Temporal Effects of Cyclic Stretching on Distribution and Gene Expression of Integrin and Cytoskeleton by Ligament Fibroblasts In Vitro

Cyclic stretching is pivotal to maintenance of the ligaments. However, it is still not clear when ligament fibroblasts switch on expression of genes related to the mechanotransduction pathway in response to cyclic stretching. This in vitro study investigated, using ligament fibroblasts, the time-dependent changes in distribution and gene expression of β1 integrin, the cytoskeleton, and collagens after the application of 6% cyclic stretching at a frequency of 0.1 Hz for 3 hr on silicon membranes. We carried out confocal laser scanning microscopy to demonstrate changes in distribution of these components as well as quantitative real-time RT-PCR to quantify levels of these gene expression both during application of cyclic stretching and at 0, 2, 6, 12, and 18 hr after the termination of stretching. Control (unstretched) cells were used at each time point. Within 1 hr of the application of stretching, the fibroblasts and their actin stress fibers became aligned in a direction perpendicular to the major axis of stretch, whereas control (unstretched) cells were randomly distributed. In response to cyclic stretching, upregulation of actin at the mRNA level was first observed within 1 hr after the onset of stretching, while upregulation of β1 integrin and type I and type III collagens was observed between 2 and 12 hr after the termination of stretching. These results indicate that the fibroblasts quickly modify their morphology in response to cyclic stretching, and subsequently they upregulate the expression of genes related to the mechanotransduction pathway mainly during the resting period after the termination of stretching.