Successful Reconstruction of the Right Ventricular Outflow Tract by Implantation of Thymus Stem Cell Engineered Graft in Growing Swine

Abstract

Graft cellularization holds great promise in overcoming the limitations associated with prosthetic materials currently used in corrective cardiac surgery. In this study, the authors evaluated the advantages of graft cellularization for right ventricular outflow tract reconstruction in a novel porcine model. After 4.5 months from implantation, improved myocardial strain, better endothelialization and cardiomyocyte incorporation, and reduced fibrosis were observed in the cellularized grafts compared with the acellular grafts. To the authors' knowledge, this is the first demonstration of successful right ventricular outflow tract correction using bioengineered grafts in a large animal model.

Keywords: CM, cardiomyocyte; Cx-43, connexin-43; DMEM, Dulbecco’s modified Eagle’s medium; EC, endothelial cell; FBS, fetal bovine serum; IL, interleukin; IsoB4, isolectin B4; MSC, mesenchymal stem cell; PBS, phosphate-buffered saline; PS, penicillin/streptomycin; RT, room temperature; RV, right ventricular; RVOT, right ventricular outflow tract; RVOT-MS, fractional area of change in the right ventricular outflow tract; SIS-ECM, small intestinal submucosa–derived extracellular matrix; T-MSC, thymus-derived mesenchymal stem cell; VMSC, vascular smooth muscle cell; cMYH, cardiac myosin heavy chain; congenital heart disease; reconstruction; right ventricular outflow swine model; tissue engineering; tract stem cells.

Graphical abstract

Figure 1

Phenotypic and Functional Characterization of Thymus-Derived Mesenchymal Stem Cells (A) Flow cytometry representative histograms illustrate the expression of CD44, CD73, CD90, and CD105 and lack of endothelial and hematopoietic markers on thymus-derived mesenchymal stem cells. (B) Bar chart showing the proportion of viable cells positive to the investigated markers (n = 4; mean ± SE). (C) Multilineage differentiation of the cells into osteocytes, adipocytes. Nontreated cells were used as negative controls.

Figure 2

Biological and Mechanical Assessment of the Unseeded and Cell-Seeded Grafts (A) Seeded graft (arrow) is mounted and cultured in a bioreactor. (B) Viable (L) and dead (D) cells attached to the seeded scaffold. Scanning electron microscopic (SEM) images illustrate the topography of the unseeded graft and a confluent layer of oriented cells growing on the seeded sample. Scale bars, 100 μm. Hematoxylin and eosin (H and E) staining shows lack of nuclei in the unseeded scaffold and a multilayer of cells in the seeded graft. Elastic van Gieson (EVG) staining confirmed the capacity of the seeded cells to produce their own extracellular matrix. Scale bars, 50 μm. (C) Young’s modulus and ultimate tensile strength of the unseeded and seeded grafts showed no significant differences between the 2 groups (n = 4; mean ± SE).

Figure 3

in Vivo Right Ventricular Outflow Tract Reconstruction and Operated Animals Follow-Up (A) Cartoon (i) and macroscopic image (ii) showing the site of the implant on the right ventricular outflow tract (RVOT). Gross analysis of the explants after 4.5 months in vivo shows the epicardial (iii) and endocardial (iv) sides of the graft (dotted line). (B) The operated pigs increased their body weight at a normal rate. (C) Doppler echocardiographic measurements of the right ventricle immediately before termination demonstrate comparable RVOT diameter and maximum velocity (Vmax) in the operated animals and unoperated control (Ctr) pigs. (D) Representative cardiac magnetic resonance images of right ventricular diastolic and systolic area (encircled in green) before surgery and 4.5 months thereafter. The patches could be visualized as a small bump protruding from the right ventricle (arrows). (E) RVOT myocardial strain measured at termination was greater in the animals implanted with seeded grafts. See Supplemental Video 1.

Figure 3

in Vivo Right Ventricular Outflow Tract Reconstruction and Operated Animals Follow-Up (A) Cartoon (i) and macroscopic image (ii) showing the site of the implant on the right ventricular outflow tract (RVOT). Gross analysis of the explants after 4.5 months in vivo shows the epicardial (iii) and endocardial (iv) sides of the graft (dotted line). (B) The operated pigs increased their body weight at a normal rate. (C) Doppler echocardiographic measurements of the right ventricle immediately before termination demonstrate comparable RVOT diameter and maximum velocity (Vmax) in the operated animals and unoperated control (Ctr) pigs. (D) Representative cardiac magnetic resonance images of right ventricular diastolic and systolic area (encircled in green) before surgery and 4.5 months thereafter. The patches could be visualized as a small bump protruding from the right ventricle (arrows). (E) RVOT myocardial strain measured at termination was greater in the animals implanted with seeded grafts. See Supplemental Video 1.

