Tissue-engineered Cardiac Patch Research Articles

Adult Bone Marrow-Derived Mesenchymal Stem Cells Seeded on Tissue-Engineered Cardiac Patch Contribute to Myocardial Scar Remodeling and Enhance Revascularization in a Rabbit Model of Chronic Myocardial Infarction.

Although the transplantation of tissue-engineered cardiac patches with adult bone marrow-derived mesenchymal stem cells (MSCs) can enhance cardiac function after acute or chronic myocardial infarction (MI), the recovery mechanism remains controversial. This experiment aimed to investigate the outcome measurements of MSCs within a tissue-engineered cardiac patch in a rabbit chronic MI model. This experiment was divided into four groups: left anterior descending artery (LAD) sham-operation group (N = 7), sham-transplantation (control, N = 7), non-seeded patch group (N = 7), and MSCs-seeded patch group (N = 6). PKH26 and 5-Bromo-2'-deoxyuridine (BrdU) labeled MSCs-seeded or non-seeded patches were transplanted onto chronically infarct rabbit hearts. Cardiac function was evaluated by cardiac hemodynamics. H&E staining was performed to count the number of vessels in the infarcted area. Masson staining was used to observe cardiac fiber formation and to measure scar thickness. Four weeks after transplantation, a remarkable improvement in cardiac functionality could be distinctly observed, which was most significant in the MSCs-seeded patch group. Moreover, labeled cells were detected in the myocardial scar, with most of them differentiated into myofibroblasts, some into smooth muscle cells, and only a few into cardiomyocytes in the MSCs-seeded patch group. We also observed significant revascularization in the infarct area implanted in either MSCs-seeded or non-seeded patches. In addition, there were significantly greater numbers of microvessels in the MSCs-seeded patch group than in the non-seeded patch group.

Abstract 12372: In-Situ Constructive Myocardial Regeneration With A 3-D Tissue-Engineered Cardiac Patch Combined Myocardial Cells and Extracellular Matrix

Objective: We developed a tissue-engineered cardiac patch derived from a myocardial cell block reinforced with extracellular matrix (ECM) to enhance in-situ myocardial regeneration. We sought to assess constructive and functional regeneration of the patch in a porcine preparation. Methods: A scaffold-free myocardial cell block was created with cardiomyocytes and embryonic fibroblast cells using a 3D Net Mold system( Fig.A ). The myocardial cell block incorporated with ECM sheets(cbECM) was surgically implanted into a right ventricular (RV) of a healthy porcine model (n=4), or Dacron patches (n=4) which served as control ( Fig.B,C ). After 60 days, the implanted patches were assessed with cardiac magnetic resonance imaging including the rest perfusion(RP) analysis, Extracellular volume fraction(ECV) analysis, and electroanatomical mapping(EAM) studies as well as post euthanasia analyses including histological analysis and Quantitative reverse transcription-polymerase chain reaction(RT-PCR). Results: RP analysis showed improved regional tissue perfusion in the cbECM patch area( Fig.D ). ECV values were much less in the cbECM patch compared to Dacron patch(36.1±3.6 vs. 72.0±13.6, p=0.012)( Fig.E ), which implied the cbECM patch had less fibrosis and edema. EAM revealed regenerated electrical conductivity in the patch as evidenced by relatively preserved voltage regions (1.11±0.8 mV) compared with the normal RV control 4.7±2.8 mV( Fig.F ). RT-PCR showed increased expression of growth factors in cbECM group( Fig.G ). Repopulation of site-specific host cells including premature cardiomyocyte and active vasculogenesis were confirmed with histology/tissue analysis( Fig.H ). Conclusions: The tissue-engineered cardiac patch facilitated in-situ constructive functional myocardial regeneration with increased regional tissue perfusion, and positive electrical activity in the porcine RV model.

Tissue-engineered cardiac patch seeded with human induced pluripotent stem cell derived cardiomyocytes promoted the regeneration of host cardiomyocytes in a rat model.

