| Tendon/ligament is a kind of connective tissue subjected to complicated mechanical microenvironment which is controlling their growth, development and regeneration. However, it is always injured in the daily life, and the cure of the injuries is a serious challenge for clinician.Mesenchymal stem cells (MSCs) are able to differentiate into a lot of cell lineages. For their advantages, such as abundant source, fast proliferation, easy to transfer exogenous gene, low immune reaction, they become one of the research focus of tissue engineering, gene therapy or cell therapy. At present, it was reported that MSCs were able to differentiate into tenocytes by mechanical stimuli, and to be used for tendon/ligament repair. It brings new hopes for the appliacation of tissue engineering for tendon/ligament repair. However, the mechanical stretch-induced tenogenic differentiation is not well understood and the mechanotransduction mechanism is still unclear. Therefore, it becomes an important research content of in the furthure.This study further investigated the effect of mechanical stretch on tenogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs), and the mechanotransduction mechanism during the process. To search for an appropriate stretch parameter, cyclic stretch device (ST-140) was used to load mechanical stretch on hMSCs which growth on febronectin-coated silicon chambers. After stretch, tendon-related gene and protein expressions were analyzed by real-time RT-PCR and western blot. After that, to examine the effect of RhoA/ROCK, cytoskeletal organization and FAK (Focal adhesion kinase) during mechanical stretch-induced tenogenic differentiation, Y-27632, ctochalasin D and PF 530228 (PF 228) were used to inhibit RhoA/ROCK, cytoskeletal organization and FAK, respectively.The main experiments and major results are as follows:?hMSCs multi-lineages differentaiton potentialTo examine the multi-lineages differentaiton potential of hMSCs used in this study, hMSCs were cultured in OS (osteogenic) induction media, AD (adipogenic) induction media or CH (chondrogenic) induction media for 21 d. Alizarin red S, oil red O and acian blue staining were conducted to analyzed the OS induction, AD induction and CH induction, respectively. The staining results suggest that hMSCs could differentiate into osteogenic, adipogenic and chondrogenic. ?Mehcanical stretch-induced tenogenic differentiation of hMSCsAimed to search for appropriate stretch parameters, hMSCs were loaded with different mechanical stretch (0.1 Hz, 15% strain, 24 h and 48 h; 0.1 Hz, 10% strain, 24 h and 48 h; 0.1 Hz, 5% strain, 24 h and 48 h; 1.0 Hz, 15% strain, 24 h and 48 h; 1.0 Hz, 10% strain, 24 h and 48 h; 1.0 Hz, 5% strain, 24 h and 48 h), gene expressions of collagen type I (Col I), collagen type III (Col III), tenascin-C (TNC) and scleraxis (SCX), and protein expressions of Col I and TNC, were anaylized by real-time RT-PCR and western blot. The results showed that only the mechanical stretch (1.0 Hz, 10% strain, 24 h and 48 h) can promote the gene expressions of Col I, Col III, TNC and SCX,and the protein expressions of Col I and TNC. It suggests that 1.0 Hz, 10% strain mechanical stretch can induce tenogenic differentiation of hMSCs. Concomitant with this process, hMSCs were induced to realign vertically to the direction of stretch, and the trend of realignment was dependent on the strain, frequence and duration of mechanical stretch.?Effects of Y-27632, cytochalsin D and PF 228 on mechanical stretch-induced changes in morphology of hMSCsY-27632, cytochalsin D and PF 228 had important effect on mechanical stretch-induced changes in cytoskeletal dynamics which defines cell shape. The changes in density of actin fibers and vertical alignment of actin fibers to the direction of stretch, are due to the adaptation to mechanical loads which can resist and reduce experienced mechanical force. Inhibition of RhoA/ROCK resulted in lacking actin fibers, failure in trailing-edge retraction and leaving a long tail behind cells, and showed stellate-like morphology. However, in the presence of Y-27632 and mechanical stretch resulted in discontinuous actin fibers and very short actin fibers in the cell edges, and no significant changes in cell shape and favorite alignment with respect to the direction of stretch. Cytochalasin D treatment resulted in disruption of actin fibers organization and circular cell shape. When the cytochalasin D treated cells were exposure to stretch, the actin fibers were further disrupted and the cell shape became more circuler. PF 228 incubation resulted in more circular cell shape and shorter actin fibers. Interestingly, PF 228 incubation with mechanical stretch resulted in stellate-like cells, shorter actin fibers and rough edges, and even resulted in generation of cell debris. In addition, there were also no specific alignments of cells and actin fibers with respect to the direction of stretch. These data suggest that mechanical stretch facilitates actin fibers formation and induces realignment of cells by inducing actin fibers perpendicular aligning to the direction of stretch which can resist and adapt to the force balance. And the stretch-induced realignment is needed activation of RhoA/ROCK, intact cytoskeltal organization and FAK phosphorylation.?Effects of Y-27632, cytochalsin D and PF 228 on mechanical stretch-induced changes in FAK phosphorylation and tenogenic differentiation of hMSCsWestern blot showed that with experience of stretch for 30 min, phosphorylation of FAK at Tyr397 was increased up to 1.966±0.167 times. When cells were treated by Y-27632, phosphorylation of FAK at Tyr397 was decreased to 1.081±0.1 times. When the assembling of F-actin was blocked by cytochalasin, phosphorylation of FAK at Tyr397 was decreased to 1.095±0.099 times. Treatment with PF 228 resulted in dramatical decrease of phosphorylation of FAK at Tyr397. It was reduced to 0.395±0.075 times. It indicates RhoA/ROCK and cytoskeletal organization are essential to mechanical stretch-activated FAK phosphorylation.Except for the influence on morphology and stretch-activated FAK phosphorylation, Y-27632, cytochalsin D and PF 228 have important effect on gene expressions and even to differentiation. Y-27632, cytochalsin D or PF 228 inhibited the mechanical stretch-induced gene expressions of Col I, Col III, TNC, SCX, EphA4, Eya2 and Six1, and the protein expressions of Col I, Col III and TNC. It suggests that RhoA/ROCK, cytoskeleton and FAK are necessary for mechanical stretch-induced tenogenic diferentiation of hMSCs. Moreover, RhoA/ROCK, cytoskeletal organization and FAK interacted with each other in the stretch-induced tenogenic differentiation of hMSCs.In summary, we can infer that RhoA/ROCK, cytoskeletal dynamics and FAK interacte with each other and compose a signaling network to sense mechanical environment. The signaling network converts mechanical stretch into biochemical signaling, and then triggers stretch-induced tenogenic differentiation. Inhibiting any component of the signaling network, it fails to convert mechanical stretching into biochemical signaling and the stretch-induced tenogenic differentiation will be aborted. It provides novel view into the mechanisms of stretch-induced tenogenesis and supports the therapeutic potential of hMSCs. |
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