Aligned Decellularized Skeletal Muscle Extracellular Matrix for Muscle Regeneration

Introduction:: Volumetric muscle loss (VML) is a condition resulting from extreme trauma to skeletal muscle tissues and is defined by the loss of more than 20% of native skeletal muscle mass. Exacerbated by the critical size in defect, VML injury perpetrates poor muscle regeneration that is associated with poor limb function, reduced muscle strength, and long-term disability. Current clinical standards that seek to fully restore and regenerate the injured muscle fail to recapitulate the native properties of the skeletal muscle niche. Therefore, regenerative treatments that focus upon reconstructing the native biophysical and biochemical properties of skeletal muscle tissues are key to supporting more robust healing and recovery from VML.



The native architecture of skeletal muscle tissues is composed of a unique blend of parallel myofibers that are supported by a collagen-based network of extracellular matrix (ECM) proteins. The structure and composition of skeletal muscle tissues contain key biophysical and biochemical cues that drive muscle remodeling and regeneration. The primary objective of this study is to develop a blueprint for muscle regeneration by mimicking the biophysical structure and biochemical composition of the native skeletal muscle ECM.



Previously, our lab has utilized a shear-based approach to nano-pattern collagen scaffolds that were shown to enhance muscle regeneration. In this study, we applied our shear-based approach to construct topographically aligned nanofibers comprised of collagen and decellularized ECM (dECM). We hypothesized that the nanofibrillar scaffolds composed of mixed collagen and dECM will enhance muscle regeneration to assist with the repair of the injured skeletal muscle niche.


Materials and Methods:: To produce dECM (Fig.1A), skeletal muscle tissues were harvested from C57BL mice and incubated in 1% Sodium Dodecyl Sulfate (SDS) for 3 days, 1% Triton X-100 for 2 days, and DNAse-1 for 2 hours (Fig.1B). The dECM is then frozen in a -80ºC freezer overnight before lyophilization for 3 days. After lyophilization, the dECM is solubilized with 0.1% pepsin suspended in 0.5 M acetic acid. The solubilized dECM is then combined with low-density (10 mg/mL) type 1 rat tail collagen in a 1:1 ratio. The dECM/collagen mixture is then dialyzed in cellulose tubing encased with polyethylene glycol to produce a dECM/collagen bioink. To construct topographically aligned scaffolds, the dECM/collagen bioink is extruded into a neutralizing solution under high shear with a blunt needle syringe (Fig.1C). To visualize skeletal muscle decellularization (Fig.1B) and scaffold fiber formation (Fig.1C), scanning electron microscope (SEM) was used.



To assess in vitro myogenesis, GFP⁺ C2C12 mouse myoblasts were seeded (5×10⁵ cells) on either collagen-only or dECM/collagen scaffolds. The myoblasts were then differentiated in low serum media for 5 days and immunofluorescently stained for myosin heavy chain (MHC) and nuclei (DAPI) (Fig.1D-F).

To assess in vivo muscle regeneration, primary GFP⁺ mouse myoblasts were isolated, seeded (5×10⁵ cells) and differentiated on either collagen-only or dECM/collagen scaffolds. The scaffolds were then implanted into a VML defect model of C57BL mice (20% ablation of the tibialis anterior (TA) muscle) for 21 days (Fig.1G-I). Myogenesis was assessed by histological analysis of laminin stained muscle cross-sections for regenerating (centrally located nuclei) myofibers.


Results, Conclusions, and Discussions:: The consecutive decellularization of native skeletal muscle tissues in SDS and Triton X-100 displayed progressive morphological changes consistent with the removal of cellular materials (Fig.1B). Hematoxylin and eosin (H&E) staining demonstrated a marked decrease in nuclear presence after the decellularization process and SEM data showed the degradation and removal of myofiber cells, leaving behind the intact dECM (Fig.1B). Shear-based extrusion was used to generate collagen-only, dECM/collagen, and dECM-only scaffolds with nanofibrillar topographical patterning (Fig.1C). Across all groups, the scaffolds exhibited highly aligned and parallel fibers (30-50 nm diameters) that were oriented along the direction of extrusion (Fig.1C). To our knowledge this is the first reporting of nano-scale patterning of dECM skeletal muscle materials.



To evaluate the regenerative capacity of the aligned dECM scaffolds, myogenesis was first assessed in vitro. The collagen-only scaffolds and the dECM/collagen scaffolds were seeded with myoblasts and were allowed to differentiate into multinucleated myotubes. The seeded scaffolds were then stained for myosin heavy chain (MHC) and quantified for myotube lengths and nuclei count (Fig.1D). The dECM/collagen scaffolds supported greater myotube differentiation when compared to the collagen only scaffolds, as demonstrated by significantly longer myotube lengths (p< 0.01) and higher nuclei count (p< 0.0001)(Fig.1E&F).



The therapeutic efficacy of the dECM was evaluated in vivo using a mouse model of VML. The collagen-only and dECM/collagen scaffolds seeded with differentiated primary GFP⁺ mouse myoblasts were implanted into the muscle defect of the VML model and were allowed to heal for 21 days. Similar to the in vitro experiments, the dECM/collagen scaffolds demonstrated improved muscle regeneration when compared to the collagen only scaffolds as shown with significantly greater percentage of regenerating myotubes (centrally located nuclei,p< 0.01); however, no significant differences were seen in muscle cross-sectional area (Fig.1H&I). Studies are underway to characterize the cytokine profile of circulating factors during the healing process, and new in vivo studies are planned to assess the contribution of the dECM/collagen scaffolds in muscle force production and dynamic weight bearing functional response. Altogether, these findings highlight the regenerative potential of nanofibrillar aligned dECM-based scaffolds to enhance myogenesis in the therapeutic treatment of severe muscle injuries.


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