Evaluating Vessel Infiltration in CAM Model

To assess the influence of the 3DP PCL framework within the CHyA-B matrix on vessel infiltration ex vivo, scaffolds were incubated within a chick chorioallantoic membrane (CAM) model as previously described (do Amaral et al., 2019a (link); Ryan et al., 2019 (link); do Amaral et al., 2019b (link)). Fertilised chicken eggs (Ovagen Group Ltd., Co. Mayo, Ireland) were purchased at day 0 of development and incubated at 37°C for 3 days. Following incubation, the eggs were cracked into 100 mm diameter petri dishes (Corning Inc., New York, United States), which in turn were then placed within larger 150 mm diameter petri dishes containing sterile PBS, to form a humidified chamber. The chicks were incubated for a further 4 days, completing 7 days of total development, viability of embryos was firstly ensured and checked at every day from this point forward; when cross-linked and sterilised CHyA-B matrices and reinforced composite CHyA-B scaffolds were placed within the chick membrane and incubated for a further 5 days, allowing for vessel ingrowth. At day 12 the samples were harvested and imaged. The vascularisation around the scaffolds was quantified using ImageJ software after treatment with the “Mexican Hat Filter” to outline the blood vessels, and then converted to 8-bit and the blood vessel area was measured.