dc.description.abstract | Salivary gland is an exocrine gland that is responsible for saliva production, absorption, and regulation. In histology, salivary is a ramified tissue formed by interconnecting branches and ducts. The arborized architecture, which is formed by the developmental process of branching morphogenesis, is essential for salivary function. Branching morphogenesis is an efficient and ubiquitous process for creating a larger cellular area for metabolic requirement in developing many glandular organs. To regenerate the glandular organ, such as salivary glands, recapitulation of branching processes may be requisite. At present, though with the progress in preserving phenotypes and promoting differentiation of salivary cells for regenerative purpose, to facilitate the morphogenesis of salivary tissue by tissue-engineering approaches has never been thoroughly explored. In this study, the possibility of promoting salivary gland morphogenesis is explored by tissue-engineering approach step by step, and the way that the chitosan-based biomaterial affects salivary tissue morphogenesis is characterized. or the purpose of recapitulating salivary gland morphogenesis, the interaction between epithelia and mesenchyme is required. During the development of ectodermal organ, the epithelium interacts with the surrounding mesenchyme to form specific phenotypes by receiving the guiding morphogenetic information. We first use murine fetal submandibular gland (SMG) model and the biomaterials which had been explored for salivary cells regeneration to study the biomaterial effects on epithelial-mesenchymal interaction and salivary morphogenesis. It is found that viability, migratory ability, and tissue interaction of salivary tissue are largely affected by different biomaterials. When salivary tissue is cultured on an appropriate substratum, the epithelial-mesenchymal interaction and the tissue-specific morphogenesis could be induced. ext, we perform global screening to find out the best cultured biomaterial which is capable of promoting morphogenesis of salivary tissue. It shows that the biomaterial effects still exist when whole salivary progenitor tissues are used in the survey. Numerous biomaterials, including synthetic, natural, biodegradable, non-biodegradable, and biological-origin polymers have been investigated. It is found that the cell behaviors as well as the tissue morphogenesis of salivary origin are biomaterial-dependent. Among them, chitosan shows a superior morphogenesis-promoting capacity, which maintains tissue viability and promotes an appropriate tissue interaction for morphogenesis. hen chitosan is prepared in the membranous form, it is capable of providing a more preferential environment for salivary gland branch formation. After culturing SMG explants on chitosan membranes, secreted extracellular matrices distribute in a reticular manner and form thicker fibers beyond the extents of cell attachment, which are not found in other biomaterials. In addition, the conditioned chitosan membranes are able to further enhance SMG branching. The fact that the promoting effects are eliminated with collagenase treatment and that type I and III collagen are identified within the adherent fibrillar extracellular matrix raise the possibility that the stimulating factors are collagen-originated. Furthermore, when chitosan is prepared in a soluble form, the morphogenesis-promoting effects are also observed. This result indicates that chitosan is a bioactive substratum for salivary tissue morphogenesis which enables active interaction between cultured salivary tissue and biomaterial. Next, the specificity of chitosan’s morphogenesis-promoting effects is further investigated. It is found that chitosan is able to promote SMG branching in a dose-dependent manner. The effect is chitosan-specific and is not reproduced by substrates with similar chemical structures or by other polymeric molecules of natural or synthetic origin. Furthermore, the branch-promoting effect is molecular weight-dependent. In addition, following digestion with lysozyme, chitinase, or chitosanase, digested chitosan is unable to reproduce the similar effects. This study clarifies the specificity and preferential activity of chitosan in enhancing branching morphogenesis of progenitor salivary tissue. ith chitosan, the morphogenesis-promoting effects of mesenchymal tissue on SMG are further enhanced. Chitosan is also competent to induce recombined SMG epithelium to form branches in the serum-free condition. In the presence of chitosan, the morphogenetic efficacy of mesenchyme-derived growth factors responsible for epithelial morphogenesis increases. The specific epithelial phenotype induced by individual growth factor is promoted by chitosan as well. Moreover, the proliferative and the chemotactic properties of these growth factors toward SMG epithelia are also reinforced by chitosan. Therefore, in orchestrating and intensifying the essential mesenchyme-derived growth factors, chitosan is versatile in mediating SMG epithelium to form a predetermined phenotype more efficiently and comprehensively. n all, the current study demonstrates that the morphogenesis of salivary tissue could be regulated by tissue-engineering approaches. It is suggested that, for salivary tissue, chitosan is a morphogenesis-regulating biomaterial. We design a novel methodology to facilitate salivary tissue morphogenesis by enhancing branch formation. The results provide a novel insight into the role of chitosan in salivary tissue morphogenesis and highlight the potential for future application in salivary tissue investigation and regeneration. | en |
