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^&lt；[0！R]&gt；，EMD)已被临床用于牙周组织的再生。虽然EMD可以被用来促进牙周病患牙周围新附着的形成，但其潜在的生物学机制尚不清楚。本研究观察了EMD对体外培养的大鼠骨髓细胞(RBMC)行为的影响，并与从人牙周膜成纤维细胞(HPLF)获得的条件培养液蛋白(CMP)进行了比较，特别是其贴壁特性、碱性磷酸酶(ALPase)活性以及ALPase、骨涎蛋白(BSP)、骨桥蛋白(OPN)和骨钙素(OCN)的mRNA表达水平。疏水培养皿以25μg/mlEMD或25μg/mlCMP4℃包被24小时。取7周龄雄性大鼠股骨，在含有10%胎牛血清和0.25 mM抗坏血酸磷酸的α-MEM中培养。红细胞贴附并在3小时内扩散。培养在EMD涂层培养皿上的RBMC，其ALPase活性和mRNA表达水平均高于CMP涂层培养皿。此外，EMD可显著增强RBMCs OPN和OCN基因的表达。EMD没有发挥趋化活性。结果表明，红细胞对EMD和CMP的反应不同，EMD刺激RBMCs向成骨细胞分化。因此，HPLF可能与包被在牙本质表面的EMD相互作用，增殖分化。间充质细胞(RBMC)可在cMP的作用下迁移并分化为成骨细胞。

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.