published_or_final_version / Dentistry / Master / Master of Orthodontics

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Thesis (M.Orth.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 137-147) Also available in print.

The in-vitro culture of human embryonic stem and keratinocyte cells has great potential to revolutionise the therapeutics industry. Indeed it is hoped that these cells will provide a superior alternative to current tissue and organ transplantation. However, both of these cell types require animal and/or donor products for their successful maintenance in-vitro. This requirement results in a significant risk of cross contamination from the animal or donor products to either the primary keratinocyte or hES cells. These potentially transplantable cells therefore need to be cultured in an environment free from animal or donor products to remove the risk of contamination to the patient. The ideal growth conditions must comprise of two attributes; firstly they must be free from animal or donor products, and secondly the culture system must be fully defined. Recently, it was discovered that an extra-cellular matrix protein, vitronectin, could be used in conjunction with growth factors and growth factor-binding proteins (VN:GF combination), to promote enhanced cell migration and growth through the co-activation of integrin and growth factor receptors. Given that growth factors and serum are clearly important in supporting the in-vitro cultivation of mammalian cells, and that vitronectin is an abundant protein in serum, I hypothesised that these VN:GF combinations could be translated into a serum-free medium that would support the serial propagation and self renewal of primary keratinocytes and hES cells. As reported in this thesis I have developed a defined, serum-free media for the culture of these cells that incorporates the VN:GF combinations. While the two media differ slightly in their compositions, both support the serial, undifferentiated expansion of their respective cells types. Together, this represents a significant advance that will ultimately facilitate the therapeutic use of these cells. However, the in-vitro expansion of these cells in these new media still required the presence of a feeder cell layer. In view of this I aimed to explore the in-vitro micro-environment of primary keratinocytes using a novel proteomic approach in an attempt to find candidate factors that could be used in conjunction with the VN:GF media to replace both serum and the feeder cells. The proteomic approach adopted examined the secretion of proteins into the defined, minimal protein content VN:GF media when the feeder cells were cultured alone, as well as in co-culture with primary keratinocytes. This strategy allowed assessment of proteins/factors that are secreted in response to both autocrine and paracrine cellular interactions and revealed a number of candidate factors that warrant further investigation. Ultimately this proteomic information and the associated new insights into the keratinocyte in-vitro culture microenvironment may lead to the development of a culture system for these cells that is not reliant on either a feeder cell layer or serum for their successful propagation. Moreover, it is likely that this will also be relevant to the feeder cell-free propagation of hES cells. This has obvious advantages for the culture of primary keratinocytes and hES cells in that it will allow a safe defined culture system for the undifferentiated propagation of these cells. This will facilitate the generation of cells and tissues free from xenogeneic and allogeneic contaminants, thus ensuring any therapeutics developed from these cell types are approved for therapeutic applications and importantly, will minimise risks to patients.

No description available.

