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

本研究的目的是通过植入细胞固定胶原膜来促进牙齿支持组织的再生。本研究的关键阶段是细胞固定膜的形成，首先研究了细胞的特性，以满足胶原膜对牙齿支持组织的再生。将从骨髓中获取的干细胞固定在胶原膜上，培养4周，分析成骨活性。通过测定碱性磷酸酶活性和检测成骨细胞特异性基因(骨钙素、骨唾液蛋白)来评价成骨活性，并识别胶原膜上的成骨活性。为了促进成骨活性，在膜中添加了碱性成纤维细胞生长因子(BFGF)的骨形态发生蛋白(BMP)。当BMP混合在胶原膜中时，成骨活性更快。然而，骨形成活性的强度并未因BMP的加入而改变。这可能与BMP促进成骨细胞分化有关。接下来，我研究了碱性成纤维细胞生长因子的作用。在胶原膜中加入高剂量的碱性成纤维细胞生长因子后，膜的总成骨活性增加。但各细胞的成骨活性均下降。这一发现可能表明bFGF促进了细胞的生长，但抑制了成骨细胞表型的表达。在胶原膜中加入低剂量的碱性成纤维细胞生长因子后，膜的成骨活性增强。然而，如果在培养液中不添加碱性成纤维细胞生长因子，这种激活就不会发生。这可能是由于胶原膜上的bFGFs丢失所致。我将创造一种新的细胞固定膜，可以长时间保持低剂量的碱性成纤维细胞生长因子。

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.