Functional Engineered Microvessels in Degradable Synthetic Elastomeric Scaffolds

Wright, Meghan 1 ; Maeda, Azusa 2 ; Yeh, Shu-Chi A 3 ; DaCosta, Ralph S 2 ; Lin, Charles P 3 ; Santerre, J Paul 1, 4

 1. Institute of Biomaterials and Biomedical Engineering, University of Toronto.; 2. Princess Margaret Cancer Center, The Campbell Family Institute for Cancer Research, University Health Network, Toronto.; 3. Harvard Medical School, Harvard University, Boston, MA.; 4. Faculty of Dentistry, University of Toronto, Toronto.

 Current strategies to engineer microvasculature in full-thickness tissue constructs often rely on the use of natural gels and/or matrix additives to promote vasculogenesis in vitro and neovascularization in vivo, despite the impractical features of these materials (1,2). In the current work, perfusion co-culture on a degradable polyurethane scaffold with optimized mechanical and surface properties for wound healing (D-PHI) (3) was used to generate micro-vascularized gingival tissue constructs. It was hypothesized that human umbilical vein endothelial cells (HUVECs) and human gingival fibroblasts (HGF) co-cultured on D-PHI under medium perfusion at a flow rate optimized for tissue production would produce constructs with a functional microvascular network, i.e. non-leaky interconnected microvessels. Cells were co-seeded onto D-PHI scaffolds, placed in a custom bioreactor, and subjected to medium flow at 0.05 mL/min for 14 days. Constructs were then either fixed for histological analysis or implanted subcutaneously into athymic mice. Surgical placement of a dorsal window at 2 weeks allowed for in vivo live imaging of the construct using confocal microscopy. HUVECs within the construct were stained using AF594-conjugated anti-human CD31 antibody administered topically. The fluorescent blood-pooling agent FITC-dextran (2000 kDa) was injected intravenously prior to imaging. At 2 weeks, some HUVEC-associated vessels were perfused with host blood (FITC-dextran and hCD31 positive); other areas in the construct showed non-perfused HUVEC vessels, while still others showed host vessels that had integrated into the construct. Prior to implantation, constructs had an average vessel lumen density of 3.05±0.57/mm2. After 2 weeks in vivo, lumen density had increased to 10.95±8.33/mm2. This was significantly greater than non-vascularized controls (density of 0.51±0.3/mm2 after 2 weeks in vivo, histological analysis, n=5, p<0.05). In conclusion, lumens generated via perfusion co-culture on D-PHI are part of a functional microvascular network that anastomose with the host vasculature system as soon as 14 days after implantation. The results validate the use of perfusion co-culture for generating pre-vascularized tissue engineered constructs on a 100% synthetic scaffold.

References: 1. Landau S et al. Biomaterials. 122, 72-82, 1993. 2. Laschke, MW et al. Biotechnol. Adv. 34, 112-121, 2016. 3. Battiston, KG et al. Acta Biomater. 24, 35-32, 2015.

Acknowledgements: NSERC Discovery, NSERC CGS D.