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

Abstract

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.

Orientation-specific responses to sustained uniaxial stretching in focal adhesion growth and turnover

“…The mechanosensitivity of FA assembly and downstream signaling is well documented, and thus they are prime candidates for mediating cellular responses to stretch (26). Indeed, the cytoskeletal reorientation that occurs in response to cyclic stretch requires several proteins that localize to FA, including integrins (27), zyxin (28), paxillin (25), Src, and p130 Crk-associated substrate (p130Cas) (29), and involves sliding reorganization of FAs (6). However, the effects of sustained stretch on FA organization and dynamics have not been explored.…”

Potential of Tissue-Engineered Ligament Substitutes for Ruptured ACL Replacement

“…Cyclic stretching is an example of biomechanical stimuli that increase collagen synthesis by cells in the tissue, thereby strengthening the resultant scaffold made of aligned fibre bundles (Goulet et al, 2000, Kaneko et al, 2009). Thus, the ACL substitute has already reached a first step towards matrix synthesis, and the process can continue to progress in response to the in vivo strains that are applied to the graft in the post-transplantation environment (Goulet et al, 2004).…”

Static and Cyclic Mechanical Loading of Mesenchymal Stem Cells on Elastomeric, Electrospun Polyurethane Meshes

“…With regard to the latter, recent studies have shown that 10 min to 3 hr of cyclic uniaxial loading with tensile strains of 2.5-15% and frequencies of 0.1-1 Hz induce the collagens I and III expression by fibroblasts and mesenchymal stem cells (MSCs) on twodimensional surfaces [22,23], in three-dimensional constructs [9][10][11], and on braided or woven constructs [21]. In addition, Teh et al-using electrospun fibers on woven mesh supports-showed that cyclic mechanical stretch and fiber orientation act synergistically to stimulate expression of the connective tissue ECM genes collagen I and III, and tenascin-C by MSCs [24].…”