specimen from Group 1 (untethered constructs) after subsequent harvest from an athymic mouse. The insert in

Figure 2.20 shows the gross tendon-to-bone enthesis construct after harvest from the athymic mouse and before processing. The unseeded PCL/PLLA control side was observed to be ruptured after tensile testing (Figure 2.20-a).

The light pink staining in the enlargement b of Figure 2.20 shows murine fibrous encapsulation tissue around the outer perimeter of the specimen. A possible area of torn or ripped tissue was noted in enlargement c and possibly indicates effects on the sample after the application of loading. Chondrocytes, however, were not observed in this specimen.

Figure 2.21 presents a light micrograph showing results from H&E staining of a tendon-to-bone allograft bone enthesis model following 20 weeks of implantation and harvest from an athymic mouse (Group 1). All aspects of the enthesis model, including the allograft bone, periosteum, cartilage, and tendon, were observed to be viable after this period of implantation in vivo. H&E staining results indicated infiltration of mouse or human cells into the PCL/PLLA control

127 scaffold alone. Hypertrophic cartilage formed from chondrocytes seeded onto

PGA sheets was observed. Resorbed bone tissue was also found in the area between periosteum and allograft bone. Aligned fibrous tissue was detected within the regenerated enthesis model. Murine fibrous tissue was observed wrapped around the tendon-to-bone enthesis specimen, forming a capsule.

Micrographs shown in Figure 2.22 provide details of an untethered tendon-to- bone insertion model after implantation in an athymic mouse for 40 weeks

(Group 1). Mouse encapsulation tissue covered the periphery of the complete construct over the implantation period as shown in the composite image.

Periosteum, tenocytes seeded onto PCL/PLLA sheets, and chondrocytes seeded onto nPGA sheets were observed viable (Figure 2.22). Aligned fibrous tissue formed at the interfaces of periosteum and chondrocyte-seeded nPGA, periosteum and tenocyte-seeded PCL/PLLA, and chondrocyte-seeded nPGA and tenocyte-seeded PCL/PLLA as shown in Figure 2.22 (highlighted by arrows).

Figure 2.23 shows light micrographs of a picrosirius red-stained tendon-to- bone enthesis model after 40 weeks of implantation in an athymic mouse (Group

1). Panels A and B (under polarized light) illustrate the presence of bone resorption where collagen fibers were absent at the interface between periosteum and allograft bone. Collagen bundles (bright red in Figure 2.23-B) were detected from the periosteal fibrous tissue in the specimen but new osteoid was not observed from this area. A small amount of collagen fibers was present in the tenocyte-seeded PCL/PLLA portion of the model (Figure 2.23-B).

128

P a

AB PCL/PLLA

P b c T ET

Figure 2.20. An H&E-stained histological section of a representative 20-week untethered tendon-to-bone enthesis specimen (Group 1) after tensile testing.

The insert shows the gross morphology after harvesting the construct from the athymic mouse. Allograft bone (AB), periosteum (P), the tenocyte-seeded

PCL/PLLA (T), and the unseeded PCL/PLLA scaffold are shown in the composite image. The tissues were viable. Periosteum was present around the periphery of allograft bone with periosteal tissue infiltration in bone cavities (formed by vascularization). Chondrocytes were not observed in this specimen. Three regions of interest of this specimen are shown in boxes. Region a: ruptured unseeded (PCL/PLLA) end of the model after stretching from tensile testing; b: mouse encapsulation tissue (ET) found at the tendon aspect of the model; c: a possible torn area of tissue observed at the interface between cell-seeded and unseeded aspects of this specimen. Scale bars = 1 cm (insert), 2 mm

(composite image).

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P ET

Resorbed T Bone C AB

Figure 2.21. An H&E-stained histological section from a representative 20-week untethered tendon-to-bone enthesis model from Group 1. Allograft bone (AB), periosteum (P), chondrocyte-seeded nPGA (cartilage, C), and tenocyte-seeded

PCL/PLLA (tendon, T) are shown. Mouse encapsulation tissue (ET) was observed covering the periphery of the specimen. Resorbed allograft bone

(white arrows) was found at its interface with the periosteum. Hypertrophic cartilage formation was observed in the chondrocyte-seeded PGA sheet. Black arrows indicate putative formation of a graded tendon-to-bone enthesis. The space between allograft bone and periosteum toward the left bottom of the image is an artifact attributable to sectioning the construct. Scale bar = 500 µm.

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A T a T P

c AB b P b AB C a B C T T

c P AB P C AB 2 mm PCL/PLLA

Figure 2.22. An H&E-stained histological section of a representative untethered

40-week tendon-to-bone enthesis model (Group 1). Allograft bone (AB), periosteum (P), chondrocyte-seeded nPGA (cartilage, C), tenocyte-seeded

PCL/PLLA (tendon, T), and unseeded PCL/PLLA are shown in the composite image. Viable periosteal tissue was observed around the periphery of the allograft bone. The empty space in the middle of the allograft bone is the medullary cavity of this specific allograft bone (fibula midshaft). Three enclosed regions (a, b, and c) are shown as enlargements in the insets. Enlargement a: aligned fibrous tissue (arrows) formed between the periosteum (P) and the tenocyte-seeded PCL/PLLA (T); b: fibrous tissue (arrowheads) formed between chondrocyte-seeded nPGA (C) and tenocyte-seeded PCL/PLLA (T); c:

131 periosteum (P) infiltration (arrows) in allograft bone (AB) with fibrous tissue

(arrowheads) forming between the periosteum and the chondrocyte-seeded nPGA (C). Scale bars = 2 mm (panel A) and 200 µm (panels a, b, c).

Figure 2.23. Light micrographs of a picrosirius red-stained 40-week untethered tendon-to-bone enthesis model specimen from Group 1. A: picrosirius red staining shows resorbed bone (RB) at the interface between the periosteum (P) and allograft bone (AB). Tenocyte-seeded PCL/PLLA (T) of the enthesis model is also apparent. New osteoid was not observed in this area. B: picrosirius red- stained section of the same area shown in A observed under polarized light. The resorbed bone (RB) tissue appears without any marked staining of collagen 132 fibers between the allograft bone (AB) and the periosteum (P); small amounts of stained collagen fibers were present in the tenocyte-seeded PCL/PLLA aspect

(T). The periosteum demonstrates regions of aligned stained collagen. Scale bars = 200 μm.

H&E staining of a representative section of a tissue-engineered tethered enthesis model (Group 2) implanted in an athymic mouse for 20 weeks is shown in Figure 2.24. Periosteum-allograft, chondrocytes and tenocytes seeded onto biodegradable scaffolds were observed viable in the specimen after tethered mechanical loading in the mouse. Cells from the fibrous layer of the periosteum appeared to have proliferated or migrated into cartilage areas to form an interesting connective tissue zone with numerous chondrocytes adjacent to the allograft bone (Figure 2.24). Aligned fibrous tissue formed at the interface between periosteum and cartilage (Figure 2.24-a) and tendon and cartilage

(Figure 2.24-b), a result indicating possible formation of a transition region from fibrocartilage to bone of a four-zone enthesis.

Specimens from tethered Group 2 implanted for 40 weeks showed viable periosteum and tenocytes that were initially seeded onto PCL/PLLA scaffolds.

As an example, the unseeded control aspect (PCL/PLLA only) of one such construct was infiltrated with mouse and possibly some human cells (from the periosteal wrap) and exhibited a relatively thinner width when compared to the tenocyte-seeded aspect (Figure 2.25). Periosteum was quite thick but cartilage was not observed in the composite micrograph of this section. Enlargements

133 from another tethered 40-week insertion model (Group 2) showed viable tenocytes seeded onto the PCL/PLLA sheet and possible new bone formation in the periosteum-wrapped allograft bone (Figure 2.26-A, B, respectively). Bone formation derived from osteoblasts in the periosteum was observed in all specimens implanted for 20 and 40 weeks (Groups 1 and 2).

Figure 2.24. Light micrographs of an H&E-stained section and enlargements of a representative enthesis specimen after application of tethered mechanical forces for 20 weeks in vivo (Group 2). A: Periosteum (P) appears intact around

134 allograft bone (AB) and viable with the presence of positive-stained cells.

Cartilage (chondrocyte-seeded nPGA, C) was observed between red-appearing periosteal fibrous tissue (FB) and tenocyte-seeded PCL/PLLA (T). Two regions

(a and b) are shown enlarged in the insets. Enlargement a: aligned fibrocartilage (arrows) formed between the chondrocyte-seeded nPGA (cartilage,

C) and periosteum (P) wrapped allograft bone (AB); b: fibrocartilage (arrows) was also visible between the cartilage (chondrocytes in lacunae, chondrocyte- seeded nPGA, C) and tenocyte-seeded PCL/PLLA scaffold (T). The round- shaped empty space in both enlargements shows the initial suture resorption site in the construct. Scale bars = 500 µm (panel A), 200 µm (panels a, b).

Figure 2.25. A light micrograph of a tensile-tested tethered 40-week tendon-to- bone enthesis model stained with H&E. Periosteum (P)-wrapped allograft bone

(AB), tenocyte-seeded PCL/PLLA (T), and unseeded PCL/PLLA were present as respective aspects of this construct were infiltrated with cells. Both unseeded

PCL/PLLA and tenocyte-seeded PCL/PLLA were ruptured from the tensile testing shown as disconnected from the periosteum-allograft bone in the composite image. Chondrocytes were not detected in this section. Staining of

135 the unseeded PCL/PLLA aspect of the model is indicative of cellular infiltration from the host mouse and/or the human periosteal wrap about the allograft bone.

Scale bar = 2 mm.

Figure 2.26. Light micrographs of an H&E-stained tethered Group 2 40-week tendon-to-bone enthesis model. A: viable tenocytes seeded onto PCL/PLLA (T);

B: periosteum (P) infiltration in allograft bone and presence of bone lining cells

(arrows) at the interface between the allograft bone and periosteum.

Chondrocytes were not detected in this section. Scale bars = 200 µm.

Type II collagen presence was indicated with IHC staining using DAB (brown).

