Gerstenfeld L C, Chipman S D, Kelly C M, Hodgens K J, Lee D D, Landis W J
Department of Orthopedic Surgery, Harvard Medical School, Boston, Massachusetts 02115.
J Cell Biol. 1988 Mar;106(3):979-89. doi: 10.1083/jcb.106.3.979.
A newly defined chick calvariae osteoblast culture system that undergoes a temporal sequence of differentiation of the osteoblast phenotype with subsequent mineralization (Gerstenfeld, L. C., S. Chipman, J. Glowacki, and J. B. Lian. 1987. Dev. Biol. 122:49-60) has been examined for the regulation of collagen synthesis, ultrastructural organization of collagen fibrils, and extracellular matrix mineralization. Collagen gene expression, protein synthesis, processing, and accumulation were studied in this system over a 30-d period. Steady state mRNA levels for pro alpha 1(I) and pro alpha 2 collagen and total collagen synthesis increased 1.2- and 1.8-fold, respectively, between days 3 and 12. Thereafter, total collagen synthesis decreased 10-fold while mRNA levels decreased 2.5-fold. In contrast to the decreasing protein synthesis after day 12, total accumulated collagen in the cell layers increased sixfold from day 12 to 30. Examination of the kinetics of procollagen processing demonstrated that there was a sixfold increase in the rate of procollagen conversion to alpha chains from days 3 to 30 and the newly synthesized collagen was more efficiently incorporated into the extracellular matrix at later culture times. The macrostructural assembly of collagen and its relationship to culture mineralization were also examined. High voltage electron microscopy demonstrated that culture cell layers were three to four cells thick. Each cell layer was associated with a layer of well developed collagen fibrils orthogonally arranged with respect to adjacent layers. Fibrils had distinct 64-70-nm periodicity typical of type I collagen. Electron opaque areas found principally associated with the deepest layers of the fibrils consisted of calcium and phosphorus determined by electron probe microanalysis and were identified by electron diffraction as a very poorly crystalline hydroxyapatite mineral phase. These data demonstrate for the first time that cultured osteoblasts are capable of assembling their collagen fibrils into a bone-specific macrostructure which mineralizes in a manner similar to that characterized in vivo. Further, this matrix maturation may influence the processing kinetics of the collagen molecule.
一种新定义的鸡颅盖成骨细胞培养系统，该系统经历成骨细胞表型的时间顺序分化并随后矿化（Gerstenfeld, L. C., S. Chipman, J. Glowacki, and J. B. Lian. 1987. Dev. Biol. 122:49 - 60），已针对胶原蛋白合成的调节、胶原纤维的超微结构组织以及细胞外基质矿化进行了研究。在该系统中，对30天内的胶原蛋白基因表达、蛋白质合成、加工和积累进行了研究。在第3天和第12天之间，前α1（I）和前α2胶原蛋白的稳态mRNA水平分别增加了1.2倍和1.8倍，总胶原蛋白合成也相应增加。此后，总胶原蛋白合成下降了10倍，而mRNA水平下降了2.5倍。与第12天后蛋白质合成减少相反，从第12天到第30天，细胞层中总积累的胶原蛋白增加了6倍。对前胶原加工动力学的研究表明，从第3天到第30天，前胶原转化为α链的速率增加了6倍，并且新合成的胶原蛋白在培养后期更有效地整合到细胞外基质中。还研究了胶原蛋白的宏观结构组装及其与培养矿化的关系。高压电子显微镜显示培养的细胞层有三到四个细胞厚。每个细胞层都与一层发育良好的胶原纤维相关联，这些纤维相对于相邻层呈正交排列。纤维具有典型的I型胶原的64 - 70纳米周期性。通过电子探针微分析确定，主要与纤维最深层相关的电子不透明区域由钙和磷组成，并通过电子衍射鉴定为结晶度很差的羟基磷灰石矿物相。这些数据首次证明，培养的成骨细胞能够将其胶原纤维组装成一种骨特异性的宏观结构，其矿化方式与体内特征相似。此外，这种基质成熟可能会影响胶原蛋白分子的加工动力学。
