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

牙周膜含有可能参与牙周膜纤维形成和维持的成纤维细胞，以及具有形成牙槽骨和牙骨质潜能的成骨细胞，但识别和分离每种细胞类型的特异性标志物尚不清楚。本研究的目的是用流式细胞术从异种人牙周膜(HPL)细胞中分离碱性磷酸酶(ALP)阳性和阴性亚群，并从有丝分裂活性、成骨潜能、核心蛋白聚糖和Biglycan的表达等方面鉴定这两个亚群之间的功能差异。我们分别从5个HPL细胞群中分离出ALP阳性和阴性细胞。碱性磷酸酶阴性细胞的有丝分裂活性高于碱性磷酸酶阳性细胞，而碱性磷酸酶阳性细胞在诱导碱性磷酸酶活性、骨特异性基因表达和成骨分化介质(ODM)培养矿化等方面明显强于碱性磷酸酶阴性细胞。在ALP阳性细胞中，Biglycan的表达水平高于ALP阴性细胞，而Decorin的表达弱于ALP阴性细胞。在碱性磷酸酶阳性细胞中外源性加入重组双聚糖可显著促进ODM诱导的成骨细胞分化。当两个亚群与ODM共同培养时，核心蛋白聚糖的基因表达增加，而大聚糖的表达没有变化。这些结果表明，ALP阳性的HPL细胞具有成骨样表型，而ALP阴性的HPL细胞为成纤维细胞群，成骨能力较弱。此外，Biglycan可能正向调节ALP阳性HPL细胞的成骨分化，而Decorin在成骨诱导后表达增加，可能对这两个亚群的矿化起负向调节作用。

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.