The Effect Of Cytoskeletal Reorganization In The Differentiation Of Tendon Stem Cells Induced By Mechanical Stretching

Tendons are constantly subjected to huge mechanical loading in vivo, so the tendon injury and tendinopathy are very common. Because of the poor regeneration ability, the normal structure and function recovery of injured tendon is a serious challenge for clinician. With the discovery of stem cells, especially the tendon stem cells(TSCs) isolated from the tendon in recent years, researchers has a new understanding to the pathogenesis of tendinopathy and the treatment of tendon injury.The same as the tendon, TSCs in tendons are also subjected to all kinds of mechanical loading in vivo. In a study in vitro, mechanically loaded TSCs underwent loading magnitude-dependent differentiation: at small mechanical stretching, TSCs differentiated into tenocytes, whereas at large mechanical stretching, TSCs differentiated into non-tenocytes(adipocytes, chondrocytes, and osteocytes) in addition to tenocytes. However, the regulation mechanism of differentiation of TSCs induced by different mechanical stretching is still not clear.Different mechanical stretching can lead to different deformation of TSCs. It is known that the maintenance of cell shape is very important for cellular function, while the key structure of maintaining the cell morphology is cytoskeleton. The cytoskeleton is a grid system composed with different protein fibers and various regulatory proteins inside cells, which plays a supporting role to the overall structure of cell. What’s more, it also plays a very important role in the following physiological processes, such as cell migration, cell growth, differention and the response to the outside environment. At present, the molecular mechanism of cell mechanical signal transduction is still lack of thorough understanding, but there is a general accepted fact that the cytoskeleton plays a crucial role in the connection and conversion of the physical-chemical signals between the internal and external environment on cell.In view of above basis, we have reason to suspect that during the differentiation of TSCs induced by mechanical stretching, cytoskeleton remodeling may play an important role. Different mechanical stretching cause different cytoskeletal changes of TSCs, and then, regulates the lineage selection of TSCs differentiation. Therefore, this study will further clarify the effect of mechanical stretching on TSCs differentiation and cytoskeleton, and the effect of cytoskeletal reorganization during the process of TSCs differentiation induced by mechanical stretching.Objective:⑴ To verify the effect of different mechanical stretching on the differentiation of TSCs.⑵ To observe the effect of cytoskeletal reorganization in the process of differentiation of TSCs induced by mechanical stretching.⑶ To investigate the role of cytoskeleton changes during the differentiation of TSCs induced by mechanical stretching.Methods:⑴ The effects of different mechanical stretching on differentiation of TSCs: setting different mechanical stretching(1 HZ, stretching load is 4% and 8%, the total cumulative stretching time is 12 h, 24 h and 60h), simulating the uniaxial cyclic stretch of TSCs in vivo, measure the mRNA expressions of tenogic differentiation-related markers(Colla 1、Tnc、Scx) and adipogenic differentiation-related markers(PPARγ、LPL、ap2) by the real-time quantitative PCR(qRT-PCR).⑵ The effects of different mechanical stretching on cytoskeletal reorganization of TSCs: setting different mechanical stretching(1 HZ, stretching load is 4% and 8%, the total cumulative stretching time is 12 h, 24 h and 60h), simulating the uniaxial cyclic stretch of TSCs in vivo, the cytoskeleton was observed through fibrous actin(F-actin) staining, and the ratio of F-actin/soluble globular actin(G-actin) was detected and calculated through Western blot.⑶ The effects of inhibition of cytoskeleton remodeling on differentiation of TSCs: After screening the applicable concentration, TSCs were treated with Cyt-D to inhibit cytoskeletal reorganization, in the presence or absence of adipogenic induction media or tenogic differentiation-induced stretching, and then dectect the m RNA and the protein expressions of tenogic differentiation-related markers and the adipogenic differentiation-related markers by qRT-PCR and Western blot.Results:⑴ The frequency of 1HZ, 4% stretching load can increase the m RNA expressions of Colla 1、Tnc and Scx on the three cumulative stretching time(P<0.05); 8% stretching load can slightly increase the mRNA expressions of above genes on the early(12h), but with the extension of stretching time(60h), it will change to decrease the m RNA expressions of above genes(P<0.05). 1HZ, 4% stretching load had no significant effect on the mRNA expressions of PPARγ、LPL、ap2 on the early(12h, 24h), but with the extension of stretching time(60h), it will significantly decrease the mRNA expressions of above genes(P<0.05); while 8% stretching load will increase the mRNA expressions of PPARγ、LPL、ap2, depending the stretching time(P<0.05).⑵ Compared to control group, 4% mechanical stimulation can induce the increasing of TSCs actin cytoskeleton polymerization, enhancement of fluorescence, and increasing of F-actin/G-actin ratio(P<0.05), depending the stretching time in a certain increase range. Drawing 12 h, 8% mechanical stimulation can enhance the F-actin fluorescence, and increase F-actin/G-actin ratio(P<0.05), but the extension of cytoskeleton F-actin decreased compared with the control group. With the extension of stretching time(24h,60h), the fluorescence intensity of cytoskeleton F-actin becomes gradually weaker, the extension of cytoskeleton F-actin decreased obviously and the shape of cell got rounder, the F-actin/G-actin ratio also decreased significantly.(P<0.05).⑶ Cyt-D can effectively inhibit the polymerization of cytoskeleton actin. In the adipogenic induction media or normal media, the inhibition of actin cytoskeleton polymerization caused by Cyt-D can increase the mRNA expressions of PPARγ、LPL、ap2(P<0.05); in the tenogic differentiation-induced stretching or normal media, The inhibition of actin cytoskeleton polymerization caused by Cyt-D can decrease the m RNA expressions of Colla 1、Tnc and Scx(P<0.05).Conclusion;⑴ Different stretching condition can make TSCs tend to different direction of differentiation. Moderate mechanical stretching load prompted tenogic differentiation of TSCs, while excessive mechanical stretching load will induce the adipogenic differentiation of TSCs.⑵Different stretching condition will lead to different cytoskeletal remodeling of TSCs. Moderate mechanical stretching load prompted polymerization of TSCs cytoskeletal actin increased, while excessive mechanical stretching load will cause the deploymerization of TSCs cytoskeletal F-actin.⑶ The inhibition of actin cytoskeleton polymerization caused by Cyt-D can promote the adipogenic differentiation of TSCs, and inhibit the tenogenic differentiation of TSCs, lead to the effect of TSCs tenogenic differentiation induced by moderate mechanical stretch disappeared.⑷ Cytoskeletal changes plays a very important role in the differentiation of TSCs induced by different mechanical stretching, which is essential for cell signal transduction between different mechanical stimulation outside the cell and the molecular changes inside the cell, and is the early signal changes regulating the mechanically induced lineage selection in TSCs. The study is not only conductive to deepen our understanding of the regulation of TSCs differentiation induced by different mechanical stretching, but also is significant for exploring the signal transduction mechanism of TSCs differentiation induced by different mechanical stretching. What’s more, it can provide experimental and theroretical basis for effective activation of TSCs differentiation. Finally, it provides a new way for the study of pathogenesis of the tendinopathy and treatment of tendon injury.