Skeletal muscle injuries that occur from traumatic incidents, such as those caused by car accidents or surgical resections, or from injuries sustained on the battlefield, result in the loss of functionality of the injured muscle. To understand skeletal muscle regeneration and to better treat these large scale injuries, termed volumetric muscle loss (VML), in vivo injury models exploring the innate mechanisms of muscle injury and repair are essential for the creation of clinically applicable treatments. While the end result of a muscle injury is often the destruction of muscle tissue, the manner in which these injuries are induced as well as the response from the innate repair mechanisms found in muscle in each animal models can vary. This targeted review describes injury models that assess both skeletal muscle regeneration (i.e., the response of muscle to myotoxin or ischemic injury) and skeletal muscle repair (i.e., VML injury). We aimed to summarize the injury models used in the field of skeletal muscle tissue engineering, paying particular attention to strategies to induce muscle damage and how to standardize injury conditions for future experiments.

Volumetric muscle loss (VML) results in the loss of large amounts of tissue that inhibits muscle regeneration. Existing therapies, such as autologous muscle transfer and physical therapy, are incapable of returning full function and force production to injured muscle. Skeletal muscle tissue constructs may provide an alternative to existing therapies currently used to treat VML. Unlike autologous muscle transplants, muscle constructs can be cultured in vitro and are not reliant on intact muscle tissue. Skeletal muscle constructs can be generated from small muscle biopsies and could be used to generate skeletal muscle tissue constructs to replace injured tissues. To serve as effective therapies, muscle constructs must be capable of generating contractile forces that can assist the function of host skeletal muscle. The contractile force of native muscle arises in part as a consequence of the highly aligned, bundled architecture of myofibers. Attempts to induce similar alignment include: applications of tension/strain across hydrogels, inducing aligned architectures within scaffolds, casting tissues in straited molds, and 3D printing. While all these methods have demonstrated efficacy towards inducing myofiber alignment, the extent of myofiber alignment, tissue formation, and force production varies. This manuscript critically reviews the advantages and limitations of these methods, and specifically discusses their ability to impart mechanical and architectural cues to induce alignment within constructs. As tissue synthesizing techniques continue to improve, muscle constructs must include more cell types than simply myoblasts, such as the addition of neuronal and endothelial cells. Higher level tissue organization is critical to the success of these constructs. Many of these technologies have yet to be implanted into host tissue to understand engraftment and how they can contribute to traumatic injury, and as such continued collaboration between surgeons and tissue engineers is necessary to ultimately result in clinical translation.

The extracellular matrix (ECM) regulates skeletal muscle development through biochemical signaling and mechanical interactions. While Matrigel supplementation is commonly used to enhance engineered muscle formation, the contribution of specific ECM proteins remain incompletely defined in 3D systems. Here, we evaluated the effects of laminin and fibronectin supplementation on myogenic differentiation in collagen type I hydrogels and assessed their influence on passive tissue compaction and alignment in 3D constructs. Two-dimensional collagen hydrogels supplemented with increasing concentrations (0–100 µg/mL) of laminin or fibronectin were screened to maximize the myoblast fusion index. These concentrations were incorporated into 3D myocyte-seeded hydrogels cultured between flexible posts to quantify passive compaction forces via cantilever mechanics. Fibronectin supplementation (10 µg/mL) resulted in significantly greater early post displacement and sustained passive compaction compared to laminin-supplemented and unsupplemented controls. Constructs cultured under tension between posts exhibited enhanced alignment, with fibronectin further increasing the proportion of fibers oriented within 0–20° of the tension axis. Together, these findings demonstrate that fibronectin enhances early passive compaction dynamics and tension-mediated alignment in collagen-based skeletal muscle constructs. These results provide insight into how specific ECM components influence 3D tissue organization and may inform the design of engineered muscle models for regenerative applications.