Myoblast -laden Injectable Hydrogel with IGF-1/Agrin Nanoparticles Ameliorates Chronic Skeletal Muscle Denervation Atrophy

Introduction: In humans, axonal regeneration in damaged nerves occurs at approximately 1 mm/day, often leading to chronic loss of innervation to target muscles during this period. Chronic denervation of muscles results in significant loss of physiological muscle cross-sectional area which precludes significant functional recovery. Previous work has demonstrated that agrin and insulin-like growth factor 1 (IGF-1) promote the formation of neuromuscular junctions, improved vascularity, and myoblast proliferation in a rat chronic denervation model1. Despite increases in innervation and vascularity of muscles treated with agrin and IGF-1, significant muscle atrophy remains an issue following a chronic injury, which has not been addressed in previous studies. Direct injection of agrin, IGF-1, or myoblasts has resulted in strong immune responses and inconsistent effects of agrin and IGF-1. Nanofibril hydrogel composites (NHC) made of polycaprolactone fibrils and hyaluronic acid have been demonstrated to improve the long-term viability of mesenchymal stem cells (MSCs) 2 and provide predictable agrin and IGF-1 release1 while limiting immune rejection of cells and promoting angiogenesis. Here we hypothesize that a combination of agrin, IGF-1, and syngeneic myoblasts delivered in NHC will promote engraftment of myoblasts, alleviate functional deficits due to chronic denervation and increase neural infiltration.

Materials and

Methods: All animal procedures were carried out as approved by institutional ACUC. Myoblast Isolation and Viability: Myoblast were isolated from GFP+ Lewis rats (F344-Tg(UBC-EGFP)F455Rrrc) and non-GFP+ Lewis rats using either serial pre-plating as described by Yoshioka et al (2020)3 (Protocol 1) or by explant expansion as described by Shahini et al (2018)4 (Protocol 2). Cells were used at passages 2 to 4 for all experiments. NHC and IGF-1 and agrin nanoparticles were synthesized as previously described2, using agrin and IGF-1 at 12.5 µg/mL and 250 µg/mL, respectively. Live/Dead assay (Invitrogen) was done on myoblasts in NHC following extrusion from a 22 G needle to determine cell viability. NHC/myoblast at 2,500 cells/µL were cultured for 6 days, with or without nanoparticles then dsDNA assay was performed on constructs. Composite Hydrogel Injection: 9-10-week-old Lewis rats received median nerve transection with 12-week delay of ulnar to median nerve transfer to create a forepaw chronic denervation model. Animals (n=5-6 per group) received 100 µL of PBS injections, myoblasts in NHC/Agrin/IGF-1 or myoblasts in PBS starting at 33 weeks post denervation, or no chronic denervation (transection with immediate transfer). Treatment was given in 3 doses, 2- and 4-week apart. Following surgeries, animals received weekly forepaw stimulated grip strength testing (SGST) (Fig. 1).

Results, Conclusions, and Discussions: Utilizing the explant protocol, more than 95% of isolated cells were desmin positive with ~75% of isolated cells being PAX7-positive satellite cells. Primary myoblast showed high viability 1-hour post extrusion from a 22G hypodermic needle. The onset of a plateau of functional recovery occurred in all groups at 16 weeks with the most remarkable improvement in SGST seen in the positive control group. Myoblasts treatment was given at 33 weeks post-injury. It showed a trend towards improvement in the functional recovery at 2 weeks (Fig. 2A), with statistically significant (p < 0.05) improvement starting at 3-4-weeks post-injection in NHC/myoblast/nanoparticles (Fig. 2B). Both myoblast treatments resulted in normalization of muscle cross-sectional area and myofiber cross-sectional area distribution (Fig. 3). Additionally, NHC/myoblast/nanoparticles caused an expansion of the satellite cell population and new fiber formation (Fig. 4). Treatment resulted in a trend towards increase in vascular density relative to controls.

It was demonstrated that primary syngeneic myoblasts resulted in significant functional improvement in middle-aged rats with chronic denervation of the forepaw. This functional recovery was coupled with a complete resolution of myofiber atrophy, satellite cell expansion, and increased vascular density.

Implanted myoblast cell populations result in histological improvement in muscle regeneration in injury models, but this has not been demonstrated with injectable hydrogels for chronic denervation-induced skeletal muscle atrophy. It is assumed that drug release and cell retention dynamics seen with MSCs 2 hold for injected myoblast and would explain the more subtle functional improvement seen from PBS injections compared to NHC-mediated injections. Given myoblast delivery in PBS resulted in the normalization of muscle morphometry, the hydrogel and nanoparticles did not significantly impact this aspect of regeneration. However, the increase in cross-sectional area did not correlate with functional improvement and was likely driven by the effects of agrin and IGF-1 on the neuromuscular junction. Histological evaluation in progress will access innervation and changes in schwann cells.

Acknowledgements (Optional):

References: 1. Hanwright, P. J. et al. Biomaterials 280, 121244 (2022). 2. Yao, Z. C. et al. Small 18, (2022). 3. Yoshioka, K. et al. Front Cell Dev Biol 8, (2020). 4. Shahini, A. et al. Stem Cell Res 30, 122–129 (2018)

ACKNOWLEDGEMENTS: This study is supported by funds from a DoD/CDMRP grant.