Investigation of cells related with ligament formation in cultured human periodontal ligament cells

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Periodontal ligament (PL) contains fibroblastic cells, which may participate in formation and maintenance of PL fibers, and osteoblastic cells with a potential to generate alveolar bone and cementum, however, the specific markers for recognition and isolation of each cell type have not been clarified. The purpose of this study was to isolate alkaline phosphatase (ALP)-positive and -negative subpopulations from heterogeneous human PL (HPL) cells using flowcytometry and to identify functional difference between the 2 subsets in terms of mitogenic activity, osteogenic potential and expression of decorin as well as biglycan. We separately isolated ALP-positive and -negative cells from each of 5 populations of HPL cells. The mitogenic activity of ALP-negative cells was higher than that of ALP-positive cells, while ALP-positive cells showed remarkably stronger osteogenic potential, such as induction of ALP activity, bone-specific gene expression and mineralization by culturing with osteogenic differentiation medium (ODM), than ALP-negative cells. In ALP-positive cells, expression level of biglycan was higher, but decorin expression was weaker, as compared with ALP-negative cells. The ODM-induced osteoblastic differentiation was significantly enhanced by adding recombinant biglycan exogenously in ALP-positive cells. When the 2 subpopulations were cultured with ODM, gene expression of decorin was increased, whereas biglycan expression had no change. These results suggest that ALP-positive HPL cells have osteoblast-like phenotype, while ALP-negative HPL cells are fibroblastic cell population with weak osteogenic potential. Further, biglycan may positively regulate osteoblastic differentiation in ALP-positive HPL cells, while decorin, whose expression is increased by osteogenic induction, may negatively regulate mineralization in both subpopulations.