GDNF-infused Biosponges for Improved Muscle Reinnervation following Volumetric Muscle Loss

Introduction: Skeletal muscle tissue has a limited capability to regenerate after volumetric muscle loss (VML), a traumatic injury defined as the loss of >20% of a muscle’s mass. VML directly reduces the number of myofibers and causes axotomy of nerves, resulting in reduced muscle function and neuromuscular junctions (NMJs). The objective of this study was to evaluate how incorporating Glial cell derived neurotrophic factor (GDNF) into a biosponge scaffold’s structure would improve NMJ quantity and morphology as well as muscle function.

Materials and

Methods: Positively charged Type A Gelatin Biosponges were fabricated as previously described [1]. After lyophilization, the sponges were disinfected and dehydrated. GDNF contains positively charged residues on its protein structure and can be electrostatically coupled with negatively charged GNPs. This overall negatively charged conjugate (GNP+GDNF) can be electrostatically bound to the positively charged biosponge [fig. 1]. This is done by rehydrating the sponges in a solution containing GDNF (1000 ng/mL) and negatively charged Type B gelatin nanoparticles (5mg/mL). To create the VML injury in male Lewis rats, 20% of muscle mass was removed from the tibialis anterior using a 6 mm biopsy punch. The VML defect was either treated with blank GNP biosponges or with GNP+GDNF loaded biosponges. Function testing was performed at 4 and 6 weeks followed by sacrifice and harvest of the TA muscle. The TA muscle was then fixed and sectioned into 60 micron thick sections. These sections were then stained with immunofluorescent antibodies to visualize Acetylcholine receptors, Neurofilament and Synaptic vesicles and analyzed using confocal microscopy.

Results, Conclusions, and Discussions: Dynamic light scattering and zeta potential measurement results [fig. 2] indicate that negatively charged GNPs can be electrostatically coupled with GDNF and loaded into positively charged biosponges. Total number of Bungarotoxin positive stains were quantified and GDNF infused biosponges showed higher number of Acetylcholine receptor clusters (BTX+ staining) [fig. 3]. The pre- and post-synaptic structures showed 75% complete overlap in uninjured muscles, 35% in the GNP loaded biosponge group, and 68% in the GNP+GDNF loaded biosponge group [fig. 4]. No significant improvements were noticed in TA muscle mass. Finally, improved muscle function was observed in week 6 compared to week 4 in the GNP+GDNF group. The GNP only group showed no significant improvements [fig. 5].
Therefore, treatment with GDNF loaded biosponges enhanced NMJ quantity, improved pre-/post-synaptic overlap and improved peak isometric torque at 6 weeks relative to 4 weeks as compared to the GNP loaded biosponges. These results indicate the biosponge mediated delivery of GDNF is an effective strategy to enhance muscle recovery post-VML. Ongoing studies are evaluating NMJ morphology (e.g., fragmentation and polyinnervation).

Acknowledgements (Optional):