The sections were counterstained with Mayer’s hematoxylin and cell nuclei appeared blue. Figure 2.27 presents images of an untethered tendon-to-bone enthesis model (Group 1) after 20 weeks of implantation. Micrographs illustrate positive staining of type II collagen in the tissue-engineered chondrocyte-seeded nPGA scaffold. The periosteum-allograft construct, tendon aspect (tenocyte- seeded PCL/PLLA), and cartilage portion (chondrocyte-seeded PGA) were viable

136 after 20 weeks of implantation. The cartilage (marked by type II collagen) was observed as a recognizable tissue developed between the periosteum and tenocyte-seeded PCL/PLLA (Figure 2.27-A). Enlargements show the detailed zone of the cartilage and chondrocytes (possibly hypertrophic) located in their lacunae (Figure 2.27-a).

An untethered 40-week tissue-engineered enthesis specimen (Group 1) was positive by IHC for type II collagen (Figure 2.28). Periosteum allograft bone, chondrocytes seeded onto nPGA, and tenocytes seeded onto PCL/PLLA were viable after the 40-week implantation time in vivo. Type II collagen was detected where the chondrocyte-seeded nPGA was initially sutured between the periosteum-allograft bone and tenocyte-seeded PCL/PLLA. The areas between chondrocyte segments contained fibrous connective tissue. Cell nuclei stained blue were observed infiltrating the nPGA scaffold (Figure 2.28).

IHC staining for a tendon-to-bone enthesis model harvested from an athymic mouse at 20 weeks (tethered, Group 2) is shown in Figure 2.29. The micrograph illustrates type II collagen presence (brown) adjacent to the periosteum-allograft bone and indicates formation of extracellular matrix by chondrocytes. The area of positive type II collagen staining was relatively smaller than that of the two untethered enthesis samples presented previously as the presence of type II collagen was reduced in this tethered specimen.

Type II collagen presence was not detected by IHC for 40-week tethered tendon-to-bone enthesis samples (data not shown). This result correlated with

137 the H&E staining results (chondrocytes in lacunae were not observed Group 2;

Figures 2.25 and 2.26).

Figure 2.27. Light micrographs of an untethered 20-week tendon-to-bone enthesis model wrapped with periosteum (Group 1) and stained with anti-type II collagen. DAB as an indicator molecule stains type II collagen brown (C) between the tenocyte-seeded PCL/PLLA aspect (T) and periosteum (P)-wrapped allograft bone (AB). A region of the tissue (a) is shown enlarged. Enlargement a: chondrocytes present in their lacunae surrounded by an extracellular matrix observed positive for type II collagen (C). The section was counterstained with

Mayer’s hematoxylin and the cell nuclei stained blue (arrows in enlargement a) in periosteal fibrous connective tissue (P) between cartilage tissue. Scale bars =

500 µm (panel A), 100 µm (panel a).

138

A a AB P P

a C C

C T

Figure 2.28. Light micrographs of an untethered 40-week tendon-to-bone enthesis model wrapped with periosteum (Group 1) and IHC-stained for type II collagen presence. DAB as an indicator molecule shows type II collagen in the cartilage portion (chondrocyte-seeded nPGA, brown, C) between the tenocyte- seeded PCL/PLLA aspect (T) and periosteum (P)-wrapped allograft bone (AB).

A region of this tissue is enclosed (a) and enlarged in the inset shows more cellular detail of the structure of the nPGA aspect. Enlargement a: chondrocytes visible in their lacunae and secreted matrix positive for type II collagen (brown).

The arrows denote cell nuclei (blue) as the section was counterstained with

Mayer’s hematoxylin. Scale bars = 500 µm (panel A), 200 µm (panel a).

139

A a a AB FB FB C

P AB

Figure 2.29. IHC (A) and H&E (a) staining results showing positive type II collagen and cartilage staining for a representative tensile-tested tethered 20- week tendon-to-bone enthesis model (Group 2). A: type II collagen presence in cartilage issue (C, brown) in the chondrocyte-seeded nPGA between periosteum

(P)-wrapped allograft bone (AB) and fibrous connective tissue (FB, arrows). The space between allograft bone and the positively stained cartilage is an artifact attributable to sectioning. Another section from the same specimen was stained with H&E (a) and shows an enlarged image of the morphology of the selected area in A. a: chondrocytes in lacunae and some larger lacunae that appear hypertrophic (chondrocyte-seeded nPGA, C) between allograft bone (AB) and aligned fibrous tissue (FB, arrows). Tenocyte-seeded PCL/PLLA is not shown in these images of the sample but was present in the specimen. Scale bars = 200

µm (panel A), 100 µm (panel B).

Scleraxis, a specific marker for tenocytes and fibroblasts, was detected by

IHC staining (brown). Sections were counterstained with Mayer’s hematoxylin

140 and cell nuclei appeared blue. Micrographs show scleraxis immunostaining results for untethered 20- and 40-week tendon-to-bone enthesis specimens

(Figures 2.30-A and B, respectively). Some cells within lacunae expressed scleraxis (Figure 2.28-A) and IHC staining of type II collagen was also detected in this specific area (Figures 2.27-b, 2.30-B). Periosteal tissue of this specimen also showed positive scleraxis staining (data not shown).

As for the tethered specimens (20 weeks, Group 2), Figures 2.31-A and B show cells that infiltrated the PCL/PLLA and were counterstained blue with

Mayer’s hematoxylin. The unseeded aspects of these constructs showed no tenocytes and only mouse host cell infiltration (Figure 2.31-A). However, the boundary of unseeded PCL/PLLA polymer-only aspect sutured to the periosteum stained positively for scleraxis, a result indicating human periosteal cell infiltration

(data not shown). Scleraxis was detected in both the seeded aspects of enthesis models and the outer fibrous layer of the periosteum (Figures 2.31-B and C, respectively). As for 40-week tethered enthesis specimens (Group 2), scleraxis was detected in the outer fibrous layer of the periosteum and the tenocyte- seeded PCL/PLLA, a result which was consistent with IHC staining from the 20- week tethered specimens (data not shown).

141

Figure 2.30. Scleraxis immunostaining results for representative untethered 20-

(A) and 40-week (B) tendon-to-bone enthesis models implanted in athymic mice

(Group 1). DAB was used as an indicator molecule showing scleraxis presence

(brown) in several cells denoted by arrows in panels A and B, respectively. A: scleraxis (arrows) detected in cells residing in lacunae of chondrocytes in cartilage (chondrocyte-seeded nPGA, C) regions of an untethered 20-week specimen. B: presence of scleraxis in nuclei of cells dispersed in the cartilage tissue (chondrocyte-seeded nPGA, C) in an untethered 40-week specimen.

Scale bars = 50 µm.

142

Figure 2.31. IHC staining results for a representative section of a tethered 20- week tendon-to-bone enthesis model (Group 2) to detect scleraxis. A: the unseeded PCL/PLLA aspect (PCL/PLLA) was negative for scleraxis and murine cells (stained blue, arrows) had infiltrated the PCL/PLLA scaffold. B: positive scleraxis staining (brown, arrows) present in most cells in the outer fibrous layer of the periosteum (P). C: scleraxis presence visible in many cell nuclei (brown, arrows) grown on the tenocyte-seeded PCL/PLLA scaffold (T). The empty spaces in this image are artifacts attributable to sectioning. The background of the tissue has nonspecific staining with DAB within the extracellular matrix of the representative section. Scale bars = 200 µm (panel A), 50 µm (panels B and C).

143

2.3.3 Discussion

The tendon-to-bone enthesis is described as a four-zone gradation of connective tissue that includes tendon followed spatially and sequentially by uncalcified cartilage, calcified cartilage, and bone. The loss of the tendon-to- bone attachment is a common and difficult problem in orthopedic injuries of the hand, elbow, shoulder, knee, heel and foot. These injuries may be treated surgically by repair, reconstruction or grafting methods, processes that may be beneficial to the patient but may also lead to complications such as adhesion formation, tenolysis, persistent tears and sub-optimal function.94, 148 Novel methodology to regenerate the four-zone gradation of tendon-to-bone enthesis would help to achieve better results in surgical therapies for enthesis defects.

A new approach to regeneration of a tissue-engineered tendon-to-bone insertion site by utilizing tenocytes/chondrocytes seeded onto biocompatible polymers as scaffolds for tendon/cartilage and human periosteum-wrapped allograft bone for new bone formation is presented in this Chapter. Such enthesis models were accomplished for the first time using the protocol detailed in the Materials and Methods section of this dissertation. Maximum loads and stiffness of the enthesis models were obtained by uniaxial tensile testing to evaluate biomechanical properties of the regenerated enthesis models. Tissue morphology and protein distribution were identified by histological and immunohistochemical (IHC) staining.

From the Elias and Saunders Laboratories, tensile testing results measured at the interface of the recreated tendon-to-bone insertion site all showed relatively

144 greater maximum loads and stiffness of tenocyte-seeded PCL/PLLA scaffolds compared to the unseeded PCL/PLLA only (Groups 1 and 2), regardless of implantation time. These results implied that the PCL/PLLA biomaterials utilized as scaffolds could support tenocyte growth and development and that the secreted ECM changed the structure of the scaffolds to sustain larger loads over time.

Mechanical testing results showed the seeded aspects of all 20- and 40-week untethered enthesis models had greater average maximum loads than those of

20- and 40-week tethered specimens, respectively. Results also showed that the seeded aspects of 20-week untethered enthesis specimens sustained greater maximum loads and stiffness compared to those of 40-week untethered specimens. In addition, the seeded aspects of 20-week tethered enthesis models had greater average maximum loads and stiffness than those of the 40- week tethered enthesis models. Concisely, the untethered enthesis models were mechanically stronger than the tethered enthesis models and the specimens

(both untethered and tethered) implanted for a shorter time (20 weeks) were respectively stronger than those implanted for a longer time (40 weeks). These results were unexpected as stronger regenerated enthesis structures were anticipated with longer implantation time periods and application of mechanical forces imparted by the host mice. Possible explanations to these data are given below.

