Abstract 716: 2D and 3D Assessment of Angiogenesis in Cardiac Engineered Tissues Implanted on Infarcted Rat Hearts

A lack of efficient vessel perfusion in cardiac engineered tissues affects cell viability and limits the success of implanted cardiac tissues. To address the need for vascular infiltration, we have developed a collagen-based cardiac construct releasing pro-angiogenic growth factors. The constructs were implanted in a rodent model of myocardial infarction four days after ischemia/reperfusion injury. Angiogenesis assessment was performed 4 weeks after implantation by 2D histological analysis with RECA-1 antibody and with Microfil® (Flow Tech, MA) perfusion followed by 3D microCT analysis (voxel dimensions 10x10x10 μm). Cross-registration of microCT z-stacks with histological images allows segmentation of remote, infarct and implant regions. The heart vasculature was reconstructed in 3D with ImageJ and Imaris software, and Image J volume calculator and skeletonization algorithm have been used to measure vessels volume, length and number of branch points in each region of interest. Results from 2D histological analysis confirmed the development of capillary-like structures in the implants, visible in both cross and longitudinal sections in the histological slides. The density of cross-sectional vessels measured in the cardiac tissues was comparable to the density of vessels present in the infarct region (118 ± 77 and 93 ±74 vessels/mm 2 counted in infarct and implant regions, respectively). Furthermore, a 1.5-fold increase in vessels density was detected in the infarct zone of the treatment group with respect to the sham rats at 4 weeks. 3D reconstruction of rat heart vasculature showed evidence of the formation of new, perfused vessels in cardiac implants and presumably host-to graft connections. However, perfused vessels volume was not very different in the implants compared to the infarct regions. To the best of our knowledge, this is the first study where a 3D reconstruction of the perfused vasculature of a repaired rat heart has been conducted with a resolution of 10 μm, and that evidence of host-to-graft connections and vascular integration were shown 4 weeks after implantation. The automated quantification proposed is this study represents a non-biased method that can be further adopted by other groups to assess angiogenesis in cardiac implants.