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

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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.