Pre-Innervated Tissue Engineered Muscle Promotes a Pro-Regenerative Microenvironment Following Volumetric Muscle Loss

Abstract

Volumetric Muscle Loss (VML) is defined as traumatic or surgical loss of skeletal muscle tissue beyond the inherent regenerative capacity of the body, generally leading to a severe functional deficit. Autologous muscle grafts remain the prevalent method of treatment whereas recent muscle repair techniques using biomaterials and tissue engineering are still at a nascent stage and have multiple challenges to address to ensure functional recovery of the injured muscle.Indeed, appropriate somato-motor innervations remain one of the biggest challenges for both autologous muscle grafts as well as tissue engineered muscle constructs. We aim to address this challenge by developing Pre-Innervated Tissue Engineered Muscle comprised of long aligned networks of spinal motor neurons and skeletal myocytes. Here, we developed methodology to biofabricate long fibrils of pre-innervated tissue engineered muscle using a coculture of myocytes and motor neurons on aligned nanofibrous scaffolds. Motor neurons lead to enhanced differentiation and maturation of skeletal myocytes in vitro. These pre-innervated tissue engineered muscle constructs when implanted in vivo in a rat VML model significantly increase satellite cell migration, micro-vessel formation, and neuromuscular junction density in the host muscle near the injury area at an acute time point as compared to non-pre-innervated myocyte constructs and nanofiber scaffolds alone. These pro-regenerative effects can potentially lead to enhanced functional neuromuscular regeneration following VML, thereby improving the levels of functional recovery following these devastating injuries.

Tissue-Engineered Skeletal Muscle Models to Study Muscle Function, Plasticity, and Disease

“…In VML injury models, injection of satellite cells in combination with muscle resident cells (which include endothelial cells and mesenchymal progenitors) attached to artificially engineered muscle fiber scaffold results in retention of SCs and functional regeneration of the injured muscle (Quarta et al, 2017). Incorporating additional cell types such as endothelial (Levenberg et al, 2005;Koffler et al, 2011;Perry et al, 2017;Choi et al, 2019), neuronal (Das et al, 2020;Kim et al, 2020), and immune (Juhas et al, 2018) cells improves the survival of transplanted 3D engineered tissues due a combination of cellular recruitment and paracrine signaling to promote vascular and neural integration. The key limitation in the clinical use of the engineered tissue replacements is neural integration with the host.…”

The effect of autologous repair and voluntary wheel running on force recovery in a rat model of volumetric muscle loss

“…There is currently no standard of care for VML that restores function to the injured skeletal muscle. Recently, research groups developing regenerative therapies have investigated the use of biological scaffolds, decellularized tissue and autologous tissue implantation in order to promote satellite cell migration into the defect site (Corona et al., 2013a; Das et al., 2020; Garg et al., 2014; Kim et al., 2020). Despite these efforts, it appears implantation alone is insufficient to elicit a robust regenerative response possibly due to lack of a proper stimulus to promote cell migration and growth factor release.…”

Innervation: the missing link for biofabricated tissues and organs

“…Intramuscular axon sprouting from transplanted neurons augmented muscle reinnervation, reduced atrophy, and restored muscle excitability 96 . In addition, we have employed the in vitro co-culture approach and developed innervated tissue-engineered muscles (InTEMs) composed of aligned neuromuscular bundles obtained by culturing spinal motor neurons with skeletal myocytes on aligned nanofibers 97 . Interestingly, these pre-innervated muscle constructs were found to promote myocyte fusion and maturation in vitro as well as augment muscle satellite cell migration, microvasculature presence, and NMJ formation near the injury following implantation in a rat model of VML 97 .…”

“…In addition, we have employed the in vitro co-culture approach and developed innervated tissue-engineered muscles (InTEMs) composed of aligned neuromuscular bundles obtained by culturing spinal motor neurons with skeletal myocytes on aligned nanofibers 97 . Interestingly, these pre-innervated muscle constructs were found to promote myocyte fusion and maturation in vitro as well as augment muscle satellite cell migration, microvasculature presence, and NMJ formation near the injury following implantation in a rat model of VML 97 . Innervation has a crucial role in development, maturation and functional regulation of the musculoskeletal system and hence it may be imperative that tissue-engineered muscle be pre-innervated during construction and/or capable of robust innervation upon implantation 98,99 .…”