Fig. 6

Whole blood perfusion of seeded and unseeded grafts for thrombogenicity testing. The dynamic cultivation setup was adapted for the whole blood perfusion experiments into a setup consisting of the vascular graft (a) immersed in Ringer Solution to prevent drying out, of the pump tubing (b), the peristaltic pump (c), the shaker (d), the blood-filled reservoir (e) and the plastic tubing (f) connecting the reservoir to the graft. The whole system was coated with heparin prior to starting the experiment and the shaker was set at 90 rpm throughout the perfusion (A). Evaluation of oxygenation prior to (t0, hollow symbol), at the first (t1, filled symbol) and second hour (t2, crossed symbol) after starting the perfusion revealed increasing oxygen levels which were significant at the second hour (B). Moreover, evaluation of hemolysis revealed extensively increasing K⁺ values, more enhanced in the flow circuit in absence of TEVG, but also significant in the other two groups (C). Evaluation of platelets as main endpoint for thrombogenicity testing indicated the seeded grafts are thromboresistant, since they induced a nonsignificant reduction in platelet while the decellularized non-seeded grafts induced a significant reduction (D). Macroscopic evaluation of the whole blood perfused decellularized graft revealed thrombus formation on the luminal surface (E), a finding that was not present in recellularized grafts (F). Staining of unseeded grafts with CD41 revealed platelets attached to the basal membrane, pointing to their thrombogenicity (G). Seeded, whole blood perfused grafts stained negative for CD41, thus histologically confirming their thromboresistance (H). Since the CD41 staining of decellularized grafts revealed numerous CD41 negative cells, we repeated the H&E staining for these grafts, which revealed several acellular areas (I) and several cellular areas (J), which corresponded with the ones seen in the CD41 staining. Staining with the leukocyte common antigen CD45 revealed a large number of these cells were leukocytes, a finding that confirmed our initial hypothesis of thrombus formation (K). Lastly, the CD31 staining we performed to make sure these cells were not cell remnants of inadequate decellularization was, as expected, negative (L). Ordinary One-Way ANOVA followed by Tukey’s Multiple Comparison Test was performed (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Scale bar represents 50 μm