Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Amsterdam, The Netherlands.
Cells Tissues Organs. 2010;192(2):116-24. doi: 10.1159/000289585. Epub 2010 Feb 24.
Stable integration of collagenous tissue-engineered constructs to surrounding solid devices can be accomplished by coating the solid surfaces with exogenous alkaline phosphatase (ALP). We showed previously that coating of culture well surfaces with the enzyme in combination with the presence of its substrate beta-glycerophosphate (beta-GP) induces mineral deposition at the interface of matrix and surface, thereby preventing matrix detachment. In this study the effect of such mineral-inducing conditions on differentiation of human periodontal ligament (PDL) fibroblasts into osteoblasts/cementoblasts was analyzed in three-dimensional collagen gels. Mineral-inducing conditions decreased collagen type I gene expression and induced dentin matrix protein 1 (DMP1; a marker of late osteoblasts/cementoblasts) gene expression by fibroblasts. DMP1 protein was detected in some fibroblasts only in mineralizing gels. Exogenous ALP released high levels of inorganic phosphate from beta-GP. Addition of inorganic phosphate alone induced DMP1 gene expression, which could be prevented by blocking phosphate entry into fibroblasts by foscarnet. We concluded that mineralizing conditions induced by exogenous ALP affect the phenotype of PDL fibroblasts. The fibroblasts are stimulated to express the late osteoblast/osteocyte marker protein DMP1, which is mediated by uptake of inorganic phosphate into the cells. The enzyme-mediated mineral deposition may thus facilitate enhanced integration of collagenous tissue-engineered constructs to devices or implants in vitro.
胶原组织工程构建体与周围实体设备的稳定整合可以通过在固体表面涂覆外源性碱性磷酸酶（ALP）来实现。我们之前曾表明，在存在其底物β-甘油磷酸（β-GP）的情况下，将酶涂覆在培养皿表面上会诱导基质与表面之间的矿物质沉积，从而防止基质脱落。在这项研究中，分析了在三维胶原凝胶中，这种诱导矿物质形成的条件对人牙周韧带（PDL）成纤维细胞分化为成骨细胞/成牙骨质细胞的影响。诱导矿物质形成的条件降低了胶原 I 型基因的表达，并诱导成纤维细胞中牙本质基质蛋白 1（DMP1；成骨细胞/成牙骨质细胞的晚期标志物）基因的表达。仅在矿化凝胶中，一些成纤维细胞中检测到 DMP1 蛋白。外源性 ALP 从β-GP 中释放出高水平的无机磷酸盐。单独添加无机磷酸盐即可诱导 DMP1 基因的表达，而通过膦甲酸酯阻断磷酸盐进入成纤维细胞可以阻止这种表达。我们得出结论，外源性 ALP 诱导的矿化条件会影响 PDL 成纤维细胞的表型。成纤维细胞被刺激表达晚期成骨细胞/成骨细胞标志物蛋白 DMP1，这是通过细胞内无机磷酸盐的摄取介导的。因此，酶介导的矿物质沉积可能有助于增强胶原组织工程构建体与体外设备或植入物的整合。
