Advancing Heart Valve Replacement: Risk Mitigation of Decellularized Pulmonary Valve Preparation for Its Implementation in Public Tissue Banks

Figure 1. Samples processing to perform uniaxial mechanical assay. (a) Decellularized pulmonary valve (b) longitudinal and transversal bone-shape samples. (c) bone-shape samples after stretching and (d) schematic diagram of bone-shaped-samples Anchorage on tensile strength tester before stretching.

Figure 2. Schematic view of test bench for hydrodynamic analysis. (a): the height of the water column is modified to adjust it to different physiological pressures for the leaking test. (b): Sketch of the closed flow circuit to assess the hemodynamic pressure response of the valves.

Figure 3. ECM biomolecules quantification. (AI) collagen pulmonary artery, (AII) elastin pulmonary artery, (AIII) GAGs pulmonary artery, (BI) collagen leaflets, (BII) elastin leaflets, (BIII) GAGs leaflets, (CI) collagen myocardium, (CII) elastin myocardium and (CIII) GAGs myocardium. Each graphic includes the results for native tissue (fresh or cryopreserved) and decellularized tissue (from fresh or cryopreserved). For the statistical analysis a non-parametric test Mann-Whitney double-tail has been performed with a minimum of N=5 and n=2. Significance level is p<0.05.

Figure 4. Biomechanical properties, obtained with uniaxial tensile test. For each heart valve group (N=5 each group) samples of two directions (longitudinal and transversal) were evaluated. (a) Comparison of different PV conditions in terms of Maximum load, Young Modulus, Elongation at rupture and Stiffness. (b) Strain-stress curve for longitudinal samples and (c) Stress-strain curves for transversal samples. Values are given as mean with standard deviation. PV, pulmonary heart valve; dPV, decellularized heart valve. For the statistical analysis a non-parametric test Mann-Whitney double-tail has been performed with a minimum of N=5. Significance level is considered for p<0.05.

Figure 5. Competence test under static pressure of cryopreserved and cryopreserved decellularized pulmonary valves at a pressure range of 10–60mmHg. Continuous line (˗˗˗˗) states for cryopreserved tissues. Dotted line (···) states for decellularized tissues from cryopreserved samples.

Figure 6. Tissue structure evaluation by histology (20x). Sections of arterial pulmonary wall and myocardium of 2 different conditions (fresh and fresh-decellularized tissue) stained with Haematoxylins-Eosin (H&E), Masson Trichrome (MS’s) and Verhoeff-Van Gieson (VH) staining. [A]Staining of pulmonary wall from two distinct conditions using H&E; [B] Staining of pulmonary wall from two distinct conditions using MT’s; [C] Staining of pulmonary wall from two distinct conditions using VH; [D] Staining of myocardium from two distinct conditions using H&E; [E] Staining of myocardium from two distinct conditions using; [F] Staining of myocardium from four distinct conditions using VH. A complete panel of tissue structure evaluation by histology of the four reported conditions (cryopreserved, cryopreserved-decellularized, fresh and fresh-decellularized) are included in supplementary information.

Figure 7. DNA quantity given in ng/mg dry tissue after decellularization of fresh pulmonary heart valves (fresh, N=5-7; freshDECEL, N = 6–9) and cryopreserved pulmonary heart valves (cryopreserved, N=5; *DECEL, N = 5–7). DNA was quantified by fluorescence detection using PicoGreen™ dye.

Figure 8. Cytotoxic assay. Cell viability of 3T3 cells (murine fibroblast) incubated for 24 hours with tissue extract prepared from fresh (N=4), decellularized (N=5), and fresh decellularized (BTB, N=3) pulmonary heart valve tissue. Cell viability is expressed as percentage relative to the negative control (culture medium) as 100% of viability. Positive cytotoxic control prepared with 0.1% Triton X-100, V/V) results to 0.8% of viability. Experiments were performed as technical triplicates using a density of 10.000 cells per well.