Schwartz Z, Carney D H, Crowther R S, Ryaby J T, Boyan B D
Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
J Cell Physiol. 2005 Feb;202(2):336-43. doi: 10.1002/jcp.20145.
A synthetic peptide representing the receptor-binding domain of human thrombin (TP508, also known as Chrysalin) accelerates fracture repair in rats via endochondral ossification and promotes repair of rabbit cartilage defects. To understand how this peptide might stimulate cartilage and bone formation, we employed an established in vitro model of growth plate cartilage regulation. Rat costochondral cartilage resting zone and growth zone chondrocytes were treated with 0, 0.07, 0.7, or 7 microg/ml TP508 or a scrambled peptide, TP508-SP. Proliferation ([3H]-thymidine incorporation) was examined in pre-confluent cultures; effects on cell number, alkaline phosphatase activity, [35S]-sulfate incorporation, and responsiveness to vitamin D metabolites were tested using confluent cultures. TP508 did not affect proliferation of resting zone cells but it caused a dose-dependent increase in cell number and DNA synthesis of growth zone cells. Alkaline phosphatase specific activity of resting zone cells was reduced by TP508, whereas [35S]-sulfate incorporation was increased. Neither parameter was affected in growth zone cell cultures. TP508 treatment for 24 h did not induce resting zone cells to respond to 1alpha,25(OH)2D3, either with respect to alkaline phosphatase activity or proteoglycan production. In contrast, TP508 treatment reduced the stimulatory effect of 24R,25(OH)2D3 on alkaline phosphatase but it did not alter the stimulatory effect of 24R,25(OH)2D3 on [35S]-sulfate incorporation. In cultures treated for 48, 72, or 140 h with TP508, 1alpha,25(OH)2D3 restored alkaline phosphatase activity to control levels but did not stimulate activity over levels observed in untreated control cultures. The stimulatory effect of TP508 on [35S]-sulfate incorporation was evident up to 48 h post-confluence but at later time points, proteoglycan production was comparable to that seen in control cultures, control cultures challenged with 1alpha,25(OH)2D3, and cultures treated with TP508 followed by 1alpha,25(OH)2D3. TP508-SP had no effect on any of the parameters tested. These results indicate that TP508 exerts maturation specific effects on chondrocytes in the endochondral lineage, promoting cartilage extracellular matrix synthesis over endochondral differentiation in resting zone cells and proliferation over differentiation of growth zone cells.
一种代表人类凝血酶受体结合域的合成肽（TP508，也称为Chrysalin）通过软骨内成骨加速大鼠骨折修复，并促进兔软骨缺损的修复。为了解该肽如何刺激软骨和骨形成，我们采用了一种已建立的生长板软骨调节体外模型。用0、0.07、0.7或7微克/毫升的TP508或一种乱序肽TP508-SP处理大鼠肋软骨静止区和生长区软骨细胞。在汇合前的培养物中检测增殖（[3H] - 胸腺嘧啶掺入）；使用汇合培养物测试对细胞数量、碱性磷酸酶活性、[35S] - 硫酸盐掺入以及对维生素D代谢物反应性的影响。TP508不影响静止区细胞的增殖，但它导致生长区细胞数量和DNA合成呈剂量依赖性增加。TP508降低了静止区细胞的碱性磷酸酶比活性，而[35S] - 硫酸盐掺入增加。生长区细胞培养物中的这两个参数均未受影响。TP508处理24小时并未诱导静止区细胞对1α,25(OH)2D3在碱性磷酸酶活性或蛋白聚糖产生方面产生反应。相比之下，TP508处理降低了24R,25(OH)2D3对碱性磷酸酶的刺激作用，但未改变24R,25(OH)2D3对[35S] - 硫酸盐掺入的刺激作用。在用TP508处理48、72或140小时的培养物中，1α,25(OH)2D3将碱性磷酸酶活性恢复到对照水平，但未刺激活性超过未处理对照培养物中观察到的水平。TP508对[35S] - 硫酸盐掺入的刺激作用在汇合后48小时内明显，但在后期时间点，蛋白聚糖产生与对照培养物、用1α,25(OH)2D3处理的对照培养物以及先用TP508处理然后用1α,25(OH)2D3处理的培养物中所见相当。TP508-SP对所测试的任何参数均无影响。这些结果表明，TP508对软骨内谱系中的软骨细胞发挥成熟特异性作用，促进静止区细胞软骨细胞外基质合成而非软骨内分化，并促进生长区细胞增殖而非分化。
