Ex vivo perfusion recellularization of acellular rat hindlimbs for vascular composite allotransplantation.

Vascular composite allotransplantation (VCA) is a promising reconstructive surgical option for severe tissue loss. Autologous reconstructive methods are limited by donor tissue availability and donor site morbidity. Long-term immunosuppression regimens have severe side effects that limit the clinical implications of VCA. Tissue engineered composite grafts using decellularization and recellularization represent a potential solution to this. We present ex vivo recellularization of decellularized rat hindlimbs using human umbilical vein endothelial cells (HUVECs) and L6 rat myoblasts. HUVECs and L6 rat myoblasts were seeded in the vascular and muscle compartments, respectively and cultured up to 7 days ex vivo. We report cell engraftment in femoral vessels, small vessels located distally in muscle, and cell clusters in muscle, with phenotypic characterization in both tissue types. Formation of a neo-endothelium and nascent myotube formation was observed. Cell survival in both vessels and muscle was also demonstrated. These findings provide a foundational basis for a proof-of-concept small animal model for recellularization of composite tissues for VCA. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-025-25223-w.