Transplantation of insulin-like growth factor-1 laden scaffolds combined with exercise promotes neuroregeneration and angiogenesis in a preclinical muscle injury model

Abstract

The regeneration of skeletal muscle can be permanently impaired by traumatic injuries, despite the high regenerative capacity of native muscle. An attractive therapeutic approach for treating severe muscle inuries is the implantation of off-the-shelf engineered biomimetic scaffolds into the site of tissue damage to enhance muscle regeneration. Anisotropic nanofibrillar scaffolds provide spatial patterning cues to create organized myofibers, and growth factors such as insulin-like growth factor-1 (IGF-1) are potent inducers of both muscle regeneration as well as angiogenesis. The aim of this study was to test the therapeutic efficacy of anisotropic IGF-1-releasing collagen scaffolds combined with voluntary exercise for the treatment of acute volumetric muscle loss, with a focus on histomorphological effects. To enhance the angiogenic and regenerative potential of injured murine skeletal muscle, IGF-1-laden nanofibrillar scaffolds with aligned topography were fabricated using a shear-mediated extrusion approach, followed by growth factor adsorption. Individual scaffolds released a cumulative total of 1244 ng ± 153 ng of IGF-1 over the course of 21 days in vitro. To test the bioactivity of IGF-1-releasing scaffolds, the myotube formation capacity of murine myoblasts was quantified. On IGF-1-releasing scaffolds seeded with myoblasts, the resulting myotubes formed were 1.5-fold longer in length and contained 2-fold greater nuclei per myotube, when compared to scaffolds without IGF-1. When implanted into the ablated murine tibialis anterior muscle, the IGF-1-laden scaffolds, in conjunction with voluntary wheel running, significantly increased the density of perfused microvessels by greater than 3-fold, in comparison to treatment with scaffolds without IGF-1. Enhanced myogenesis was also observed in animals treated with the IGF-1-laden scaffolds combined with exercise, compared to control scaffolds transplanted into mice that did not receive exercise. Furthermore, the abundance of mature neuromuscular junctions was greater by approximately 2-fold in muscles treated with IGF-1-laden scaffolds, when paired with exercise, in comparison to the same treatment without exercise. These findings demonstrate that voluntary exercise improves the regenerative effect of growth factor-laden scaffolds by augmenting neurovascular regeneration, and have important translational implications in the design of off-the-shelf therapeutics for the treatment of traumatic muscle injury.

Figure 1.

Characterization and quantification of cellular interactions on IGF-1 laden scaffolds. (A) Low magnification image of 3D collagen scaffold with SEM expansion to show aligned nanofiber organization within the scaffold. (B) Release of IGF-1 from scaffolds laden with IGF-1 over a 21-day period measured by ELISA. (C, D) Immunofluorescence staining of C2C12 myoblasts seeded on scaffolds, without IGF-1 and with IGF-1, respectively. Double headed arrow denotes orientation of aligned nanofibers. (E) The average length of MHC⁺ myotubes containing nuclei ≥3 (n=4). (F) Quantification of the average number of nuclei per myotube. (G) Distribution of myotube lengths on scaffolds represented in panels C & D, with and without IGF-1. * denotes a statistically significant relationship (p<0.05).

Figure 2.

Volumetric muscle loss in a murine model with voluntary exercise. (A) Schematic overview of the experimental design depicting induction of VML, introduction of voluntary running wheel exercise, and collection of tissues for histological analysis. (B) VML surgical procedure depicting (1) 20% muscle ablation, (2) scaffold implantation with scaffold denoted by white arrow, (3) muscle closure over the implanted scaffold and (4) skin incision closure with vicryl suture. (C) Hematoxylin and eosin (H&E) staining of transverse cross section of the tibialis anterior (TA) muscle with collagen scaffold implant (denoted by the black dashed line), and (D) High magnification view of the scaffold implant from panel C to closely visualize the scaffold morphology within the muscle bundles 21 days after implant.

Figure 3.

Myofiber regeneration and tissue revascularization in the TA muscle. (A) Confocal microscopy of a representative transverse cross section of the TA muscle 21 days following injury and scaffold +IGF-1 implantation. Immunofluorescence staining of laminin used to delineate myofiber boundaries. Inset: Regenerating myofibers with centrally located nuclei adjacent to the scaffold (outlined by dashed white line) (B) Quantification of regenerating myofibers located within a 500 μm area from the scaffold region. (C) Immunofluorescence staining of mature neuromuscular junctions co-stained with synaptophysin+/alpha-bungarotoxin+. (D) Quantification of mature neuromuscular junctions within a 500 μm radial area from the scaffold region. (n=4 for each group except n=6 for the scaffold+IGF-1 with exercise). * denotes a statistically significant relationship (p<0.05) and ** (p<0.01).

Figure 4.

Vessel regeneration and perfusion. (A) Immunofluorescence staining of co-stained CD31+/Isolectin+ anastomotic vessels in the TA muscle (B) Quantified density of perfused vessels within a 500 μm area from the scaffold region. Dotted line represents control non-regenerating tissue values (C) Characterize of the vascular network along the myofiber bundles using immunofluorescence staining of Isolectin perfused vessels in longitudinal muscle fibers adjacent to scaffold implant. (n=4 for each group except n=6 for the scaffold+IGF-1 with exercise). * denotes a statistically significant relationship (p<0.05).