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Surgical techniques for the management of recalcitrant osteochondral lesions of the talus have improved; however, the poor healing potential of cartilage may impede long-term outcomes. Repair (microfracture) or replacement (osteochondral transplants) is the standard of care. Reparative strategies lead to production of fibrocartilage, which, compared with the native type II articular cartilage, has decreased mechanical and wear properties. The success of osteochondral transplants may be hindered by poor integration between grafts and host that results in peripheral cell death and cyst formation. These challenges have led to the investigation of biologic adjuvants to augment treatment. In vitro and in vivo models have demonstrated promise for cartilage regeneration by decreasing inflammatory damage and increasing the amount of type II articular cartilage. Further research is needed to investigate optimal formulations and time points of administration. In addition, clinical trials are needed to investigate the long-term effects of augmentation.

Objectives: Individuals who participate in sports have an increased risk of osteoarthritis (OA), characterized by articular cartilage degeneration. Currently, there is no cure for OA with treatment aimed at symptom relief and improved function. Muscle-derived stem cells (MDSCs) have been shown to exhibit long-term proliferation, high self-renewal, and multipotent differentiation capabilities in vitro. Previously, we have demonstrated that murine MDSCs retrovirally transduced to express chondrogenic proteins (BMPs) differentiate into chondrocytes and enhance cartilage repair in vivo. Direct injection of therapeutic proteins can promote cartilage healing; however, they have relatively short half-lives requiring muitiple injections of high dosages. This presents a challenge in terms of maintaining adequate local BMP levels and could negatively affect both injured and normal structures and lead to side effects such as osteophyte formation. Gene therapy is a promising approach that addresses this problem; however, its utilization in clinical applications is much further down the road. In order to circumvent viral transduction of cells for cartilage regeneration, we developed a unique growth factor delivery platform comprised of native heparin and a synthetic polycation, poly(ethylene argininylaspartate diglyceride) (PEAD) incorporated with BMP2 (BMP2 coacervate). In this study, we show that sustained delivery of BMP2 via a BMP2 coacervate can induce the differentiation of MDSCs to a chondrocyte lineage for in vivo cartilage regeneration and healing in a Monoiodoacetate (MIA)-induced osteoarthritis model. Methods: mMDSCs were isolated from muscle biopsies via a modified pre-plated technique. The BMP2 coacervates were prepared as previously described. The release profiles of BMP2 coacervate were tested by ELISA. The chondrogenic effects that delivery of BMP2 had on MDSCs were evaluated by RT-PCR. The efficacy of MDSC with BMP2 coacervate were evaluated in vivo in a MIA-induced OA model in C57B6 mice. Mice received an intraarticular injection of 20μl of MIA to induce osteoarthritic lesions. Two weeks after MIA injection, mice were injected with PBS (control), MDSC + free BMP2, or MDSC + BMP2 coacervate. Histologic evaluations of cartilage regeneration were conducted at week 4. Institutional Animal Care and Use Committee approval was obtained prior to all animal studies. Results: Release profiles of BMP2 showed sustained release for more than 28 days demonstrating sustained release. The chondrogenic differentiation of MDSCs following BMP2 delivery was assayed using cell culture. The mRNA expression of Aggrecan and Col2A were significantly higher in each BMP2 group compared to control or vehicle only (PConclusion: This study demonstrates that sustained growth factor delivery (BMP2) is a potential therapeutic option for muscle-derived stem cell based cartilage regeneration for the treatment of osteoarthritis. Our results demonstrate an effective method for prolonged exposure to BMP2 via a non-gene therapy approach which is preferred and clinically translatable.

