Graft alignment impacts the regenerative response of skeletal muscle after volumetric muscle loss in a rat model.

A key event in the etiology of volumetric muscle loss (VML) injury is the bulk loss of structural cues provided by the underlying extracellular matrix (ECM). To re-establish the lost cues, there is broad consensus within the literature supporting the utilization of implantable scaffolding. However, while scaffold based regenerative medicine strategies have shown potential, there remains a significant amount of outcome variability observed across the field. We suggest that an overlooked source of outcome variability is differences in scaffolding architecture. The goal of this study was to test the hypothesis that implant alignment has a significant impact on genotypic and phenotypic outcomes following the repair of VML injuries. Using a rat VML model, outcomes across three autograft implant treatment groups (aligned implants, 45° misaligned, and 90° misaligned) and two recovery time points (2 weeks and 12 weeks) were examined (n = 6-8/group). At 2 weeks post-repair there were no significant differences in muscle mass and torque recovery between the treatment groups, however we did observe a significant upregulation of MyoD (2.5 fold increase) and Pax7 (2 fold increase) gene expression as well as the presence of immature myofibers at the implant site for those animals repaired with aligned autografts. By 12 weeks post-repair, functional and structural differences between the treatment groups could be detected. Aligned autografts had significantly greater mass and torque recovery (77 ± 10% of normal) when compared to 45° and 90° misaligned autografts (64 ± 10% and 61 ± 11%, respectively). Examination of tissue structure revealed extensive fibrosis and a significant increase in non-contractile tissue area fraction for only those animals treated using misaligned autografts. When taken together, the results suggest that implant graft orientation has a significant impact on in-vivo outcomes and indicate that the effect of graft alignment on muscle phenotype may be mediated through genotypic changes to myogenesis and fibrosis at the site of injury and repair. STATEMENT OF SIGNIFICANCE: A key event in the etiology of volumetric muscle loss injury is the bulk loss of architectural cues provided by the underlying extracellular matrix. To re-establish the lost cues, there is broad consensus within the literature supporting the utilization of implantable scaffolding. Yet, although native muscle is a highly organized tissue with network and cellular alignment in the direction of contraction, there is little evidence within the field concerning the importance of re-establishing native architectural alignment. The results of this study suggest that critical interactions exist between implant and native muscle alignment cues during healing, which influence the balance between myogenesis and fibrosis. Specifically, it appears that alignment of implant architectural cues with native muscle cues is necessary to create a pro-myogenic environment and contractile force recovery. The results also suggest that misaligned cues may be pathological, leading to fibrosis and poor contractile force recovery.

Development of a biological scaffold engineered using the extracellular matrix secreted by skeletal muscle cells.

The performance of implantable biomaterials derived from decellularized tissue, including encouraging results with skeletal muscle, suggests that the extracellular matrix (ECM) derived from native tissue has promising regenerative potential. Yet, the supply of biomaterials derived from donated tissue will always be limited, which is why the in-vitro fabrication of ECM biomaterials that mimic the properties of tissue is an attractive alternative. Towards this end, our group has utilized a novel method to collect the ECM that skeletal muscle myoblasts secrete and form it into implantable scaffolds. The cell derived ECM contained several matrix constituents, including collagen and fibronectin that were also identified within skeletal muscle samples. The ECM was organized into a porous network that could be formed with the elongated and aligned architecture observed within muscle samples. The ECM material supported the attachment and in-vitro proliferation of cells, suggesting effectiveness for cell transplantation, and was well tolerated by the host when examined in-vivo. The results suggest that the ECM collection approach can be used to produce biomaterials with compositions and structures that are similar to muscle samples, and while the physical properties may not yet match muscle values, the in-vitro and in-vivo results indicate it may be a suitable first generation alternative to tissue derived biomaterials.