BackgroundThousands of babies are born with congenital heart defects that require surgical repair involving a prosthetic implant. Lack of growth in prosthetic grafts is especially detrimental in pediatric surgery. Cell seeded biodegradable tissue engineered grafts are a novel solution to this problem. The purpose of the present study is to evaluate the feasibility of seeding human induced pluripotent stem cell derived cardiomyocytes (hiPS-CMs) onto a biodegradable cardiac patch.MethodsThe hiPS-CMs were cultured on a biodegradable patch composed of a polyglycolic acid (PGA) and a 50:50 poly (l-lactic-co-ε-caprolactone) copolymer (PLCL) for 1 week. Male athymic rats were randomly divided into 2 groups of 10 animals each: 1. hiPS-CM seeded group, and 2. Unseeded group. After culture, the cardiac patch was implanted to repair a defect with a diameter of 2 mm created in the right ventricular outflow tract (RVOT) wall. Hearts were explanted at 4 (n = 2), 8 (n = 2), and 16 (n = 6) weeks after patch implantation. Explanted patches were assessed immunohistochemically.ResultsSeeded patch explants did not stain positive for α-actinin (marker of cardiomyocytes) at the 4 week time point, suggesting that the cultured hiPS-CMs evacuated the patch in the early phase of tissue remodeling. However, after 16 weeks implantation, the area fraction of positively stained α-actinin cells was significantly higher in the seeded group than in the unseeded group (Seeded group: 6.1 ± 2.8% vs. Unseeded group: 0.95 ± 0.50%, p = 0.004), suggesting cell seeding promoted regenerative proliferation of host cardiomyocytes.ConclusionsSeeded hiPS-CMs were not present in the patch after 4 weeks. However, we surmise that they influenced the regeneration of host cardiomyocytes via a paracrine mechanism. Tissue-engineered hiPS-CMs seeded cardiac patches warrant further investigation for use in the repair of congenital heart diseases.

Electroactive polyurethane/siloxane derived from castor oil as a versatile cardiac patch, part I: Synthesis, characterization, and myoblast proliferation and differentiation.

Tissue-engineered cardiac patch aims at regenerating an infarcted heart by improving cardiac function and providing mechanical support to the diseased myocardium. In order to take advantages of electroactivity, a new synthetic method was developed for the introduction of an electroactive oligoaniline into the backbone of prepared patches. For this purpose, a series of electroactive polyurethane/siloxane films containing aniline tetramer (AT) was prepared through sol-gel reaction of trimethoxysilane functional intermediate polyurethane prepolymers made from castor oil and poly(ethylene glycol). Physicochemical, mechanical, and electrical conductivity of samples were evaluated and the recorded results were correlated to their structural characteristics. The optimized films were proved to be biodegradable and have tensile properties suitable for cardiac patch application. The embedded AT moieties in the backbone of the prepared samples preserved their electroactivity with the electrical conductivity in the range of 10-4 S/cm. The prepared films were compatible with proliferation of C2C12 and had potential for enhancing myotube formation even without external electrical stimulation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 775-787, 2016.

Functional Effects of a Tissue-Engineered Cardiac Patch From Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in a Rat Infarct Model

A tissue-engineered cardiac patch provides a method to deliver cardiomyoctes to the injured myocardium with high cell retention and large, controlled infarct coverage, enhancing the ability of cells to limit remodeling after infarction. The patch environment can also yield increased survival. In the present study, we sought to assess the efficacy of a cardiac patch made from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to engraft and limit left ventricular (LV) remodeling acutely after infarction. Cardiac patches were created from hiPSC-CMs and human pericytes (PCs) entrapped in a fibrin gel and implanted acutely onto athymic rat hearts. hiPSC-CMs not only remained viable after in vivo culture, but also increased in number by as much as twofold, consistent with colocalization of human nuclear antigen, cardiac troponin T, and Ki-67 staining. CM+PC patches led to reduced infarct sizes compared with myocardial infarction-only controls at week 4, and CM+PC patch recipient hearts exhibited greater fractional shortening over all groups at both 1 and 4 weeks after transplantation. However, a decline occurred in fractional shortening for all groups over 4 weeks, and LV thinning was not mitigated. CM+PC patches became vascularized in vivo, and microvessels were more abundant in the host myocardium border zone, suggesting a paracrine mechanism for the improved cardiac function. PCs in a PC-only control patch did not survive 4 weeks in vivo. Our results indicate that cardiac patches containing hiPSC-CMs engraft onto acute infarcts, and the hiPSC-CMs survive, proliferate, and contribute to a reduction in infarct size and improvements in cardiac function. In the present study, a cardiac patch was created from human induced pluripotent stem cell-derived cardiomyocytes and human pericytes entrapped in a fibrin gel, and it was transplanted onto infarcted rat myocardium. It was found that a patch that contained both cardiomyocytes and pericytes survived transplantation and resulted in improved cardiac function and a reduced infarct size compared with controls.