Although mechanical force stimulates tenocyte growth, it also promotes polymer degradation.149, 150 Further, it is possible that the tenocytes altered their

145 phenotype to chondrocytic (fibrocartilage) when mechanical forces were applied on tethering the constructs to the animal model.149 As a cell-seeded biodegradable polymer, a PCL/PLLA scaffold might lose its strength during its degradation over implantation time. The overall strength would depend on ECM matrix production and integrity over the same period as the polymer were to disappear. Therefore, if the rate of ECM development (new matrix formation) were slower than that of the polymer degradation, the cell-seeded polymer scaffolds would sustain smaller loads. In addition, the Nylon 5-0 sutures utilized to tether both unseeded and tenocyte-seeded construct aspects to the trapezius and the gluteus muscles of the nude mice could have caused muscle relaxation in the animals. A consequence of this possibility might be a failure of continuous mechanical loading during implantation time periods. Further, it may be that the mechanical forces exerted on the implanted tethered constructs by the mice during their cage activity were too great and caused these constructs to suffer premature and unanticipated degradation leading to inconsistent loading data.

These potential reasons above could all result in stronger mechanical properties of the untethered enthesis specimens compared to their counterpart tethered enthesis specimens (20 and 40 weeks) and stronger mechanical properties of the 20-week enthesis models compared to their counterpart 40-week specimens

(Groups 1 and 2).

The capability of periosteum to induce bone regeneration in a periosteum- allograft bone as described in Chapter I of this dissertation is further confirmed by detection of bone resorption and formation in the periosteum-allograft bone

146 portion of the enthesis models harvested after 20 and 40 weeks of implantation.

Histological H&E staining showed that chondrocytes seeded onto PGA and tenocytes seeded onto PCL/PLLA were viable in 20- (untethered and tethered) and 40-week (untethered) enthesis models implanted subcutaneously in athymic mice. Potential fibrocartilage, observed in tethered tendon-to-bone enthesis specimens after a 20-week implantation time period, could be a marker for possible formation of a normal fibrocartilage-to-bone enthesis gradation. The appearance of fibrocartilage in these specimens could be explained by the mice activity on walking or running about their cages. Here, compression forces from the dorsal skin of the mice could be applied to the chondrocyte-seeded nPGA while both PCL/PLLA aspects with or without tenocytes were stretched under tension by being tethered to the trapezius and the gluteus muscles of the animals.

IHC staining results provided clear evidence for the presence of type II collagen and scleraxis in the enthesis specimens. Type II collagen was detected in 20-week (untethered and tethered) and 40-week (untethered) tendon-to-bone insertion models, and these data confirmed the presence of chondrocytes

(cartilage) in these specimens. However, type II collagen was not detected for chondrocyte-seeded nPGA sheets in 40-week tethered enthesis models (as was noted in the previous paragraph). In this regard, the articular chondrocytes utilized for the 40-week tethered enthesis models were obtained from a 77-year- old male cadaveric donor. It is well known that the metabolism of articular chondrocytes decreases with age and results in formation of an impaired

147 cartilage matrix.151-153 Therefore, one possible reason for the absence of type II collagen (cartilage) in tethered 40-week enthesis models could be the likely senescence of the chondrocytes available for this particular set of experiments and their failure to secrete a robust cartilage matrix.

It is perhaps valuable to consider scleraxis and its implications in this study.

The basic helix-loop-helix transcription factor, scleraxis, has been suggested as a regulator for formation of collagen-rich tissues in embryonic and limb tendon development.118, 154-156 Cultured tenocytes can dedifferentiate to fibroblasts, which are known to express scleraxis.157 Scleraxis was detected in cells within lacunae by IHC and also in the tenocyte-seeded aspects of the tissue- engineered enthesis models, regardless of application of mechanical load (Group

1 and 2) and implantation time periods. Possible tenocytes or fibroblasts and the tenocytes seeded onto PCL/PLLA scaffolds (Group 1 and 2) would secrete ECM to support the original scaffolds. Given that these cells produce ECM in the constructs, it may be understandable that the tenocyte-seeded PCL/PLLA aspects expressed greater average maximum loads and stiffness than those loads of the unseeded aspects in specimens from both Groups. Scleraxis was observed in tissue areas where type II collagen was also positive (20-week untethered specimens), a result which might be a potential sign for enthesis formation or development.

With application of mechanical loading, the specimens (Group 2) at 40 weeks were anticipated to have positively scleraxis-stained tenocytes/fibroblasts residing within parallel arrays of collagen fibers closely packed together, as found

148 in normal tendon. However, there was no qualitative staining difference in scleraxis appearance observed in the seeded aspects of all constructs with or without a mechanical framework (tethering) over all implantation time, an unexpected result. Here again, as possible explanations to the observations and measurements for tensile testing results, this aspect of the constructs (40-week tethered, Group 2) was seeded with aged tenocytes (77-year-old-male donor), and these cells may have been changed in tenocyte phenotype. There may also have been failure of continuous loading of this specimen during implantation.

One other factor that might contribute to regeneration of the tissue-engineered enthesis models is presence of the periosteum. Both the chondrocyte-seeded nPGA and tenocyte-seeded PCL/PLLA aspects of constructs were sutured to periosteum that was wrapped around the allograft bone, and cartilage and potential fibrocartilage were each observed adjacent to the fibrous layer of the periosteum. Moreover, the penetration of the periosteum into the cartilage through the fibrous layer also demonstrated a possible formation of a transition

(fibrocartilage to bone) to the four-zone enthesis in the models. As has been detailed in Chapter I, the cambium and the fibrous layer of normal periosteum are comprised of osteo/chondroprogenitor cells and fibroblasts aligned and intermingled with collagen fibrils. An earlier published study implied potential functions of osteoblast-fibroblast and chondrocyte-fibroblast interactions in enthesis development.158 Since periosteum is chondrogenic and osteogenic, chondrocytes and osteoblasts originating from the periosteum may both affect the neoformation of the enthesis model over time of the implantation. In this

149 respect, the effect of the periosteum in regenerating a fibrous and fibrocartilaginous entheses is not negligible.135

Future experiments can be performed in various aspects to validate the potential function of periosteum in reproducing a tendon-to-bone enthesis.

Genetic studies regarding enthesis-related gene expression, application of growth factors (for example, transforming growth factor-beta and platelet-derived growth factor) to maintain tenocyte phenotype in culture, different ages of patients for tissue and cell procurement, and utilization of customized shaped graded polymer scaffolds to replace the suturing technique for PCL/PLLA and nPGA sheets could all lead to a better tendon-to-bone enthesis model and its regeneration.

To summarize, this pilot study provides novel insight into the regeneration of a tendon-to-bone enthesis by utilizing chondrocytes, tenocytes, and periosteum from cadaveric donors to fabricate an enthesis model. Despite the fact that cadaveric periosteum and isolated cells were used in this study, initial data support the concept that tissue engineering approaches could advance to fabricating a tendon-to-bone insertion site for repair of a damaged enthesis.

Methods developed here could be applied in a broad translational sense for treatment and repair of isolated tendon injuries such as tears of the flexor tendon,

Achilles tendon, and rotator cuff.

150

CHAPTER

III. SILICON-FUNCTIONALIZED POLY(LACTIC ACID)

3.1 Introduction

The previous two Chapters described tissue-engineered models using human allograft bone biomaterials for regeneration new bone and tendon-to-bone enthesis for healing of segmental bone defects and enthesis defects. Although the treatments of utilizing allografts and autografts have been lifesaving and contemporary, risks in harvesting autografts and transplanting allografts exist.

Autograft harvesting from a patient is painful and costly, and the patients might develop infections and a hematoma. An allograft, similarly, has problems in collecting enough tissue to meet the massive needs of patients, possible diseases may be transmitted from the donor to the patient, and there are risks of allograft rejection by the immune system of the patient. Therefore in place of allografts, alternative biomaterials, such as materials derived from ceramics, polymers, metallic components, and composite materials, have also been widely adapted as scaffolds in medical applications. Among those polymer biomaterials, poly(lactic acid) (PLA), well known as a biodegradable thermoplastic aliphatic polyester, has been functionalized with silicon in this

151 dissertation study and utilized as a scaffold for human periosteal cell growth and development.

Ultimately, this project intends to grow bone cells on a silicon-functionalized scaffold. A scaffold with silicon can be synthesized through a bromine- functionalized initiator. In order to determine possible toxicity of brominated PLA

(poly[(lactic acid)-co-(2-bromo-3-hydroxypropionic acid)], PLB), the initiator used in the synthesis of silicon-functionalized PLA (Si-PLA), a cytotoxicity assay was performed in this study. For compatibility, the bromine concentration in PLB should mimic the silicon concentration in vivo. This Chapter includes the description and details of function of silicon in bone growth, bromine toxicity, applications of synthetic biomaterials, other than natural human allografts, synthesis of PLB, a cytotoxicity study on human osteoblast-seeded PLB scaffolds, results and discussion.

3.1.1 Silicon and Bone Health

This content contains introduction to silicon in bone health, bromine toxicity, and scaffolding in tissue engineering.

3.1.1.a Chemistry of Silicon

Silicon (Si) is a chemical element with atomic weight 28. It is the second most abundant chemical element in the crust of the Earth by mass (~26%), just after oxygen (~46%), but it rarely occurs in the form of a pure free element.159

Generally, silicon is present in the form of a silicate or silicon dioxide (silica, an

152 oxide of silicon with the chemical formula SiO2) comprising various rocks, clays, and sands in nature.

Silica is naturally a nontoxic transparent solid and is insoluble in water. Silica is commonly presented in quartz and is the predominant constituent of sand. As has been widely recognized and applied in industry, silica is known as one of the most complicated materials with a wide variety of related family members. Silica- based materials have a broad range of industrial applications including their use as a food additive (applied as an anti-caking agent, thickener, and clarifying agent), a drink-clearing agent (to improve the quality of drinks by removal of polymer impurities), and a pharmaceutical flow agent (to help powder dispersion and flow in the formation of tablets).159 It is of note that any remaining silica is removed completely through a filtration system from a food product even though silica is not toxic.

