Myocardial Scaffolds Research Articles

Advancing Myocardial Infarction Treatment: Harnessing Multi-Layered Recellularized Cardiac Patches with Fetal Myocardial Scaffolds and Acellular Amniotic Membrane.

Myocardial infarction (MI) is a leading cause of irreversible functional cardiac tissue loss, requiring novel regenerative strategies. This study assessed the potential therapeutic efficacy of recellularized cardiac patches, incorporating fetal myocardial scaffolds with rat fetal cardiomyocytes and acellular human amniotic membrane, in adult Wistar rat models of MI. Decellularized myocardial tissue was obtained from 14 to 16 week-old human fetuses that had been aborted. Chemical detergents (0.1% EDTA and 0.2% sodium dodecyl sulfate) were used to prepare the fetal extracellular matrix (ECM), which was characterized for bio-scaffold microstructure and biocompatibility via scanning electron microscopy (SEM) and MTT assay, respectively. Neonatal cardiomyocytes were extracted from the ventricles of one-day-old Wistar rats' littermates and characterized through immunostaining against Connexin-43 and α-smooth muscle actin. The isolated cells were seeded onto decellularized tissues and covered with decellularized amniotic membrane. Sixteen healthy adult Wistar rats were systematically allocated to control and MI groups. MI was induced via arterial ligation. Fourteen days post-operation, the MI group was received the engineered patches. Following a two-week post-implantation period, the animals were euthanized, and the hearts were harvested for the graft evaluation. Histological analysis, DAPI staining, and ultra-structural examination corroborated the successful depletion of cellular elements, while maintaining the integrity of the fetal ECM and architecture. Subsequent histological and immunohistochemichal (IHC) evaluations confirmed effective cardiomyocyte seeding on the scaffolds. The application of these engineered patches in MI models resulted in increased angiogenesis, reduced fibrosis, and restricted scar tissue formation, with the implanted cardiomyocytes remaining viable at graft sites, indicating prospective in vivo cell viability. This study suggests that multi-layered recellularized cardiac patches are a promising surgical intervention for myocardial infarction, showcasing significant potential by promoting angiogenesis, mitigating fibrosis, and minimizing scar tissue formation in MI models. These features are pivotal for enhancing the therapeutic outcomes in MI patients, focusing on the restoration of the myocardial structure and function post-infarction.

Evaluation of Different Decellularization Protocols for Obtaining and Characterizing Canine Cardiac Extracellular Matrix.

Cardiovascular diseases are considered the leading cause of mortality globally; even with low mortality in dogs, such diseases are described in the same way in companion animals and humans. This study aimed to devise an effective decellularization protocol for the canine myocardium through the association of physical, chemical, and enzymatic methods, assessing resultant alterations in the myocardial extracellular matrix to obtain a suitable scaffold. Two canine hearts were collected; the samples were sectioned into ±1 cm2 fragments, washed in distilled water and 1× PBS solution, and followed by treatment under four distinct decellularization protocols. Sodium Dodecyl Sulfate (SDS) 1% 7 days + Triton X-100 1% for 48 h (Protocol I); Sodium Dodecyl Sulfate (SDS) 1% 5 days + Triton X-100 1% for 48 h (Protocol II); Trypsin 0.05% for 1 h at 36 °C + freezing -80 °C overnight + Sodium Dodecyl Sulfate (SDS) 1% for 3 days, Triton-X-100 for 48 h hours (Protocol III); 0.05% trypsin for 1 h at 36 °C + freezing at -80 °C overnight + 1% Sodium Dodecyl Sulfate (SDS) for 2 days + 1% Triton-X-100 for 24 h (Protocol IV). After analysis, Protocols I and II showed the removal of cellular content and preservation of extracellular matrix (ECM) contents, unlike Protocols III and IV, which retracted the ECM and removed essential elements of the matrix. In theory, although Protocols I and II have similar results, Protocol II stands out for the preservation of the architecture and components of the extracellular matrix, along with reduced exposure time to reagents, making it the recommended protocol for the development of a canine myocardial scaffold.

Diastolic function in chronic kidney disease.

