Interactions between cell adhesion and siganaling molecules in mechanical stress response of chondrocytes

生物力学力是决定骨骼组织的形成和分化的主要表观遗传因素，并且可以通过细胞粘附到细胞外基质（ECM）来转导。为了验证逐步张力在早期软骨形成过程中被转换成分子信号的假设，我们开发了一种研究软骨细胞增殖和分化的培养系统。将大鼠胚胎第12天的肢芽显微解剖并解离成细胞，然后将其在硅树脂膜上微团培养并维持长达6天。从第3天开始对这些培养物施加逐步张力。分析软骨特异性标志物和非软骨形成标志物的表达模式和水平的时间过程，并与非拉伸对照培养物中的那些进行比较。在张力条件下，阿辛蓝染色显示软骨形成的速率和程度明显降低，共聚焦激光扫描显微镜显示细胞明显拉伸。定量，II型胶原蛋白和聚集蛋白聚糖的表达显着抑制20至30%和10至20%，分别在12小时的张力负荷后，并通过3天的培养保持这种差异。相反，I型胶原和纤连蛋白的表达在整个实验期间保持相对恒定。当GRGDSPK（Gly-Arg-Gly-Asp-Ser-Pro-Lys）肽抑制细胞-ECM附着时，软骨形成标记物表达的这种下调被完全拯救。我们的结论是，逐步张力抑制软骨形成通过整合素在胚胎肢芽间充质，并提出，从生物力学刺激的信号转导可能介导的细胞-ECM粘附。

Biomechanical forces are major epigenetic factors that determine the form and differentiation of skeletal tissues, and may be transduced by cell adhesion to the extracellular matrix (ECM). To test the hypothesis that stepwise tension forces are transduced into molecular signals during early chondrogenesis, we developed a culture system studying the proliferation and differentiation of chondrocytes. Rat embryonic day-12 limb buds were microdissected and dissociated into cells which were then micro-mass cultured on a silicone membrane and maintained for up to 6 days. Stepwise tension was applied to these cultures from day 3. The time course of the expression pattern and level of cartilage-specific markers and non-chondrogenic markers were analyzed and compared with those in non-stretched control cultures. Under tension conditions, Alcian blue staining showed an apparent decrease in the rate and extent of chondrogenesis, and confocal laser scanning microscopy demonstrated apparent stretching of the cells. Quantitatively, type II collagen and aggrecan expression were significantly inhibited by 20 to 30% and 10 to 20%, respectively, after 12 hrs of tension force loading, and this difference was maintained through 3 days of culture. In contrast, the expression of type I collagen and fibronectin remained relatively constant throughout the experimental period. This down-regulation in the expression of chondrogenic markers was completely rescued when cell-ECM attachment was inhibited by GRGDSPK (Gly-Arg-Gly-Asp-Ser-Pro-Lys) peptide. We conclude that stepwise tension inhibits chondrogenesis through integrins in embryonic limb bud mesenchyme, and propose that signal transduction from biomechanical stimuli may be mediated by cell-ECM adhesion.