Human cell-derived tissue-engineered heart valve with integrated Valsalva sinuses: towards native-like transcatheter pulmonary valve replacements

Figures

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  Fig. 1. Qualitative evaluation of hTEM patches cultured with hDF and quantitative tissue composition after 4 weeks culture at different TGF-β1 concentrations. a Representative image of the polyglycolic acid/poly-4-hydroxybutyrate scaffold sutured onto a stainless metal ring before in vitro culture. b Representative image of a hTEM after 4 weeks culture showing shiny neo-tissue deposition and homogeneous matrix distribution. c GAGs content showed slightly higher amount in patches cultured at 5 ng/ml TGF-β1. d HYP content showed similar expression values among all the different groups. e–h Representative images of H&E staining show more pronounced neo-tissue formation for hTEM cultured with TGF-β1 in comparison with the control group. i–l VGEL staining did not show any expression of elastic fibers at 4 weeks follow-up. Col-1 m–p and Col-3 q–t deposition is enhanced in hTEM cultured in the presence of TGF-β1 (scale bars 100 μm). Dot plot graphs present mean ± standard deviation (center line and bounds of error bars, respectively)

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  Fig. 2. Manufacturing process of a hTESV. a PGA scaffold is sutured and integrated into the nitinol stent using continuous sutures. b, c PGA scaffold is coated with 1% P4HB in tetrahydrofuran. d–e hTESVs show dense, shiny, and homogeneous tissue formation laterally d, distally e, and proximally f, which was retained after decellularization procedure

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  Fig. 3. In vitro characterization of the hTESVs after 4 weeks culture. a H&E staining shows the general hTESV morphology comprising a dense collagenous matrix and the scaffold core (scale bar: 5000 μm). b–d Magnification pictures of the hinge region b, middle of the leaflet and wall c, and tip of the leaflet d marked by the dotted rectangles in a (scale bars: 500 μm). e Immunofluorescence staining shows the presence of Collagen 3 (green) and Collagen 1 (red) fibers in the newly formed matrix (scale bars: 100 μm). f Quantitative tissue composition demonstrates the slightly higher amount of GAGs and HYP in hTESV cultured in presence of 5 ng/ml TGF-β1 (white points) in comparison with control hTESV (black points). g Sequence of the opening and closing behavior of hTESVs tested in a valve pulse duplicator system for 1 hour at pulmonary pressure conditions. h Pressure and flow values for a representative hTESV after 1 hour in vitro valve testing; in the left panel, blue and red indicate pulmonary pressure and ventricular pressure, respectively; in the right panel, red indicates flow rate over time. Dot plot graphs present mean ± standard deviation (center line and bounds of error bars,…

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  Fig. 4. Transapical hTESV delivery under fluoroscopy guidance. a Imaging of the native pulmonary root. b, c Insertion and alignment of the delivery system to the implantation site. d, e Controlled positioning and deployment of the hTESV. f Final evaluation of the deployed hTESV confirming sufficient positioning and good valve functionality

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  Fig. 5. Echocardiographic assessment of valve functionality in vivo. a–f 2D images of hTESV show good leaflet motion during systole a–c and diastole d–f. b, e 2D-Doppler of hTESV demonstrates absence of anomalies in the forward flow, no regurgitation nor paravalvular leakage. c, f 3D representation of hTESV showing proper leaflet mobility during systole c and diastole f. g–i 3D-CT reconstruction performed pre-operatively shows the adult sheep heart and the valve implantation site from a proximal g, distal h, and lateral i perspective, confirming the correct positioning of the hTESV in the right ventricular outflow tract, over the native valve. AV = aortic valve, MV = mitral valve

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  Fig. 6. Post-mortem macroscopical and histological evaluation of hTESVs after 4 hours follow-up. All three hTESVs were correctly positioned at the entrance of the pulmonary root without signs of stent migration. Representative images of the H&E staining were performed on the valve cross-section. a, b Correct positioning of the hTESV confirmed by the presence of native leaflets entrapped in the stent struts. c, d hTESV showing thrombus-free smooth surfaces and absence of tears or ruptures. e H&E staining shows retained valve morphology after 4 hours in vivo follow-up (scale bar: 5000 μm). f–h H&E staining of the region marked by the dotted rectangles in e (1–3) shows host cellular infiltration predominantly at the surface and in the hinge region of the hTESVs (black arrows) (scale bars: 200 μm). i–k 60X magnification pictures of region marked by the dotted rectangles in f–h present an organized and packed collagenous matrix together with infiltrated leukocytes (black asterisks, I) and erythrocytes (triangle, k) (scale bars: 25 μm). p = polymer remnants