In situ constructive myocardial remodeling of extracellular matrix patch enhanced with controlled growth factor release

ObjectiveIn an effort to expand treatment for advanced heart failure, we sought to develop a tissue-engineered cardiac patch for constructive and functional in situ myocardial regeneration. MethodsAn extracellular matrix patch derived from porcine small intestine submucosa was incorporated with a controlled release of basic fibroblast growth factor. The patch was surgically implanted into the porcine right ventricle (group B, n = 5). Untreated extracellular matrix (group U) and Dacron (group D) patches served as control (n = 5/group). Cardiovascular magnetic resonance was performed in all 3 groups 60 days postsurgery to evaluate regional contractility with peak longitudinal strain, perfusion with relative maximum upslope, and extent of fibrosis/edema with extracellular volume fraction. Electrophysiologic-anatomic mapping was performed in group B. Histology and quantitative reverse transcription-polymerase chain reaction were performed for further tissue characterization. ResultsCardiovascular magnetic resonance–derived parameters were significantly better in group B compared with groups U and D (strain: group B = −16.6% ± 1.8%, group U = −14.7% ± 1.2%, group D = −9.0% ± 1.5%, P < .001; upslope: group B = 13.7% ± 1.1%, group U = 10.8% ± 1.3%, group D = 6.4% ± 1.8%, P < .001; extracellular volume: group B = 45% ± 7%, group U = 54% ± 10%, group D = 70% ± 10%, P = .003). Histology in group B showed a homogenous distribution of host cells, including tropomyosin and α-sarcomeric actinin–positive maturing cardiomyocytes. Group B demonstrated the greatest degree of vasculogenesis as determined by capillary density analysis (group B = 19.5 ± 6.2/mm3, group U = 12.7 ± 2.5/mm3, group D = 6.9 ± 3.7/mm3, P < .001). Quantitative reverse transcription-polymerase chain reaction supported the histologic findings. Electrophysiologic-anatomic mapping in group B indicated positive electrical conductivity in the patch area. ConclusionsThe extracellular matrix patch enhanced with controlled release of fibroblast growth factor facilitated in situ constructive repopulation of the host cells, including cardiomyocyte and functional regeneration, increased regional contractility and tissue perfusion, and positive electrical activity in a porcine preparation.

Fibrin patch-based insulin-like growth factor-1 gene-modified stem cell transplantation repairs ischemic myocardium.

Bone marrow mesenchymal stem cells (BMSCs), tissue-engineered cardiac patch, and therapeutic gene have all been proposed as promising therapy strategies for cardiac repair after myocardial infarction. In our study, BMSCs were modified with insulin-like growth factor-1 (IGF-1) gene, loaded into a fibrin patch, and then transplanted into a porcine model of ischemia/reperfusion (I/R) myocardium injury. The results demonstrated that IGF-1 gene overexpression could promote proliferation of endothelial cells and cardiomyocyte-like differentiation of BMSCs in vitro. Four weeks after transplantation of fibrin patch loaded with gene-modified BMSCs, IGF-1 overexpression could successfully promote angiogenesis, inhibit remodeling, increase grafted cell survival and reduce apoptosis. In conclusion, the integrated strategy, which combined fibrin patch with IGF-1 gene modified BMSCs, could promote the histological cardiac repair for a clinically relevant porcine model of I/R myocardium injury.

