Functional evaluation of prevascularization in one-stage versus two-stage tissue engineering approach of human bio-artificial muscle.

A common shortcoming of current tissue engineered constructs is the lack of a functional vasculature, limiting their size and functionality. Prevascularization is a possible strategy to introduce vascular networks in these constructs. It includes among others co-culturing target cells with endothelial (precursor) cells that are able to form endothelial networks through vasculogenesis. In this paper, we compared two different prevascularization approaches of bio-artificial skeletal muscle tissue (BAM) in vitro and in vivo. In a one-stage approach, human muscle cells were directly co-cultured with endothelial cells in 3D. In a two-stage approach, a one week old BAM containing differentiated myotubes was coated with a fibrin hydrogel containing endothelial cells. The obtained endothelial networks were longer and better interconnected with the two-stage approach. We evaluated whether prevascularization had a beneficial effect on in vivo perfusion of the BAM and improved myotube survival by implantation on the fascia of the latissimus dorsi muscle of NOD/SCID mice for 5 or 14 d. Also in vivo, the two-stage approach displayed the highest vascular density. At day 14, anastomosis of implanted endothelial networks with the host vasculature was apparent. BAMs without endothelial networks contained longer and thicker myotubes in vitro, but their morphology degraded in vivo. In contrast, maintenance of myotube morphology was well supported in the two-stage prevascularized BAMs. To conclude, a two-stage prevascularization approach for muscle engineering improved the vascular density in the construct and supported myotube maintenance in vivo.