Silicon is also present in the human body where its most concentrated locations reported are bone, cartilage, and other connective tissues.160 Silicon has a relative higher concentration than other trace elements in the body as a physiologically essential element, similar to iron, copper, and zinc.161 Ingestion of silicon comes from several sources including whole grain cereals, fruits, rice, vegetables, and drinking water.162-164

Water soluble forms of silica, silicic acids (SiOx[OH]4-2x, metasilicic acid, orthosilicic acid, disilicic acid), exist in well water. Orthosilicic acid (H4SiO4), among these three forms, is stable in dilute aqueous solutions.165 Silicon is

153 mainly absorbed by the body through the form of orthosilicic acid which can be found in bone, tendons, liver, and kidney.159, 166

Recently, silica and silicic acid were shown to play a crucial role in bone metabolism.167-171 Bioactive silica-based nanoplatelets and nanoparticles were reported to promote osteoblast proliferation and differentiation while inhibiting osteoclast activity.167, 168 Gene expression of osteocalcin and alkaline phosphatase (important markers for bone formation) in osteoblasts and MSCs can be induced by silicic acid.169-171

3.1.1.b The Function of Silicon in Animal Growth

Silicon is found to perform an important metabolic role in the human body and it directly affects most aspects of the development of connective tissues and the skeletal system.172, 173 Silicon is associated with bone mineralization, collagen synthesis, growth of cartilage, tendon, ligaments, and skin, as well as the health of nails. Currently, the exact means or mechanism of silicon function in connective tissues is not well understood but previous studies have shown that, in order to repair bone and cartilage deformities, deficiencies, or impairments, silicon is necessary in both collagen and glycosaminoglycan (GAG) formation.160

The functions of silicon in animal skeletal systems have been described and defined through a number of studies.

A series of well known basic experiments was initially performed by Carlisle to investigate the function of silicon in the bone growth of chickens.174-176 One of first of these experiments was designed to feed young chicks only an amino acid

154 diet or a silicon-supplemented diet and to observe the outward appearance and growth of the animals. Over a 3-week time period, the growth curves of chicks fed with and without silicon in their diet showed that chicks fed a Si-deficient diet

(amino acids) had observable slower growth compared to silicon supplementation (Figure 3.1).175

Figure 3.1. Growth curves of chicks fed with/without silicon in their diet. The upper curve shows the diet supplemented with silicon ( ). The lower curve was a silicon-deficient basal diet ( ). Twenty-four animals were observed in each group. The figure has been reproduced with permission from the American

Association for the Advancement of Science.175

A subsequent experiment was modified from the earlier chick studies to demonstrate the essential quality of silicon in the growth and development of the

155 animals.175, 177 Here, a Si-deficient basal diet with (Si+) or without (Si-) supplemental silicon was utilized to feed one-day old cockerels. With twelve chicks in each group, the growth rates and the appearance of the animals (both

Si+ and Si-) were recorded intermittently. No significant difference was found in the appearance between the two groups after four weeks. Further experiments to examine the structures of the skulls of the animals from both groups were performed (Figure 3.2). For the animals fed with the Si-deficient basal diet, skulls were narrower and shorter with structural abnormalities that had less calcification and bone trabeculae compared to the control group (Figure 3.2). A possible reason for Si-deficient animals having abnormal skull structure was that silicon affected the collagen content in the cranial bone matrix.177

Figure 3.2. Macropathologic examination results from 4-week-old chick skulls without and with silicon supplements. A: Light photographs of the ventral surface of skull halves of Si-deficient basal diet fed chicks (Si-) and the control

156 group (Si+). The frontal area of the Si- group was narrower and shorter than that of the Si+ group. B and C: X-ray photographs of the ventral surface from the skulls of 4-week-old chicks (Si-/Si+). Less calcification and trabecular bone tissue were detected in the Si-deficient group. The figure has been reproduced with permission from the American Society for Nutrition.

A similar experiment utilizing a silicon-depleted diet to feed weanling rats was reported by Schwarz and Milne.178 Two groups of rats were fed a silicon- deficient amino acid diet or a diet supplemented with silicon. Over 26 days, the outer appearance of the rats was recorded. For the Si-deficient rats, the structure of the bone tissue around their eye sockets was deformed while the pigmentation of the incisors was delayed compared to the rats fed a normal diet.178 In this study, the researchers made a conclusion that, as an essential trace element, silicon could possibly participate in the formation of the organic matrix affecting bone mineralization. But the exact mechanism of the changes was not clear.178

Recent studies conducted with modified methodologies, however, were unable to reproduce the significant results reported by Carlisle and by Schwarz and Milne.179-183 One of the recent experiments, performed by Jugdaohsingh et al., was designed to determine the long-term effects of silicon on 21-day-old female Sprague–Dawley rats.184 Three groups of animals including animals fed with Si-deprived diets, silicon-deprived diets plus silicon supplemented drinking water, and rodent stock feed plus tap water as a control group. After 26 weeks,

157 the growth and development of the three groups of animals were compared in different aspects. Compared to the other two groups, the Si-deficient group of rats showed less fasting serum silicon concentration, lower urinary silicon excretion, and lower tibia silicon level. In addition, increased chondrocyte density and reduced bone growth plate thickness were observed in the Si-deficient group. Nonetheless, increased longitudinal body growth at week 18 and bone length at necropsy were detected in the Si-depleted group. As the researchers failed to reproduce the results Carlisle reported of decreased bone formation in

Si-deficient animals,174, 175 it was thought that different experimental conditions led to the different results.184 Urinary excretion of silicon was used to monitor the silicon level in the rats. Low circulating silicon levels inhibited growth plate closure that resulted in increased longitudinal bone growth of the Si-depleted group of rats.184

3.1.1.c The Function of Silicon in Bone Formation

As an essential trace element, Si was reported as a crucial element associated with early bone formation. Experimental reports from Carlisle illustrated the possible relationship between silicon and calcium based on the fact

Si is formed in the active region of newly formed osteoid.176 In this study, Si increased along with calcium in young bone tissue, while it decreased in developed bone areas where calcium increased and hydroxyapatite (HA) was detected.176 Carlisle also proposed that Si had an accelerating effect on the rate of bone mineralization in a manner similar to that of vitamin D.185-187

158

In 1983, Carlisle conducted further experiments about the function of Si and vitamin D in chick growth.172 Comparisons of the growth of three groups of one- day-old cockerels fed with a silicon-deprived diet (± vitamin D2/D3) or with a Si- supplemented diet (± vitamin D2/D3) were recorded. In 4 weeks, the Si-depleted chicks (with or without vitamin D2 or D3) showed abnormal skull development and lighter body weights. No significant differences were found in Si-deficient and supplemented groups with the same levels of vitamin D.172 Interestingly, chicks fed with the Si-deprived diet (+ vitamin D3) and the Si-supplemented diet (+ vitamin D3) had almost three times the body weight compared to the other two groups. The skulls of the Si-deficient chicks independent of vitamin D level all showed distorted structure and a shorter and narrower shape. These findings implied that silicon, regardless of vitamin D, had important effects during early stages of bone formation.172

Additional animal studies were undertaken to identify the function of Si in bone metabolism.188-190 Hott et al. reported that a Si-adequate diet (diet treated with silanol) fed rats resulted in greater bone formation, improved trabecular bone volume, and reduced bone resorption in metaphyseal bone in comparison to Si-deficient rats.190 Seaborn and Nielsen reported in 1994 that Si-deprived rats had reduced alkaline and acid phosphatase in their femurs, decreased copper concentration in their tibiae, and reduced uptake of 45Ca in their ectopic bone tissue.189 These results implied that dietary silicon affected bone formation through its effects on osteoblast and osteoclast enzyme activities (a difference in acid and alkaline phosphatase). Moreover, the decreased copper concentration

159 in rat tibiae and reduced 45Ca uptake in ectopic bone provided evidence that lack of dietary silicon resulted in insufficient bone metabolism.189, 191 In a later publication, Seaborn and Nielsen indicated that arginine, as an amino acid involved in collagen synthesis and bone formation, affected the physiological role of dietary silicon in bone metabolism.188

The macroscopic physiological functions of silicon on animal growth and bone formation have been demonstrated in various experiments.160, 172, 174, 175, 185-191

The microscopic response of bone cells to silicon will be discussed in the following paragraphs.

Nanocrystalline thin film coatings of hydroxyapatite (HA) substituted with different silicon contents [0 wt% (titanium coated as a control-Ti), 0.8 wt% (S1),

2.2 wt% (S2), and 4.9 wt% (S3)] were applied in an experiment designed by

Thian et al. to determine the effect of Si on human osteoblast-like (HOB) cell growth in vitro.192 HA was utilized as an ideal biomaterial as it has a similar structure to bone mineral, it leads to osteoconductivity, and it has compatibility for bone cells that then proliferate and spread while attached to its surface. Over a

14-day period, the HOB cells seeded on silicon-substituted surfaces exhibited enhanced cell adhesion, broadened cell spreading, and further development of their cytoskeletal structure (abundant actin stress fibers presented in cell membranes) with increasing silicon content (Figure 3.3).192

160

Figure 3.3. Cell growth of HOB cells over a 14-day period of culture in vitro. ap<0.05: Cell growth activity was significantly higher on uncoated Ti between groups; bp<0.05: Cell growth activity was significantly higher on S1 between groups; cp<0.05: Cell growth activity was significantly higher on S2 between groups; dp<0.05: Cell growth activity was significantly higher on S3 between groups; ep<0.05: Cell growth activity was significantly higher on S1, S2, and S3 than uncoated Ti within groups; fp<0.05: Cell growth activity was significantly higher on S3 than S1 within groups; gp<0.05: Cell growth activity was significantly higher on S2 than S1 within groups. The figure has been reproduced with permission from Elsevier.192

Silicon also enhances bone formation once substituted in calcium phosphate bioceramics.193-195 When incorporated into calcium phosphate, silicon generates a special microstructure, induces the solubility of the bioceramics by changing

161 the materials to a biologically equivalent apatite, and produces a more electronegative surface.194 In another study, Si-incorporated calcium phosphate was ectopically implanted into the paraspinal muscles of sheep to determine the osteoinductivity of silicon.195 In a 12-week time period, both silicon-substituted calcium phosphate and stoichiometric calcium phosphate showed osteoinductivity but the former exhibited significantly increased newly formed bone tissue as well as increased bone mass associated with the implant.195

3.1.2 Bromine Toxicity

The ultimate research goal for this project is to conduct a cytoviability study of silicon-functionalized PLA scaffolds. However, as mentioned in previous paragraphs, the design of silicon-functionalized scaffolds is dependent on a bromine-functionalized initiator. Since some bromine may remain attached to

PLB precursor scaffolds, the toxicity of bromine on PLB is of importance.

