Valvulogenesis of a living, innervated pulmonary root induced by an acellular scaffold

Figures

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  Fig. 1. Design and characterisation of the HCCV. A CT scanned image of a normal aortic root used as a template for designing the HCCV a; Segmentation of the root region b; 2D drawing of the components of the HCCV (all units are in mm) c; 3D computer-assisted design (CAD) of the external geometry d; valve assembly where one-third of the HCCV was modelled to the shape of a single sinus, leaflet and one third of ascending artery and 3 of these sinus models were assembled to create the complete HCCV e. A holder was designed to house the 3 sinus models and to create the ascending artery. A counter mould was designed to complementarily fit the sinuses to create the interleaflet triangles and the annulus e. Three jet sprayed sinus moulds and housed into the holder (top) with the counter mould (bottom) f. Whole mould assembly jet sprayed g with the knitted support h. Pictures showing the design of the knitted support with 2 sinus view (top) and one sinus view (bottom) with regions of magnification of the different regions i. Final HCCV after the dissolution of the PVA moulds j showing top view k and annular view l. SEM through the wall of the sinus showing the knitted PCL support (bottom fibres)…

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  Fig. 2. Macroscopic images of explanted HCCV with increases in sinus wall thickness. The functional implanted view of HCCV immediately after implantation a. Graphs showing mean sinus thicknesses b, and mean leaflet lengths c at 26 weeks post-implantation, n = 4 (mean ± SD) biologically independent samples) using one-way ANOVA followed by Tukey’s multiple comparison test. The arterial view of HCCV at 1 week post-implantation d. Open views of 4 HCCV at 26 weeks post-implantation ordered anterior, right posterior, and left posterior for each HCCV e–h. Leaflets showing the position of tears at 9 weeks i, 12 weeks j, k, and 26 weeks l post-implantation.

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  Fig. 3. 1, 2, and 3 weeks post-implantation sections of HCCV. 1-week post-implantation of HCCV showing representative sinus wall images for CD45 immunostaining a, collagen by EVG (*) on luminal side b; 2 weeks post-implantation (ABSR and haematoxylin staining) c, magnified boxes in c d. Cells expressing α-SMA e, TEM showing fibroblast f and myofibroblast g morphology, ECs (CD31) lining neo-vessels (*) h, M1 macrophage expression i, M2 macrophage expression j, neo-vessel (*) and adjacent pericyte k, TEM showing pericytes adjacent to ECs l, m with peg/socket connections (red circles). Collagen n and elastogenesis in neo-vessels (*) o, neo-intima (arrow indicating elastin) p, neo-media q, incomplete capsule (*) r, adipogenesis in the neo-adventitia (*) s by EVG. 3 weeks post-implantation showing lamellae in neo-vessels (EVG) t, α-SMA u, and SMM v by immunostaining, collagen EVG w in the knitted region, elastin sheath on the outer knit (EVG) x, nerve bundle stained with neurofilament protein y, and FBGCs (*) z. # Indicates adventitial side. Scale bars: 250 µm on a, b, h, m, q, v; 500 µm in c, I; 100 µm in d, e, r, s, t, u; 1 µm in f, g, l; 5 µm in k; 25 µm in j, p; 50 µm in n, o, x, y and…

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  Fig. 4. 9 and 12 weeks post-implantation sections of HCCV wall. HCCV showing collagen deposition stained by EVG a, nerve bundles immunostained stained with neurofilament protein b, embedded within expanding adipose tissue stained with FABP4 on the adventitial side c at 9 weeks post-implantation and leptin d at 12 weeks post-implantation. Complete cellularisation of the sinus wall stained with H&E showing a layer of multicellular FBGCs (*) e, cuboidal ECs on the sinus wall f, longitudinal ECs on the arterial side of the leaflets g stained with CD31, SEM of ECs on the sinus wall h, i, TEM of EC j at 9 weeks post-implantation, incomplete elastin capsule stained with EVG k at 9 weeks post-implantation, nerve bundles stained with VIP l, TH m, NPY n, and neurofilament protein o at 12 weeks post-implantation. Scale bars: 500 µm in a, b and k; 250 µm in l, m, n, o; 100 µm in c, d, e, f, g; 10 µm on panel h; 5 µm in i and 0.5 µm in j.

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  Fig. 5. 26 weeks post-implantation sections of HCCV. HCCV showing maturation of the vessels residing in the adventitial adipose layer adjacent to a nerve bundle stained with EVG a; tyrosine hydroxylase immunostained nerve bundle b, neuropeptide Y c, neurofilament protein d, silver stained nerve bundle e, ultrastructure of nerve bundles by TEM f, g, VwF immunostaining on sinus wall h, arterial side of leaflet i, EVG stained EC basement membrane j, elastin immunostaining k, α-SMA l, SMM m, co-expression of CD31 (red) and α-SMA (green) n, full root section showing adipose tissue, elastin and collagen staining by EVG o, magnified panels showing adipose tissue with nerve bundles and vessels p, collagen creeping on ventricular aspect of leaflet q. Retracted leaflet r, normal pulmonary root s showing adipose tissue housing a nerve bundle and vessels in close proximity with magnified image t. HCCV leaflet immunostained with α-SMA u, CD163 v. Upper side in u and v is leaflets’ arterial side. Scale bars: 100 µm in a–c; 50 µm in d, e, h, i, j, k, l, m; 2 µm in f; 5 µm in g; 20 µm in n; 10 µm in o; 1 mm in p, q; 2.5 mm in s; 500 µm in u and 250 µm in v.

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  Fig. 6. In vivo function and degradation of the HCCV. First row shows the echocardiograms of the HCCV at implant during systole a and diastole b, followed by Doppler ultrasound showing laminar flow during systole c and minimal regurgitation during diastole d. The second row shows the HCCV at 2 months during systole e and diastole f, followed by Doppler ultrasound during systole g, and diastole h. Third row shows the HCCV at 6 months during systole i and diastole j, followed by Doppler ultrasound showing systole k and trace regurgitation during diastole l. MRI generated root geometry of 3 HCCVs and one control at 26 weeks post-implantation m. MRI generated ventriculogram showing right (yellow) and left (red) ventricles in 3 HCCV and one control n, instantaneous flow of HCCV at annulus (PS, peak systole; LD, late diastole: ED, early diastole) o, CFD showing the pattern of flow through the pulmonary artery bifurcation of one HCCV and 2 opposing vortices p, CFD showing parallel velocity streamlines in one control (normal sheep) and through 2 HCCVs at peak systole at 6 months post-implantation q. Molecular weight of original and 26 weeks implanted PCL nanofibers in leaflets, sinuses, and…