Inosculation of preformed microvessels with the host microvasculature represents a promising approach to accelerate vascularization of tissue constructs. Herein, we analyzed whether cultivation of prevascularized tissue constructs promotes inosculation by reducing the perivascular cell coverage of the preformed microvessels. Poly(ester-urethane) scaffolds were implanted into FVB/N-TgN (Tie2/green fluorescent protein [GFP]) 287 Sato mice to generate prevascularized tissue constructs with GFP-positive microvessels. These constructs were then cultivated for 3 or 10 days before implantation into dorsal skinfold chambers of FVB/N mice to analyze inosculation and vascularization by intravital fluorescence microscopy and immunohistochemistry. Noncultivated tissue constructs served as controls. Cultivation reduced the number of α-smooth muscle actin-positive preformed microvessels within the constructs and increased the production of vascular endothelial growth factor. After 3 days of cultivation, tissue constructs still exhibited good cell viability, whereas apoptotic cell death was massively increased in the 10-day-cultivated group. After implantation, inosculation of preformed microvessels was accelerated in the 3-day-cultivated constructs. This resulted in an improved vascularization, as indicated by an increased functional microvessel density and blood perfusion. Immunohistochemical detection of GFP-positive microvessels revealed that internal and external inosculation occurs in parallel. In conclusion, this study demonstrates that inosculation of in situ prevascularized tissue constructs can be easily accelerated by destabilization of preformed microvessels and angiogenic activation during short-term cultivation.
