Diffusion limits of an in vitro thick prevascularized tissue

Although tissue engineering promises to replace or restore lost function to nearly every tissue in the body, successful applications are currently limited to tissue less than 2 mm in thickness. in vivo capillary networks deliver oxygen and nutrients to thicker (> 2 mm) tissues, suggesting that introduction of a preformed in vitro vascular network may be a useful strategy for engineered tissues. This article describes a system for generating capillary-like networks within a thick fibrin matrix. Human umbilical vein endothelial cells, growing on the surface of microcarrier beads, were embedded in fibrin gels a known distance (Delta = 1.8-4.5 mm) from a monolayer of human dermal fibroblasts. The distance of the growth medium, which contained vascular endothelial growth factor and basic fibroblast growth factor, from the beads, C, was varied from 2.7 to 7.2 mm. Capillaries with visible lumens sprouted in 2-3 days, reaching lengths that exceeded 500 microm within 6-8 days. On day 7, capillary network formation was largely independent of C; however, a strong inverse correlation with Delta was observed, with the maximum network formation at Delta = 1.8 mm. Surprisingly, the thickness of the gel was not a limiting factor for oxygen diffusion as these tissue constructs retained a relatively high oxygen tension of > 125 mmHg. We conclude that diffusion of oxygen in vitro is not limiting, allowing the development of tissue constructs on the order of centimeters in thickness. In addition, diffusion of fibroblast-derived soluble mediators is necessary for stable capillary formation, but is significantly impeded relative to that of nutrients present in the medium.