| 研究実績の概要 |
For dental implant placement a certain minimum amount of bone is required; however, critical-size bone defects are challenging and current treatment approaches still require much improvement. Recently, a novel procedure for tissue regeneration involves the use of microRNAs to regulate cell differentiation. miRNA play an important role regulating local cell to cell communication in situ and also at distant sites.
To elucidate the role of extracellular miRNAs in bone regeneration, bone marrow mesenchymal stem cells (BMCs) were isolated from 4-week-old Wistar rat’s femur and tibia; then expanded in 10 cm culture dish until 80% confluence. Osteogenic differentiation of BMCs in two different conditions, “Control” and “Induced”, was performed as described before. To assess cell differentiation, Alkaline phosphatase (ALP) activity, using a solution of fast violet B and Naphthol AS-MX and Alizarin red staining, to observe the calcium deposits in the cultures, were performed.
To further determine the effect of miRNA in cell differentiation, gene expression analysis using a quantitative realtime PCR using selected primer-probes were perform. Also, miRNAs profiling of the MSCs in Control and Induced conditions were performed in order to determine the relevant miRNA associated with osteogenic differentiation.
A selection of miRNA will be added to collagen gel scaffolds and its effect on bone regeneration, angiogenesis and endogenous stem cell homing will be analyzed
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
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理由
Microarray-based expression analysis is a strategy for identifying candidate miRNAs that correlate with biological pathways.
A screening of osteogenic-related miRNAs in MSC is required. Presently, miRNA Microarray Screening results are insufficient to properly select relevant osteogenic related miRNA.
Osteogenic gene expression of selected miRNA is required to analyze, compared and determine differences in gene expression.
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| 今後の研究の推進方策 |
To further investigate the effect of specific miRNAs in bone regeneration; either miRNA transfected or non-treated control cells will be prepared. Angiogenesis and endogenous stem cell homing will be analyzed by calvarial bone defect model. A circular defect of 5 mm in diameter will be performed on calvarias of 8-week old male Wistar rats.
Cell containing collagen gel as scaffolds will be transplanted to the defects and analyze after 1, 2, 4 and 8 weeks.
Analysis of the samples will be performed according to the guidelines using a CosmoScan GX MicroCT and Quantum FX uCT imaging system (Rigaku) machine to obtain images of the defect area. 3D reconstruction and analysis will be done using a specialized software (Tri-Bon software, Ratoc, Japan).
Samples will be then cut into histological section using a microtome to a thickness of 5 um and placed onto glass slides.
Angiogenesis will be analyzed on histological preparations using anti-Endomucin and anti-CD31 antibody, while MSC recruitment using anti-CD29, anti-CD90 and anti-Stro-1 antibodies.
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