The present study used a novel micro-strain stress loading system designed by our research group[13–15]. The osteoarthritic chondrocyte proliferation and functional marker expression were detected after subjecting cells to varying micro-strain stress during culture. The results showed that the application of adequate micro-strain stress could promote osteoarthritic chondrocyte proliferation and functional marker expression. The cyclin D1 and collagen II mRNA and protein expression increased significantly under micro-strain stress and peaked in the 10% micro-strain group. With increasing micro-strain stress, the cytoskeleton exhibited rearrangement. However, the above-described changes were suppressed when the Rock inhibitor Y-27632 was used before the application of micro-strain stress.
Articular cartilage is in a complex physiological and mechanical environment in the body[16, 17]. The mechanical stimuli are important factors in maintaining the normal structure and function of articular cartilage. Xu et al. applied intermittent cyclic mechanical tension (0.5 Hz, 10% deformation, 4h/d, 6d/week) to rat endplate chondrocytes, and showed that tension stimulation promotes the proliferation of endplate chondrocytes. Thomopoulos et al. applied cyclic tensile loading tension (1 Hz, 10% deformation, 7d) to bone marrow stromal cells in a 3D in vitro model, showing that the cyclic tensile strain could promote spindle cell formation, increase collagen I and glycosaminoglycan synthesis. Therefore, we believe that an appropriate micro-strain stress could promote the proliferation and matrix anabolism of chondrocytes, and also maintain the normal structure and function of chondrocytes. However, micro-strain stress beyond the chondrocytes bearing range might inhibit cell proliferation, damage structural function and integrity of cells, and further damage the articular cartilage. Such changes weaken the ability of damaged cartilage tissue to withstand mechanical stimulation and may further aggravate the effects of mechanical stimulation leading to a vicious cycle of complete loss of cartilage tissue structure and function.
The experimental results showed that proliferative index values, mRNA, and cyclin D1 and collagen II protein expression increased gradually with increasing micro-strain stress, and peaked at 10% micro-strain stress and then decreased gradually. However, after pretreatment of cells with the Rock inhibitor Y-27632, the proliferative index values, mRNA, and cyclin D1 and collagen II protein expression decreased. The above results indicate that micro-strain stress affects the proliferation and functional marker expression of osteoarthritic chondrocytes, and 10% micro-strain stress might offer the best mechanical stimulation. Rock inhibitor Y-27632 inhibits this process.
After experiencing mechanical stimulation, the cells convert mechanical signals to chemical signals through specific signal transduction mechanisms resulting in changes in the biological function[21, 22]. In this series of signal transduction processes, the cytoskeleton plays a crucial role as the hub across the cell. Cytoskeleton, a critical component of cells, is composed of a large number of actin filaments and is the internal framework of cells. It consists of microtubules, microfilaments, and intermediate filaments, which are interlinked with protein-lipid molecules of the cytoplasmic side of the cell membrane to form the structural basis for cell movement, cell morphology, and transmembrane information transmission. It was shown earlier that cytoskeletal rearrangements occur during periodic mechanical stress and space microgravity. Phalloidin specifically binds to actin fibers in the cytoskeleton. The results of this experiment confirmed that under appropriate micro-strain stress, the osteoarthritic chondrocyte cytoskeletal microfilaments changed in structure and arrangement. However, too much mechanical stimulation can suppress the above changes.
Studies have shown that Rho GTPases can regulate the structure and function of the cytoskeleton in several ways under biomechanical stimulation, thus playing a critical role in biomechanical signal transduction[28, 29]. Rock is the downstream signaling molecule of the Rho GTP family and plays a crucial role in the Rho signaling pathway. Whether the Rho/Rock signaling pathway is involved in the regulation of osteoarthritic chondrocyte proliferation and functional expression of markers induced by micro-strain stress is still unclear. Western blot results showed that with increasing micro-strain stress, the Rock level increased gradually, reached a peak at 10% micro-strain, and decreased later. These results showed activation of the Rho/Rock signaling pathway in osteoarthritic chondrocytes by micro-strain stress, leading to cytoskeletal reorganization, and promoting the proliferation and functional expression of osteoarthritic chondrocyte markers.
In summary, adequate micro-strain stress can activate the Rho/Rock signaling pathway in osteoarthritic chondrocytes, which leads to the transmission of mechanical signals to the cytoskeleton. The above processes cause the cytoskeletal reorganization and transmit the mechanical signals, leading to the promotion of proliferation and functional expression of osteoarthritic chondrocyte markers.