Critical-Size Muscle Defect Regeneration Using an Injectable Cell-Laden Nanofibrous Matrix: An Ex Vivo Mouse Hindlimb Organ Culture Study

Musculoskeletal injuries involving volumetric muscle loss remain difficult to treat due to limited regenerative capacity and the lack of physiologically relevant experimental models. This study introduces a computer-controlled ex vivo mouse hindlimb culturing platform that applies dynamic mechanical loading to evaluate muscle regeneration in a critical-size tibialis anterior (TA) defect. The defect was treated with an injectable myoblast-laden nanofibrous scaffold composed of polycaprolactone nanofibers and collagen (PNCOL). The ex vivo mouse hindlimb culturing platform maintained tissue viability and transmitted physiological strain across bone and muscle without disrupting the unity of the bone-muscle structure. PNCOL treatment under mechanical loading enhanced muscle fiber organization, extracellular matrix regeneration, and anti-inflammatory responses (CD206) while upregulating <i>paired box 7 (PAX7)</i>, <i>myogenic factor 5 (MYF5)</i>, <i>myogenic regulatory factor 4 (MRF4)</i>, <i>and transforming growth factor beta1 (TGFβ1)</i> expression. Cytokine profiling revealed an anabolic shift involving wingless/integrated (WNT) and insulin-like growth factor-1 (IGF-1) signaling, indicating a pro-regenerative microenvironment. Overall, the combination of mechanical stimulation and biomaterial-based therapy significantly improved muscle regeneration within a controlled ex vivo model. This multidimensional approach provides a reproducible and ethical platform that advances musculoskeletal regenerative research while reducing animal use.