Fabrication of a myocardial patch with cells differentiated from human-induced pluripotent stem cells.

The incidence of cardiovascular disease represents a significant and growing health-care challenge to the developed and developing world. The ability of native heart muscle to regenerate in response to myocardial infarct is minimal. Tissue engineering and regenerative medicine approaches represent one promising response to this difficulty. Here, we present methods for the construction of a cell-seeded cardiac patch with the potential to promote regenerative outcomes in heart muscle with damage secondary to myocardial infarct. This method leverages iPS cells and a fibrin-based scaffold to create a simple and commercially viable tissue-engineered cardiac patch. Human-induced pluripotent stem cells (hiPSCs) can, in principle, be differentiated into cells of any lineage. However, most of the protocols used to generate hiPSC-derived endothelial cells (ECs) and cardiomyocytes (CMs) are unsatisfactory because the yield and phenotypic stability of the hiPSC-ECs are low, and the hiPSC-CMs are often purified via selection for expression of a promoter-reporter construct. In this chapter, we describe an hiPSC-EC differentiation protocol that generates large numbers of stable ECs and an hiPSC-CM differentiation protocol that does not require genetic manipulation, single-cell selection, or sorting with fluorescent dyes or other reagents. We also provide a simple but effective method that can be used to combine hiPSC-ECs and hiPSC-CMs with hiPSC-derived smooth muscle cells to engineer a contracting patch of cardiac cells.

Magnetically actuated alginate scaffold: a novel platform for promoting tissue organization and vascularization.

Among the greatest hurdles hindering the successful implementation of tissue-engineered cardiac patch as a therapeutic strategy for myocardial repair is the know-how to promote its rapid integration into the host. We previously demonstrated that prevascularization of the engineered cardiac patch improves cardiac repair after myocardial infarction (MI); the mature vessel networks were generated by including affinity-bound angiogenic factors in the patch and its transplantation on the blood vessel-enriched omentum. Here, we describe a novel in vitro strategy to promote the formation of capillary-like networks in cell constructs without supplementing with angiogenic factors. Endothelial cells (ECs) were seeded into macroporous alginate scaffolds impregnated with magnetically responsive nanoparticles (MNPs), and after pre-culture for 24 h under standard conditions the constructs were subjected to an alternating magnetic field of 40 Hz for 7 days. The magnetic stimulation per se promoted EC organization into capillary-like structures with no supplementation of angiogenic factors; in the non-stimulated constructs, the cells formed sheets or aggregates. This chapter describes in detail the preparation method of the MNP-impregnated alginate scaffold, the cultivation setup for the cell construct under magnetic field conditions, and the set of analyses performed to characterize the resultant cell constructs.

Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide a promising source for cell therapy and drug screening. Several high-yield protocols exist for hESC-CM production; however, methods to significantly advance hESC-CM maturation are still lacking. Building on our previous experience with mouse ESC-CMs, we investigated the effects of 3-dimensional (3D) tissue-engineered culture environment and cardiomyocyte purity on structural and functional maturation of hESC-CMs. 2D monolayer and 3D fibrin-based cardiac patch cultures were generated using dissociated cells from differentiated Hes2 embryoid bodies containing varying percentage (48–90%) of CD172a (SIRPA)-positive cardiomyocytes. hESC-CMs within the patch were aligned uniformly by locally controlling the direction of passive tension. Compared to hESC-CMs in age (2 weeks) and purity (48–65%) matched 2D monolayers, hESC-CMs in 3D patches exhibited significantly higher conduction velocities (CVs), longer sarcomeres (2.09 ± 0.02 vs. 1.77 ± 0.01 μm), and enhanced expression of genes involved in cardiac contractile function, including cTnT, αMHC, CASQ2 and SERCA2. The CVs in cardiac patches increased with cardiomyocyte purity, reaching 25.1 cm/s in patches constructed with 90% hESC-CMs. Maximum contractile force amplitudes and active stresses of cardiac patches averaged to 3.0 ± 1.1 mN and 11.8 ± 4.5 mN/mm2, respectively. Moreover, contractile force per input cardiomyocyte averaged to 5.7 ± 1.1 nN/cell and showed a negative correlation with hESC-CM purity. Finally, patches exhibited significant positive inotropy with isoproterenol administration (1.7 ± 0.3-fold force increase, EC50 = 95.1 nm). These results demonstrate highly advanced levels of hESC-CM maturation after 2 weeks of 3D cardiac patch culture and carry important implications for future drug development and cell therapy studies.

