The PEDIAVALVE project – tissue-engineered pulmonary valves for paediatric valvular replacement

Figures

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  FIGURE 1. Design and implementation of the decellularization system. (A,B) The system was first designed in AutoCAD (Autodesk, CA, USA), and then all components were printed on a 3D printer. The system contains mounts for 5 valves and is fixed on a glass container. (C) Lamb hearts were brought to the laboratory. (D) The valves were dissected and mounted in the system, after which the container was connected to a perfusion pump with pressure and fluid flow measurement via a LabView (NI, TX, USA) interface (E).

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  FIGURE 2. Mounting the valves in the bioreactor–(A) Decell valve, (B) fresh valve, (C) heart valve bioreactor, (D) image captured by the high-speed camera, (E) image processed with the ImageJ Software.

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  FIGURE 3. Validation of decellularization of ovine pulmonary valves. Samples from cusps, wall and sinus. DAPI staining (A,B,E,F,I,J), H&E staining (C,D,G,H,K,L), cell nuclei present in fresh tissue (left columns), nuclei absent after decellularization (right columns).

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  FIGURE 4. The same tissue samples were used for DNA extraction, and the extracts were analyzed on agarose (left) and by spectrophotometry (right). Both tests show a decrease in the amount of DNA below the detection limit after decellularization. Inset in left: 100% sterility for all samples. Legend for the left image: C = DNA standard, F = fresh pulmonary valves, D = decellularized pulmonary valves, L = leaflet, W = wall, S = sinus.

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  FIGURE 5. Frame-by-frame opening area (mm2) of a decellularized pulmonary valve over six consecutive cycles in the bioreactor. Orifice area was measured on individual frames extracted from high-speed camera recordings and processed in ImageJ. Peak opening values ranged between 47 and 51 mm2, with consistent behaviour across all cycles.

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  FIGURE 6. Mean Doppler parameters measured over 12 cycles for the four valve types evaluated in the bioreactor. The decellularized valve performed similarly to the biological prosthesis across all parameters, and differences relative to the fresh valve did not reach statistical significance for Vmax (p = 0.053), Pmax (p = 0.158), or VTI. Vmax—maximal velocity (m/s); Vmed—mean velocity (m/s); Pmax—maximal pressure gradient (mmHg); Pmed—mean pressure gradient (mmHg); VTI—velocity-time integral (cm).

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  FIGURE 7. Biocompatibility testing of decellularized pulmonary valves, Live/Dead staining after seeding with fibroblasts and endothelial cells. Cells appeared viable 14 days after seeding.

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  FIGURE 8. Ex vivo implantation of the decellularized pulmonary valve (decell). (A) A sheep heart with right ventricular outflow tract (RVOT) before “surgery”; a decellularized valve was placed next to the RVOT for comparison. (B) The native valve was extracted and the decellularized valve was sutured to the pulmonary valve annulus. (C) After complete suturing, the decellularized valve shows good ex vivo dimensional fit in the RVOT orifice.