Regeneration of tooth supporting tissue by the implantation of cell fixed collagen membrane

本研究旨在通过植入细胞固定胶原膜促进牙支持组织再生。本研究的关键阶段是细胞固定膜的制备，首先研究了细胞的特性，以满足胶原膜再生牙齿支持组织的需要。将从骨髓中收获的干细胞固定在胶原膜中，并将膜培养四周以分析骨形成活性。通过碱性磷酸酶活性的测量和成骨细胞特异性基因（骨钙素、骨唾液酸蛋白）的检测来估计骨形成活性，并且I识别胶原膜中的骨形成活性。为了促进骨形成活性，在膜中加入碱性成纤维细胞生长因子（bFGF）的骨形态发生蛋白（BMP）。当BMP与胶原膜混合时，骨形成活性出现更快。然而，骨形成活性的强度并没有通过添加BMP而改变。这可能与BMP促进成骨细胞分化有关。接下来，我研究了bFGF的作用。高浓度bFGF与胶原膜混合后，膜的总成骨活性增加。但各细胞的成骨活性降低。这一发现可能表明bFGF促进细胞生长，但抑制成骨细胞表型的表达。低浓度bFGF与胶原膜混合后，胶原膜的成骨活性增强。然而，如果在培养基中不添加bFGF，则不会发生这种激活。这可能是由于bFGF从胶原膜上丢失所致。本实验旨在研制一种能长期维持低浓度bFGF的细胞固定膜。

The aim of this study is to promote the regeneration of tooth supporting tissue by the implantation of'cell fixed collagen membrane. The crucial stage in this study is creation of cell fixed membrane, and I first investigated features of cells to satisfy the regeneration of tooth supporting tissue by the collagen membrane. The stem cells harvested from bone marrow were fixed in the collagen membrane, and the membrane was cultured for four weeks to analyze bone forming activity. The bone forming activity was estimated by the measurement of alkaline phosphatase activity and detection of osteoblast-specific gene (osteocalcin, bone sialoprotein), and I recognized bone forming activity in the collagen membrane. To promote the bone forming activity, Bone Morphogenetic Protein (BMP), of basic Fibroblast Growth Factor (bFGF) was added in the membrane. When BMP was mixed in the collagen membrane, the bone forming activity appeared faster. However, the intensity of bone forming activity did not change by the addition of BMP. This might be due to the enhancement of osteoblastio differentiation by BMP. Next I examined the effect of bFGF. The total bone forming activity of the membrane increased when high dose of bFGF was mixed in the collagen membrane. However the bone forming activity of each cell decreased. This finding might indicate that bFGF accelerated cell growth, but suppressed the expression of osteoblastic phenotype. When low dose of bFGF was mixed in the collagen membrane, the bone forming activity of membrane increased. However this activation did not occurred if bFGF was not added in the culture medium. This might be due to the loss of bFGFfrom the collagen membrane. I am going to create the new cell fixed membrane which could maintain the low dose of bFGF for a long time.