Objectives: Little is known about the regenerative mechanism of human periodontal ligament (PDL) owning to the absence of cell lines and the fact that the PDL tissue consists of heterogeneous cell populations. Therefore, to understand this mechanism, first we have established human PDL cell lines immortalized by the transduction of hTERT and SV40T-antigen, named as STPLF, and reported in 83^rd IADR meeting that they maintain the characteristics of primary human PDL cells. In this study, we have developed clonal cell lines from STPLF, and characterized them. Methods: Twenty clonal human PDL cell lines were obtained by limiting dilution of STPLF. Three of them, 1-4, 1-11, and 1-24, were used in this study. These cell lines were examined on gene expressions, the expression of STRO-1 and CD146, calcification, and adipogenesis in vitro, and the phenotype of clones transplanted into SCID mouse.Results: 1-4, 1-11, and 1-24 expressed RUNX2, Col I, ALP, OPN, OCN, RANKL, OPG, Scx, Periostin, Col XII, and a-SMA mRNA, comparable to primary human normal PDL cells. Immunocytochemical data showed that CD146 was expressed in 1-4 and 1-11, and STRO-1 was expressed in 1-11 and 1-24. All of these cell lines produced calcified matrices in the presence of ascorbic acid and b-glycerophosphate. 1-11 cultured in the adipogenic induction medium showed adipocytic differentiation. When these cell lines were separately transplanted in SCID mice with b-TCP for four weeks, only 1-11 transplant produced bone-like structures around b-TCP. Conclusion: The present results suggest that a clonal human PDL cell line, 1-11, would be derived from the immature cell present in PDL, and that 1-11 could be very helpful for the study of regeneration of human PDL.

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