by Cheng Sze Lok, Alfred. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 104-109). / Abstract also in Chinese. / DECLARATION --- p.i / ABSTRACT --- p.ii / ACKNOWLEDGEMENT --- p.vii / ABBREVIATIONS --- p.ix / LIST OF FIGURES --- p.x / LIST OF TABLES --- p.xii / TABLE OF CONTENTS --- p.xiii / Chapter CHAPTER ONE 226}0ؤ --- INTRODUCTION / Chapter 1.1 --- The Growth Plate / Chapter 1.1.1 --- "Function, Structure and Biochemistry of the Growth Plate" --- p.1 / Chapter 1.1.2 --- Extracellular Matrix of the Growth Plate Cartilage --- p.4 / Chapter 1.1.3 --- Vascular Supply to the Growth Plate --- p.9 / Chapter 1.1.4 --- Endochondral Ossification --- p.10 / Chapter 1.2 --- Growth Plate Damage and the Contemporary Reconstruction Models --- p.13 / Chapter 1.3 --- The 3-D Chondrocyte Pellet Culture --- p.15 / Chapter 1.4 --- The Study Plan --- p.16 / Chapter 1.5 --- The Objectives of the Study --- p.18 / Chapter CHAPTER TWO 一 --- METHODOLOGY / Chapter 2.1 --- Biosynthesis of Artificial Growth Plate using 3-D Chondrocyte Pellet Culture / Chapter 2.1.1 --- Isolation of Rabbit Costal Resting Chondrocytes --- p.19 / Chapter 2.1.2 --- Chondrocyte Monolayer Culture --- p.20 / Chapter 2.1.3 --- Three-dimensional Chondrocyte Pellet Culture --- p.20 / Chapter 2.1.4 --- Optimization of 3-D Chondrocyte Pellet Culture System --- p.20 / Chapter 2.2 --- Characterization of the 3-D Chondrocyte Pellet Culture and Monolayer Culture / Chapter 2.2.1 --- Histomorphology --- p.22 / Chapter 2.2.2 --- Alkaline Phosphatase Histochemistry --- p.22 / Chapter 2.2.3 --- Collagen Typing --- p.23 / Chapter 2.2.3.1 --- Labeling and extraction of newly synthesized collagen / Chapter 2.2.3.2 --- SDS-PAGE and autoradiography / Chapter 2.2.4 --- Growth Rate --- p.25 / Chapter 2.2.4.1 --- Total DNA content determination / Chapter 2.2.4.2 --- Thymidine incorporation assay / Chapter 2.3 --- Implantation of Artificial Growth Plate and Assessment / Chapter 2.3.1 --- Implantation of Artificial Growth Plate into Partial Growth Plate Defect Model --- p.27 / Chapter 2.3.1.1 --- Animals / Chapter 2.3.1.2 --- Surgical procedure / Chapter 2.3.1.3 --- Experimental groups / Chapter 2.3.2 --- Histology --- p.30 / Chapter 2.3.3 --- Metabolism of Artificial Growth Plate In Vivo --- p.31 / Chapter 2.3.3.1 --- Radio sulfate labeling / Chapter 2.3.3.2 --- Liquid emulsion and autoradiography / Chapter CHAPTER THREE 一 --- RESULTS / Chapter 3.1 --- Biosynthesis of Artificial Growth Plate using 3-D Chondrocyte Pellet Culture / Chapter 3.1.1 --- Morphology of the Isolated Rabbit Chondrocyte --- p.32 / Chapter 3.1.2 --- Three-dimensional Chondrocyte Pellet Culture --- p.32 / Chapter 3.1.3 --- Optimization of 3-D Chondrocyte Pellet Culture System --- p.35 / Chapter 3.2 --- Characterization of the 3-D Chondrocyte Pellet Culture and Monolayer Culture / Chapter 3.2.1 --- Histomorphology --- p.38 / Chapter 3.2.2 --- Alkaline Phosphatase Histochemistry --- p.43 / Chapter 3.2.3 --- Collagen Typing --- p.47 / Chapter 3.2.4 --- Growth Rate --- p.50 / Chapter 3.2.4.1 --- Total DNA content determination / Chapter 3.2.4.2 --- Thymidine incorporation assay / Chapter 3.3 --- Implantation of Artificial Growth Plate and Assessment / Chapter 3.3.1 --- Histology --- p.54 / Chapter 3.3.2 --- Metabolism of Artificial Growth Plate In Vivo --- p.65 / Chapter CHAPTER FOUR 一 --- DISCUSSION / Chapter 4.1 --- Optimal Condition for 3-D Chondrocyte Pellet Culture System --- p.67 / Chapter 4.1.1 --- Some Critical Characteristics of the Growth Plate --- p.68 / Chapter 4.1.2 --- Selection of Animal Model --- p.69 / Chapter 4.1.3 --- Optimization of Culturing Conditions 226}0ؤ Screening Based on Morphological Studies --- p.69 / Chapter 4.2 --- Characterization of the 3-D Chondrocyte Pellet Culture and Monolayer Culture --- p.73 / Chapter 4.2.1 --- Development of the 3-D Chondrocyte Pellet Culture --- p.73 / Chapter 4.2.2 --- Development of the Chondrocyte Monolayer Culture --- p.78 / Chapter 4.2.3 --- Comparing the 3-D Chondrocyte Pellet Culture and Monolayer Culture --- p.79 / Chapter 4.2.3.1 --- Cellular organization / Chapter 4.2.3.2 --- Terminal differentiation of chondrocytes / Chapter 4.2.3.3 --- Cell division potential / Chapter 4.2.3.4 --- Production of cartilaginous matrix / Chapter 4.3 --- Resumption of Physeal Characteristics by Artificial Growth Plate In Vivo --- p.86 / Chapter 4.3.1 --- Three Stages of In Vivo Development of the Artificial Growth Plate --- p.86 / Chapter 4.3.1.1 --- Incorporation of artificial growth plate with host tissues / Chapter 4.3.1.2 --- Growth of the artificial growth plate invivo / Chapter 4.3.1.3 --- Resumption of endochondral ossification in the artificial growth plate / Chapter 4.3.2 --- Significance of Development of the 3-D Pellet Culture on its In Vivo Development --- p.89 / Chapter 4.3.2.1 --- 3-D pellet culture processes similar extracellular matrix with host / Chapter 4.3.2.2 --- 3-D pellet culture acquires growth plate-like cellular organization and differentiation pattern / Chapter 4.3.3 --- Effect of Host Microenvironment on Artificial Growth Plate Development --- p.90 / Chapter 4.3.3.1 --- Orientation of artificial growth plate implants / Chapter 4.3.3.2 --- Evidence from development of 3-D pellet culture in longer period of culture / Chapter 4.4 --- Comparison with other Growth Plate Reconstruction Models --- p.93 / Chapter 4.4.1 --- Implantation of Biologic or Inert Fillers --- p.93 / Chapter 4.4.2 --- Physeal Transplantation --- p.94 / Chapter 4.4.3 --- Transplantation of Cartilage Allografts --- p.95 / Chapter 4.4.4 --- Transplantation of High-density Chondrocyte Culture --- p.96 / Chapter CHAPTER FIVE 一 --- SUMMARY AND CONCLUSION --- p.98 / Chapter CHAPTER SIX 一 --- FURTHER STUDIES --- p.102 / REFERENCES --- p.104

