Gerstenfeld L C, Landis W J
Department of Orthopaedic Surgery, Harvard Medical School, Boston, Massachusetts.
J Cell Biol. 1991 Feb;112(3):501-13. doi: 10.1083/jcb.112.3.501.
Conditions were defined for promoting cell growth, hypertrophy, and extracellular matrix mineralization of a culture system derived from embryonic chick vertebral chondrocytes. Ascorbic acid supplementation by itself led to the hypertrophic phenotype as assessed by respective 10- and 15-fold increases in alkaline phosphatase enzyme activity and type X synthesis. Maximal extracellular matrix mineralization was obtained, however, when cultures were grown in a nutrient-enriched medium supplemented with both ascorbic acid and 20 mM beta-glycerophosphate. Temporal studies over a 3-wk period showed a 3-4-fold increase in DNA accompanied by a nearly constant DNA to protein ratio. In this period, total collagen increased from 3 to 20% of the cell layer protein; total calcium and phosphorus contents increased 15-20-fold. Proteoglycan synthesis was maximal until day 12 but thereafter showed a fourfold decrease. In contrast, total collagen synthesis showed a greater than 10-fold increase until day 18, a result suggesting that collagen synthesis was replacing proteoglycan synthesis during cellular hypertrophy. Separate analysis of individual collagen types demonstrated a low level of type I collagen synthesis throughout the 21-d time course. Collagen types II and X synthesis increased during the first 2 wk of culture; thereafter, collagen type II synthesis decreased while collagen type X synthesis continued to rise. Type IX synthesis remained at undetectable levels throughout the time course. The levels of collagen types I, II, IX, and X mRNA and the large proteoglycan core protein mRNA paralleled their levels of synthesis, data indicating pretranslational control of synthesis. Ultrastructural examination revealed cellular and extracellular morphology similar to that for a developing hypertrophic phenotype in vivo. Chondrocytes in lacunae were surrounded by a well-formed extracellular matrix of randomly distributed collagen type II fibrils (approximately 20-nm diam) and extensive proteoglycan. Numerous vesicular structures could be detected. Cultures mineralized reproducibly and crystals were located in extracellular matrices, principally associated with collagen fibrils. There was no clear evidence of mineral association with extracellular vesicles. The mineral was composed of calcium and phosphorus on electron probe microanalysis and was identified as a very poorly crystalline hydroxyapatite on electron diffraction. In summary, these data suggest that this culture system consists of chondrocytes which undergo differentiation in vitro as assessed by their elevated levels of alkaline phosphatase and type X collagen and their ultrastructural appearance.(ABSTRACT TRUNCATED AT 400 WORDS)
已确定了促进源自胚胎鸡椎体软骨细胞的培养系统中细胞生长、肥大及细胞外基质矿化的条件。单独补充抗坏血酸会导致肥大表型，这可通过碱性磷酸酶活性和X型胶原合成分别增加10倍和15倍来评估。然而，当培养物在富含营养的培养基中生长，并补充抗坏血酸和20 mMβ-甘油磷酸时，可实现最大程度的细胞外基质矿化。为期3周的时间研究表明，DNA增加了3 - 4倍，同时DNA与蛋白质的比例几乎保持恒定。在此期间，总胶原蛋白从细胞层蛋白质的3%增加到20%；总钙和磷含量增加了15 - 20倍。蛋白聚糖合成在第12天前达到最大值，但此后下降了四倍。相比之下，总胶原蛋白合成在第18天前增加了10倍以上，这一结果表明在细胞肥大过程中胶原蛋白合成正在取代蛋白聚糖合成。对各型胶原蛋白的单独分析表明，在整个21天的时间进程中，I型胶原蛋白合成水平较低。II型和X型胶原蛋白合成在培养的前2周增加；此后，II型胶原蛋白合成下降，而X型胶原蛋白合成继续上升。IX型胶原蛋白合成在整个时间进程中一直处于检测不到的水平。I型、II型、IX型和X型胶原蛋白mRNA以及大蛋白聚糖核心蛋白mRNA的水平与其合成水平平行，这些数据表明存在合成的转录前调控。超微结构检查显示细胞和细胞外形态与体内发育中的肥大表型相似。陷窝内的软骨细胞被排列良好的细胞外基质所包围，该基质由随机分布的II型胶原纤维（直径约20纳米）和大量蛋白聚糖组成。可检测到许多囊泡结构。培养物可重复性地矿化，晶体位于细胞外基质中，主要与胶原纤维相关。没有明确证据表明矿物质与细胞外囊泡有关。电子探针微分析显示矿物质由钙和磷组成，并通过电子衍射鉴定为结晶度很差的羟基磷灰石。总之，这些数据表明，该培养系统由软骨细胞组成，通过碱性磷酸酶和X型胶原水平的升高及其超微结构外观评估，这些软骨细胞在体外发生分化。（摘要截断于400字）
