PGE2 and WNT3a Promote Skeletal Muscle Regeneration after Barium Chloride Damage In‐vitro

Abstract

IntroductionAlthough skeletal muscle has a remarkable regenerative capacity, certain traumatic injuries are beyond the normal physiologic repair and require extensive regenerative therapy. Musculoskeletal injury (MSI) afflicts more than 2.3 billion people globally and ~30 million Americans leading to nearly one‐trillion dollars in medical care costs (~ 6% of our GDP). In the tissue engineering field, muscle regeneration to overcome the compromised regeneration after traumatic injuries has emerged. Tissue engineering introduces novel in‐vitro models that simulate in‐vivo models that aid in reducing/eliminating the use of animal models, allow rapid screening of potential therapies and clinical interventions, reduce the associated cost and complexity, and are broadly available for research laboratories. Following our previous results showing that PGE2 and WNT3a enhance muscle cell differentiation, this study intended to validate in‐vitro muscle damage model induced by barium chloride (BaCl2) and screen PGE2 and WNT3a as potential molecules for muscle regeneration after injuries.Materials and MethodsC2C12 skeletal muscle cells under proliferation were firstly used to determine the concentration of BaCl2 for this study, then differentiation studies were performed to test chemical damage in muscle and regeneration under PGE2 and WNT3a treatment. In the first part of study, C2C12 cells were seeded in 12 well‐plates and cultured in growth medium (GM) with different concentrations of BaCl2 for 24 and 48 hr. and their effects on cell viability were monitored. After adjusting the BaCl2 concentration, C2C12 cells were seeded in 12 well‐plates and cultured in GM till 70% confluency, then allowed to differentiate in differentiation medium (DM) for 48 hr. before damage introduced using OPTI‐MEM containing BaCl2 for 6 hours, followed by treatment with condition media (DM + PEG2, WNT3a) for 48 hrs. Cells were finally stained with live/dead assay kit and imaged directly, or RNA was extracted for our custom‐built muscle specific PCR array.Results and ConclusionOur preliminary data indicated that 25 mM BaCl2 significantly (p<0.0007) decreased the cells viability and increased the number of dead cells as observed at 24 and 48 hr. under proliferative condition. Post‐treatment with 50nM PGE2 or 10ng/ml WNT3a significantly enhanced the regeneration as indicated by the significant increase in the percentage area of live cells (myotubes/myoblast) and significant decrease in the number of dead cells. Results of the custom‐built muscle specific PCR array indicated that 6 and 3 genes were differentially expressed after WNT3a and PGE2 treatment, respectively, compared to the BaCl2 group. Several of detected genes (e.g., Tnnc2, Actc1, Il6, Myh1, Nppa, Itpr1, etc.) are main contributors to the contractile machinery, while others are associated with inflammation and the extracellular matrix. This study concluded that BaCl2 affects both mononuclear and multinuclear C2C12 cells and PGE2 and Wnt3a could enhance the regeneration after the chemical damaged introduced by BaCl2, and provides initial validation of our new in vitro model