Chronic kidney disease (CKD) is characterized by clustered age-independent concentric left ventricular (LV) geometry, geometry-independent systolic dysfunction and age and heart rate-independent diastolic dysfunction. Concentric LV geometry is always associated with echocardiographic markers of abnormal LV relaxation and increased myocardial stiffness, two hallmarks of diastolic dysfunction. Non-haemodynamic mechanisms such as metabolic and electrolyte abnormalities, activation of biological pathways and chronic exposure to cytokine cascade and the myocardial macrophage system also impact myocardial structure and impair the architecture of the myocardial scaffold, producing and increasing reactive fibrosis and altering myocardial distensibility. This review addresses the pathophysiology of diastole in CKD and its relations with cardiac mechanics, haemodynamic loading, structural conditions, non-haemodynamic factors and metabolic characteristics. The three mechanisms of diastole will be examined: elastic recoil, active relaxation and passive distensibility and filling. Based on current evidence, we briefly provide methods for quantification of diastolic function and discuss whether diastolic dysfunction represents a distinct characteristic in CKD or a proxy of the severity of the cardiovascular condition, with the potential to be predicted by the general cardiovascular phenotype. Finally, the review discusses assessment of diastolic function in the context of CKD, with special emphasis on end-stage kidney disease, to indicate whether and when in-depth measurements might be helpful for clinical decision making in this context.

Acellular Myocardial Scaffolds and Slices Fabrication, and Method for Applying Mechanical and Electrical Simulation to Tissue Construct.

Cardiac tissue engineering/regeneration using decellularized myocardium has attracted great research attention due to its potential benefit to myocardial infarction (MI) treatment. Here, we described an optimal decellularization protocol to generate 3D porcine myocardial scaffolds with well-preserved cardiomyocyte lacunae, myocardial slices as a biomimetic cell culture and delivery platform, and a multi-stimulation bioreactor that is able to provide coordinated mechanical and electrical stimulations for facilitating cardiac construct development.

Tunable biomaterials for myocardial tissue regeneration: promising new strategies for advanced biointerface control and improved therapeutic outcomes.

Following myocardial infarction (MI) and the natural healing process, the cardiac mechanostructure changes significantly leading to reduced contractile ability and resulting in additional pressure on the heart muscle thereby increasing the risk of heart failure (HF). The application of cardiac scaffolds in the form of epicardial patches or injectable hydrogels at the infarcted region of the myocardium helps to mechanically reinforce the ventricular walls and allows control over the various stages of the healing process, reducing pathological remodeling and fibrosis and eventually restoring cardiac function. Recent progress in the field of biomaterials engineering allows tuning of cardiac scaffold properties for more effective tissue-biomaterial interaction leading to improved therapeutic outcomes. Nanoscaffold characteristics required for myocardial tissue engineering (TE) including mechanical property, pore size/porosity, immunomodulation, bioactivity, electroconductivity, injectability (for hydrogels) and thickness (for cardiac patches) are herewith reviewed. Strategies for controlling each of these properties via blending, scaffold fabrication, degree of crosslinking, and incorporation of bioactive molecules, amongst others are also discussed. The mechanism of myocardial restoration via enhanced angiogenesis, stem cell homing and mechanical support is further detailed. Finally, key novel innovative strategies with high promise for clinical translation are presented; in particular, the use of extracellular vesicle-loaded scaffolds, integration of electronics within scaffolds for real time monitoring of the engineered tissue performance as well as the possibility of refilling scaffolds with drugs/cells/proteins via a subcutaneous port are highlighted.

DYNAMIC BIOREACTOR MODEL TO MIMIC EARLY CARDIAC FIBROSIS IN DIABETES

In clinical diabetic cardiomyopathy, hyperglycemia and dyslipidemia induce tissue injury, activation of cardiac fibroblasts and interstitial and perivascular fibrosis. Myofibroblasts repair the injured tissue by increasing collagen deposition in the cardiac interstitium and suppressing the activity of matrix metalloproteinases. The goal of this study was to find an ideal model to mimic the effect of high glucose concentration on human cardiac fibroblast activation. The profibrotic role of the transforming growth factor-[Formula: see text] (TGF-[Formula: see text]) and the protective modulation of nitric oxide were examined in two-dimensional and three-dimensional cell culture models, as well as tissue engineering models, that involved the use of cardiac fibroblasts cultured within myocardial matrix scaffolds mounted in a bioreactor that delivered biochemical and mechanical stimuli. Results showed that high glucose levels were potent pro-fibrotic stimuli. In addition, high glucose levels in concert with TGF-[Formula: see text] constituted very strong signals that induced human cardiac fibroblast activation. Cardiac fibroblasts cultured within decellularized myocardial scaffolds and exposed to biochemical and mechanical stimuli represented an adequate model for this pathology. In conclusion, the bioreactor platform was instrumental in establishing an in vitro model of early fibrosis; this platform could be used to test the effects of various agents targeted to mitigate the fibrotic processes.

