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

釉质基质蛋白（釉质基质衍生物，Emdogain）>， EMD)已在临床上用于牙周组织再生。虽然EMD被认为可以促进牙周病牙齿周围新的附着体的形成，但其潜在的生物学机制尚不清楚。我们已经证明，HPLF分泌的蛋白具有促进组织再生的细胞附着和扩散活性因子（CASF）的效力。本研究研究了EMD对体外培养大鼠骨髓细胞（RBMC）行为的影响，并与对人牙周韧带源性成纤维细胞（HPLF）条件培养基蛋白（CMP）的影响进行了比较，特别是对其附着特性、碱性磷酸酶（ALPase）活性以及ALPase、骨唾液蛋白（BSP）、骨桥蛋白（OPN）和骨钙素（OCN） mRNA水平的影响。疏水培养皿包被EMD （25μg/ml）或CMP (25μg/ml)，在4℃下包被24小时。用热灭活牛血清白蛋白处理。从7周龄雄性大鼠股骨中获得rbmc，在含有10% FCS和0.25mM抗坏血酸磷酸的α-MEM中培养，在涂有EMD或CMP的疏水培养板上培养。红细胞在3小时内附着并扩散。与CMP包被的RBMCs相比，EMD包被培养皿培养的RBMCs表现出较高的ALPase活性和mRNA水平。此外，EMD的存在显著增强了RBMCs中OPN和OCN mrna的表达。EMD不具有趋化活性。CMP刺激HPLF的迁移活性。我们的研究结果表明，RBMCs对EMD或CMP的反应不同，EMD刺激了RBMCs的成骨分化。因此，HPLF可能与包裹在牙本质表面的EMD相互作用，增殖分化。间充质细胞（RBMC）可通过CMP迁移分化为成骨细胞。

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.