Novel Combination Therapeutics for Enhanced Functional Recovery and Neuromuscular Regeneration Following Volumetric Muscle Loss Injuries

Volumetric muscle loss (VML) injuries, the loss of tissue beyond the endogenous regenerative capacity of skeletal muscle, produce permanent functional and cosmetic deficits. There are no current therapeutics that can satisfactorily restore full muscle function after VML injury. To address this unmet medical need, we have previously designed and evaluated two therapeutics with favorable regenerative potential in preclinical animal models. The first therapeutic is the Tissue Engineered Muscle Repair (TEMR) construct, which consists of a monolayer of muscle progenitor cells seeded on both sides of a sheet-like bladder acellular matrix and preconditioned in a bioreactor. The second is a tunable, biologically-inspired hyaluronic acid-based (HyA) hydrogel designed for enhanced cell-dependent remodeling and vascularization. In this dissertation, we investigated the two therapeutics, alone and in combination. We hypothesized that the combination of the enhanced vascularization and void-filling properties of the HyA-based hydrogel with the cell-based and tissue-building properties of the TEMR technology platform would further enhance the rate and/or magnitude of functional recovery post-VML injury. In Chapter 2, we evaluated these treatments, along with additional HyA formulations, using a well-established rat tibialis anterior (TA) VML injury model. In Chapter 3, we evaluated the early wound healing mechanisms of the TA model on a cellular level through development of a random forest classifier for histological analysis, and in Chapter 4, we upscaled our injury to a novel and more clinically relevant injury model, the gastrocnemius (GN). As a potential alternative hydrogel to combine with the TEMR, we also tested the KeraGenics® keratin-based hydrogel in the GN model. This dissertation yielded several key findings, including: (1) accelerated functional recovery and elimination of the TEMR “nonresponder” for the HyA+TEMR combination in the TA VML, (2) functional improvement consistent with HyA hydrogel (alone) results across multiple TA studies, (3) successful development of random forest classifier for macrophage phenotype classification, (4) increased immune cell counts, especially for M2-like macrophages, and dispersion of the cells throughout the injured region for the TEMR-containing groups, and (5) improved functional recovery for the HyA+TEMR in the larger GN VML model. Collectively, the dissertation provided critical insight into the efficacy of combinatorial strategies for VML treatment, and it identified the HyA+TEMR as a promising candidate for translation into larger-scale models and eventual clinical applications.