| BackgroundPeriodontitis is the main factor leading to the mobility and loss of tooth. How to regenerate the alveolar bone, periodontal membrance and cementum and rebuild the normal anatomic structure and physiological function of periodontal tissue is always being an area of considerable interest. The technology of tissue engineering has been introduced into the field of periodontology which brings on a novel way to periodontal regeneration. Seed cells, growth factors and scaffold materials are three main factors in periodontal tissue engineering. But some problems are still unsolved such as the resource and potential ability of differentiation of seed cells, the bioactivity and release rate of growth factor and the biocompatibility of scaffold materials.Chitosan thermosensitive hydrogel is a novel injectable hydrogel which emerged after 2000. It has so many excellent bionomics such as physiological neutrality, transformating into semisolid hydrogel at 37℃, degradation, slow-release of macromolecule drugs, vehicle of live cells and etc. Bone marrow stromal cells are hopeful to be the seed cells of periodontal tissue engineering, because of the priorities such as multi-directional differentiation, adequate resource and easily amplified. Enamel matrix proteins are secreted by Hertwig epithelial root shealth during the development of dental germ. It has been widely used in basic research of periodontal regeneration and clinic therapy. aimThis study will attempt to prepare chitosan thermosensitive hydrogel and check its slow-release property. To study the character of compatibility, absorbability and cytocompatiblility of the chitosan thermosensitive hydrogel in vitro and vivo.methods and results1 Preparation of chitosan thermosensitive hydrogel and its slow-release property of EMPs.1mL 56%β-glycerophosphate solution was carefully added drop by drop into 9mL 2% chitosan solution. Clear and homogeneous liquid solution was obtained after tiring 10 minutes. 0.1 mL different concentration of EMPs were added into 0.9 mL chitosan/β-GP solutions. Its slow-releasing effect of EMPs was checked by coomassiebluestaining kit in vitro. It clearly indicated that the liquid solution took eight~ten minutes to become a semi-solid gel in the vicinity of 37°C. The release of EMPs in chitosan thermosensitive hydrogel lasted for more than three weeks.2 The proliferation rates and ALP activity of rat BMSCs when loaded with EMPs in chitosan thermosensitive hydrogelRat BMSCs were obtained from rat bone marrow aspiration and its multiple differention potentiasl were checked by Von Kossa and Oil Red O staining. Rat BMSCs were exposed to various concentrations of EMPs and their proliferation rates were assessed by cell cycle analysis and MTT assay. The proliferation rates and ALP activity of rat BMSCs were examined by MTT assay and ALP kit when BMSCs cultured on the scaffolds of chitosan thermosensitive hydrogel loading with 100μg/mL EMPs or without.The results showed that 50μg/mL EMPs significantly enhanced BMSCs proliferation from day 3 over the experiment. In chitosan thermosensitive hydrogel scaffolds loading 100μg/mL EMPs, both the proliferation and the ALP activity dof BMSCs were promoted.3 The biocompatibility of chitosan thermosensitive hydrogel in vivoChitosan/β-GP solutions loaded with 100μg/mL EMPs or without were subcutaneously injected into the rat. The chitosan thermosensitive hydrogel loaded with BMSCs was injected into the dorsum of nude mice.This animal experiment demonstrates that the chitosan thermosensitive hydrogel has the character of biocompatibility and absorbability and cytocombability in vivo.ConclusionEMPs loaded on the chitosan thermosensitive hydrogel exhibits significant effects on proliferation and ALP activity of rat BMSCs in vitro. Chitosan thermosensitive hydrogel has the character of cytocompability, biocompatibility and absorbability in vivo and is likely to be a new slow-release scaffold material in the regeneration of periodontal tissue.
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