Critical-Size Defect Tibialis Anterior (TA) Muscle Regeneration using Ex-Vivo Mice Hindlimbs Culturing under Dynamic Mechanical Loading

In this study, we introduced an innovative computer-controlled ex vivo mice hindlimb culturing platform operating under dynamic loading, coupled with injectable cell-laden nanofibrous matrix (PNCOL), to investigate tissue response and therapeutic outcomes in critical size defect tibialis anterior (TA) muscle regeneration. The combination of mechanical stimulation and cell therapy offers a distinctive opportunity to delve into the regenerative rehabilitation field and create sustainable solutions in musculoskeletal (MSK) tissue regeneration. The application of mechanical loading on the whole mice hindlimbs increased total bone area and marrow area suggesting an increase in periosteal bone formation and resorption on the endosteal surface. Viability assessments confirmed the sustained culturing of the samples throughout the study. Then, the effect of mechanical loading and PNCOL injection on muscle regeneration at the TA defect site was evaluated. Histological analyses revealed enhanced muscle regeneration in PNCOL-treated hindlimbs. Structural analysis of the defect area through scanning electron microscopy (SEM) showed regeneration of ECM fibers at the defect site in PNCOL-treated groups. An analysis of cytokine levels in conditioned media at the end experiment showed changes in the number of proteins with the role in wound healing, muscle regeneration WNT, and IGF-1 signaling suggesting an anabolic effect of mechanical stimulation on muscle and bone. Similarly, gene expression analysis showed a significant upregulation of PAX7, Mrf4, MYF5, and TGF{beta}1 mRNA levels, indicating enhanced muscle regeneration after coupled mechanical loading and PNCOL treatments. Lastly, immunostaining showed an increase in tissue regeneration and anti-inflammatory response (CD206) in PNCOL-treated groups. Overall, the ex vivo hindlimb organ culturing platform- maintained tissue functions under mechanical loading, while PNCOL treatment promoted muscle tissue regeneration and reduced inflammation. These findings demonstrated the potential of multidimensional approaches for enhancing therapeutic outcomes in MSK disorders. In addition, this study aligns with the growing emphasis on minimizing the number of animals used in research and developing a robust sense of responsible animal experimentation through introducing dynamic ex-vivo muscle organ culturing platform.