Elemental bromine is a halogen with chemical symbol Br and atomic number

35. Bromine as Br2 is a deep-red, oily liquid with a sharp smell. It does not exist freely in nature. Bromine (Br2) is toxic as it can be rapidly absorbed by the lungs or absorbed by the intestine if liquid bromine is ingested. Bromine (Br2) spreads widely to most tissues, and it cannot be metabolized by the body.

A previous experiment investigating the toxicity effects of sodium hypobromide on human osteoblasts has been reported.196 In this study, the cytotoxicity of sodium hypochlorite, sodium bromide, and sodium hypobromite were compared by evaluating human osteoblast morphology and cell proliferation

162 for 6 days of cell culture. ACTI-BROM 7342 (containing 42.8% of sodium bromide) and Clorox Ultra Mountain (containing 5.25% sodium hypochlorite) were utilized to obtain controlled Br content solutions. The sodium hypochlorite was mixed with ACTI-BROM at different molar ratios to obtain controlled hypobromous acid and hypochlorous acid as the bromine was converted to form hypobromous acid and hypobromite ions with a hypochlorous acid byproduct.

Sodium bromide resulted in greater cytotoxicity than sodium hypochlorite or activated sodium hypobromite (Figure 3.4 with the group design shown in the caption). The bromide alone was more cytotoxic compared to the mixture of bromide and hypochlorite. In addition, at higher bromine concentration (100%

BR and 80% BR), when bromide was mixed with media, the solution turned acidic and resulted in decreased cell proliferation.196 From this report196 and the known effects of bromine in the body, cells are clearly very sensitive to bromine and even trace levels, such as those that may be present in PLB scaffolds intended for supporting cell growth and development in this dissertation, must be assessed for cytoxicity.

163

Figure 3.4. Cell counts at day 6. Each well was originally seeded at a density of

50,000 cells/well. (Control = no test chemicals; 100% CL = 1.5 µL of Clorox in 2 ml of media; 100% BR = 1.5 µL of ACTI-BROM in 2 mL of media; 80% BR = 1.5

µL of test solutions [20% Clorox = 80% ACTI-BROM] in 2 mL of media; 50% BR

= 1.5 µL of test solutions [50% ACTI-BROM = 50% Clorox] in 2 mL of media;

20% BR = 1.5 µL of test solutions [80% Clorox = 20% ACTI-BROM] in 2 mL of media). Statistical analysis was performed by a one-way ANOVA. * = p < 0.05.

The figure has been reproduced with permission from Elsevier.196

3.1.3 Scaffolding in Tissue Engineering

Tissue engineering is a fascinating methodology of combining the applications of cells, suitable biochemical factors, and scaffolds in the treatment of damaged or lost tissues or organs. Clinically, the approach utilizes cells isolated from 164 patients and those cells are seeded or deposited onto the scaffolds to regenerate tissues, organs, or parts of organs. This study utilized polymer scaffolding techniques to fabricate scaffolds for cytoviability assays. Application of polymer scaffolds in cell/clinical studies would avoid human autografts or allograft harvests/potential surgeries.

The most appropriate and ultimately most successful scaffolds for tissue engineering are replicates of the extracellular matrix (ECM) of those cells being developed. This conclusion rests with the fact that the normal cells reside and function in their own native solid matrix. ECM functions can be categorized into five aspects.197 First of all, the ECM provides physical support for cells so that they may adhere and move (although some cells, such as osteocytes, do not move) and be guided to proliferate and differentiate. Second, the ECM defines and maintains scaffolding structure and mechanical stability to the cells. For example, type I collagen, the dominant protein in bone tissue, provides bone cell rigid structure by interacting with bone mineral.198 The alignment of type I collagen may vary as the response to different types of mechanical forces applied to the bone tissue. Third, the ECM may navigationally supply the cells with signaling to guide the activity of the cells. Fourth, the ECM components can bind growth factors to stabilize and maintain the bioactivity of the growth factors.

And last, the ECM is the dynamic physical environment where remodeling and vascularization formation are taking place.197

The scaffolds provide structural support and a physical environment for attachment and migration, physically and mechanically support cell growth,

165 maintain both biocompatibility and biodegradability, and have desired porosity to produce variable surface/volume ratios suitable for cell growth and development.199 Thus, the scaffolds are able to guide the cells to grow in the shape of new organs, stimulate cell proliferation and/or differentiation, and degrade normally while developing new tissue and organs. Typically for biodegradable scaffolds, the rate of polymer degradation needs to match the rate of new tissue formation on the scaffolds.

In this study, polymer scaffolds in the shape of small disks and having a porosity of 70% were designed to test PLB cytotoxicity in vitro. Human osteoblasts from cadaveric donors were seeded onto the PLB scaffolds to evaluate the scaffold biocompatibility.

3.2 Materials and Methods

This section includes experimental materials and methods that applied in the thesis study.

3.2.1 Experimental Materials

This section includes experimental materials utilized in polymer synthesis and cell culture.

3.2.1.a Materials for Synthesis

D,L-lactic acid (LA, 80-85%, w/v aq, Alfa Aesar, Ward Hill, MA), diphenylether

(DPE, 99%, Acros Organics, Morris Plains, NJ), and p-toluenesulfonic acid monohydrate (pTSA·H2O, 98.5%, Sigma-Aldrich, St. Louis, MO) were utilized in synthesis as received from the suppliers. Poly(D,L-lactic acid) (PLA, Mn = 75-85,

166 kDa, Đ = 1.30, Akita, Inc., West Lafayette, IN) was applied in this project as the control sample. 2-Bromo-3-hydroxypropionic acid (BrA) was synthesized by Dr.

Colin Wright (laboratory of Dr. Coleen Pugh, Department of Polymer Science, the

University of Akron) through a scheme developed previously.200

3.2.1.b Materials for Cell Toxicity Assay

Fresh periosteal strips (1 cm × 1 cm) were harvested within 24 hours of donor death from the knees of a 61-year-old female (obtained from the Gift of Hope

Tissue & Organ Donor Network, Itasca, IL, through Rush University, Chicago, IL).

Minimum essential medium eagle (MEM, Mediatech, Inc., Manassas, VA) supplemented with 10% Fetal Bovine Serum (HyClone/Thermo Scientific Co.,

Waltham, MA), 1% antibiotic/antimycotic (Mediatech, Inc.), and 0.2% primocin

(InvivoGen, San Diego, CA) constituted the complete feeding or medium used to culture the periosteum. Cell detachment was conducted with trypsin (0.05%

Trypsin, 0.53 Mm EDTA, Mediatech, Inc.). Glycerol (Fisher Scientific, Fair Lawn,

NJ) was used for the cell freezing process. PrestoBlue® cell viability reagent

(Life Technologies, Frederick, MD) was utilized to perform the cytotoxicity study.

3.2.2 Experimental Methods

This section includes experimental methods that applied in this dissertation study.

3.2.2.a PLB Synthesis and Analysis

This polymerization was conducted on a Schlenk line in a N2 atmosphere.

Typically, an aqueous solution was prepared with BrA (3.830 g, 22.4 mmol), LA

167

(7.658 g, 84.8 mmol), catalyst pTSA (1.071 g, 5.6 mmol), and 12 mL DPE measured and transferred to a 50 mL round bottom flask at 95 °C with reduced pressure (1-3 mm Hg) for 72 hours (BrA:LA = 20:80). To stop the reaction, the vacuum was released to atmosphere. CH2Cl2 (10 mL) was added to dissolve the polymer in the flasks. The polymer contained in solution was precipitated in cold stirred methanol (100 mL) in a beaker followed with filtration through a fritted glass funnel. The procedure was repeated four times to ensure removal of DPE and small molecules. To remove water and other solvent residue in the polymer product, the PLB sample was then dried in a vacuum oven for 48 hours. The resulting polymer yielded 66% (1.3 g) as a white solid powder and it was stored in a -20 °C freezer until usage.

Scheme3.1. Synthesis of PLB.

1H nuclear magnetic resonance (NMR) and gel permeation chromatography

(GPC) were applied to determine the bromine content in PLB and the average molecular weight of PLB. NMR spectroscopy (δ, ppm) was recorded utilizing a

Varian Mercury 300 spectrometer (300 MHz, Varian, Inc., Palo Alto, CA). All

NMR samples were dissolved in deuterated chloroform (CDCl3) while the resonances were measured relative to tetramethylsilane (0.00 ppm).

Number average molecular weights (Mn), weight average molecular weights

(Mw), and polydispersities (PDI, Đ = Mw/Mn) of all PLB samples were determined

168 by testing the samples through GPC relative to standard linear polystyrene

(GPCPSt) calibration curves of log Mn vs. elution volume at 35 °C using tetrahydrofuran (THF) as the solvent (1.0 mL/min). A GPC sample was first dissolved in THF (~1.0 g/L) followed by filtration of the mixture solution through a

0.45 µm polytetrafluoroethylene (PTFE) filter prior to the assay. Samples were tested by GPC using a set of 50 Å, 100 Å, 500 Å, 104 Å, and linear 50-104 Å styragel 5 µm columns, a Waters 410 differential refractometer (Waters Corp.,

Milford, MA), and Millennium Empower 3 data analysis software (Waters Corp.).

