The Treatment of Volumetric Muscle Loss in a Clinically Relevant Large Animal Model

Abstract

Volumetric muscle loss (VML) is the traumatic or surgical loss of skeletal muscle comprising 20-30% or more of the muscle volume. By definition, VML exceeds the muscle’s capacity for self-repair and results in persistent functional deficits. Significantly, no treatment options exist that can fully restore native structure and function. To address the limitations of current treatments, our laboratory has developed tissue-engineered skeletal muscle units (SMUs) as a novel treatment for VML repair. SMUs have shown promising regenerative potential in a rat VML model; however, limitations of rodent models necessitated transitioning our technology to a large animal (sheep) model.

Despite substantial heterogeneity of muscle progenitor cell populations obtained from craniofacial, trunk, and limb muscle, engineered skeletal muscle tissues are almost exclusively fabricated from cells derived from hindlimb muscle, making the effects of cell source on engineered muscle tissue unknown. Thus, we conducted a comparison of the development (myogenesis), structure (histology), and function (biomechanics) of SMUs fabricated from muscle cells isolated from both craniofacial and hindlimb muscle sources. Specifically, we showed that the semimembranosus muscle was best suited for the fabrication of sheep-derived SMUs.

We also sought to develop a method to scale our SMUs to clinically relevant sizes. We developed a modular fabrication method that combines multiple smaller SMUs into a larger implantable graft. Consequently, we successfully fabricated of one of the largest engineered skeletal muscle tissues to date while avoiding the formation of a necrotic core. To treat peripheral nerve injuries that accompany VML, we also developed engineered neural conduits (ENCs) to bridge gaps between native nerve and the injury site. We used scaled-up SMUs and ENCs to treat a 30% VML in the ovine peroneus tertius muscle. After a 3-month recovery, we performed in situ biomechanical testing and histological analyses on explanted muscles. Results showed that SMU-treated groups restored muscle mass and force production to a level that was statistically indistinguishable from the uninjured contralateral muscle.

Lastly, we evaluated the efficacy of SMUs in repairing craniofacial VML. Despite reported differences in the regenerative capacity of craniofacial muscle compared to limb muscle, prior to the work described herein, there were no models of craniofacial VML in either large or small animal models. Thus, we introduced the first model of craniofacial VML and evaluated the ability of SMUs to treat a 30% VML in the zygomaticus major muscle. Despite using the same injury and repair model in both implantation studies, results showed differences in pathophysiology between craniofacial and hindlimb VML. The fibrotic response was greater in the facial muscle model, and there was tissue tethering and intramuscular fat deposition that was not observed in the hindlimb study. The craniofacial model was also confounded by concomitant denervation and ischemia injuries which were too severe for our SMUs to repair.

Overall, this work significantly contributed to the field of skeletal muscle tissue engineering by evaluating the effects of muscle source on the structure and function of SMUs, creating a modular fabrication method for tissue scale-up, and introducing a new large animal model and a craniofacial model of VML. The success of this technology demonstrates its potential for treating clinical VML in the future.