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

牙周膜韧带（periodicalligament，PL）中含有成纤维细胞和成骨细胞，前者可能参与PL纤维的形成和维持，后者可能参与牙槽骨和牙骨质的形成，但目前尚未明确识别和分离每种细胞类型的特异性标志物。本研究的目的是使用流式细胞术从异质性人PL（HPL）细胞中分离碱性磷酸酶（ALP）阳性和阴性亚群，并鉴定2个亚群之间在促有丝分裂活性、成骨潜能和核心蛋白聚糖以及双糖链蛋白聚糖表达方面的功能差异。我们分别从5个HPL细胞群中分离出ALP阳性和阴性细胞。ALP阴性细胞的促有丝分裂活性高于ALP阳性细胞，而ALP阳性细胞在成骨分化培养基（ODM）中诱导ALP活性、骨特异性基因表达和矿化等成骨潜能明显强于ALP阴性细胞。在ALP阳性细胞中，双糖链蛋白聚糖的表达水平较高，但核心蛋白聚糖的表达较弱，与ALP阴性细胞相比。在ALP阳性细胞中外源性加入重组双糖链蛋白聚糖可显著增强ODM诱导的成骨细胞分化。当两个亚群与ODM一起培养时，核心蛋白聚糖的基因表达增加，而双糖蛋白聚糖的表达没有变化。这些结果表明，ALP阳性的HPL细胞具有成骨细胞样表型，而ALP阴性的HPL细胞是成纤维细胞群，具有弱的成骨潜力。此外，双糖链蛋白聚糖可以积极调节成骨细胞分化的ALP阳性HPL细胞，而核心蛋白聚糖，其表达增加成骨诱导，可能负调节矿化在两个亚群。

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.