Vascularized Atrial Tissue Patch Cardiomyoplasty With Omentopexy Improves Cardiac Performance After Myocardial Infarction

The tissue-engineered cardiac patch can alleviate ventricular remodeling and improve functional recovery in experimental myocardial infarction. However, the size of the engineered patch is limited due to insufficient vascularization. This study evaluated the effects of autologous atrial tissue patch cardiomyoplasty and omentopexy in rats with myocardial infarction. Myocardial infarction was induced by left coronary artery ligation in Sprague-Dawley rats. Three weeks later, either a patch of left atrium (A group) or omentum (O group) or both (OA group) were placed over the infarct zone. The atrial tissue patch was harvested from the autologous left atrial appendage along its long axis. The rats in the Control group received rethoracotomy only. After 4 weeks, the survival of the transplanted atrial tissue patch, ventricular remodeling, and cardiac performance were examined. After 4 weeks, surviving myocardium was only detected in the OA group, as indicated by immunolabeling of cardiac troponin-I. Compared with the Control group, only animals in the OA group showed improved heart function assessed by left ventricular ejection fraction (57.9% ± 5.8% vs 47.5% ± 4.5%, p < 0.05) and left ventricular fractional shortening (25.2% ± 3.6% vs 20.7% ± 2.0%, p < 0.05). The histologic analysis demonstrated increased scar thickness in the OA group. This was accompanied by increased angiogenesis of the border zone but decreased expression and activity of matrix metalloproteinase and endothelin-1 levels. The omentopexy supported the survival of the autologous atrial tissue patch, which resulted in attenuated ventricular remodeling and restoration of heart function in rats with myocardial infarction. Our findings might represent a novel therapeutic strategy for heart failure.

Cardiac tissue engineering using stem cells [Cellular/Tissue Engineering

The limited clinical success of stem-cell injections for the treatment of myocardial infarction has been mainly attributed to the low retention and survival of injected cells. Alternative methods for improving the efficiency of cell delivery include the following: 1) injection of a mixture of bioactive hydrogels and cells followed by cell-hydrogel polymerization in situ and 2) the epicardial implantation of a tissue-engineered cardiac patch. Although surgically more complex than cell or cell-hydrogel injection, the patch implantation is also expected to yield improved survival of delivered cells, and potentially, a more efficient structural and functional tissue reconstruction at the infarct site.

Evaluation of a Peritoneal-Generated Cardiac Patch in a Rat Model of Heterotopic Heart Transplantation

Tissue engineering holds the promise of providing new solutions for heart transplant shortages and pediatric heart transplantation. The aim of this study was to evaluate the ability of a peritoneal-generated, tissue-engineered cardiac patch to replace damaged myocardium in a heterotopic heart transplant model. Fetal cardiac cells (1 x 10(6)/scaffold) from syngeneic Lewis rats were seeded into highly porous alginate scaffolds. The cell constructs were cultured in vitro for 4 days and then they were implanted into the rat peritoneal cavity for 1 week. During this time the peritoneal-implanted patches were vascularized and populated with myofibroblasts. They were harvested and their performance in an infrarenal heterotopic abdominal heart transplantation model was examined (n = 15). After transplantation and before reperfusion of the donor heart, a 5-mm left (n = 6) or right (n = 9) ventriculotomy was performed and the patch was sutured onto the donor heart to repair the defect. Echocardiographical studies carried out 1-2 weeks after transplantation showed normal LV function in seven of the eight hearts studied. After 1 month, visual examination of the grafted patch revealed no aneurysmal dilatation. Microscopic examination revealed, in most of the cardiac patches, a complete disappearance of the scaffold and its replacement by a consistent tissue composed of myofibroblasts embedded in collagen bundles. The cardiac patch was enriched with a relatively large number of infiltrating blood vessels. In conclusion, cardiac patches generated in the peritoneum were developed into consistent tissue patches with properties to seal and correct myocardial defects. Our study also offers a viable rat model for screening and evaluating new concepts in cardiac reconstruction and engineering.

