Decellularized allogeneic heart valves demonstrate self-regeneration potential after a long-term preclinical evaluation

Abstract

Tissue-engineered heart valves are proposed as novel viable replacements granting longer durability and growth potential. However, they require extensive in vitro cell-conditioning in bioreactor before implantation. Here, the propensity of non-preconditioned decellularized heart valves to spontaneous in body self-regeneration was investigated in a large animal model. Decellularized porcine aortic valves were evaluated for right ventricular outflow tract (RVOT) reconstruction in Vietnamese Pigs (n = 11) with 6 (n = 5) and 15 (n = 6) follow-up months. Repositioned native valves (n = 2 for each time) were considered as control. Tissue and cell components from explanted valves were investigated by histology, immunohistochemistry, electron microscopy, and gene expression. Most substitutes constantly demonstrated in vivo adequate hemodynamic performances and ex vivo progressive repopulation during the 15 implantation months without signs of calcifications, fibrosis and/or thrombosis, as revealed by histological, immunohistochemical, ultrastructural, metabolic and transcriptomic profiles. Colonizing cells displayed native-like phenotypes and actively synthesized novel extracellular matrix elements, as collagen and elastin fibers. New mature blood vessels, i.e. capillaries and vasa vasorum, were identified in repopulated valves especially in the medial and adventitial tunicae of regenerated arterial walls. Such findings correlated to the up-regulated vascular gene transcription. Neoinnervation hallmarks were appreciated at histological and ultrastructural levels. Macrophage populations with reparative M2 phenotype were highly represented in repopulated valves. Indeed, no aspects of adverse/immune reaction were revealed in immunohistochemical and transcriptomic patterns. Among differentiated elements, several cells were identified expressing typical stem cell markers of embryonic, hematopoietic, neural and mesenchymal lineages in significantly higher number and specific topographic distribution in respect to control valves. Following the longest follow-up ever realized in preclinical models, non-preconditioned decellularized allogeneic valves offer suitable microenvironment for in vivo cell homing and tissue remodeling. Manufactured with simple, timesaving and cost-effective procedures, these promising valve replacements hold promise to become an effective alternative, especially for pediatric patients.

Figure 1. Macroscopic appearance of TRICOL allogeneic aortic valve before and after implantation.

Allogeneic substitutes demonstrated similar gross morpho-anatomic structure to native valves without signs of leaflet fenestration, rupture or degeneration both after decellularization (C–D) and explant at 15 months (C–D).

Figure 2. Histologic evaluation of explanted allografts.

Panoramic (A and G: H&E, B and H: Elastic van-Gieson, C and I: Azan’s Heidenhain trichrome, magnification: 5 cm) and close-up (D–F and J–L) allograft views respectively at 6 and 15 months after surgery: note trilaminated arrangement with recipient’s repopulating cells on both leaflet sides, i.e. ventricularis (v) and fibrosa (f) (D–E, J–K: H? F and L: Elastic van-Gieson). Magnifications: (A–C and G–I) 1 cm; (D) 500 µm; (E, F) 100 µm; (J) 700 µm; (K, L) 200 µm.

Figure 3. Immunohistochemical characterization of allograft walls at 15 months.

Vimentin- and MyHC_Apla1-positive fibroblasts (A–F) with broad SMA, rare smoothelin and calponin detection (G–O). Re-endothelialization was observed at the intimal layer (G). Novel mature arterioles and capillaries were identified in adventitia and media (H, I, K, L, N, O, Q and R). Magnification: 100 µm.

Figure 4. Immunohistochemical profile of recellularized leaflets at 6 months.

Undetectable calcifications (B, C) or immune rejections against allogeneic tissues (D, E). Conserved trilaminated ECM architecture (A, G and H). Native-like EC (I) and VIC phenotypes (J–N). Stem cell markers of mesenchymal (O) and embryonic (P and Q) lineages, mainly expressed in ventricularis. In (F), PH3-positive leaflet-colonizing cells. V = ventricularis; f = fibrosa. Magnification: 200 µm.

