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

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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.