Além das funções mecânicas, sabe-se que o colágeno do tipo I é um dos componentes mais ativos na modulação das atividades biológicas que ocorrem na matriz extracelular (MEC) tais como a adesão celular, a coagulação e a mineralização do osso. Estes eventos ocorrem nos limites do período D, que é provavelmente o domínio funcional que mais prevalece em relação à transferência da informação extracelular para o interior da célula. O objetivo deste trabalho foi estudar as correlações existentes entre as atividades biológicas da MEC e as características estruturais do colágeno do tipo I, principalmente em relação à distribuição topográfica dos aminoácidos (aa) ácidos, básicos e hidrofóbicos. A metodologia empregada foi baseada em informações já existentes sobre a estrutura colágeno do tipo I e suas atividades relacionadas com a MEC. Os resultados mostraram que aa ácidos, básicos e hidrofóbicos formam duas regiões distintas. Em uma delas, aa ácidos e básicos se alojam em áreas delimitadas por aa hidrofóbicos; na outra, aa básicos, ácidos e hidrofóbicos formam aglomerados localizados principalmente nas proximidades dos limites Overlap:Gap. Exceto para os eventos de natureza estrutural são as regiões do colágeno do tipo I mais envolvidas na modulação das atividades da MEC, com a interface Overlap:Gap funcionando como um divisor do controle destas atividades. Enquanto 83,2% das atividades de adesão estão localizadas na zona do Overlap, 93,3% das atividades de natureza estruturais ocorrem a partir da zona limite Overlap:Gap e em direção à zona Gap. Embora nenhuma correlação possa ter sido estabelecida para a resultante de carga, sugere-se que a participação do colágeno do tipo I nas atividades da MEC, possa estar associada a diferenças de interações dos ligantes (carga, massa molecular e hidrofobicidade) com gradientes eletrostático e hidrofóbico existentes ao longo do período D. / Besides mechanical function, it is known today that type I collagen plays a major role in the modulation of many of the activities occurring in the extra-cellular matrix (ECM) such as cell adhesion, coagulation cascade and mineral deposition during bone formation. All these events occur by interaction of cells and ligands within the D-period of collagen microfibrill assembly, which is perhaps the most prevalent module of ECM information in the body. The purpose of this work was the study of correlations between the biological activities of ECM and the structural features of collagen associated with the distribution of acidic, basic and hydrophobic amino acids (aa) within the D period. The methodology used was based on existing information with respect to the structure of type I collagen and its function with respect to ECM activities. The results showed that acidic, basic and hydrophobic aa are found in two major distinct regions: one in which acidic and basic aa are located in areas defined by hydrophobic aa barriers; the other in which acidic, basic and hydrophobic aa forming clusters located preferentially in the boundaries of the Overlap:Gap zones. Except for the structural event, these clusters are the major sites for the interaction involving cell adhesion. The results also suggest that the Overlap:Gap interface function as a borderline for collagen interactions with ECM since 83,2% of the activities related to cell adhesion are located in the Overlap region while 93,3% of the activities of structural nature are enclosed within the limiting zone of Overlap:Gap to the direction of the Gap zone. Although no correlation could be established for net charge along the D period it is suggested that type I collagen participation in the activities of the ECM may also be associated to differences in ligand interactions (charge, molecular mass and hydrophobicity) with the electrostatic and the hydrophobic gradients that exists within the D period.

Development of the secondary palate (SP) is a complex event despite the small area it encompasses. Problems with SP development can lead to a cleft palate, which is one of the most common birth disorders. The matrix metalloproteinases (MMPs) are required for proper SP development, but a functional role for any one of them remains unknown. MMP-25 is a candidate MMP to have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the Mmp-25 gene. The purpose of this thesis is to investigate gene expression of Mmp-25 in the developing mouse SP, whether it has a functional role in mouse SP development and begin to identify factors potentially upstream of Mmp-25 expression.<p> Mmp-25 mRNA and protein is found at all SP developmental stages in mice with highest expression at embryonic day (E) 13.5 when analyzed by quantitative real-time PCR and western blotting. Immunohistochemistry localizes MMP-25 protein primarily to the plasma membranes of palate shelf epithelial cells with secondary expression in apical mesenchymal cells. Mmp-25 knockdown with siRNA in palatal cultures resulted in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium in vitro. Mmp-25 mRNA and protein levels are significantly decreased in vitro when cultured palate shelves are incubated in growth medium with 5 ìg/ml of a TGFâ3-neutralizing antibody. Mmp-25 gene expression is highest at E12.5 and E13.5, which corresponds to increasing palate shelf growth downward alongside the tongue. Immunohistochemistry localized MMP-25 protein expression predominantly in the epithelium of the palate shelves, but also in areas of the mesenchyme that were immediately adjacent to the epithelium and apical in location. Knockdown of Mmp-25 expression resulted in palate shelf fusion being impaired and significant medial edge epithelium remaining in contacted areas. Bioneutralization of TGFâ3 resulted in a significant decrease in Mmp-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development by removing extra-cellular matrix barriers to increased palate shelf growth and place its expression downstream of TGF-â3 signaling. This is the first research to present a role for a single MMP in mouse SP development.