Cheung Wing-hoi. / "December 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Osteoarthritis (OA) is a disease that has significant individual, social, and economic impact worldwide. Although many etiologies lead to the eventual development of OA, one potentially treatable cause is the acute articular cartilage (AC) injury. These injuries are common and have a poor inherent healing capacity, leading to the formation of OA. In an effort to repair AC injuries several treatment strategies have been developed but none have proven completely successful. Studies examining AC tissue-engineering strategies have suggested that those with the most potential for success involve the introduction of autogenous or allogenous cells to the site of injury. These strategies are designed to encourage creation of a matrix with the appropriate characteristics of normal AC. However, development of a completely successful repair method has proven difficult because the biomechanical properties of normal AC are not easy to replicate, a cell source with the appropriate functional characteristics has not been optimized, and the problem of effective incorporation of a repair construct into the host tissue remains unresolved. In an effort to more fully understand the cartilage repair process, this work first focused on the development and utilization of an in vitro human explant model of AC to study the ability of seeded human chondrocytes to integrate into an AC defect. Further work elucidated the gene expression patterns of cultured adult human chondrocytes and human mesenchymal stem cell (MSC)-derived chondrocytes. Results from this work determined that cultured human chondrocytes were able to adhere to articular cartilage defects in a viable in vitro explant model and produce a matrix containing collagen type II. However, further work with the in vitro expanded chondrocytes revealed that these cells have increased expression of collagen type I which promotes the formation of a less durable fibrocartilagenous tissue. This unfavorable expression persisted despite placing the chondrocytes in an environment favoring a chondrocytic phenotype. Further work with MSC-derived chondrocytes demonstrated a similar and unfavorable production of collagen type I. This work represented an important first step towards a treatment for acute AC lesions but it is clear that further work to optimize the culture microenvironment is still required. / Experimental Surgery