3.2.2.b PLB Scaffold Fabrication

The cytotoxicity assay was conducted on PLB scaffolds with controlled Br content to modify the ultimate sample. PLA (Akita, Inc.) was ground and mixed with PLB (BrA:LA = 20:80) to yield 10%, 5%, and 1% molar Br content PLB scaffolds (Table 3.1).

Table 3.1. Weight amounts of designated controlled Br content polymer blends.

169

To fabricate the scaffolds for cell toxicity studies, for instance, PLB with 10%

Br, 44 mg PLB was ground and mixed with 6 mg PLA and 150 mg sodium chloride (NaCl) for 1 minute to obtain a fine powder. This procedure was to assure a 70% porosity in the scaffolds to study in vitro.201 The mixture was then transferred to a stainless steel scaffolding mold (Figure 3.5). To produce scaffold disks (diameter = 1 cm, thickness = 2 mm), samples were compressed in a mold under 5,000 pounds pressure with a laboratory press for 3 minutes.

The compressed scaffold disks were immersed in distilled water with a stirring bar in a beaker for 12 hours to leach out the salt.201 The scaffolds were then transferred to a vacuum oven to dry out water residue for 24 hours. Before usage, the scaffolds were placed in 12-well plates to be sterilized with ethyl oxide.

Figure 3.5. A schematic for fabrication of a PLB scaffold. Ground and mixed

NaCl (white particles) and PLB (blue particles) powders were transferred to a stainless steel scaffold mold. The sample in the mold was then pressed under

170

5,000 pounds pressure to retrieve a scaffold disk (diameter = 1 cm, thickness = 2 mm). Scale bars = 1 cm.

3.2.2.b Periosteal Cell Expansion, Freezing, and Thawing

Periosteum strips (1 cm x 4 cm) harvested from a 43-year-old male or a 61- year-old human female cadaveric limb (protocol described in Chapter I) were dissected with a surgical scalpel into 1 cm x 1 cm pieces. Twelve dissected periosteum pieces were then placed onto two 6-well plates with the cambium layer of the periosteum facing the bottom of the wells. Approximately 1-1.5 mL

MEM media were added to each well to ensure the attachment of periosteum strip to the bottom of the well. While the periosteum piece attached to the plate, the primary periosteal cells from the cambium layer migrated to the plate and started to proliferate. Periosteum strips in 6-well plates were cultured in a humidified 5% CO2 incubator at 37 °C for 2 weeks until removal from each well when the primary cell culture (P0) reached confluency (Figure 3.6).

171

Figure 3.6. Light photomicrographs of cadaveric primary human periosteal cell growth from the periosteum tissue cambium layer and expansion in vitro. A:

Human periosteal cell growth after day 2 of culture. Cell begin to migrate from the periosteal (P) tissue to the culture plate. B: Human periosteal cell growth after day 7 of culture. Migrated cells from the periosteum are numerous on the culture plate, have developed spindle shapes with large interconnecting extensions, and are beginning to intercommunicate with each other. Scale bars

= 200 µm; images were taken with an Olympus inverted microscope, model IX70.

172

To increase cell expansion, the periosteal cells were trypsinized from the 6- well plates to T75 or T175 flasks based on their cell growth condition. When the cells in flasks were growing well and in their exponential growth phase, cells were harvested for cryopreservation. Approximately 15 x 106 cells (Passage 1,

P1) were harvested from the T175 cell culture flasks. The cells were diluted with

12 mL MEM media in a 15 mL Falcon tube and then 3 mL of glycerol (~ 20%) were slowly pipetted into the cell suspension. Before aliquoting cells to 2 mL sterile cryo tubes, the cell samples were vortexed carefully. Each 2 mL cryo tube contained 1.5 x 106 cells in 1.5 mL solution. The cryo tubes were labelled and placed in a -80 °C freezer for storage.

Upon usage, the cryo tubes were first rapidly thawed in a 37 °C water bath for 1 minute before gently transferring the cells to a 15 mL Falcon tube. Pre- warmed MEM (10 mL) was pipetted dropwise to the Falcon tube to dilute the thawed cells. The cell solution was centrifuged at 2000 rpm for 8 minutes and the cells were then resuspended in an appropriate amount of MEM for plating them at high density in T175 flasks to assure cell viability. Freezing and thawing cells were performed under aseptic conditions in a laminar flow hood. When they had reached confluency, periosteal cells were detached from the cell culture flasks for further cytotoxicity testing.

3.2.2.c Cell Seeding

Sterilized PLA and PLB (mole bromine content 1%, 5%, and 10%) scaffolds were transferred to new 12-well plates with four scaffolds of each type of

173 polymer. Each scaffold was rinsed three times with PBS (3 mL, 15 minutes each), followed by three rinses of 3 mL MEM for 15 minutes each.

Periosteal cells were harvested and counted when they reached confluency.

For each type of scaffold, three of the disks were carefully seeded with 20,000 cells using a micropipettor (N = 3), and the fourth disk was placed in MEM cell culture medium without cells as a reference (Figure 3.7). The seeded samples were first placed in a 37 °C incubator for 2 hours for cell attachment before addition of 3 mL of MEM medium. At the same time, tissue culture polystyrene plates (TCPS) with only cells seeded to the bottom of the wells were used as controls. The medium was changed every other day.

174

A 1 2 3 Ctrl

PLA

10% Br

5% Br

B 1 2 3 Ctrl

PLA

10% Br

5% Br

Figure 3.7. PLA and PLB (10% and 5% Br) scaffolds before (A) and after (B) seeding with human periosteal cells. Note that the control samples (Ctrl) were not seeded with human periosteal cells. PLB (1% Br) is not shown in the figure.

Scale bars = 2 cm.

3.2.2.d Cytotoxicity Assay

The Prestoblue® live cell viability assay was performed and sample data were quantitatively normalized to a customized standard curve. Prestoblue® solution

(Life Technologies) was freshly mixed with cell culture medium at a ratio of 1:9.

Prior to applying the Prestoblue® reagent, the culture medium was aspirated

175 from each well. A 1 mL mixture containing 100 µL Prestoblue® reagent and 900

µL cell culture medium was gently pipetted into each well using a micropipettor.

Samples were incubated for 25 minutes at 37 °C following pipetting 100 µL

Prestoblue® mixture into a sterile 96-well plate in triplicate. The 96-well plate was then covered with aluminum foil and transferred to a fluorescence plate reader to obtain cell count data points. The results from a SynergyTM MX plate reader (BioTek Instruments, Inc., Winooski, VT) were normalized to a standard curve and plotted as intensity over cell number. A standard curve was generated from serial dilutions in MEM of periosteal cells (1.0x106, 0.5x106, 0.25x106,

1.25x105, 6.25x104, 3.1x104, 1.5x104, 7.8x103, 3.9x103 cells). Higher fluorescence intensity values correlate to greater gross metabolic activity of cells.

A 21-day (day 1, 3, 5, 7, 10, 14, and 21) cell viability assay was reported. The experiment was repeated three times with periosteal cells from the same patient on PLB scaffolds.

3.3 Results and Discussion

Known as a vital connective tissue that forms about the outer lining of long bones and contains undifferentiated osteo-precursors, periosteum has been widely utilized in bone-regeneration related studies.10, 81, 83, 202, 203 Cell culture studies of human periosteal cells demonstrate that they remain osteogenic for up to 10 passages.203 The periosteal cells utilized in this study were cultured up to passage 3 and were seeded onto polymer scaffolds. Four 21-day Prestoblue®

(PB) metabolic assays on PLB (varied properties) scaffolds seeded with frozen-

176 preserved and cultured human periosteal cells obtained from a 43-year-old male and a 61-year-old female patient were performed (Table 3.2). Periosteal cell growth curves obtained from each individual PB assay were analyzed to optimize the content of bromine in PLB for subsequent synthesis of Si-functionalized PLA.

Table 3.2. Average molecular weight and PDI (Đ) of PLB samples, donor information and cell passage of human periosteal cells, and initial cell seeding density for all four PB assays conducted in the study.

PB PLB Human Periosteal Cells Seeding Density Assay (Mn [kDa], Đ) (Donor Information, Cell Passage) (Cells/Scaffold) I 20 kDa, 2.03 43-year-old male, P3 2.00E+04 II 25 kDa, 1.37 43-year-old male, P3 2.00E+04 III 25 kDa, 1.37 61-year-old female, P3 2.00E+04 IV 17 kDa, 2.17 61-year-old female, P3 2.00E+04

3.3.1 PLB Synthesis Results

PLB (poly[(lactic acid)-co-(2-bromo-3-hydroxypropionic acid)]) was synthesized by acid-catalyzed melt polycondensation in the presence of diphenyl ether reported by Wright et al.204 Figure 3.8 represents the 300 MHz 1H NMR spectrum of a representative PLB copolymer sample supplied from Haidong Zhu

(Department of Polymer Science, the University of Akron). The resonances at δ

= 5.17 ppm (parts per million) and δ = 1.57 ppm refer to methine and methyl groups from lactic acid, respectively. The resonance at δ = 4.51 ppm can be attributed to the overlap of the hydrogens from CHBr and the methylene group on the β position of the BrA portion of the PLB copolymer (Figure 3.8). The ratio of

177 the integrals of δ = 5.17 ppm and δ = 4.51 ppm determines the content of functional Br groups in the copolymer. The NMR results indicated that the composition of the copolymer matches the feeding ratio of the two monomers

([BrA]:[LA] = 20:80).

Figure 3.8. A 300 MHz 1H NMR spectrum of a poly[(lactic acid)-co-(2-bromo-3- hydroxypropionic acid)] (PLB) copolymer, [BrA]:[LA] = 20:80. The sample was synthesized through acid-catalyzed melt polycondensation in the presence of diphenyl ether. The asterisk denotes the resonance signal for solvent CDCl3.

Conventional GPC was applied to determine the average molecular weight of the PLB samples. The number average molecular weight of the PLB sample is

1.68 × 104 g/moL with a PDI of 2.17.