Figure 5. Morphological and immunophenotypical analyses of primary cultures from 15-month regenerated allogeneic walls.

I. Two main morphologies could be observed during culturing medial cells: a cell line more elongated reaching confluence with hill and valley distribution (A, B and C) and another one characterized by spindle-shaped cells growing in clusters (E, F and G). Such morphological aspects revealed a fine analogy of these two primary cells respectively to mature artery SMCs (D) and bone marrow mesenchymal stem cells (H), both of porcine origin. Magnifications: (A, E) 150 µm, (B, F) 100 µm, (C, D, G, H) 50 µm. II. Immunophenotype of primary cultures from 15-month regenerated allogeneic walls. Wide FITC-conjugated SMA-positivity (green), less usual co-immunodetection with smoothelin or SM-MyHC (in red) (A–E), rare OC positivity (F), extensive CD29 expression (J) in contrast to SSEA4, OCT4 and CD90 (G–I) (in green). Magnification: 30 µm.

Figure 6. Scanning electron microscopy on allograft cusps at 6 and 15 implantation months.

Almost complete endothelial coverage (blue), progressive EC acquisition of surface microvilli (purple) and no platelet aggregation onto fibrosa (green; at 6 months in A–B, after 15 months in E–F) and ventricularis (green; at 6 months in C–D, after 15 months in G–H). Note the absence of fibrin deposition, as well as no red cell (red) entrapment in H. Magnifications: (A) 20 µm; (B, D, F, H) 5 µm; (C, E, G) 10 µm.

Figure 7. Transmission electron microscopy on allograft walls.

ECs covering intimal surface (A); underneath the intima, fibroblasts (Fbs) with dilated endoplasmic reticulum (B; asterisks), synthesizing collagen fibrils from fibril-forming channels (C) and elastin fibers (e) (D) or with abundant myofilaments (mf) (E). Erythrocytes (Er)-containing capillaries coated by ECs joined by tight junctions (opposite arrowhead in inset) (F). Amyelinic nerve fibers (NF) encapsulated by Schwann cells (SC) (G). Blood vessels (arrows) in media and adventitia after trichrome staining (H). Neural marker PGP9.5-expressing nerve fiber (arrows) in media (I). Magnifications in I: A 3 µm; B 1 µm; C, D, G 0,5 µm; E, F inset 0,25 µm; F 2 µm; H, I 250 µm. Transmission electron microscopy on allograft cusps. ECs onto intimal surface and sub-intimal Fbs (A). Immature intercellular junction (opposite arrowheads) between ECs adhering elauninic fibers (ef) surrounded by fibrillin microfibrils (encircled area) (B). Interstitial myofibroblasts showing intercellular junctions (opposite arrowheads) (C). Interstitial SMCs containing microfilaments (Mf) (D). Fbs with multiple fibril-forming channels (arrow) and adjacent efs (E). Abundant efs in interstitium (F). Magnifications in II: A 3 µm; B, C, E 0,5 µm; D, F 1 µm.

Figure 8. Gene expression profiles at 15 months post-implantation.

Heat map of differentially expressed genes: Colors based on logarithmic against averaged gene expressions. Green: down-regulated genes; red: up-regulated ones. A.L.: Allograft Leaflet; B.L.: Autograft Leaflet; A.W.: Allograft wall; L.A.: Left Atrium; L.V.: Left Ventricle.

Figure 9. Gene expression profiles at 15 months post-implantation.

Histograms representing enriched Gene Ontology terms for clusters 1, 3, 4 (green and red bars) and 2 (blue bars). P-value ≤0,05 (A). Gene interaction network derived from STRING database analysis of up- and down-regulated genes on A.L. tissues (clusters 1, 3, 4). Pink nodes with grey border connect elements between up- or down-regulated nodes in analyzed clusters. Central color node: gene expression in A.L. tissues; border: B.L. tissues. Yellow edges link connected nodes, while blue edges those altered in analyzed clusters. Purple gene names represent relevant nodes according to Centiscape algorithm. *Genes discussed in the text (B).