Effects of Mechanical Stress on Proliferation and Differentiation of Chondrocytes

机械应变是调节软骨细胞增殖和分化的重要因素。软骨细胞对机械应变的反应可能具有分化依赖性。软骨生成ATDC 5细胞响应于胰岛素分化为肥大软骨细胞，并调节II型、X型胶原、聚集蛋白聚糖、ALP和PTH/PTHrP受体mRNA表达。本研究以1×10^6个细胞/板的密度将ATDC 5细胞接种于塑料板上，在含5%FBS和10 μg/ml胰岛素的F12/DMEM培养基中培养，观察ATDC 5细胞对机械应力的反应。ATDC 5甲苯胺蓝染色观察形态学变化。我们使用了一个。使用四点弯曲的体外细胞加载系统。应变量为4200 μ应变，频率为0.5Hz。为了评估机械应变对ATDC 5细胞的影响，使用北方印迹分析测定II型、X型胶原、聚集蛋白聚糖、ALP和PTH/PTHrP受体mRNA的表达。在第4天至第28天开始加载后6至24小时，提取总RNA并与各种cDNA探针杂交。ATDC 5细胞如之前报道的那样开始软骨形成分化。II型胶原mRNA在第4天首次表达，X型胶原和聚集蛋白聚糖在第14天首次表达。在第4天或第7天没有观察到机械应变的影响，但机械应变上调II型，X型胶原和聚集蛋白聚糖mRNA的时间依赖性的方式之后。然而，ALP和PTH/PTHrP受体的表达没有增加。这些结果表明，分化的，但不是未分化的ATDC 5细胞上调II型，X型胶原蛋白，和聚集蛋白聚糖的转录在响应机械应变。

Mechanical strain is an important factor to regulate chondrocyte proliferation and differentiation. Response of chondrocyte to mechanical strain could be differentiation dependent. Chondrogenic ATDC5 cells differentiate into hypertrophic chondrocytes in response to insulin, and regulates type II, X collagen, aggrecan, ALP, and PTH/PTHrP receptor mRNA expression. In the present study, we examined whether ATDC5 cells respond to mechanical strain in differentiation specific manner.ATDC5 cells were cultured on plastic plates at 1×10^6 cell/plate in the medium of F12/DMEM containing 5% FBS and 10 μg/ml insulin. ATDC5 were stained with toluidine-blue to observe the morphologic change. We utilized an. in vitro cell loading system using four-point bending. The magnitude of strain was 4200 μstrain and the frequency was 0.5 Hz. To assess the effects of mechanical strain on ATDC5 cells, the expression of type II, X collagen, aggrecan, ALP, and PTH/PTHrP receptor mRNAs were determined using northern blot analysis. Six to 24 hours after beginning of loading on day 4 through day 28, total RNA was extracted and hybridized with various cDNA probe.ATDC5 cells initiated chondrogenic differentiation as reported before. Type II collagen mRNA expression was firstly observed on day 4, type X collagen and aggrecan on day 14. The effects of mechanical strain were not observed on day 4 or 7, but mechanical strain up-regulates type II, X collagen, and aggrecan mRNA in a time-dependent manner thereafter. However, there were no increase in ALP and PTH/PTHrP receptor expression.These results indicate that differentiated, but not undifferentiated ATDC5 cells up-regulates type II, X collagen, and aggrecan transcripts in response to mechanical strain.