Transcriptome profiling of a synergistic volumetric muscle loss repair strategy.

Volumetric muscle loss overwhelms skeletal muscle's ordinarily capable regenerative machinery, resulting in severe functional deficits that have defied clinical repair strategies. In this manuscript we pair the early in vivo functional response induced by differing volumetric muscle loss tissue engineering repair strategies that are broadly representative of those explored by the field (scaffold alone, cells alone, or scaffold + cells) to the transcriptomic response induced by each intervention. We demonstrate that an implant strategy comprising allogeneic decellularized skeletal muscle scaffolds seeded with autologous minced muscle cellular paste (scaffold + cells) mediates a pattern of increased expression for several genes known to play roles in axon guidance and peripheral neuroregeneration, as well as several other key genes related to inflammation, phagocytosis, and extracellular matrix regulation. The upregulation of several key genes in the presence of both implant components suggests a unique synergy between scaffolding and cells in the early period following intervention that is not seen when either scaffolds or cells are used in isolation; a finding that invites further exploration of the interactions that could have a positive impact on the treatment of volumetric muscle loss.

In vivo testing of an injectable matrix gel for the treatment of shoulder cuff muscle fatty degeneration.

INTRODUCTION: Extracellular matrix (ECM) gels have shown efficacy for the treatment of damaged tissues, most notably cardiac muscle. We hypothesized that the ECM gel prepared from skeletal muscle could be used as a treatment strategy for fatty shoulder cuff muscle degeneration. METHODS: We conducted experiments to (1) evaluate host biocompatibility to ECM gel injection using a rat model and (2) examine the effect of ECM gel injection on muscle recovery after delayed repair of a released supraspinatus (SSP) tendon using a rabbit model. RESULTS: The host biocompatibility to the ECM gel was characterized by a transient rise (first 2 weeks only) in several genes associated with macrophage infiltration, matrix deposition, and inflammatory cytokine production. By 8 weeks all genes had returned to baseline levels and no evidence of fibrosis or chronic inflammation was observed from histology. When gel injection was combined with SSP tendon repair, we observed a significant reduction (7%) in SSP muscle atrophy (24 + 3% reduction from uninjured) when compared with treatment with tendon repair only (31 + 7% reduction). Although fatty degeneration was elevated in both treatment groups, fat content trended lower (2%) in response to combined tendon repair and intramuscular ECM injection (4.1 + 2.1%) when compared with tendon repair only (6.1 + 2.9%). Transcriptome analysis revealed adipogenesis and osteoarthritis pathway activation in the repair only group. These key pathways were abrogated in response to treatment using combined repair plus gel. DISCUSSION: The findings suggest that ECM injection had a modest but positive effect on muscle mass, fatty degeneration, and key cellular signaling pathways.

Nandrolone supplementation does not improve functional recovery in an aged animal model of volumetric muscle loss injury.

Aging hinders the effectiveness of regenerative medicine strategies targeting the repair of volumetric muscle loss (VML) injury. Anabolic steroids have been shown to improve several factors which contribute to the age-related decline in muscle's regenerative capacity. In this study, the impact of exogenous nandrolone decanoate (ND) administration on the effectiveness of a VML regenerative repair strategy was explored using an aged animal model. Unilateral tibialis anterior VML injuries were repaired in 18-month-aged animal models (male Fischer 344 rat) using decellularized human skeletal muscle scaffolds supplemented with autologous minced muscle. The contralateral limb was left untreated/uninjured. Following repair, ND(+) or a carrier control (ND-) was delivered via weekly injection for a period of 8 weeks. At 8 weeks, muscle isometric torque, gene expression, and tissue structure were assessed. ND(+) treatment did not improve contractile torque recovery following VML repair when compared to carrier only ND(-) injection controls. Peak isometric torque in the ND(+) VML repair group remained significantly below contralateral uninjured control values (4.69 ± 1.18vs. 7.46 ± 1.53 N mm/kg) and was statistically indistinguishable from carrier only ND(-) VML repair controls (4.47 ± 1.18 N mm/kg). Gene expression for key myogenic genes (Pax7, MyoD, MyoG, IGF-1) were not significantly elevated in response to ND injection, suggesting continued age related myogenic impairment even in the presence of ND(+) treatment. ND injection did reduce the histological appearance of fibrosis at the site of VML repair, and increased expression of the collagen III gene, suggesting some positive effects on repair site matrix regulation. Overall, the results presented in this study suggest that a decline in regenerative capacity with aging may present an obstacle to regenerative medicine strategies targeting VML injury and that the delivery of anabolic stimuli via ND administration was unable to overcome this decline.

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.

Regenerative Repair of Volumetric Muscle Loss Injury is Sensitive to Age.

In this study, the influence of age on effectiveness of regenerative repair for the treatment of volumetric muscle loss (VML) injury was explored. Tibialis anterior (TA) VML injuries were repaired in both 3- and 18-month-old animal models (Fischer 344 rat) using allogeneic decellularized skeletal muscle (DSM) scaffolds supplemented with autologous minced muscle (MM) paste. Within the 3-month animal group, TA peak contractile force was significantly improved (79% of normal) in response to DSM+MM repair. However, within the 18-month animal group, muscle force following repair (57% of normal) was not significantly different from unrepaired VML controls (59% of normal). Within the 3-month animal group, repair with DSM+MM generally reduced scarring at the site of VML repair, whereas scarring and a loss of contractile tissue was notable at the site of repair within the 18-month group. Within 3-month animals, expression of myogenic genes (MyoD, MyoG), extracellular matrix genes (Col I, Col III, TGF-ß), and key wound healing genes (TNF-α and IL-1ß) were increased. Alternatively, expression was unchanged across all genes examined within the 18-month animal group. The findings suggest that a decline in regenerative capacity and increased fibrosis with age may present an obstacle to regenerative medicine strategies targeting VML injury. Impact Statement This study compared the recovery following volumetric muscle loss (VML) injury repair using a combination of minced muscle paste and decellularized muscle extracellular matrix carrier in both a younger (3 months) and older (18 months) rat population. Currently, VML repair research is being conducted with the young patient population in mind, but our group is the first to look at the effects of age on the efficacy of VML repair. Our findings highlight the importance of considering age-related changes in response to VML when developing repair strategies targeting an elderly patient population.