Changes in DNA methylation accompany changes in gene expression during chondrocyte hypertrophic differentiation in vitro.

During osteoarthritis (OA), articular chondrocytes undergo phenotypic changes that resemble developmental patterns characteristic of growth plate chondrocytes. These phenotypic alterations lead to a hypertrophy-like phenotype characterized by altered production of extracellular matrix constituents and increased collagenase activity, which, in turn, results in cartilage destruction in OA disease. Recent studies have shown that the phenotypic instability and dysregulated gene expression in OA are associated with changes in DNA methylation patterns. Subsequent efforts have aimed to identify changes in DNA methylation with functional impact in OA disease, to potentially uncover therapeutic targets. Here, we paired an in vitro 3D/pellet culture system that mimics chondrocyte hypertrophy with RNA sequencing (RNA-Seq) and enhanced reduced representation of bisulfite sequencing (ERRBS) to identify transcriptomic and epigenomic changes in murine primary articular chondrocytes undergoing hypertrophy-like differentiation. We identified hypertrophy-associated changes in DNA methylation patterns in vitro. Integration of RNA-Seq and ERRBS datasets identified associations between changes in methylation and gene expression. Our integrative analyses showed that hypertrophic differentiation of articular chondrocytes is accompanied by transcriptomic and epigenomic changes in vitro. We believe that our integrative approaches have the potential to uncover new targets for therapeutic intervention.

Mouse Models of Osteoarthritis: Surgical Model of Post-traumatic Osteoarthritis Induced by Destabilization of the Medial Meniscus.

The surgical model of destabilization of the medial meniscus (DMM) has become a gold standard for studying the onset and progression of post-traumatic osteoarthritis (OA). The DMM model mimics clinical meniscal injury, a known predisposing factor for the development of human OA, and permits the study of structural and biological changes over the course of the disease. In addition, when applied to genetically modified or engineered mouse models, this surgical procedure permits dissection of the relative contribution of a given gene to OA initiation and/or progression. This chapter describes the requirements for the surgical induction of OA in mouse models, and provides guidelines and tools for the subsequent histological, immunohistochemical, and molecular analyses. Methods for the assessment of the contributions of selected genes in genetically modified strains are also provided.

Local and Systemic Effects of Blood Flow Restriction Therapy in an Animal Model.

BACKGROUND: Blood flow restriction therapy (BFRT) has been increasingly applied to improve athletic performance and injury recovery. Validation of BFRT has lagged behind commercialization, and currently the mechanism by which this therapy acts is unknown. BFRT is one type of ischemic therapy, which involves exercising with blood flow restriction. Repetitive restriction of muscle blood flow (RRMBF) is another ischemic therapy type, which does not include exercise. HYPOTHESIS/PURPOSE: The purpose was to develop a rat model of ischemic therapy, characterize changes to muscle contractility, and evaluate local and systemic biochemical and histologic responses of 2 ischemic therapy types. We hypothesized that ischemic therapy would improve muscle mass and strength as compared with the control group. STUDY DESIGN: Controlled laboratory study. METHODS: Four groups of 10 Sprague-Dawley rats were established: control, stimulation, RRMBF, and BFRT. One hindlimb of each subject underwent 8 treatment sessions over 4 weeks. To simulate exercise, the stimulation group underwent peroneal nerve stimulation for 2 minutes. The RRMBF group used a pneumatic cuff inflated to 100 mm Hg with a 48-minute protocol. The BFRT group involved 100-mm Hg pneumatic cuff inflation and peroneal nerve stimulation for a 5-minute protocol. Four methods of evaluation were performed: in vivo contractility testing, histology, immunohistochemistry, and ELISA. Analysis of variance with post hoc Tukey test and linear mixed effects modeling were used to compare the treatment groups. RESULTS: There was no difference in muscle mass among groups (P = .40) or between hindlimbs (P = .73). In vivo contractility testing showed no difference in maximum contractile force among groups (P = .64) or between hindlimbs (P = .30). On histology, myocyte cross-sectional area was not different among groups (P = .55) or between hindlimbs (P = .44). Pax7 immunohistochemistry demonstrated no difference in muscle satellite cell density among groups (P = .06) or between hindlimbs (P = .046). ELISA demonstrated the RRMBF group as eliciting elevated GH levels as compared with the other groups (P < .001). CONCLUSION: Ischemic therapy did not induce gains in muscle mass, contractility strength, fiber cross-sectional area, or satellite cell density locally or systemically in this model, although the RRMBF group did have elevated GH levels on ELISA. CLINICAL RELEVANCE: This animal model does not support ischemic therapy as a method to improve muscle mass, function, or satellite cell density.

Ischemic Therapy in Musculoskeletal Medicine.

BACKGROUND: The competitive environment of athletics has promoted the exploration of any technology application that may offer an edge with performance and recovery from injury. Ischemic therapy is one such technology that has rapidly been incorporated into training rooms and physical therapy clinics worldwide. This therapy modality is reported to increase an athlete's ability to improve muscle mass, strength, and endurance. PURPOSE: To provide the sports medicine physician with an understanding of the current state of ischemic therapy technology, including treatment specifications, known physiological effects, hypothesized mechanisms, biochemical effects, athletic applications, medical applications, animal models, and future research recommendations. STUDY DESIGN: Literature review. METHODS: A computer-based search of the PubMed database was used to perform a comprehensive literature review on musculoskeletal ischemic therapy. RESULTS: The current research on ischemic therapy is largely composed of case series with varying equipment, methods, and therapy specifications. The publication of case series has value in identifying this technology for future research, but the results of these studies should not be justification for application to athletes without validation of safety and effectiveness. CONCLUSION: To date, ischemic therapy remains unvalidated, and the mechanism by which it improves muscle performance is not clear.

Insulin-like growth factor 1 signaling in tenocytes is required for adult tendon growth.

Tenocytes serve to synthesize and maintain collagen fibrils and other extracellular matrix proteins in tendon. Despite the high prevalence of tendon injury, the underlying biologic mechanisms of postnatal tendon growth and repair are not well understood. IGF1 plays an important role in the growth and remodeling of numerous tissues but less is known about IGF1 in tendon. We hypothesized that IGF1 signaling is required for proper tendon growth in response to mechanical loading through regulation of collagen synthesis and cell proliferation. To test this hypothesis, we conditionally deleted the IGF1 receptor (IGF1R) in scleraxis (Scx)-expressing tenocytes using a tamoxifen-inducible Cre-recombinase system and caused tendon growth in adult mice via mechanical overload of the plantaris tendon. Compared with control Scx-expressing IGF1R-positive (Scx:IGF1R+) mice, in which IGF1R is present in tenocytes, mice that lacked IGF1R in their tenocytes [Scx-expressing IGF1R-negative (Scx:IGF1RΔ) mice] demonstrated reduced cell proliferation and smaller tendons in response to mechanical loading. Additionally, we identified that both the PI3K/protein kinase B and ERK pathways are activated downstream of IGF1 and interact in a coordinated manner to regulate cell proliferation and protein synthesis. These studies indicate that IGF1 signaling is required for proper postnatal tendon growth and support the potential use of IGF1 in the treatment of tendon disorders.-Disser, N. P., Sugg, K. B., Talarek, J. R., Sarver, D. C., Rourke, B. J., Mendias, C. L. Insulin-like growth factor 1 signaling in tenocytes is required for adult tendon growth.