Asahina I, Sampath T K, Nishimura I, Hauschka P V
Children's Hospital Medical Center, Boston, Massachusetts.
J Cell Biol. 1993 Nov;123(4):921-33. doi: 10.1083/jcb.123.4.921.
Osteogenetic protein-1 (OP-1), a member of the TGF-beta superfamily, induces endochondrial bone formation at subcutaneous sites in vivo and stimulates osteoblastic phenotypic expression in vitro. Primary cultures of newborn rat calvarial cells contain a spectrum of osteogenic phenotypes ranging from undifferentiated mesenchymal osteoprogenitor cells to parathyroid hormone (PTH)-responsive osteoblasts. We examined whether treatment of this cell population with recombinant human osteogenic protein-1 could induce chondrogenesis in vitro. Markers of chondroblastic versus osteoblastic differentiation included alcian blue staining at pH 1, alkaline phosphatase-specific activity, osteocalcin radioimmunoassay, and expression of collagen mRNAs. 6 d of treatment (culture days 1-7) with 4-100 ng OP-1/ml caused dose-dependent increases in alcian blue staining intensity and alkaline phosphatase activity (4.7- and 3.4-fold, respectively, at 40 ng/ml), while osteocalcin production decreased twofold. Clusters of round, refractile, alcian blue-stained cells appeared by day 3, increased in number until day 7, and then became hypertrophic and gradually became less distinct. Histochemically, the day 7 clusters were associated with high alkaline phosphatase activity and became mineralized. mRNA transcripts for collagen types II and IX were increased by OP-1, peaking at day 4, while type X collagen mRNA was detectable only on day 7 in OP-1-treated cultures. Delay of OP-1 exposure until confluence (day 7) amplifies expression of the normal osteoblastic phenotype and accelerates its developmental maturation. In contrast, early OP-1 treatment commencing on day 1 strongly amplifies chondroblastic differentiation. In the same protocol, TGF-beta 1 alone at 0.01-40 ng/ml fails to induce any hypertrophic chondrocytes, and in combination with OP-1, TGF-beta 1 blocks OP-1-dependent chondroinduction. OP-1 is believed to act on a subpopulation of primitive osteoprogenitor cells to induce endochondrial ossification, but does not appear to reverse committed osteoblasts to the chondrocyte phenotype.
骨形成蛋白-1（OP-1）是转化生长因子-β超家族的成员之一，可在体内皮下部位诱导软骨内成骨，并在体外刺激成骨细胞表型表达。新生大鼠颅骨细胞的原代培养物包含一系列成骨表型，范围从未分化的间充质骨祖细胞到甲状旁腺激素（PTH）反应性成骨细胞。我们研究了用重组人骨形成蛋白-1处理该细胞群体是否能在体外诱导软骨形成。成软骨细胞与成骨细胞分化的标志物包括pH 1时的阿尔新蓝染色、碱性磷酸酶特异性活性、骨钙素放射免疫测定以及胶原蛋白mRNA的表达。用4 - 100 ng OP-1/ml处理6天（培养第1 - 7天）导致阿尔新蓝染色强度和碱性磷酸酶活性呈剂量依赖性增加（40 ng/ml时分别增加4.7倍和3.4倍），而骨钙素产生减少两倍。到第3天出现圆形、折光性强、阿尔新蓝染色的细胞簇，数量持续增加直至第7天，然后变得肥大并逐渐变得不那么明显。组织化学上，第7天的细胞簇与高碱性磷酸酶活性相关并开始矿化。II型和IX型胶原蛋白的mRNA转录本被OP-1增加，在第4天达到峰值，而X型胶原蛋白mRNA仅在OP-1处理的培养物中的第7天可检测到。将OP-1暴露延迟至汇合（第7天）可增强正常成骨细胞表型的表达并加速其发育成熟。相反，从第1天开始早期OP-1处理强烈增强成软骨细胞分化。在相同方案中，单独使用0.01 - 40 ng/ml的转化生长因子-β1未能诱导任何肥大软骨细胞，并且与OP-1联合使用时，转化生长因子-β1会阻断OP-1依赖性软骨诱导。OP-1被认为作用于原始骨祖细胞亚群以诱导软骨内骨化，但似乎不会使已分化的成骨细胞逆转为软骨细胞表型。
