Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

No description available.

Tissue engineering approaches have attempted to address some of the problems associated with articular cartilage defect repair, but grafts with sufficient functional properties have yet to reach clinical practice. Mechanical loads are properly controlled in the body to maintain the functional properties of articular cartilage. This inspires the inclusion of mechanical stimulation in any in vitro production of tissue engineered constructs for defect repair. This mechanical stimulation must improve the functional properties (both biochemical and structural) of engineered articular cartilage tissue. Only a few studies have applied more than two loading types to mimic the complex in vivo load/flow conditions. The general hypothesis of the present thesis proposes that the generation of functional articular cartilage substitute tissue in vitro benefits from load and fluid flow conditions similar to those occurring in vivo. It is specifically hypothesized that application of compression, shear and perfusion on chondrocyte-seeded constructs will improve their properties. It is also hypothesized that protein production of the cell-seeded constructs can be improved in a depth-dependent manner with some loading combinations. Thus, a hydromechanical stimulator system was developed that was capable of simultaneously applying compression, shear and perfusion. Functionality of system was tested by series of short-term pilot studies to optimize some of the system parameters. In these studies, agarose-chondrocytes constructs were stimulated for 2 weeks. Then, longer-term (21- 31 days) studies were performed to examine the effects of both mechanical (compression and dynamic shear) and hydromechanical (compression, dynamic shear and fluid flow) stimulation on glycosaminoglycan and collagen production. The effects of these loading conditions were also investigated for three layers of construct to find out if protein could be localized differently depth-wise. In one of the longer-term studies, the chosen mechanical and hydromechanical stimulation conditions increased total collagen production, with higher amount of collagen for hydromechanical compared with mechanical loading condition. However, their effectiveness in increasing total glycosaminoglycan production was inconclusive with the current loading regimes. The hydromechanically stimulated construct could localize higher collagen production to the top layer compared with middle and bottom layers. Some effectiveness of hydromechanical stimulation was demonstrated in this thesis. Future studies will be directed towards further optimization of parameters such as stimulation frequency and duration as well as fluid perfusion rate to produce constructs with more glycosaminoglycan and collagen.

No description available.

In order to validate the appropriate functional characteristics of cartilage, we need to systematically study and understand what constitutes normality and degradation in cartilage. This thesis provides an important step in this direction. To understand the mechanical repercussions of disruption to the matrix properties, cartilage is often artificially degraded using common enzymes. Although the process of artificial degradation does not provide an accurate representation of osteoarthritis, it can provide insight into the biomechanical properties of single matrix components by examining the behaviour of the tissue following its removal. Through histological analysis utilising the optical absorbance measurements of Safranin O stain, this work has demonstrated that for a given time and enzyme concentration, the action of Trypsin on proteoglycans is highly variable and is dependent on: * The initial distribution and concentration of proteoglycans at different depths * The intrinsic sample depth * The location in the joint space, and * The medium type. These findings provide initial data towards a mathematical model which researchers can use to optimise Trypsin treatment of articular cartilage, and therefore model degeneration in vitro with a better degree of certainty. The variability noted in the distribution and concentration of proteoglycans, and most likely the collagen network, creates a large variation in the compressive and tensile stiffness of all samples, and total failure strain energy. The average values for each of these tests indicate that a loss of proteoglycan through Trypsin treatment results in decreased compressive stiffness, increased tensile stiffness, and little change to the failure strains or total failure strain energy. Conversely, disruption to the collagen network shows increased compressive and tensile stiffness, as well as failure strain and total failure strain energy. Due to the large variation in the results for each treatment group, the average values for the treated samples fall within the range of results for normal cartilage. These values cannot therefore be used as dependable parameters to benchmark cartilage, since the parameters for artificially degraded cartilage are within the normal levels. The Yeoh and Polynomial hyperelastic laws were found to best represent the material characteristics of cartilage across the range of tested samples, regardless of differences in health and strength. The results presented here provide important insight into the biomechanical outcomes of artificial degradation and provide direction for future research in this area.

Articular cartilage disorders are a leading cause of human disability in many countries around the world. In this work, new techniques and strategies were developed to improve the quality of cartilage produced in vitro by methods of tissue engineering. Chondrocytes were isolated from the hip and knee joints of aborted human foetuses. The cells were expanded and seeded into scaffolds and the seeded scaffolds were cultured in perfusion bioreactors. The quality of the final cartilage constructs was assessed biochemically by measuring their content of glycosaminoglycan (GAG), total collagen and collagen type II and histologically by staining cross-sections of the constructs for GAG, collagen type I and collagen type II. The amount of proteoglycan released in the culture medium was also measured at regular intervals. Proteoglycans from tissue-engineered cartilage and spent culture medium were compared and analysed for degradation and capability of aggregation. During monolayer expansion, the chondrocyte differentiation indices decreased, the cell size increased and the percentage of cells present in G2/S??M phase decreased with the greatest changes occurring during the first passage. Expanding chondrocytes in PGA or PGA??alginate scaffolds produced cells with a higher level of differentiation than monolayer-expanded cells. However, PGA and PGA??alginate could not be justified as suitable systems for the routine expansion of chondrocytes mainly because of the relatively low cell proliferation obtained. Two new methods for seeding of cells into scaffolds were investigated using PGA and PGA??alginate as scaffold materials. Both methods produced high seeding efficiencies and homogeneous distribution of cells. When seeded PGA??alginate scaffolds were cultured in perfusion bioreactors, they produced good quality constructs with higher concentrations of extracellular matrix (ECM) components compared with previously described methods. However, when seeded PGA scaffolds were cultured in perfusion bioreactors, they produced small constructs of poor quality. Investigation of the effect of medium flow rate on the PGA scaffolds showed that a low flow rate was needed at the beginning of the culture to enable the cells to form a framework onto which other synthesised elements could deposit. Applying a gradual increase in medium flow rate to PGA scaffolds cultured in perfusion bioreactors solved the shrinkage problem and produced constructs with quality similar to those produced using PGA??alginate scaffolds. A novel compression bioreactor that mimicked the physiological stimulation of cartilage by joint movement was constructed. Using this bioreactor, compressed constructs showed significantly higher wet weight and higher concentrations of GAG, total collagen and collagen type II compared with non-compressed constructs.

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.

Bibliography: leaves 266-303 / xviii, 303 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 1985

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.