Effects Of Mechano-growth Factor On The Motility Of Rat Tenocyte And The Repair Of Rat Injured Tendon

Tendon is a tough band of fibrous connective tissue, which connects muscle to bone and allow transmission of forces generated by muscle to bone, resulting in joint movement. However, inappropriate physical training or excessive repetitive stretch often results in overuse injuries. Unfortunately, due to their poor blood supply and modest metabolic rate, the tendon injuries are difficult to treat and result in slow/incomplete healing, long-term functional disability and pain after injury. Therefore, how to manage the damaged tendon is still one of the most challenging problems in orthopedics.Mechano-growth factor(MGF) is an alternative splice variant of the igf-1 gene. Because the first study linked this variant with mechanical stimuli in muscle and therefore named this isoform mechano-growth factor. The igf-1 gene generates three isoforms in human, IGF-1 Ea, IGF-1 Eb and IGF-1 Ec, but only two isoforms in rat, IGF-1 Ea and IGF-1 Eb. Rodent IGF-1 Eb and human IGF-1 Ec are named MGF. Although the MGF and IGF-1 differ only in the C-terminal E-peptide, their physiological functions are significantly distinct. MGF has been shown to activate satellite cells in muscle resulting in hypertrophy or regeneration, and functions as a neuroprotectant in brain ischemia. Moreover, the previous study demonstrated that MGF promoted bone-defect healing and induced more blood vessels in bone regeneration around the defective areas. However, the effect of MGF on tendon repair is not understood.Directional motility is a fundamental cellular process essential for tendon healing. In the early stages of tendon healing, tenocytes gradually move to the wound and proliferate while secreting collagens and glycoproteins to fulfill the regeneration. The aim of this study was to elucidate the possible beneficial effects of MGF-C25E(a synthetic mechano-growth factor E peptide) on tenocyte motility, and further study the effect of MGF-C25 E on tendon healing. The detailed results are listed as follows:① MGF-C25 E promoted rat tenocyte motility via the FAK-ERK1/2 signaling pathwayTenocyte migration and invasion were evaluated using the scratch wound assay and transwell system, respectively. The results showed that MGF-C25 E promoted the migration and invasion of rat tenocytes in a concentration dependent manner. Western blot was used to detect the expressions of focal adhesion kinase(FAK) and extracellular signal-regulated kinases 1/2(ERK1/2), and the results showed that MGF-C25 E elevated the expression of signal molecules FAK and ERK1/2. Inhibitor of FAK or ERK1/2 spoiled MGF-C25E-induced tenocyte migration and invasion, indicating that MGF-C25 E promoted tenocyte motility through the FAK-ERK1/2 signaling pathway.Gelatin zymograms showed that MGF-C25 E promotes secretion of matrix metalloproteinase 2(MMP-2), but have no effect on the secretion of matrix metalloproteinase 9(MMP-9). The MMP-2 inhibitor OA-Hy blocked MGF-C25E-promoted tenocyte invasion. Inhibitor of FAK or ERK1/2 also spoiled MGF-C25E-promoted MMP-2 secretion. In terms of the mechanical properties, Young’s modulus of tenocytes was decreased by treatment of MGF-C25 E, and obvious formation of pseudopodia and F-actin was also observed in MGF-C25E-treated tenocytes by immunofluoresence staining. Inhibitor of FAK or ERK1/2 restored the decrease of Young’s modulus and deprived the formation of pseudopodia and F-actin. Overall, our study demonstrated that MGF-C25 E promotes rat tenocyte motility by lessening cell stiffness, increasing pseudopodia formation and MMP-2 secretion via the FAK-ERK1/2 signaling pathway.② MGF-C25 E promoted rat tenocyte migration by increasing nuclear stiffness via FAK-ERK1/2 signaling pathwayUsing atomic force microscopy(AFM), we detected the stiffness of nucleus directly. The result showed that MGF-C25 E increases the nuclear stiffness by FAK-ERK1/2 signaling pathway. The MGF-C25 E has no effect on laminA/C expression, but the chromatin condensation may contribute to nuclear stiffness. Moreover, MGF-C25 E can promote DNA methylation, and inhibition of the DNA methylation inhibited the elevation in chromatin condensation, nuclear stiffness, and cell migration induced by MGF-C25 E. The FAK-ERK1/2 signaling pathway also plays and important role in MGF-C25E-promoted DNA methyaltion. Therefore, we speculate that MGF-C25E-promoted tenocyte migration may increase the nuclear stiffness and chromatin concensation by FAK-ERK1/2 signaling pathway.③ MGF-C25 E reduced the expression of inflammatory factor in tenocytesTo construct the model of tenocyte damage by cyclic stretching or tumor necrosis factor α(TNF-α), and the expression of Collagen I, Collagen III, cyclooxygenase 2(COX-2) and prostaglandin E2(PGE2) were then detected using qRT-PCR. The results showed that cyclic stretching decreases the mRNA expression of Collagen I and Collagen III, but has no effect on COX-2 and PGE2 mRNA levels. TNF-α increases the mRNA expression of COX-2 and PGE2, and decreases the mRNA expression of Collagen I and Collagen III. After MGF-C25 E treatment, the TNF-α-increased expression of COX-2 and PGE2 were significantly decreased, but MGF-C25 E has not affected the mRNA expression of Collagen I and Collagen III, indicating that MGF-C25 E may play an important role in tendinitis.④ Role of MGF-C25 E on tendon healing in a rat Achilles tendon rupture modelTo construct the animal model of Achilles tendon injury by surgery, and the Achilles functional index(AFI), biomechanical testing, macroscopical observation, histological examination, macroscopical and histological scoring system were used to evaluate the efficiency of tendon healing. The results showed that MGF-C25 E improves the functional properties of the repaired tendon and increases the stiffness of MGF-C25E-treated tendon. To further determine the relationship between tissue mechanical properties and structural features, histological examination of the repaired tendons was also performed. Hematoxylin and eosin(H&E) staining showed that higher MGF-C25E-treated tendons have denser connective tissue than that of NS-treated tendons, with a lesser number of cells exhibiting a spindle-shaped morphology aligned/organized along the longitudinal axis of the tendon. Moreover, masson’s thricome(MT) staining showed that more collagen fibers were formed in higher MGF-C25E-treated tendons. Histology score of higher-MGF-C25E-treated tendons were also significantly higher than the NS-treated tendons.In our study, we demonstrated that MGF-C25 E promotes rat tendon repair by enhancing tenocyte motility, reducing inflammatory action, increasing synthesis of extracellular matrix and so on. This study suggests that MGF could be used as a potential therapeutic factor in application for tendon repair and regeneration, and provides a theoretical reference for improving development of growth factor on tendon repair.