dc.description.tableofcontents | 中文摘要 1bstract 6hapter 1. Introduction 10-1. Salivary gland 10-2. Xerostomia and treatment of salivary gland dysfunction 12-3. Branching morphogenesis and salivary gland development 13-4. Tissue engineering of salivary gland 15-5. Aims and study design of the dissertation 16hapter 2. The effect of biomaterials on epithelial-mesenchymal interaction and morphogenesis of progenitor salivary tissue 19-1. Introduction 19-2. Materials and Methods 22-2-1. Preparation and characterization of biomaterials 22-2-2. Culture of isolated salivary epithelia and mesenchyme 22-2-3. 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide (MTT) assay 23-2-4. Scanning electron microscopy 24-2-5. Salivary tissue recombination assay 24-2-6. Immunohistochemistry of type III collagen expression 25-2-7. Immunofluorescence of collagen expression and basement membrane formation 26-3. Results 28-3-1. Characterization of membranes 28-3-2. The biomaterial effects on the isolated salivary epithelia and mesenchyme 28-3-3. The interaction between biomaterial surface and salivary tissue 32-3-4. Type III collagen expression in the salivary epithelial-mesenchymal interface 34-3-5. De novo basement membrane synthesis in the salivary epithelial-mesenchymal interface 36-3-6. The morphogenesis of salivary tissue recombinants on biomaterials 38-4. Discussion 40hapter 3. Screening of biomaterials for branch enhancement of salivary gland 45-1. Introduction 45-2. Materials and Methods 47-2-1. Preparation and characterization of membranes 47-2-2. Organotypic culture of salivary gland 47-2-3. Degradation test 48-2-4. 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide (MTT) assay 49-2-5. Immunohistochemistry of collagen deposition 49-3. Results 51-3-1. Salivary gland morphogenesis on different biomaterials 51-3-2. Substrates degradation and associated effects on salivary gland morphogenesis 56-3-3. Salivary gland viability on different biodegradable biomaterials 58-3-4. Cell migration of salivary cells on biomaterials 60-3-5. Deposition of extracellular matrix of salivary gland on different biomaterials 62-4. Discussion 64hapter 4. The effects of chitosan on salivary gland branching morphogenesis 68-1. Introduction 68-2. Materials and Methods 70-2-1. Preparation and characterization of membranes 70-2-2. Medium and Reagents 70-2-3. Ex vivo organ culture of SMG explants 71-2-4. Scanning electron microscopy 72-2-5. Preparation and culture of SMG explants on conditioned membranes 72-2-6. Immunohistochemistry and quantification of collagen deposition 73-3. Results 75-3-1. Branching morphogenesis of SMG explants on PVA, chitosan and PC membranes 75-3-2. Extracellular matrix deposition on substrates after cultured with SMG explants 77-3-3. SMG branching on conditioned membranes 79-3-4. SMG branching on conditioned membranes treated with collagenase 81-3-5. Expression and quantification of collagen on conditioned membranes 83-3-6. Soluble chitosan promotes branching morphogenesis of SMG explants 86-4. Discussion 88hapter 5. The specificity of chitosan in promoting branching morphogenesis of salivary gland 93-1. Introduction 93-2. Materials and Methods 95-2-1. SMG ex vivo organ culture 95-2-2. Preparation of chitosan-containing culture medium 95-2-3. Preparation of chitosan monomers, analogues, and related polymeric substrates 96-2-4. Enzyme digestion assay 96-3. Results 98-3-1. Chitosan promotes SMG branching morphogenesis in a dose-dependent manner 98-3-2. Effects of GAGs on SMG branching morphogenesis 100-3-3. Effects of chitosan monomers and analogues on SMG branching morphogenesis 101-3-4. The effects of natural and synthetic polymers on SMG explant branching 103-3-5. The effect of chitosan molecular weight on SMG explants branching 105-3-6. The effect of lysozyme, chitinase, and chitosanase on the chitosan-mediating SMG branching morphogenesis 107-4. Discussion 112hapter 6. Mechanism of chitosan branch-promoting effects in salivary gland morphogenesis 116-1. Introduction 116-2. Materials and Methods 118-2-1. Submandibular glands ex vivo organ culture 118-2-2. Antisense oligodeoxynucleotides assay and the rescue experiments with fresh medium 119-2-3. Supplement of exogenous branching morphogens 119-2-4. Culture of SMG epithelium 120-2-5. Mesenchyme recombination assay 121-2-6. Cell proliferation assay 121-2-7. Chemotactic assay with HGF soaked beads 122-3. Results 124-3-1. Chitosan effects on the SMG morphogenesis with morphogen down-regulation 124-3-2. Synergism of chitosan and exogenous morphogens on SMG morphogenesis 127-3-3. The branching-promoting effects of mesenchyme were enhanced by chitosan 130-3-4. Chitosan effects on SMG epithelium morphogenesis with homotypic mesenchymal recombination in serum-free culture 132-3-5. Chitosan effects on SMG epithelial morphogenesis induced by FGF7, FGF10, and HGF 135-3-6. Chitosan effects on SMG cell proliferation induced by FGF7, FGF10, and HGF 140-3-7. The chemotactic capacity of HGF beads with chitosan 142-4. Discussion 145hapter 7. Conclusion and Perspective 150-1. Conclusion 150-2. Perspective 151eferences 153ppendix 169 | en |