Supplemental Video 1

Figure 4

Examination of the Explanted Grafts (A,B) Hematoxylin and eosin (H and E) and Masson’s trichrome staining of the explants illustrates the collagen-rich grafts well integrated with the right ventricle (RV). (C) Higher magnification images of the samples. H and E staining shows little remains of the scaffold (arrows) 4.5 months after implantation (i,v). Masson’s trichrome staining demonstrates the presence of new muscle tissue (arrows) generated from the implanted grafts (ii,vi). Elastic van Gieson staining illustrates an elastin-rich endothelium laying the inner side of the explants (iii,vii). No calcification was detected as shown by von Kossa stain (iv,viii). (D) More abundance of collagen was detected in the unseeded samples in comparison with the seeded group. (E) Immunostaining for matrix metalloproteinase 1 (MMP1) confirmed that the extracellular matrix was undergoing remodeling processes, and no differences were observed between the 2 groups. (F) Immunostaining and quantification of the fibrotic marker discoidin domain-containing receptor 2 (DDR2) (green fluorescence) showed more fibrosis in the unseeded explants compared with the seeded ones. (G) Cells expressing cardiac myosin heavy chain (cMYH) were present in explants from both groups, though more abundant in the seeded one. In addition, only the seeded graft contained cells double positive for cMYH and connexin-43 (Cx-43). Scale bars, 50 μm. Dapi = 4′,6-diamidino-2-phenylindole; Iso = isolectin B4.

Figure 4

Examination of the Explanted Grafts (A,B) Hematoxylin and eosin (H and E) and Masson’s trichrome staining of the explants illustrates the collagen-rich grafts well integrated with the right ventricle (RV). (C) Higher magnification images of the samples. H and E staining shows little remains of the scaffold (arrows) 4.5 months after implantation (i,v). Masson’s trichrome staining demonstrates the presence of new muscle tissue (arrows) generated from the implanted grafts (ii,vi). Elastic van Gieson staining illustrates an elastin-rich endothelium laying the inner side of the explants (iii,vii). No calcification was detected as shown by von Kossa stain (iv,viii). (D) More abundance of collagen was detected in the unseeded samples in comparison with the seeded group. (E) Immunostaining for matrix metalloproteinase 1 (MMP1) confirmed that the extracellular matrix was undergoing remodeling processes, and no differences were observed between the 2 groups. (F) Immunostaining and quantification of the fibrotic marker discoidin domain-containing receptor 2 (DDR2) (green fluorescence) showed more fibrosis in the unseeded explants compared with the seeded ones. (G) Cells expressing cardiac myosin heavy chain (cMYH) were present in explants from both groups, though more abundant in the seeded one. In addition, only the seeded graft contained cells double positive for cMYH and connexin-43 (Cx-43). Scale bars, 50 μm. Dapi = 4′,6-diamidino-2-phenylindole; Iso = isolectin B4.

Figure 4

Examination of the Explanted Grafts (A,B) Hematoxylin and eosin (H and E) and Masson’s trichrome staining of the explants illustrates the collagen-rich grafts well integrated with the right ventricle (RV). (C) Higher magnification images of the samples. H and E staining shows little remains of the scaffold (arrows) 4.5 months after implantation (i,v). Masson’s trichrome staining demonstrates the presence of new muscle tissue (arrows) generated from the implanted grafts (ii,vi). Elastic van Gieson staining illustrates an elastin-rich endothelium laying the inner side of the explants (iii,vii). No calcification was detected as shown by von Kossa stain (iv,viii). (D) More abundance of collagen was detected in the unseeded samples in comparison with the seeded group. (E) Immunostaining for matrix metalloproteinase 1 (MMP1) confirmed that the extracellular matrix was undergoing remodeling processes, and no differences were observed between the 2 groups. (F) Immunostaining and quantification of the fibrotic marker discoidin domain-containing receptor 2 (DDR2) (green fluorescence) showed more fibrosis in the unseeded explants compared with the seeded ones. (G) Cells expressing cardiac myosin heavy chain (cMYH) were present in explants from both groups, though more abundant in the seeded one. In addition, only the seeded graft contained cells double positive for cMYH and connexin-43 (Cx-43). Scale bars, 50 μm. Dapi = 4′,6-diamidino-2-phenylindole; Iso = isolectin B4.

