Basic Research for Tissue Engineering of Periodontal Tissue Regeneration : Validity of Enamel Matrix Proteins and Periodontal Ligament Fibroblast Synthesized Proteins.

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Enamel matrix proteins (enamel matrix derivative, Emdogain^<【○!R】>, EMD) have been clinically used regenerate the periodontal tissues. Although it is clamed that EMD can be used to promote new attachment formation around periodontally invoved teeth, the underlying biological mechanism is not understood. We have demonstrated that the secreted proteins of HPLF have a potency for the cell attachment and spreading activity factor (CASF) promoting tissue regeneration.The present study investigated the effect of EMD on the behavior of rat bone marrow cells(RBMC) in vitro and compared it with that for the conditioned medium protein(CMP) obtained from human periodontal ligament-derived fibroblasts (HPLF), especially their attachment property, alkaline phosphatase(ALPase) activity and the mRNA levels for ALPase, bone sialoprotein(BSP), Osteopontin(OPN), and osteocalcin(OCN). The hydrophobic dishes were coated EMD (25μg/ml) or CMP (25μg/ml) for 24 hrs at 4℃. The dishes were treated with heat inactivated BSA.RBMCs were obtained from the femora of 7-week-old male rats and cultured in α-MEM containing 10% FCS and 0.25mM ascorbic acid phosphate on hydrophobic culture plates coated with EMD or CMP. RBMCs attached and spread within 3 hours. RBMCs cultured onto EMD coated dishes expressed high ALPase activity and mRNA levels when compared with CMP coated ones. Furthermore, the expression of OPN and OCN mRNAs of RBMCs was significantly enhanced by the presence of EMD. EMD did not play a chemotactic activity. CMP stimulate the migration activity of HPLF.Our results indicated that RBMCs responded differently to EMD or CMP, and that EMD stimulated osteogenic differentiation of RBMCs. Therefore, HPLF may interacted with EMD coated on dentin surface, proliferate and differentiate. And mesenchymal cells (RBMC) couled be migrated by CMP and differentiated to osteogenic cells.