The development of periodontal ligament substitutes of fibroblast sheets and their applications for periodontal regenerative therapy

为了开发用于牙周再生治疗的牙周膜替代物，我们研究了细胞外基质(ECM)在多层成纤维细胞片生产中的作用。将正常人牙龈成纤维细胞接种于含有转化生长因子-β、抗坏血酸和10%胎牛血清的多层培养液中，或仅含10%胎牛血清的多层形成液中培养6天。通过DNA含量测定检测细胞数量。用BrdU标记处于细胞周期S期的细胞。免疫组织化学方法检测ECM蛋白的表达。在单独含血清的培养中，纤维连接蛋白(FN)基质中几乎没有TN-C积聚，融合后细胞仍处于生长停滞状态。在含有MFM的培养中，密度受阻的细胞恢复DNA合成，并在沉积越来越多的TN-C时形成多层。加入针对TN-C第四和第五结构域(Tnfn4-5)的抗体可阻断融合细胞的再生长。已知Thfn4-5有助于Tn-C与Fn的结合。FN受体整合素α-5-β-1抗体的加入阻断了融合细胞的再生长。这些结果表明，TN-C促进密度依赖的生长受阻成纤维细胞向多层细胞的再增殖。这种TN-C效应似乎与细胞与FN相互作用的调节有关。本研究对牙周膜替代物的工程化具有一定的指导意义。

To develop the periodontal ligament substitutes useful for periodontal regenerative therapy, we investigated the role of the extracellular matrix(ECM) in the production of multilayered fibroblast sheets. Normal human gingival fibroblasts were grown to confluence and were then transferred to either the multilayer formation medium(MFM) containing TGF-β, ascorbic acid and 10% FBS or the medium with 10% FBS alone for 6 days. Cell numbers were assayed by measurement of DNA content. Cells in the Sphase of cell cycle were labeled with BrdU. The ECM proteins were assayed by immunochemistry. In cultures with serum alone, little tenascin-C(TN-C) accumulated in the fibronectin(FN) matrix and the cells remained growth-arrested after confluence. In cultures with MFM, the density-arrested cells resumed DNA synthesis and formed multilayers as they deposited increasing amounts of TN-C. The re-growth ofconfluent cells was blocked by the addition of antibody against the fourth and fifth domains of TN-C(TNfn4-5). Thfn4-5 is known to contribute to the binding of TN-C to FN. The addition of antibodies against the FN receptor integrin α5β1 blocked the re-growth of confluent cells. These results indicate that TN-C promotes the re-proliferation of density-dependent growth-arrested fibroblasts into multiple cell-layers. This TN-C's effect appears to relevant to the modulation of cell interactions with FN. Our study has implications in engineering the periodontal ligament substitutes.