Dynamic Aspect of Mechanical Stretching on the Synthesis of Cell Attachment and Spreading Factors of Human Periodontium Derived Cells.

牙周膜是一种典型的非钙化结缔组织，位于牙骨质和牙槽骨之间，起着支持牙齿的作用。PL参与了牙周组织的发育、组织再生以及咬合和咀嚼等物理应力的重塑过程。从人牙周组织中分离出人牙周膜成纤维细胞（HPLF），在含5%FCS和0.25mM抗坏血酸-2-P的D-MEM培养基中进行传代培养。将HPLF与不含FCS的MCDB 107培养24小时。收集HPLF的条件培养基（HPLF-CM），通过超滤法浓缩，并通过阴离子交换层析（IEC-DEAE柱）进行分级。SDS-PAGE显示，IEC-DEAE的一个组分中含有一个分子量为40 kDa的蛋白质作为主要成分。使用蛋白质测序系统的氨基酸序列分析将40 kDa蛋白质鉴定为骨粘连蛋白（ON）（BNT/BM-40）。拉伸可增加ON的表达。在胶原凝胶中培养的HPLF通过凝胶收缩显著降低ON的合成。包被在疏水孔上的ON具有CASF活性。用0.28M NaCl（IEC-DEAE）洗脱的级分含有与Octyl-Sepharose柱结合的CASF。将1%CHAPS洗脱组分用于SDS-PAGE，并使用单克隆抗ON抗体通过Western印迹法鉴定。羟基磷灰石是牙骨质的主要矿物成分，ON与羟基磷灰石有很强的相互作用。将釉基质蛋白和HPLF-CM包被在疏水孔中，直接与HPLF相互作用，可增加HPLF中ON mRNA的表达，HPLF产生的ON可能是CASF的一种，在牙周组织再生中起重要作用。

The periodontal ligament (PL) is a typical noncalcified connective tissue that functions on tooth support and lies between cementum and alveolar bone which are mineralized tissues. PL is involved in the remodeling processes with the development of periodontium, tissue regeneration and physical stresses such as occlusion and mastication. Human periodontal ligament derived fibroblasts (HPLF), which were obtained by the explants of human periodontal tissue, were subcultured in D-MEM supplemented with 5% FCS and 0.25mM ascorbic acid-2-P.At the confluent condition. HPLF were cultured with FCS-free MCDB107 for 24 hrs. The conditioned medium of HPLF (HPLF-CM) was collected, condensed by an ultrafiltration method and fractionated with an anion exchange chromatography (IEC-DEAE column). The cell attachment and spreading factor assay was performed by using a hydrophobic well coated with proteins in HPLF-CM.SDS-PAGE revealed that one of the fraction of IEC-DEAE contained a protein with molecular weight of 40kDa as a major component. Amino acid sequence analysis using a protein sequencing system identified 40kDa protein as osteonectin (ON) (SPARC/BM-40). The expression of ON was increased by stretching. HPLF cultured in a collagen gel dramatically decreased the synthesis of ON by gel contraction. ON coated on a hydrophobic well has CASF activity. The fraction eluted with 0.28M NaCl (IEC-DEAE) contained CASFs which binded to Octyl-Sepharose column. 1%CHAPS eluted fraction was applied to SDS-PAGE and identified by the Western blotting method using monoclonal anti-ON antibody. ON was strongly interacted with hydroxyapatite which is major mineral component of cementum. The expression of ON mRNA HPLF was increased by the enamel matrix protein and HPLF-CM which were coated on a hydrophobic well and directly interacted with HPLF.ON produced by HPLF could be one of CASF and play an important role of in periodontal regeneration.