Development of the secondary palate (SP) is a complex event despite the small area it encompasses. Problems with SP development can lead to a cleft palate, which is one of the most common birth disorders. The matrix metalloproteinases (MMPs) are required for proper SP development, but a functional role for any one of them remains unknown. MMP-25 is a candidate MMP to have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the Mmp-25 gene. The purpose of this thesis is to investigate gene expression of Mmp-25 in the developing mouse SP, whether it has a functional role in mouse SP development and begin to identify factors potentially upstream of Mmp-25 expression.<p> Mmp-25 mRNA and protein is found at all SP developmental stages in mice with highest expression at embryonic day (E) 13.5 when analyzed by quantitative real-time PCR and western blotting. Immunohistochemistry localizes MMP-25 protein primarily to the plasma membranes of palate shelf epithelial cells with secondary expression in apical mesenchymal cells. Mmp-25 knockdown with siRNA in palatal cultures resulted in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium in vitro. Mmp-25 mRNA and protein levels are significantly decreased in vitro when cultured palate shelves are incubated in growth medium with 5 ìg/ml of a TGFâ3-neutralizing antibody. Mmp-25 gene expression is highest at E12.5 and E13.5, which corresponds to increasing palate shelf growth downward alongside the tongue. Immunohistochemistry localized MMP-25 protein expression predominantly in the epithelium of the palate shelves, but also in areas of the mesenchyme that were immediately adjacent to the epithelium and apical in location. Knockdown of Mmp-25 expression resulted in palate shelf fusion being impaired and significant medial edge epithelium remaining in contacted areas. Bioneutralization of TGFâ3 resulted in a significant decrease in Mmp-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development by removing extra-cellular matrix barriers to increased palate shelf growth and place its expression downstream of TGF-â3 signaling. This is the first research to present a role for a single MMP in mouse SP development.

Various members of the insulin-like growth factor (IGF) family of growth factors are highly expressed in bone tissue and are vitally important for the normal development and function of bone. Recent studies have shown that IGF-I can associate with the extra-cellular matrix proteins vitronectin (VN) and fibronectin (FN) via IGF binding protein-5 (IGFBP-5). Furthermore, when these complexes are pre-bound to a tissue culture surface they can stimulate enhanced responses in epithelial cell types in vitro. More recently, transforming growth factor-beta 1 (TGF-β1), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) have also been shown to interact with VN and to elicit functional responses in various cell types. Taken together, these findings indicate that exploitation of the adhesive properties of these ECM proteins might allow immobilisation of various growth factors at the culture surface. This may provide a novel means of coating engineered biomaterial constructs with agents which can elicit specific functional effects in therapeutically important cells, such as those used in cell-based therapeutics for the replacement and / or regeneration of damaged bone tissue. Since both VN and FN are also important matrix components of bone, this study sought to investigate the hypothesis that select pre-bound combinations of these matrix proteins and growth factors could also stimulate functional responses in bone cells and the therapeutically important so called mesenchymal stem cells. Thus it is reported here that pre-bound combinations of VN, IGFBP-5 and IGF-I or FN IGFBP-5 and IGF-I significantly stimulate cell migration in the osteoblast-like SaOS-2 cells. While, VN, IGFBP-5 and IGF-I stimulated cell proliferation over 72 hr, FN, IGFBP-5 and IGF-I did not. Moreover, I found that VN, IGFBP-5 and IGF-I could facilitate alkaline phosphatase (ALP) expression in SaOS-2 cells. VN, FN and EGF on the other hand could sustain SaOS-2 cells for up to 12 days in culture, but could not sustain ALP expression; hence it is possible that these cells may have entered a state of quiescence in response to this treatment. Extending these studies to cells derived from clinical samples, pre-bound combinations of VN / IGFBP-5 / IGF-I were not able to support initiation of human mesenchymal stem cell (hMSC) cultures. Nevertheless, VN alone in serum free media stimulated substantial metabolic activity and protein synthesis in hMSCs once the cultures were established. Moreover, the addition of IGFBP-3 or -5 together with IGF-I can enhance the response to levels equivalent to that observed with 10% FCS. I also report that the responses to VN and TGF-β1 are synergistic and stimulate greater hMSC metabolic activity than 10% FCS. Interestingly, hMSCs cultured in IGF-I or TGF-β1 and low concentrations of VN aggregated, an effect that was not observed when higher concentrations of VN were used. I hypothesise that this aggregation effect was due to endogenous protease activity, and therefore examined MMP-2 and 9 activity in hMSC conditioned media. Both pro-MMP-2 and pro-MMP-9 were constitutively expressed by hMSCs but there was no evidence of the active forms in the conditioned media, indicating that neither IGF-I nor TGF-β1 affect MMP-2 or -9 expression or activation in serum-free media. However, hMSC conditioned media could degrade IGFBP-5, suggesting that there is proteolytic activity within the conditioned media which may impact on the function of ECM / growth factor components in serum-free media settings. Thus, while ECM and growth factors may stimulate desirable responses in therapeutically important cells in serum-free culture, the role that endogenously expressed proteases have on the efficacy of such media supplements needs to be examined closely. Taken together, the studies reported in this thesis provide proof of principle data indicating that select combinations of ECM proteins and growth factors could be utilised in bone tissue engineering applications. This may be achieved for example, as a biomaterial coating, or could form the basis of a viable alternative media supplement for the serum-free culture of hMSCs.