Reproduction de : Thèse de doctorat : Odontologie. Biologie cellulaire et ingénierie tissulaire : Nantes : 2007. / Bibliogr.

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. I present a novel, single-step method of generating murine ESC (mESC)-derived chondrocytes in monolayer cultures in chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, Activin A or Wnt3a. Prolonged culture with sustained Activin A, TGFβ3 or BMP4 supplementation led to robust chondrogenic induction. A short pulse of Activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with Activin A or with Activin A followed by TGFβ3 may specifically promote articular cartilage formation. Thus, I devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. I am also in the process of creating a Col2a1 promoter-controlled, Cre-inducible reporter cell line to be used in my SF culture system using the Multisite Gateway® cloning technology. ESCs undergoing chondrogenic differentiation can be identified and quantified in HTS via the expression of fluorescent proteins. In addition, this transgenic line can be used to isolate ESC-derived chondrocytes as well as their progeny via cell sorting or antibiotic selection for in-depth characterization. The modular design of my construct system allows transgenic lines to be generated using various promoters of chondrogenic marker genes to perform parallel HTS analyses.

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. I present a novel, single-step method of generating murine ESC (mESC)-derived chondrocytes in monolayer cultures in chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, Activin A or Wnt3a. Prolonged culture with sustained Activin A, TGFβ3 or BMP4 supplementation led to robust chondrogenic induction. A short pulse of Activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with Activin A or with Activin A followed by TGFβ3 may specifically promote articular cartilage formation. Thus, I devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. I am also in the process of creating a Col2a1 promoter-controlled, Cre-inducible reporter cell line to be used in my SF culture system using the Multisite Gateway® cloning technology. ESCs undergoing chondrogenic differentiation can be identified and quantified in HTS via the expression of fluorescent proteins. In addition, this transgenic line can be used to isolate ESC-derived chondrocytes as well as their progeny via cell sorting or antibiotic selection for in-depth characterization. The modular design of my construct system allows transgenic lines to be generated using various promoters of chondrogenic marker genes to perform parallel HTS analyses.

No description available.