Head-to-head comparison of two engineered cardiac grafts for myocardial repair: From scaffold characterization to pre-clinical testing

Cardiac tissue engineering, which combines cells and supportive scaffolds, is an emerging treatment for restoring cardiac function after myocardial infarction (MI), although, the optimal construct remains a challenge. We developed two engineered cardiac grafts, based on decellularized scaffolds from myocardial and pericardial tissues and repopulated them with adipose tissue mesenchymal stem cells (ATMSCs). The structure, macromechanical and micromechanical scaffold properties were preserved upon the decellularization and recellularization processes, except for recellularized myocardium micromechanics that was ∼2-fold stiffer than native tissue and decellularized scaffolds. Proteome characterization of the two acellular matrices showed enrichment of matrisome proteins and major cardiac extracellular matrix components, considerably higher for the recellularized pericardium. Moreover, the pericardial scaffold demonstrated better cell penetrance and retention, as well as a bigger pore size. Both engineered cardiac grafts were further evaluated in pre-clinical MI swine models. Forty days after graft implantation, swine treated with the engineered cardiac grafts showed significant ventricular function recovery. Irrespective of the scaffold origin or cell recolonization, all scaffolds integrated with the underlying myocardium and showed signs of neovascularization and nerve sprouting. Collectively, engineered cardiac grafts -with pericardial or myocardial scaffolds- were effective in restoring cardiac function post-MI, and pericardial scaffolds showed better structural integrity and recolonization capability.

P4464An acellular myocardial scaffold optimal for cardiac recovery: proteomic, structural and mechanical characterization

P4464An acellular myocardial scaffold optimal for cardiac recovery: proteomic, structural and mechanical characterization

Preparation of acellular myocardial scaffolds with well-preserved cardiomyocyte lacunae, and method for applying mechanical and electrical simulation to tissue construct.

Cardiac tissue engineering/regeneration using decellularized myocardium has attracted great research attention due to its potential benefit for myocardial infarction (MI) treatment. Here we describe an optimal decellularization protocol to generate 3D porcine myocardial scaffolds with well-preserved cardiomyocyte lacunae and a multi-stimulation bioreactor that is able to provide coordinated mechanical and electrical stimulation for facilitating cardiac construct development.

Myocardial Scaffold-Based Cardiac Tissue Engineering: Application of Coordinated Mechanical and Electrical Stimulations

Recently, we developed an optimal decellularization protocol to generate 3D porcine myocardial scaffolds, which preserve the natural extracellular matrix structure, mechanical anisotropy, and vasculature templates and also show good cell recellularization and differentiation potential. In this study, a multistimulation bioreactor was built to provide coordinated mechanical and electrical stimulation for facilitating stem cell differentiation and cardiac construct development. The acellular myocardial scaffolds were seeded with mesenchymal stem cells (10(6) cells/mL) by needle injection and subjected to 5-azacytidine treatment (3 μmol/L, 24 h) and various bioreactor conditioning protocols. We found that after 2 days of culturing with mechanical (20% strain) and electrical stimulation (5 V, 1 Hz), high cell density and good cell viability were observed in the reseeded scaffold. Immunofluorescence staining demonstrated that the differentiated cells showed a cardiomyocyte-like phenotype by expressing sarcomeric α-actinin, myosin heavy chain, cardiac troponin T, connexin-43, and N-cadherin. Biaxial mechanical testing demonstrated that positive tissue remodeling took place after 2 days of bioreactor conditioning (20% strain + 5 V, 1 Hz); passive mechanical properties of the 2 day and 4 day tissue constructs were comparable to those of the tissue constructs produced by stirring reseeding followed by 2 weeks of static culturing, implying the effectiveness and efficiency of the coordinated simulations in promoting tissue remodeling. In short, the synergistic stimulations might be beneficial not only for the quality of cardiac construct development but also for patients by reducing the waiting time in future clinical scenarios.

