| 研究課題/領域番号 |
18K09680
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| 研究機関 | 新潟大学 |
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研究代表者 |
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| 研究分担者 |
加来 賢 新潟大学, 医歯学系, 准教授 (30547542)
魚島 勝美 新潟大学, 医歯学系, 教授 (50213400)
井田 貴子 新潟大学, 医歯学総合病院, 医員 (60790285)
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| 研究期間 (年度) |
2018-04-01 – 2021-03-31
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| キーワード | Methylcellulose / scaffolds / Tissue Regeneration |
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| 研究実績の概要 |
Cell-based therapy for bone augmentation requires the transplant of cells into the damaged site, generally a collagen base scaffold is used. However, due to its low biomechanical stiffness and rapid biodegradation, Methylcellulose (MC) scaffolds are an alternative. During this time, we prepare the MC-based scaffolds. MC 500mg mixed with 50 mg of gelatin (G) 50 mg agarose (AG) in 25 mL dimethyl sulfoxide (DMSO) then 5g of ground-NaCl was added followed by vigorous stirring. After mixing, a cross-linking agent carbonyldiimidazole (CDI) was added into the mix.
We observe that MC can completely dissolve in DMSO with a gentle oscillation. G and AG require a stronger oscillation and longer time to completely dissolve. MC at 2% solution gels at approximately 48 °C, by adding G and GA the gelation temperature can be adjusted. NaCl however, does not completely dissolve in DMSO. NaCl crystals showed 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. We found that when pouring the solution, equal distribution of NaCl in the wells is difficult. Due to the high concentration of salt, freezing point is low, therefore Salt, DMSO and unreacted chemicals need to be removed with distilled water before freezing at -80C. Lyophilization overnight produced porous scaffolds, however, thickness was considerably reduced compared with the gel, more trials with different thickness are required. As a result a porous MC scaffold was achieved.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
The Lyophilization equipment has been damaged and a new replacement is been considered.
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| 今後の研究の推進方策 |
Microstructure of the scaffolds will study by scanning electron microscope (SEM) images to determine pore size and architecture. For cell isolation and culture, MSC’s cells will be isolated from Wistar rat’s 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. Cell Migration assay; were cells will be seeded in Cellulose scaffolds for 1, 3 and 7 days and migration area per unit length will be determine. Cell differentiation; in which cells will be seeded in the cellulose scaffolds and cultured under 3 different conditions, Control medium (Cont), Ostoblast (OB) medium, and MSC’s medium to observe differentiation onto osteogenic cells. Alkaline phosphatase (ALP) activity; cells will be cultured for 7, 14 and 21 days, fixed and stained with fast violet B and naphthol AS-MX ALP solution. 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.
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| 次年度使用額が生じた理由 |
Due to the damage of the Lyophilization equipment, currently, a replacement of the equipment is been considered. We would like to invest the remaining amount in the purchase of the replacement.
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