178

Figure 3.9. A GPC chromatogram of poly[(lactic acid)-co-(2-bromo-3- hydroxypropionic acid)] (PLB) copolymers. The sample was synthesized through acid-catalyzed melt polycondensation in the presence of diphenyl ether,

4 [BrA]:[LA] = 20:80. GPCPSt Mn = 1.68 × 10 g/mol, Đ = 2.17. The sample was eluted in THF.

3.3.2 Cytotoxicity Study Results

All four PB assays were repeated in triplicate. Figure 3.10 shows the growth curves of human periosteal cells (43-year-old male, P3) on a TCPS control, a

PLA scaffold, and PLB ([BrA]:[LA] = [20:80], Mn = 20 kDa, Đ = 2.03) scaffolds that contain 10 mol%, 5 mol%, and 1 mol% Br (PB assay I). After cell seeding of

2 x 104 cells/scaffold on culture day 0, on day 1, there were ~ 3.3 x 104, 8.0 x 103,

179 and 1.5 x 103 cells detected from the TCPS, PLA, and PLB (1 mol% Br) scaffolds, respectively. After 21 days, cell counts increased to 5.0 x 105 for

TCPS and 5.3 x 104 for PLA scaffolds. For all brominated PLB scaffolds, the cells failed to proliferate after day 5. Some of the PLB scaffolds failed to maintain their structural integrity in culture on day 21 (Figure 3.11).

Periosteal Cell Viability on Polymer Scaffolds over a 21-Day Period (PB Assay I) 6.0E+05 5.0E+05 4.0E+05 TCPS 3.0E+05 PLA 2.0E+05 10% Br 5% Br Cell Number Cell 1.0E+05 0.0E+00 1% Br 1 3 5 7 10 14 21 -1.0E+05 Time (days)

Figure 3.10. A chart of growth curves for PB assay I of human periosteal cells

(43-year-old male, P3) on tissue culture polystyrene (TCPS) control, PLA scaffolds, and PLB ([BrA]:[LA] = [20:80], Mn = 20 kDa, Đ = 2.03, 10 mol%, 5 mol%, or 1 mol% Br) scaffolds over a 21-day period. Cells were cultured in triplicate on each type of scaffold. Cell growth was only found on TCPS and PLA scaffolds and was not detected with any bromine-containing PLB scaffold (all

PLB plots overlap in the figure). Error bars represent standard error mean of cell number, but errors are too small to be shown on the plots of the respective scaffolds.

180

Figure 3.11. A representative light photograph of a human-periosteal-cell- seeded PLB (10 mol% Br) scaffold in cell culture media on day 21. The scaffold failed to maintain its original disk structure. Scale bar = 1 cm.

Cell growth curves for PB assay II of human periosteal cells (43-year-old male, P3) on TCPS control, PLA scaffolds, PLB ([BrA]:[LA] = [20:80], Mn = 25 kDa, Đ = 1.37) scaffolds that contain 10 mol%, 5 mol%, and 1 mol% Br are shown in Figure 3.12. It is of note that the value of Mn of the PLB sample used in this PB assay II was larger and had narrower dispersity than the value applied in

PB assay I. Cell growth for PB assay II was observed on TCPS controls, PLA scaffolds, and PLB scaffolds with 1 mol% Br. On the first day of this PB assay, there were ~ 3.0 x 104 cells growing on TCPS and ~ 6.0 x 103 and 5.3 x 103 cells growing respectively on PLA and PLB (1 mol% Br) scaffolds. By 21 days, there were 1.2 x 106 cells growing on TCPS, 3.8 x 105 cells on PLA scaffolds, and 4.8 x

181 105 cells on PLB (1 mol% Br) scaffolds. Cell growth was effectively not detected on 10 or 5 mol% bromine PLB scaffolds over the full extent of culture times.

Periosteal Cell Viability on Polymer Scaffolds over a 21-Day Period (PB Assay II) 1.4E+06 1.2E+06 1.0E+06 TCPS 8.0E+05 PLA 6.0E+05 10% Br 4.0E+05 5% Br Cell Number Cell 2.0E+05 1% Br 0.0E+00 -2.0E+05 1 3 5 7 10 14 21 Time (days)

Figure 3.12. A chart of growth curves for PB assay II of human periosteal cells

(43-year-old male, P3) on tissue culture polystyrene (TCPS) control, PLA scaffolds, and PLB ([BrA]:[LA] = [20:80], Mn = 25 kDa, Đ = 1.37, 10 mol%, 5 mol%, or 1 mol% Br) scaffolds over a 21-day period. Cells were cultured in triplicate on each type of scaffold. Cell growth was detected on TCPS controls,

PLA scaffolds, and PLB (1 mol% Br) scaffolds. Little cell growth was detected on

PLB scaffolds with 10 or 5 mol% Br. Error bars represent standard error of mean cell number, but they are too small to be shown on the plots of the respective scaffolds.

The growth curves for PB assay III of human periosteal cells (61-year-old female, P3) on TCPS control, PLA scaffolds, PLB ([BrA]:[LA] = [20:80], Mn = 25

182 kDa, Đ = 1.37) scaffolds that contain 10 mol%, 5 mol%, and 1 mol% Br are presented in Figure 3.13. The PLB sample utilized in PB assay III was the same as that used in PB assay II. Cell proliferation was observed on TCPS controls,

PLA scaffolds, and PLB scaffolds with 1 mol% Br. At day 1, 4.2 x 104 cells were detected growing on TCPS and 1.5 x 104 and 1.4 x 104 cells were observed growing respectively on PLA and PLB (1 mol% Br) scaffolds. After 20 days, cell counts were 2.1 x 105 on TCPS, 3.8 x 104 on PLA scaffolds, and 5.5 x 104 on

PLB (1 mol% Br) scaffolds. Little cell growth was detected on 10 mol% or 5 mol% bromine PLB scaffolds.

Periosteal Cell Viability on Polymer Scaffolds over a 21-Day Period (PB Assay III) 2.4E+05

1.9E+05 TCPS 1.4E+05 PLA 9.0E+04 10% Br

Cell Number Cell 5% Br 4.0E+04 1% Br -1.0E+04 1 3 5 7 10 14 21 Time (days)

Figure 3.13. A chart of growth curves for PB assay III of human periosteal cells

(61-year-old female, P3) on tissue culture polystyrene (TCPS) control, PLA scaffolds, and PLB ([BrA]:[LA] = [20:80], Mn = 25 kDa, Đ = 1.37, 10 mol%, 5 mol%, or 1 mol% Br) scaffolds over a 21-day period. Cells were cultured in triplicate on each type of scaffold. Cell proliferation was detected on TCPS

183 controls, PLA scaffolds, and PLB (1 mol% Br) scaffolds. Minimal cell growth was detected on PLB scaffolds with 10 or 5 mol% Br. Error bars represent standard error of mean cell number, but errors are too small to be shown on the plots of the respective scaffolds.

PB assay IV was conducted with human periosteal cells (61-year-old female,

P3) on TCPS control, PLA scaffolds, PLB ([BrA]:[LA] = [20:80], Mn = 17 kDa, Đ =

2.17) scaffolds that contain 10 mol%, 5 mol%, and 1 mol% Br and is shown in

Figure 3.14. The Mn of the PLB sample was smaller than the one used in PB assays I, II, and III, with a PDI value close to that of the PLB used in PB assay I.

Cell proliferation was observed on TCPS controls, PLA scaffolds, and PLB scaffolds with 1 mol% Br. At day 1, 3.6 x 104 cells were detected growing on

TCPS and 1.2 x 104 and 1.0 x 104 cells were observed growing respectively on

PLA and PLB (1 mol% Br) scaffolds. By day 21, cell counts were 1.7 x 105 for

TCPS, 2.4 x 104 for PLA scaffolds, and 2.2 x 104 on PLB (1 mol% Br) scaffolds.

Very little cell growth was identified on 10 mol% or 5 mol% bromine PLB scaffolds.

184

Periosteal Cell Viability on Polymer Scaffolds over a 21-Day Period (PB Assay IV)

1.9E+05

1.5E+05 TCPS 1.1E+05 PLA 7.0E+04 10% Br Cell Numer Cell 5% Br 3.0E+04 1% Br

-1.0E+04 1 3 5 7 10 14 21 Time (days)

Figure 3.14. A chart of growth curves for PB assay IV of human periosteal cells

(61-year-old female, P3) on tissue culture polystyrene (TCPS) control, PLA scaffolds, and PLB ([BrA]:[LA] = [20:80], Mn = 17 kDa, Đ = 2.17, 10 mol%, 5 mol%, or 1 mol% Br) scaffolds over a 21-day period. Cells were cultured in triplicate on each type of scaffold. Cell proliferation was observed on TCPS controls, PLA scaffolds, and PLB (1 mol% Br) scaffolds. Very little cell growth was observed on PLB scaffolds with 10 or 5 mol% Br. Error bars represent standard error of mean cell number, but errors are too small to be shown on the plots of the respective scaffolds.

Cell viability results of the four PB assays examined on TCPS, PLA, and PLB

(1 mol%) are combined in Table 3.3. The numbers of cells growing on day 1 and day 21 of the assays are listed (Table 3.3). Comparing cell counts at day 21 to those at day 1 in PB assay II, III, and IV, all the listed samples expressed and

185 maintained increasing cell growth. PB assay I, however, only gave increased cell counts for TCPS and PLA scaffolds from day 1 to day 21. For PB II, III, and IV, student’s t-tests were applied to determine if the cell growth on PLA and PLB (1 mol% Br) from day 1 to day 21 was significantly different. The statistical results showed that the periosteal cell growth traces between PLA and PLB (1 mol% Br) over the 21-day culture period were not significantly different (p = 0.90 [PB assay

II], p = 0.73 [PB assay III], and p = 0.58 [PB assay IV]).

