| 研究実績の概要 |
Currently, there has been an increase interest in the use of cell-based therapy to facilitate bone regeneration in the treatment of dental implant and alveolar bone defects. Scaffold in particular, has proven to further influence bone formation, delivery and organization of the cells in bone defects. During this period, we further prepare Methylcellulose (MC) based scaffolds. In short: MC (500mg) was mixed with gelatin (50 mg) and agarose (50 mg) in 25 mL dimethyl sulfoxide (DMSO) and 5g ground-NaCl by vigorous stirring for 4 h. Cross-linking agent (carbonyl diimidazole, CDI) was added and vigorously agitated for 15 min. The solutions will be incubated in ice bath for scaffold formation, and then washed with distilled water 5 times to remove NaCl, DMSO and unreacted reagents. Finally, these samples were frozen and lyophilized. During preparation, we observed that NaCl crystals to influence the scaffold porosity. Samples with no salt showed no porosity and remained in a gel like state. 35mm, 24 and 96 well dishes were used as molds to obtain consistent sizes and shapes. Moreover, the high concentration of salt, influenced the freezing point, therefore Salt, DMSO and unreacted chemicals had to be removed with distilled water before freezing at -80C. MC scaffold were achieved, but it reduced thickness is an issue to be addressed and invitro cell culture is in progress.
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| 今後の研究の推進方策 |
For in-vitro cell culture MSC’s cells will be isolated from mice femur for Bone Marrow cells (BMC’s), and Calvarial site for Periosteum derived cells (PDC’s); then cultured until 80% confluency and expanded to perform the following assays.
In-vitro: Cell Migration assay; were cells will be seeded in the scaffolds and migration area per unit length will be determine. Cell differentiation; under 3 different conditions, Control medium (Cont), Ostoblast (OB) medium, and MSC’s medium to observe differentiation onto osteogenic cells. Alkaline phosphatase (ALP) activity. Picrosirius red staining; after 7, 14 and 21 days of culture, cells will be fixed with 4% formaldehyde and stained with picrosirius red solution to observe collagen formation. Alizarin red staining; after 3 weeks culture in the different culture conditions, cell seeded cellulose scaffolds will be fixed with 4% formaldehyde and stained with alizarin red solution for quantitative measurement of mineralization.
In-vivo: To investigate the extent of MSC’s transplanted to MC-scaffold in bone regeneration; angiogenesis and endogenous stem cell homing will be analyzed by utilizing calvarial bone defect model.
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| 次年度使用額が生じた理由 |
The incurring amount was planned to be used for international presentations, however, due to the constraints and restrictions of the coronavirus state of emergency and travel restrictions, presentations had to be canceled. The remaining amount will be used on other international presentations next year.
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