Investigating neovascularization in rat decellularized intestine - an in vitro platform for studying angiogenesis

One of the main challenges currently faced by tissue engineers is the loss of tissues post implantation due to delayed neovascularization. Several strategies are under investigation to create vascularized tissue but none have yet overcome this problem. In this study we produced a decellularized natural vascular scaffold from rat intestine to use as an in vitro platform for neovascularization studies for tissue engineered constructs. Decellularization resulted in almost complete (97%) removal of nuclei and DNA, while collagen, glycosaminoglycans and laminin content was preserved. Decellularization did, however, result in the loss of elastin and fibronectin. Some proangiogenic factors were retained, as fragments of decellularized intestine were able to stimulate angiogenesis in the chick chorioallantoic membrane assay. We demonstrated that decellularization left perfusable vascular channels intact, and these could be repopulated with human dermal microvascular endothelial cells. Optimization of reendothelialisation of the vascular channels showed this was improved by continuous perfusion of the vasculature and further improved by infusion of human dermal fibroblasts into the intestinal lumen, from where they invaded into the decellularized tissue. Finally we explored the ability of the perfused cells to form new vessels. In the absence of exogenous angiogenic stimuli, Dll4, a marker of endothelial capillary-tip cell activation during sprouting angiogenesis was absent, indicating the reformed vasculature was largely quiescent. However, after addition of VEGFA, Dll4 positive endothelial cells could be detected, demonstrating this engineered vascular construct maintained its capacity for neovascularization. In summary we have demonstrated how a natural xenobiotic vasculature can be used as an in vitro model platform to study 3 neovascularization and provide information on factors that are critical for efficient reendothelialisation of decellularized tissue.