To demonstrate cell proliferation on 1 mol% brominated PLB scaffolds from day 1 to day 21, cell growth-fold change was obtained by normalizing cell counts on day 7, 14, and 21 to cell counts on day 1 (Table 3.3). For PB assay II, III, and

IV, cell growth-fold change curves for cell-seeded PLB (1 mol% Br) are presented in Figure 3.15-A, B, and C, respectively. Linear trend lines indicating overall traces of related fold changes of cell growth on PLB scaffolds (1 mol% Br) from day 1 to day 21 in PB assay II, III, and IV were automatically generated with

Microsoft Excel 2013 (Microsoft Corp., Redmond, WA; Figure 3.15-A, B, and C, respectively). Trend lines of all cell growth-fold change curves increased in the

21-day culture time period. In assay II and III, the growth-fold changes on 1 mol% brominated PLB were observed to decrease after 14 days of culture

(Figure 3.15-A and B).

Table 3.3. Human periosteal cell viability on TCPS, PLA scaffolds, and PLB (1 mol% Br) scaffolds detected with the PB assay. The numbers of cells growing on

186

TCPS, PLA, and PLB (1 mol% Br) scaffolds are listed below.

Human Seeding PLB (Mn Periosteal Cells Day 1 Day21 PB Assay Density Sample [kDa], Đ) (Donor Info, (Cells) (Cells) (Cells/Scaffold) Cell Passage) TCPS 3.3E+04 5.0E+05 20 kDa, 43-year-old PLA 8.6E+03 5.3E+04 I 2.0E+04 2.03 male, P3 PLB 1.6E+03 0.0E+00 (1% Br) TCPS 3.0E+04 1.2E+06 25 kDa, 43-year-old PLA 5.9E+03 3.9E+05 II 2.0E+04 1.37 male, P3 PLB 5.4E+03 4.8E+05 (1% Br) TCPS 4.2E+04 2.1E+05 25 kDa, 61-year-old III 2.0E+04 PLA 1.5E+04 3.8E+04 1.37 female, P3 PLB 1.4E+04 5.6E+04 TCPS 3.6E+04 1.7E+05 17 kDa, 61-year-old PLA 1.2E+04 2.4E+04 IV 2.0E+04 2.17 female, P3 PLB 1.0E+04 2.2E+04 (1% Br)

187

A Cell Growth-Fold Change to Day (PB Assay II) 100.0

80.0

60.0

40.0

20.0

0.0 Fold Change to Day Day to 1 Change Fold 1 7 14 21 -20.0 Time (Days)

B Cell Growth-Fold Change to Day 1 (PB Assay III) 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Fold Change to Day Day to 1 Change Fold 1 7 14 21 Time (Days)

C Cell Growth-Fold Change to Day 1 (PB Assay IV) 3.0 2.5 2.0 1.5 1.0 0.5 Fold Change to Day Day to 1 Change Fold 0.0 1 7 14 21 Time (Days)

188

Figure 3.15. Human periosteal cell growth-fold change from day 1 to day 21

(solid lines) on PLB scaffolds (1 mol% Br) from PB assay II (A), III (B), and IV

(C), respectively. Linear trend lines (dotted) indicate overall traces of related fold changes of cell growth on PLB scaffolds (1 mol% Br) from day 1 to day 21. The three trend lines all show increased fold changes of cell growth over the 21-day culture period (A, B, and C). N = 3. Error bars represent standard error of mean fold change.

3.3.3 Discussion

As mentioned in the Introduction to this dissertation, this project ultimately intends to grow bone cells on silicon-functionalized PLA scaffolds. The synthesis of Si-PLA requires utilization of PLB as the initiator in the reaction. As reported cytotoxic to osteoblasts, the content of bromine residues on the backbone of Si-

PLA is of importance for cell viability. For the first time, four PB cell viability assays were performed to determine the toxicity of PLB with different bromine content in order to optimize the amount of bromine in Si-PLA. Also, for the first time human periosteal cells from the cambium layer of periosteum tissue from cadaveric donors were utilized in this study.

Cryopreserved human periosteal cells from both a 43-year-old male and a 61- year old female were able to proliferate when seeded on TCPS, PLA, and PLB (1 mol% Br) scaffolds over a 21-day culture in vitro. Periosteal cells obtained from the younger donor (43-year-old male, PB assay I and II) exhibited higher cell counts on TCPS, PLA, and PLB scaffolds compared to the cells from the 61-

189 year-old female (PB assay III and IV). This observation is in agreement with previous studies that cells isolated from younger patients yielded greater cell proliferation compared to aged patients.205, 206 Moreover, the gender of the donors has been reported to affect human bone-derived cells from females vs. males in being more susceptible to a loss in proliferation as a function of increasing age.207 Though periosteal cells remain viable after cryopreservation and achieve high cell density in culture, cryopreservation of human donor periosteal cells and the age and the gender of those donors are several possible reasons for the differences in cell proliferation between PB assay I and II and PB assay III and IV.202

Cell counts from PB assays II, III, and IV increased on TCPS, PLA, and PLB

(1 mol% Br) over the full 21-day extent of culture time. While identical numbers of cells were seeded at day 0 of culture on the different scaffolds, initial cell attachment at day 1 varied based on these TCPS, PLA, and PLB substrates.

Cell growth curves of the human donor periosteal cell-seeded TCPS controls from PB assay I, II, III, and IV all expressed typical cell growth curve features such as a lag phase and an exponential phase. Cells on TCPS continued to proliferate on culture day 21. TCPS had an average 3 x 104 cells growing at day

1, a value 1.5 times the number of the cells growing on PLA and 1 mol% brominated PLB scaffolds. Though the periosteal cells were gently seeded onto the surfaces of the PLA and PLB scaffolds, only about a quarter of the seeded 2 x 105 cells were detected growing on the scaffolds at day 1. Low initial cell attachment on PLA and PLB scaffolds is one of the possible reasons for

190 decreased cell proliferation compared to TCPS controls in the 21-day culture period.

PLA scaffolds were utilized as reference controls in the study as PLA has been reported to be one of the most promising biodegradable polymers for tissue engineering and biomedical use.199 The PLA scaffolds tested in all four PB assays were fabricated from same batch of PLA materials (Mn = 75-85 kDa, Đ =

1.30). In all four PB assays, human periosteal cells were observed proliferating on PLA scaffolds in the 21-day culture period. At culture day 21, cell counts from

PLA scaffolds were only ~10-30% of that of TCPS (PB assay I, II, III, and IV), a result that indicates the standard control TCPS has greater biocompatibility than

PLA scaffolds.

In PB assay II, III, and IV, human periosteal cells on PLB with 1 mol% Br showed cell growth curves similar to those of PLA scaffolds. This observation indicates that the human periosteal cells exhibited similar cell viability when seeded to PLB scaffolds with 1 mol% bromine. The plots of cell growth-fold change of the 1 mol% brominated PLB scaffolds from PB assay II, III, and IV further confirmed an increasing cell growth on these scaffolds. These results imply PLB with 1 mol% Br is nontoxic and has biocompatibility similar to that of

PLA scaffolds when seeded with human periosteal cells and cultured (21-day assay). The initial (day 1) cell attachment number for 10 and 5 mol% PLB scaffolds was approximately the same as that for PLA and 1 mol% brominated

PLB scaffolds (~5 x 103), but the cells on these scaffolds failed to proliferate within the 21-day culture time. This result would suggest that 10 mol% and 5

191 mol% bromine for the PLB scaffolds is cytotoxic to the human donor periosteal cells. Additionally, possible postulation to be made here is that the differences in molecular weight and PDI (which determine the mechanical properties of the polymer) of the PLB samples utilized in all four PB assays might also provide different physical environments for human donor periosteal cells growth. To be specific, the PLB samples (1 mol% Br) with higher molecular weight and narrow distribution of polymer (Mn = 25 kDa, Đ = 1.37, PB assay II and III) showed relative greater cell count values compared to those of the PLB samples (1 mol%) applied in PB assay I (Mn = 20 kDa, Đ = 2.03) and IV (Mn = 17 kDa, Đ =

2.17), when seeded with cells from the same donor.

For assay I, the cells failed surviving from even the 1 mol% brominated PLB scaffolds as these scaffolds could not maintain their original structural integrity. It is of importance that the scaffolds preserve the initial properties of their structure such as pore size to provide structural support and a physical environment for the cultured cells. Some scaffolds from assay I were observed partially disintegrating in the 21-day culture period. Additionally, the cell growth obtained from 1 mol% brominated PLB scaffolds in PB assay III and IV decreased after day 14, results that were very different from the increased cell growth from control TCPS in both assays. One possible explanation to these data is that the

PLB scaffolds lost their structural and biomechanical integrity as the polymer began to degrade after day 14 of culture. Thus, any cells on this type of unstable scaffold had difficulty proliferating.

192

The average molecular weight (17-25 kDa) and polydispersity (1.4-2.2) of the

PLB samples were within an applicable range for scaffolding fabrication, cell seeding, and culture. To obtain a more consistent and intact scaffolding structure, other scaffold fabrication methods can be explored such as the use of salt-leaching sponges and three-dimensional (3D) printing. Polymer scaffolds may also be fabricated by methodologies that maintain structure by connecting their constituent polymer chains chemically. A physical approach (pressure) was utilized used in this study. Moreover, the addition of appropriate coatings on the surface of scaffolds to increase their hydrophilicity or the application of growth factors that enhance cell proliferation might result in better cell attachment to the scaffolds at the point of cell seeding. A higher molecular weight (~20-25 kDa) and a narrower polydispersity (~1.37) of the PLB could also provide desired physical properties for scaffold fabrication, cell seeding, and cell culture.

This study presents for the first time a cytotoxic study of PLB scaffolds with different bromine content when seeded with human donor periosteal cells.

Conclusions can be drawn from this study that PLB scaffolds containing greater than 5 mol% bromine are cytotoxic for frozen-preserved and cultured human periosteal cells from cadaveric donors investigated here. PLB with 1 mol% bromine can be considered nontoxic when seeded with human periosteal cells and cultured for 21 days. For subsequent experimental designs, the cytotoxicity study of Si-PLA, silicon-functionalized PLA (1-5 mol% Si) scaffolds should be tested follow the protocol described in this chapter. In addition, advanced

193 fabrication techniques (salt-leaching sponges, 3D printing, and surface-coating) may also be explored to support Si-PLA materials.

194

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APPENDIX

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