A Tissue Engineering Approach to Progenitor Cell Delivery Results in Significant Cell Engraftment and Improved Myocardial Remodeling

Cell replacement therapy has become an attractive solution for myocardial repair. Typical cell delivery techniques, however, suffer from poor cell engraftment and inhomogeneous cell distributions. Therefore, we assessed the hypothesis that an epicardially applied, tissue-engineered cardiac patch containing progenitor cells would result in enhanced exogenous cell engraftment. Human mesenchymal stem cells (hMSCs) were embedded into a rat tail type I collagen matrix to form the cardiac patch. Myocardial infarction was induced by left anterior descending coronary artery ligation in immunocompetent male cesarean-derived fischer rats, and patches with or without cells were secured to hearts with fibrin sealant. After patch formation, hMSCs retained a viability of >90% over 5 days in culture. In addition, >75% of hMSCs maintained a high degree of potency prior to patch implantation. After 4 days in culture, patches were applied to the epicardial surface of the infarct area and resulted in 23% +/- 4% engraftment of hMSCs at 1 week (n = 6). Patch application resulted in a reduction in left ventricle interior diameter at systole, increased anterior wall thickness, and a 30% increase in fractional shortening. Despite this improvement in myocardial remodeling, hMSCs were not detectable at 4 weeks after patch application, implying that improvement did not require long-term cell engraftment. Patches devoid of progenitor cells showed no improvement in remodeling. In conclusion, pluripotent hMSCs can be efficiently delivered to a site of myocardial injury using an epicardial cardiac patch, and such delivery results in improved myocardial remodeling after infarction. Disclosure of potential conflicts of interest is found at the end of this article.

A clinically relevant large-animal model for evaluation of tissue-engineered cardiac surgical patch materials

Extracellular matrix (ECM) scaffolds may be useful as a tissue engineering approach toward myocardial regeneration in the infarcted heart. An appropriate large-animal model for testing the utility of biologically derived ECM in this application is needed. The purpose of this study was to develop such a model for optimal procedural success during and after patch implantation surgery. Myocardial infarction (MI) was created by embolization of the diagonal artery (DA) branch of the left anterior descending coronary artery with collagen suspension. After 4 to 6 weeks, 14 pigs received patch implant (ECM or expanded polytetrafluoroethylene). Six pigs were infarcted in the first DA and seven pigs in the second DA. Electrophysiology study was performed within 3 days before surgery. During surgery, the size and location of the infarct were measured. Infarcted myocardium (1.5-cm diameter) was transmurally excised under partial cardiopulmonary bypass. Patches (3-cm diameter) were sutured to the endomyocardial defect. Four pigs died postoperatively. After 1 month, 10 pigs were euthanized and the locations of patches were examined. Success rate of patch implant in the second DA (85.7%) was higher than the first DA (50%) group. Infarct size in the second DA was smaller than in the first DA (4.6+/-1.2 vs. 10.8+/-2.4 cm(2), P<.05). The second DA was more anteriorly positioned, which enabled easier access from the midsternal thoracotomy. However, the first DA was more laterally located requiring more manipulation of the heart during surgery. Electrophysiology revealed no ventricular tachyarrhythmia in the second DA but 33.3% in the first DA group (P<.05). At necropsy, the endocardial position of the first DA-infarct patches was anteroapical, whereas the second DA-infarct patches were more basolateral and often involved the anterior papillary muscle. The success rate of patch implant was associated with infarction size and location, and may be related to arrhythmic substrate. Experimental MI created by the second DA embolization is a feasible model for investigation of tissue-engineered cardiac patch implantation. This large-animal model is also suitable for study of cell therapy via endocardial catheter-based approaches or open surgical methods.