The formation of fibrous tissue during the healing of skeletal muscle injuries leads to incomplete recovery of the injured muscle. Platelet-rich-plasma (PRP) contains beneficial growth factors for skeletal muscle repair; however, it also contains deleterious cytokines and growth factors, such as TGF-β1, that can cause fibrosis and inhibit optimal muscle healing. Objective To test if neutralizing TGF-β1's action within PRP, through neutralization antibodies, could improve PRP's beneficial effect on skeletal muscle repair. Methods PRP was isolated from in-bred Fisher rats. TGF-β1 neutralization antibody (Ab) was used to block the TGF-β1 within the PRP prior to injection. The effects of customized PRP (TGF-β1 neutralized PRP) on muscle healing was tested on a cardiotoxin (CTX) induced muscle injury model. Results A significant increase in the numbers of regenerative myofibers was observed in the PRP and customized PRP groups compared to the untreated control. A significant decrease in collagen deposition was observed in customized PRP groups when compared to the control and PRP groups. Significantly enhanced angiogenesis and more Pax-7 positive satellite cells were found in the PRP and customized PRP groups compared to the control group. Macrophage infiltration was increased in the customized PRP groups when compared with the PRP group. More M2 macrophages were recruited to the injury site in the customized PRP groups when compared with the PRP and control groups. Conclusion Neutralizing TGF-β1 within PRP significantly promotes muscle regeneration while significantly reducing fibrosis. Not only did the neutralization reduce fibrosis, it enhanced angiogenesis, prolonged satellite cell activation, and recruited a greater number of M2 macrophages to the injury site which also contributed to the efficacy that the customized PRP had on muscle healing.

Objectives: Skeletal muscle injuries are among the most common sports-related trauma. Current treatment strategies result in formation of fibrous tissue that hinders the healing process before complete recovery. Incomplete recovery impairs muscle function and predisposes to re-injury. Platelet-Rich-Plasma (PRP) contains a multitude of growth factors and is an autologous source of growth factors for various tissue repairs. It is well established that PRP contains beneficial growth factors for muscle repair; however, it also contains high concentrations of deleterious growth factors for optimal muscle healing, such as transforming growth factor-beta 1 (TGF-β1). TGF-β1 leads to increased fibrosis impeding muscle healing. We therefore hypothesized that neutralization of TGF-β1’s action within PRP could improve PRP’s beneficial effect on skeletal muscle repair. Methods: Sixteen week old in-bred Fisher rats were used. Three rats were used for PRP isolation. 10 ml of blood were extracted from abdominal aorta and mixed with citrate phosphate dextrose solution. PRP were isolated by twice centrifugation. 24 rats were randomly assigned to four groups. A small incision was made along the tibialis anterior (TA) muscle; 50 µl cardiotoxin (CTX) (0.15ug/ul) was injected intramuscularly to the TA. One day after CTX injection, the animals were treated with PBS (control), plain PRP (PRP group), customized PRP+Ab-1x, and PRP+Ab-5x. Animals were sacrificed, and TA muscles were dissected on week 1 and 2 for assessment of muscle regeneration, fibrosis, macrophage infiltration, and satellite cell activation. Results: We observed significantly more regenerative myofibers in the PRP and customized PRP groups compared to control (Fig 1A-C). Collagen deposition (fibrosis) was detected in all groups at week 1 and week 2 after injury; while customized PRP group showed significantly decreased collagen deposition at week 1 and week 2 when compare to control and PRP groups (Fig. 1D-F). PRP was also shown to activate satellite cells at day 7 while customized PRP extended satellite cell activation through day 14 (Fig. 1G, H). Macrophage infiltration was significantly decreased in PRP group when compared to control at week 1 after injury; however the macrophage infiltration was increased in customized PRP group when compare with PRP group, specifically, we observed an increased in the percentage of the M2 macrophage subtype in the customized PRP group at week 1 but not week 2(Fig. 1I, J). Conclusion: This study demonstrates that substantial fibrosis occurs in skeletal muscle following CTX injury. PRP treatment significantly increases muscle regeneration by promoting angiogenesis, satellite cell activation, and reducing the infiltration of inflammatory macrophages. However, augmented fibrosis was also observed. Neutralizing TGF-β1 within PRP also significantly promotes muscle regeneration while significantly reducing fibrosis. It has been suggested that M2 macrophages are involved in the suppression, resolution and reversal of fibrosis. Hence, the increase in M2 macrophages in the customized PRP group may have contributed to the observed decrease in fibrosis. Our results demonstrate that neutralization of TGF-β1 within PRP is a promising approach to improve PRP’s beneficial effect on skeletal muscle repair while decreasing deleterious fibrosis.