Além das funções mecânicas, sabe-se que o colágeno do tipo I é um dos componentes mais ativos na modulação das atividades biológicas que ocorrem na matriz extracelular (MEC) tais como a adesão celular, a coagulação e a mineralização do osso. Estes eventos ocorrem nos limites do período D, que é provavelmente o domínio funcional que mais prevalece em relação à transferência da informação extracelular para o interior da célula. O objetivo deste trabalho foi estudar as correlações existentes entre as atividades biológicas da MEC e as características estruturais do colágeno do tipo I, principalmente em relação à distribuição topográfica dos aminoácidos (aa) ácidos, básicos e hidrofóbicos. A metodologia empregada foi baseada em informações já existentes sobre a estrutura colágeno do tipo I e suas atividades relacionadas com a MEC. Os resultados mostraram que aa ácidos, básicos e hidrofóbicos formam duas regiões distintas. Em uma delas, aa ácidos e básicos se alojam em áreas delimitadas por aa hidrofóbicos; na outra, aa básicos, ácidos e hidrofóbicos formam aglomerados localizados principalmente nas proximidades dos limites Overlap:Gap. Exceto para os eventos de natureza estrutural são as regiões do colágeno do tipo I mais envolvidas na modulação das atividades da MEC, com a interface Overlap:Gap funcionando como um divisor do controle destas atividades. Enquanto 83,2% das atividades de adesão estão localizadas na zona do Overlap, 93,3% das atividades de natureza estruturais ocorrem a partir da zona limite Overlap:Gap e em direção à zona Gap. Embora nenhuma correlação possa ter sido estabelecida para a resultante de carga, sugere-se que a participação do colágeno do tipo I nas atividades da MEC, possa estar associada a diferenças de interações dos ligantes (carga, massa molecular e hidrofobicidade) com gradientes eletrostático e hidrofóbico existentes ao longo do período D. / Besides mechanical function, it is known today that type I collagen plays a major role in the modulation of many of the activities occurring in the extra-cellular matrix (ECM) such as cell adhesion, coagulation cascade and mineral deposition during bone formation. All these events occur by interaction of cells and ligands within the D-period of collagen microfibrill assembly, which is perhaps the most prevalent module of ECM information in the body. The purpose of this work was the study of correlations between the biological activities of ECM and the structural features of collagen associated with the distribution of acidic, basic and hydrophobic amino acids (aa) within the D period. The methodology used was based on existing information with respect to the structure of type I collagen and its function with respect to ECM activities. The results showed that acidic, basic and hydrophobic aa are found in two major distinct regions: one in which acidic and basic aa are located in areas defined by hydrophobic aa barriers; the other in which acidic, basic and hydrophobic aa forming clusters located preferentially in the boundaries of the Overlap:Gap zones. Except for the structural event, these clusters are the major sites for the interaction involving cell adhesion. The results also suggest that the Overlap:Gap interface function as a borderline for collagen interactions with ECM since 83,2% of the activities related to cell adhesion are located in the Overlap region while 93,3% of the activities of structural nature are enclosed within the limiting zone of Overlap:Gap to the direction of the Gap zone. Although no correlation could be established for net charge along the D period it is suggested that type I collagen participation in the activities of the ECM may also be associated to differences in ligand interactions (charge, molecular mass and hydrophobicity) with the electrostatic and the hydrophobic gradients that exists within the D period.