Structural Characters of Cell Attachment and Spreading Factors Deribed from Human Periodontal Ligament Fibroblasts

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Recently, considerable attention has been directed toward extracellular and cell growth factors to understand the processes of periodontal disease as well as the function of the cells periodontium. The cellular events of regeneration might be performed by 1) cell chemotaxis/migration, 2) cell attachment and spreading, 3) cell proliferation and differentiation, and 4) synthesis of extracellular matrix proteins. The purpose of this research projest was to clarify the characterization of cell attachment and spreading factors (CASFs) synthesized by human periodontal ligament derived fibroblast-like cells (HPLF) which were migrated from explantsby the method of Kawase T.et al (Adv.Dent.Res.1988). The confluent HPLF were cultured in the FCS-free MCDB107 medium supplemented with 0.2mM ascorbic acid-2P for 24 hours. The conditioned medium of quiencent HPLF (HPLF-CM) was collected, condenced by a ultrafiltration and applied to IEC-DEAE column eluted with 0 to 0.5M NaCl in 20mM Tris-HCl pH.8.0. … More The HPLF-CM was applied to a hydroxyapatite column eluted with 0.5M phosphate buffer pH.7.0. The CASF assay of intact and fractionated HPLF-CM were performed by the method by Kawase T.et al, using antibodies against fibronectin and vitronectin, and their receptors. The purification of CASFs were demonstrated by SDS-PAGE on fractions which were separated by IEC-DEAE column. In order to confirm that HPLF syntheze the CASFs, HPLF-CM of HPLF incubated with cycloheximide (10ug/ml) inhibited the CASF activity. GRGDS peptide containing RGD cell attachment signal peptide inhibited only approximately 30% of total CASF activity in HPLF-CM.It is suggested that CASFs may have another sequence which could be a binding domain for receptor. Trypsin cleaved the activity of CASF of HPLF-CM.Antibodies didn't alter the CASF activity of HPLF-CM.And heat treatment (80ﾟC,15 min) completely inhibited CASF activity of HPLF-CM.Thus, main domain for CASF activity should be peptide. There were several CASFs in HPLF-CM which were recognized by DEAE column and SDS-PAGE.The CASF eluted with 0.25M NaCl was estimated approximately 43KDa protein stained with a silver staining. And, one of another CASFs which was estimated 70KDa proteinn can bind to hydroxyapatite, which is essential component of calcified tissue such as cementum.Therefore, it was suggested that CASFs could be acidic proteins which may be rich of acidic amino acids and/or posttranslational modification such as phosphorylation. Less