Rehabilitative exercise and spatially patterned nanofibrillar scaffolds enhance vascularization and innervation following volumetric muscle loss

Abstract

Muscle regeneration can be permanently impaired by traumatic injuries, despite the high regenerative capacity of skeletal muscle. Implantation of engineered biomimetic scaffolds to the site of muscle ablation may serve as an attractive off-the-shelf therapeutic approach. The objective of the study was to histologically assess the therapeutic benefit of a three-dimensional spatially patterned collagen scaffold, in conjunction with rehabilitative exercise, for treatment of volumetric muscle loss. To mimic the physiologic organization of skeletal muscle, which is generally composed of myofibers aligned in parallel, three-dimensional parallel-aligned nanofibrillar collagen scaffolds were fabricated. When implanted into the ablated murine tibialis anterior muscle, the aligned nanofibrillar scaffolds, in conjunction with voluntary caged wheel exercise, significantly improved the density of perfused microvessels, in comparison to treatments of the randomly oriented nanofibrillar scaffold, decellularized scaffold, or in the untreated control group. The abundance of neuromuscular junctions was 19-fold higher when treated with aligned nanofibrillar scaffolds in conjunction with exercise, in comparison to treatment of aligned scaffold without exercise. Although, the density of de novo myofibers was not significantly improved by aligned scaffolds, regardless of exercise activity, the cross-sectional area of regenerating myofibers was increased by > 60% when treated with either aligned and randomly oriented scaffolds, in comparison to treatment of decellularized scaffold or untreated controls. These findings demonstrate that voluntary exercise improved the regenerative effect of aligned scaffolds by augmenting neurovascularization, and have important implications in the design of engineered biomimetic scaffolds for treatment of traumatic muscle injury.

Fig. 1

Characterization of aligned nanofibrillar collagen scaffold aggregates. a Schematic diagram of three-dimensional randomly oriented scaffold bundle and scanning electron microscopy (SEM) image of nanofibril organization. b Schematic diagram of three-dimensional parallel-aligned scaffold bundle and SEM image of nanofibril organization. c, d Mechanical characterization of stiffness by Young’s Modulus c and maximum load d between randomly oriented scaffolds (random) and aligned scaffolds (n = 5). e Cellular attachment of human endothelial cells to aligned nanofibrillar scaffolds. Scale bars: 400 µm a, b, 50 µm e. Arrow denotes the direction of nanofibril alignment. Statistically significant comparisons: * P < 0.05, ***P < 0.001. Error bars denote standard deviation

Fig. 2

Therapeutic effect of aligned nanofibrillar scaffold with exercise on revascularization in the ablated muscle. a Confocal microscopy images adjacent to the site of scaffold implantation depict CD31 (red) and isolectin (green). b Perfused vascular density was quantified based on the density of CD31⁺/isolectin⁺ vessels. c Total capillary density was quantified as the density CD31⁺ vessels. Shown are mean ± SD aligned scaffold without exercise (n = 6), randomly oriented scaffold with exercise (n = 6), and all other groups (n = 4). Statistically significant comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. a Scale bar: 100 µm. Error bars denote standard deviation

Fig. 3

Effect of aligned nanofibrillar scaffold and exercise on myogenesis. a Confocal microscopy images adjacent to the site of scaffold implantation depicting regenerated myofibers based on antibodies against skeletal muscle myosin heavy chain (MHC, red) and laminin (green). b Quantification of myogenesis based the density of MHC⁺ myofibers with centrally located nuclei. c Quantification of myofiber cross-sectional area among regenerated myofibers. Shown are mean ± SD (aligned scaffold with or without exercise (n = 6), randomly oriented scaffold with exercise (n = 5), and all other groups (n = 4)). Statistically significant comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. a Scale bar: 100 µm. Error bars denote standard deviation

Fig. 4

Effect of aligned nanofibrillar scaffold and exercise on re-innervation. a, b Confocal microscopy images adjacent to the site of scaffold implantation depicting re-innervation, based on α-bungarotoxin (α-BTX) and synaptophysin (green). c Quantification of re-innervation by the total number of neuromuscular junctions (α-bung⁺) at 500 µm or 1000 µm distance surrounding the scaffold. d Quantification of mature neuromuscular junctions (α-bung⁺/ synaptophysin⁺) at 500 µm or 1000 µm distance surrounding the scaffold. Shown are mean ± SD (n = 4 all groups). * Statistically significant comparisons (*P < 0.05). a, b Scale bar: 50 µm. Error bars denote standard deviation