Decellularization of Rat Gracilis Muscle Flap as a Potential Scaffold For Skeletal Muscle Composite Allotransplantation

There are limited biomaterials for skeletal muscle regeneration. This study aimed to apply a decellularization protocol in a muscle flap model and investigate its patency. Twenty-six gracilis-muscle (GM) flaps were harvested from 13 rats. GMs were divided into groups of either 1) normal (control), 2) perfusion with 1% sodium dodecyl sulfate or SDS for 48h, followed by Triton X-100 or TX100, or lastly, 3) perfusion with SDS for 72h, followed by TX100. The morphology, microcirculatory network patency, and residual DNA content (DNAC) were evaluated. Decellularized muscle (DM) for 72h was more translucent than DM-48h. Despite longer decellularization, the DM-72h microcirculatory network maintained its integrity, except when the dye infiltrated from the muscle edges. Compared to normal, all DM had significantly lower DNAC (normal of 1.44 μg/mg vs . DM-48h of 0.37 μg/mg vs . DM-72h of 0.089 μg/mg; P < 0.001). The DNAC of the DM-72h group was significantly lower than DM-48h (P< 0.001). We report successful GM flap decellularization. Longer decellularization led to lower DNAC, which did not compromise circulation. Our protocol may be applicable as a free-flap scaffold model for transplantation in the future. Statement of clinical significance The impact of our work involves a reproducible skeletal muscle decellularization protocol to later apply in translational research. ### Competing Interest Statement The authors have declared no competing interest.