Introduction: Previous studies have shown that the number of myoblastically differentiated fibroblasts known as myofibroblasts (MFs) is significantly increased in stiff joint capsules, indicating their crucial role in the pathogenesis of post-traumatic joint stiffness. Although the mode of MFs' function has been well defined for different diseases associated with tissue fibrosis, the underlying mechanisms of their regulation in the pathogenesis of post-traumatic joint capsule contracture are largely unknown.Methods: In this study, we examined the impact of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) on cellular functions of human joint capsule MFs. MFs were challenged with different concentrations of TNF-α with or without both its specifically inactivating antibody infliximab (IFX) and cyclooxygenase-2 (COX2) inhibitor diclofenac. Cell proliferation, gene expression of bothalpha-smooth muscle actin (α-SMA)andcollagen type I, the synthesis of prostaglandin derivates E₂, F_1A, and F_2A, as well as the ability to contract the extracellular matrix were assayed in monolayers and in a three-dimensional collagen gel contraction model. The α-SMA and COX2 protein expressions were evaluated by immunofluorescence staining and Western blot analysis.Results: The results indicate that TNF-α promotes cell viability and proliferation of MFs, but significantly inhibits the contraction of the extracellular matrix in a dose-dependent manner. This effect was associated with downregulation ofα-SMAandcollagen type Iby TNF-α application. Furthermore, we found a significant time-dependent upregulation of prostaglandin E₂synthesis upon TNF-α treatment. The effect of TNF-α on COX2-positive MFs could be specifically prevented by IFX and partially reduced by the COX2 inhibitor diclofenac.Conclusions: Our results provide evidence that TNF-α specifically modulates the function of MFs through regulation of prostaglandin E₂synthesis and therefore may play a crucial role in the pathogenesis of joint capsule contractures.

Recent clinical and animal studies have shown that extracorporeal shock wave therapy has a promoting influence on the healing process of musculoskeletal disorders. However, the underlying biological effects of extracorporeal shock wave therapy on human skeletal muscle cells have not yet been investigated. In this study, we investigated human skeletal muscle cells after exposure to radial extracorporeal shock waves in a standardized in vitro setup. Cells were isolated from muscle specimens taken from adult patients undergoing spine surgery. Primary muscle cells were exposed once or twice to radial extracorporeal shock waves in vitro with different energy flux densities. Cell viability and gene expression of the paired box protein 7 (Pax7), neural cell adhesion molecule (NCAM), and myogenic factor 5 (Myf5) and MyoD as muscle cell markers were compared to non-treated muscle cells that served as controls. Isolated muscle cells were positive for the hallmark protein of satellite cells, Pax7, as well as for the muscle cell markers NCAM, MyoD, and Myf5. Exposure to radial extracorporeal shock waves at low energy flux densities enhanced cell viability, whereas higher energy flux densities had no further significant impact. Gene expression analyses of muscle specific genes (Pax7, NCAM, Myf5, and MyoD) demonstrated a significant increase after single exposure to the highest EFD (4 bar, 0.19 mJ/mm2) and after double exposure with the medium EFDs (2 and 3 bar; 0.09 and 0.14 mJ/mm2, respectively). Double exposure of the highest EFD, however, results in a significant down-regulation when compared to single exposure with this EFD. This is the first study demonstrating that radial extracorporal shock wave therapy has the potential to modulate the biological function of human skeletal muscle cells. Based on our experimental findings, we hypothesize that radial extracorporal shock wave therapy could be a promising therapeutic modality to improve the healing process of sports-related structural muscle injuries.

The leading cause of training interruption in sport is a muscle injury, for which the standard treatment is nonsteroidal anti‐inflammatory drugs (NSAIDs). To find alternative treatments, we investigated whether the radial extracorporeal shockwave application (rESWT) could stimulate muscle regeneration. A lesion with complete rupture (grade III muscle tear) was set in the musculus rectus femoris of 12‐week‐old Wistar rats, and the NSAID diclofenac, rESWT, or a combined therapy were applied on day 0, 3, and 5 directly following the surgery. Rats were euthanized at 2, 4, and 7 days after surgery and the area of muscle lesion was excised for histological and gene expression analysis to determine the progress in the healing of damaged fibers and tissue regeneration. The best effect on muscle regeneration was observed in the group treated with rESWT alone. Monotherapy by diclofenac showed a smaller but still positive effect and lowest effects were detected when both therapies were applied. rESWT alone demonstrated a significant upregulation of the muscle markers MyoD and myosin. The presence of myosin gene expression indicated newly formed muscle fibers, which was confirmed by hematoxylin and eosin staining. Seven days after injury the amount of mononucleated cell decreased and regenerating fibers could be detected. This effect is most pronounced in the group treated with rESWT alone. In our study, shockwaves demonstrated the best effect on muscle regeneration. Therefore, we recommend prospective clinical studies to analyze the effect of rESWT after sports trauma to improve muscle regeneration and to shorten the rehabilitation.

Skeletal muscle injuries in competitive sports cause lengthy absences of athletes from tournaments. This is of tremendous competitive and economic relevance for both the athletes and their respective clubs. Therapy for structural muscle lesions aims to promote regeneration and fast-track return-to-play. A common clinical treatment strategy for muscle injuries is the intramuscular injection of calf blood compound and the homeopathic drug, Tr14. Although the combination of these two agents was reported to reduce recovery time, the regulatory mechanism whereby this occurs remains unknown. In this in vivo study, we selected a rat model of mechanical muscle injury to investigate the effect of this combination therapy on muscle regeneration. Gene expression analysis and histological images revealed that this combined intramuscular injection for muscle lesions can enhance the expression of pro-myogenic genes and proteins and accelerate muscle regeneration. These findings are novel and depict the positive effects of calf blood compound and the homeopathic drug, Tr14, which are utilized in the field of Sports medicine.

Post-traumatic joint contracture was reported to be associated with elevated numbers of contractile myofibroblasts (MFs) in the healing capsule. During the physiological healing process, the number of MFs declines; however, in fibroconnective disorders, MFs persist. The manifold interaction of the cytokines regulating the appearance and persistence of MFs in the pathogenesis of joint contracture remains to be elucidated. The objective of our current study was to analyze the impact of the anti-inflammatory cytokine interleukin (IL)-4 on functional behavior of MFs. Cells were isolated from human joint capsule specimens and challenged with three different concentrations of IL-4 with or without its neutralizing antibody. MF viability, contractile properties, and the gene expression of both alpha-smooth muscle actin (α-SMA) and collagen type I were examined. Immunofluorescence staining revealed the presence of IL-4 receptor (R)-alpha (α) on the membrane of cultured MFs. The cytokine IL-4 promoted MF viability and enhanced MF modulated contraction of collagen gels. Moreover, IL-4 intervened in gene expression by up-regulation of α-SMA and collagen type I mRNA. These effects could be specifically lowered by the neutralizing IL-4 antibody. On the basis of our findings we conclude that the anti-inflammatory cytokine IL-4 specifically regulates viability and the contractile properties of MFs via up-regulating the gene expression of α-SMA and collagen type I. IL-4 may be a helpful target in developing anti-fibrotic therapeutics for post-traumatic joint contracture in human. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1290–1298, 2017.