Cartilage development relies on the coordinated presentation of biological signals to direct chondrocyte morphology and function. This is largely controlled by perlecan, a heparan sulfate proteoglycan (HSPG). Understanding the role of perlecan and its pendant glycosaminoglycan chains (GAG) in cartilage development is essential for advances in tissue engineered cartilage replacement strategies. Perlecan was immunolocalised to the pericellular matrix of prehypertrophic and hypertrophic chondrocytes in human fetal feet. Human fetal chondrocytes were isolated and cultured in 3-dimensional (3D) scaffolds for a period of 4 weeks. Their chondrogenic phenotype, based on extracellular matrix (ECM) components, was assessed and compared to 2D cultures. Chondrocyte perlecan was immunopurified from human fetal chondrocytes grown in vitro and fetal cartilage tissue and characterised using a combination of antibody-based techniques (ELISA, Western blotting) and gel electrophoresis. The biological function of chondrocyte perlecan was determined by its ability to form ternary complexes with fibroblast growth factors (FGF) and their receptors (FGFR) using an antibody-based technique as well as a cell proliferation assay using cells expressing FGFR isotypes. Perelcan was restricted to the prehypertrophic and hypertrophic zones of cartilage. This zonal organisation of chondrocytes and chondrogenic properties, determined by their morphology and PG deposition, was recapitulated in the 3D constructs while 2D cultures displayed dedifferentiated chondrocytes. Exogenous FGF2 promoted chondrocyte proliferation, while FGF18 stimulated the synthesis of perlecan, reflecting chondrocyte hypertrophy. Chondrocyte perlecan (630kDa) contained HS, chondroitin sulfate (CS) and keratan sulfate (KS) chains. Chondrocyte perlecan formed HS dependent ternary complexes with FGF2-FGFR1c and FGF18-FGFR3c, while FGF18-FGFR3c binding to perlecan protein core was also observed. Binding of FGF18-FGFR3c to chondrocyte perlecan HS was more promiscuous than FGF2-FGFR1c. Furthermore, chondrocyte perlecan HS mediated biological activity with FGF18 via FGFR3c, which was modulated by mammalian heparanase, while no biological activity was elicited by FGF2-FGFR1c. The findings underline how perlecan and its GAGs interact with FGF and FGFR in a spatio-temporal manner to promote signalling, effecting chondrocyte behaviour and morphology in cartilage development. This insight can be utilised in tissue engineering to improve the development of biologically functional cartilage replacements.

Cartilage development relies on the coordinated presentation of biological signals to direct chondrocyte morphology and function. This is largely controlled by perlecan, a heparan sulfate proteoglycan (HSPG). Understanding the role of perlecan and its pendant glycosaminoglycan chains (GAG) in cartilage development is essential for advances in tissue engineered cartilage replacement strategies. Perlecan was immunolocalised to the pericellular matrix of prehypertrophic and hypertrophic chondrocytes in human fetal feet. Human fetal chondrocytes were isolated and cultured in 3-dimensional (3D) scaffolds for a period of 4 weeks. Their chondrogenic phenotype, based on extracellular matrix (ECM) components, was assessed and compared to 2D cultures. Chondrocyte perlecan was immunopurified from human fetal chondrocytes grown in vitro and fetal cartilage tissue and characterised using a combination of antibody-based techniques (ELISA, Western blotting) and gel electrophoresis. The biological function of chondrocyte perlecan was determined by its ability to form ternary complexes with fibroblast growth factors (FGF) and their receptors (FGFR) using an antibody-based technique as well as a cell proliferation assay using cells expressing FGFR isotypes. Perelcan was restricted to the prehypertrophic and hypertrophic zones of cartilage. This zonal organisation of chondrocytes and chondrogenic properties, determined by their morphology and PG deposition, was recapitulated in the 3D constructs while 2D cultures displayed dedifferentiated chondrocytes. Exogenous FGF2 promoted chondrocyte proliferation, while FGF18 stimulated the synthesis of perlecan, reflecting chondrocyte hypertrophy. Chondrocyte perlecan (630kDa) contained HS, chondroitin sulfate (CS) and keratan sulfate (KS) chains. Chondrocyte perlecan formed HS dependent ternary complexes with FGF2-FGFR1c and FGF18-FGFR3c, while FGF18-FGFR3c binding to perlecan protein core was also observed. Binding of FGF18-FGFR3c to chondrocyte perlecan HS was more promiscuous than FGF2-FGFR1c. Furthermore, chondrocyte perlecan HS mediated biological activity with FGF18 via FGFR3c, which was modulated by mammalian heparanase, while no biological activity was elicited by FGF2-FGFR1c. The findings underline how perlecan and its GAGs interact with FGF and FGFR in a spatio-temporal manner to promote signalling, effecting chondrocyte behaviour and morphology in cartilage development. This insight can be utilised in tissue engineering to improve the development of biologically functional cartilage replacements.

Diss. (sammanfattning) Stockholm : Karol. Inst., 2002. / Härtill 6 uppsatser.