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

成纤维细胞片牙周膜替代物的研制及其在牙周再生治疗中的应用

基本信息

批准号：
15592192
负责人：
ABE Tatsuya
金额：
$ 1.92万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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.

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产生中的作用。将正常人牙龈成纤维细胞生长至汇合，然后转移到含有TGF-β、抗坏血酸和10%FBS的多层形成培养基（MFM）或仅含有10%FBS的培养基中6天。通过测量DNA含量测定细胞数量。BrdU标记细胞周期S期细胞。免疫组化法检测细胞外基质蛋白。在仅含血清的培养物中，很少的腱生蛋白-C（TN-C）在纤维连接蛋白（FN）基质中积累，并且细胞在汇合后保持生长停滞。在含有MFM的培养物中，随着TN-C沉积量的增加，密度停滞的细胞恢复DNA合成并形成多层膜。加入抗TN-C第四和第五结构域的抗体（TNfn 4 -5）可阻断融合细胞的再生长。已知Thfn 4 -5有助于TN-C与FN的结合。添加针对FN受体整联蛋白α5β1的抗体可阻断融合细胞的再生长。这些结果表明TN-C促进密度依赖性生长停滞的成纤维细胞再增殖成多个细胞层。这种TN-C的作用似乎与调节细胞与FN的相互作用有关。我们的研究对牙周膜替代物的工程化具有一定的意义。