Role of Matrix Bound Nanovesicles on Macrophage Polarization and Myoblast Differentiation During Skeletal Muscle Repair

Summary: Background: Volumetric Muscle Loss (VML) is a significant injury to skeletal muscle, leading to chronic long-term disability. An appropriately timed shift from a pro-inflammatory to a pro-healing immune response is essential for functional repair in acute muscle injuries. This regenerative capacity is lost following VML. The spatiotemporal dynamics of immune/myoblast crosstalk are crucial for functional skeletal muscle regeneration. We have shown that an Extracellular Matrix (ECM) scaffold can facilitate a type II immune response, leading to functional tissue deposition and myogenesis in VML patients. Recently, we have identified matrix bound nanovesicles (MBV) as the major signaling moiety within ECM that promotes a type II response and downstream constructive remodeling. We showed that MBV are a rich source of polyunsaturated fatty acids (PUFA) crucial for macrophage polarization and stem cell differentiation. Thus, we hypothesize that the MBV phospholipid membrane may serve as a hydrolytic platform of pro-resolving lipid mediators activated by different phospholipases dependent on the pro-/anti-inflammatory context of the extracellular environment. Herein, we evaluated the role of MBV, MBV lipids or their hydrolyzed lipid mediator products on macrophage polarization and myoblast differentiation. Methods: MBV were extracted from decellularized porcine small intestine via enzymatic digestion and ultracentrifugation, with subsequent lipid mediator release achieved through PAF-hydrolase treatment and Folch-based lipid extraction. Extracted lipids were evaluated in-vitro for their ability to promote macrophage polarization and myogenic activation, and in-vivo using a VML murine model. Results: Results showed that in-vitro, hydrolyzed MBV lipids significantly increased expression of CD206 in macrophages, and MBV increased expression level of Desmin in myoblasts and increased number of myotubes. In-vivo, MBV treatment significantly increased the number of F4/80+CD206+ macrophages at day 3 and 7 compared to no treatment. Importantly, the timing and frequency of MBV administration was shown to have a significant effect on outcomes. Conclusions: The present study showed that the MBV lipids play an important role in immunomodulation and myogenic activation. The release of hydrolyzed MBV lipids by hydrolases suggests that MBV biological activity is regulated by the pro- or anti-inflammatory context of the wound environment and is dependent on an appropriately timed release of lipid mediators. Zachary Clemens, Jagruti Kosaraju, Amrita Sahu, George S. Hussey