Neuromuscular Regeneration in Response to Agrin and Exercise post-Volumetric Muscle Loss Injury

Introduction: Skeletal muscle is well-adapted to regeneration, as satellite cells differentiate upon injury and replace damaged tissue. However, volumetric muscle loss (VML) is a critical sized defect in which the healing mechanism is overwhelmed, and loss of function persists. Transplantation of tissue engineered muscle scaffolds have been evaluated as a potential treatment. In past work, we characterized human primary myogenic cells (hPMCs) on electrospun fibrin scaffolds, and demonstrated that the cells are able to align, fuse, and myogenically mature on the scaffold. We successfully enabled muscle regeneration using these cell seeded-scaffolds upon transplantation into a mouse model of VML injury. However, regeneration of the connection between nerves and muscle is also critical to achieving functional recovery, as long term denervation causes muscle atrophy. A promising avenue is the use of rehabilitative exercise to promote nerve regeneration, via the induction of nerve growth factor secretion from muscle. We previously identified a gender-appropriate exercise regimen that improved AChR clustering and neural ingrowth into the injured muscle, but was not sufficient to increase force output or muscle weight. In this study, we attempt to further promote neuromuscular regeneration by the use of agrin-treated scaffolds. Agrin is a proteoglycan secreted by motor nerve terminals to induce AChR clustering and the formation of neuromuscular junctions. Various groups have suggested that the agrin-induced AChR clustering mechanism may be influenced by mechanical strain, but this has not been well-evaluated. In this study, we evaluated the synergistic effect of rehabilitative exercise and agrin-treated cell-seeded scaffolds on neuromuscular regeneration.

Materials and

Methods: Electrospun scaffolds: solutions of 1% fibrinogen and 0.75% alginate in 0.2% Poly(ethylene oxide) are co-extruded through a Y-piece connector into a 27-G blunt needle with applied voltage of 3-5 kV. The electrospun solution is collected in a grounded spinning metal collection plate for 5.75 minutes, containing bath solution of 50mM calcium chloride and 20 U/mL thrombin. The extruded threads of hydrogel are wrapped around 1.5 × 1.5 cm frames 4 times. An overnight incubation in 250mM sodium citrate dissolves the alginate, leaving a porous structure to encourage cell infiltration and nutrient diffusion. In Tethered Agrin groups, electrospun scaffolds were cross-linked with agrin at 100ug/mL using EDC/SNHS chemistry, and seeded with hPMCs at 600,000 cells/fiber or 1,000,000 cells/fiber. ‘Soluble Agrin’ groups instead received agrin in media at 300ng/mL for the last 2 days of culture. VML defects were created by 40-50% resection of the tibialis anterior (TA) in male NSG mice, followed by transplantation of ‘No Agrin’ or ‘Tethered Agrin’ muscle scaffolds (approved by ACUC protocol). Treadmill regime: continuous (1 hour daily @ 10m/min) for 7 weeks. Muscles were cryosectioned, and muscle sections & cell-seeded scaffolds underwent IHC staining. Quantification in FIJI. N6 for all groups of mice. Statistical significance determined by t-test, one-way or two-way ANOVA.

Results, Conclusions, and Discussions: A primary human myogenic cell source (hMP) was successfully induced into aligned, multi-nucleated myotubes on electrospun fibrin scaffolds. hMP-seeded scaffolds implanted into the defect not only survive for a period of 8+ weeks, but induce almost complete regeneration (90% of contralateral) of the muscle mass. In previous work, we also developed a successful model of exercise-induced regeneration by shortening the recovery period and lengthening the exercise period, and comparing high-intensity interval vs. low-intensity continuous running. In this study, we evaluated the combinatorial effect of agrin treatment and exercise. To characterize the effects of agrin on the hMP-seeded scaffolds, two different cell seeding concentrations were evaluated: 500,000 cells/fiber and 1,000,000 cells/fiber. In the lower cell seeding group, the addition of agrin significantly increased the proliferation of myogenic cells, and agrin-treated fibers trended towards increased myogenic maturity (Fig 1A). In the higher cell-seeding group, the fiber was saturated with cells and the agrin treatment produced no additional benefit in cell proliferation or myogenic maturity (Fig 1B). In both groups, the addition of agrin trended towards increased numbers of AChR clusters. In the subsequent study, No Agrin and Tethered Agrin fibers seeded with hMPs were implanted into a mouse model of VML injury (Fig 2A). These animals were further separated into groups with and without rehabilitative exercise. The implantation of Tethered Agrin fibers significantly increased the weight of the injured muscle, as compared to an untreated control (Fig 2B). Functional testing revealed a trend towards increasing force output with the Agrin group (although not significant, Fig 2C-D). The addition of exercise to the No Agrin group trended towards increasing force output, but produced no change with the Tethered Agrin group. Considering that the average weight of the injured muscle in the Agrin groups was close to 100% of the contralateral muscle weight, it is likely that these treatment methods have maximized neuromuscular recovery in this injury model (highly regenerative young mice with acute VML injuries). To better evaluate the combinatorial effects of rehabilitative exercise and agrin treatment, a model of chronic injury with young and aged mice will be utilized in the future.

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