Muscle Function Improvement in Injured Mice with Combination Treatment

INTRODUCTION: Loss of skeletal muscle from direct injury presents debilitating effects to an individual. Current treatments addressing muscle loss are limited by insufficient reconstitution of functioning muscle. Novel regenerative medicine technologies include the application of Urinary Bladder Matrix (UBM) and mesenchymal stem cells (MSCs) to restore functional muscle tissue. In our previous studies, we found that UBM increased muscle myoblast cell proliferation. Therefore, we examined whether co-treatment with MSCs would further augment regeneration as compared to individual treatments. METHODS: Twenty C57BL/6 male adult mice received bilateral laceration injuries on the gastrocnemius muscle under anesthesia, and were randomly grouped to a designed treatment applied 14 days after injury. Treatment groups were 1) DMEM culture medium, 2) UBM only (150μg), 3) MSCs only (1 million mouse derived cells), and 4) UBM+MSCs. 4 additional mice served as a control baseline not receiving injury. Efficacy of treatment was analyzed through isometric muscle force testing as well as histomorphologic examination at 50 days after injury. Two-way ANOVA was applied for statistical analysis. RESULTS: Isometric muscle force was measured, including twitch (Pt), tetanic (Po), and fatigue isometric functions with the muscle stretched to optimal length (Lo). Muscle twitch (Pt) significantly decreased in the DMEM group compared to the non-injured group at day 50 (p < 0.05). Furthermore, twitch significantly increased with UBM treatment, but not with MSC treatment. Regenerating myofiber nuclei were counted and myofiber cross sectional area was measured with histology. New myotubes were identified as having centrally located nuclei. Further, Ki-67 nuclear immunofluorescence staining was performed to demonstrate proliferating satellite cells. The myofiber cross sectional area and the number of Ki-67/DAPI overlapping stained nuclei significantly increased in the DMEM group compared to the non-injured group (p < 0.05). No differences were observed with other treatments in injured mice at day 50. CONCLUSION: We observed a significant improvement in muscle function with combination treatment and single UBM treatment applied 50 days post-injury. The current animal model provides a tool to study muscle regeneration, and is feasible for clinical translation to address impairment in skeletal muscle function after burn injury.