Figure 5

Evaluation of the Endothelialization and Vascularization of the Explants (A) Scanning electron microscopic images show that the topography of the endocardial surface of the explants is composed of aligned and compacted cells. Similar cell confluency to the native tissue was detected in the unseeded and seeded patches. (B) By measuring the thickness of the endocardium of the sections stained for isolectin B4 (Iso), we observed that the seeded group developed a thicker layer than the unseeded group (C). The explanted grafts generated a fine and rich vascularization shown by isolectin B4 and alpha–smooth muscle actin (αSMA) staining. However, no differences were observed between the 2 groups in terms of capillaries and arteriole density. Scale bars, 50 μm. Dapi = 4′,6-diamidino-2-phenylindole; RV = right ventricle.

Figure 6

Y Chromosome Fluorescent in Situ Hybridization of the Explanted Grafts The male seeded cells (Y chromosome [Y chr] positive) were detected over the scaffold before implantation and after 24 h, 1 week, and 2 weeks. The number of seeded cells decreased with time in vivo. No seeded cells were observed at 4.5 months. Scale bars, 50 μm. Dapi = 4′,6-diamidino-2-phenylindole.

Figure 7

Evaluation of Proliferating Cells in Vivo (A) Immunostaining for Ki67 and isolectin B4 (Iso), smooth muscle myosin heavy chain (sm-MYH), α–sarcomeric actinin (αSA), and vimentin (Vim) in the seeded grafts explanted at 24 h, 1 week, and 2 weeks. Coexpression of Ki67 with Iso and Vim suggests that the proliferating cells at early stages consist of endothelial cells (ECs) and fibroblasts. Scale bars, 50 μm. (B) The numbers of proliferating ECs and fibroblasts increase after 1 week in vivo, with the fibroblasts showing an increased proliferation also after 2 weeks. (C) Double staining for Ki67 and Iso, sm-MYH, αSA, and Vim in the unseeded and seeded grafts explanted at 4.5 months. Cells coexpressing Ki67 with Iso, sm-MYH, and Vim were detected in the grafts, suggesting that the proliferating cells are ECs, vascular smooth muscle cells (VSMCs), and fibroblasts. Scale bars, 50 μm. (D) The number of Ki67-positive cells seemed to be the same between the unseeded and seeded groups 4.5 months after implantation.

Figure 7

Evaluation of Proliferating Cells in Vivo (A) Immunostaining for Ki67 and isolectin B4 (Iso), smooth muscle myosin heavy chain (sm-MYH), α–sarcomeric actinin (αSA), and vimentin (Vim) in the seeded grafts explanted at 24 h, 1 week, and 2 weeks. Coexpression of Ki67 with Iso and Vim suggests that the proliferating cells at early stages consist of endothelial cells (ECs) and fibroblasts. Scale bars, 50 μm. (B) The numbers of proliferating ECs and fibroblasts increase after 1 week in vivo, with the fibroblasts showing an increased proliferation also after 2 weeks. (C) Double staining for Ki67 and Iso, sm-MYH, αSA, and Vim in the unseeded and seeded grafts explanted at 4.5 months. Cells coexpressing Ki67 with Iso, sm-MYH, and Vim were detected in the grafts, suggesting that the proliferating cells are ECs, vascular smooth muscle cells (VSMCs), and fibroblasts. Scale bars, 50 μm. (D) The number of Ki67-positive cells seemed to be the same between the unseeded and seeded groups 4.5 months after implantation.

Figure 8

in Vitro Assessment of the Paracrine Effect of the Thymus-Derived Mesenchymal Stem Cells (A) Evaluation of the proliferation and apoptosis of rat cardiomyocytes (rCMs) cultured in the presence of thymus-derived mesenchymal stem cells (T-MSCs) or with their conditioned medium showed that the presence of the stem cells stimulates the proliferation of the cardiomyocytes (CMs), without affecting apoptosis (n = 3). (B) A migration assay of the rCMs either cultured with the conditioned medium or cocultured with the T-MSCs demonstrated that the stem cells can promote the migration of the CMs. The proportion of gap closure 14 h following the scratch was greater in the cocultured condition compared with the control. (C) Analysis of the cytokines and growth factors released by the T-MSCs showed secretion of interleukin (IL)–1ra, IL-2, and IL-6 and a high level of IL-8. Absence of interferon (INF)-γ and tumor necrosis factor (TNF)–α was observed in the porcine T-MSC (pT-MSC)–conditioned medium.