Stem Cell Regenerative Potential Combined with Nanotechnology and Tissue Engineering for Myocardial Regeneration

The stem cell-based therapy for post-infarction myocardial regeneration has been introduced more than a decade ago, but the functional improvement obtained is limited due to the poor retention and short survival rate of transplanted cells into the damaged myocardium. More recently, the emerging nanotechnology concepts for advanced diagnostics and therapy provide promising opportunities of using stem cells for myocardial regeneration. In this paper will be provided an overview of the use of nanotechnology approaches in stem cell research for: 1) cell labeling to track the distribution of stem cells after transplantation, 2) nanoparticle-mediated gene delivery to stem cells to promote their homing, engraftment, survival and differentiation in the ischemic myocardium and 3) obtaining of bio-inspired materials to provide suitable myocardial scaffolds for delivery of stem cells or stem cell-derived factors.

The Acellular Myocardial Flap: A Novel Extracellular Matrix Scaffold Enriched with Patent Microvascular Networks and Biocompatible Cell Niches

There is a great need for acellular, fully vascularized, and biocompatible myocardial scaffolds that provide agreeable biological, nutritional, and biomechanical niches for reseeded cells for in vitro and in vivo applications. We generated myocardial flap scaffolds comprising porcine left-anterior ventricular myocardium and its associated coronary arteries and veins and investigated the combinatorial effects of sodium dodecyl sulfate (SDS) and sodium hydroxide (NaOH) perfusion on both the myocardial extracellular matrix (ECM) and the vascular ECM. Results showed that all scaffolds displayed a fully intact and patent vasculature, with arterial burst pressures indistinguishable from native coronary arteries and perfusion to the level of capillaries. Scaffolds were free of cellular proteins and retained collagen and elastin ECM components, exhibited excellent mechanical properties, and were cytocompatible toward relevant seeded cells. SDS perfusion preserved collagen IV, laminin, and fibronectin well, but only reduced DNA content by 33%; however, this was further improved by post-SDS nuclease treatments. By comparison, NaOH was very effective in removing cells and eliminated more than 95% of tissue DNA, but also significantly reduced levels of laminin and fibronectin. Such constructs can be readily trimmed to match the size of the infarct and might be able to functionally integrate within host myocardium and be nourished by direct anastomotic connection with the host's own vasculature; they might also be useful as physiologically accurate models for in vitro studies of cardiac physiology and pathology.

Structural and biomechanical characterizations of porcine myocardial extracellular matrix

Extracellular matrix (ECM) of myocardium plays an important role to maintain a multilayered helical architecture of cardiomyocytes. In this study, we have characterized the structural and biomechanical properties of porcine myocardial ECM. Fresh myocardium were decellularized in a rotating bioreactor using 0.1 % sodium dodecyl sulfate solution. Masson's trichrome staining and SEM demonstrated the removal of cells and preservation of the interconnected 3D cardiomyocyte lacunae. Movat's pentachrome staining showed the preservation of cardiac elastin ultrastructure and vascular elastin distribution/alignment. DNA assay result confirmed a 98.59 % reduction in DNA content; the acellular myocardial scaffolds were found completely lack of staining for the porcine α-Gal antigen; and the accelerating enzymatic degradation assessment showed a constant degradation rate. Tensile and shear properties of the acellular myocardial scaffolds were also evaluated. Our observations showed that the acellular myocardial ECM possessed important traits of biodegradable scaffolds, indicating the potentials in cardiac regeneration and whole heart tissue engineering.

The Quest for an Optimized Protocol for Whole-Heart Decellularization: A Comparison of Three Popular and a Novel Decellularization Technique and Their Diverse Effects on Crucial Extracellular Matrix Qualities

Decellularized cardiac extracellular matrix (ECM) has been introduced as a template for cardiac tissue engineering, providing the advantages of a prevascularized scaffold that mimics native micro- and macroarchitecture to a degree difficult to achieve with synthetic materials. Nonetheless, the decellularization protocols used to create acellular myocardial scaffolds vary widely throughout the literature. In this study we performed a direct comparison of three previously described protocols while introducing and evaluating a novel, specifically developed fourth protocol, by decellularizing whole rat hearts through software-controlled automatic coronary perfusion. Although all protocols preserved the macroarchitecture of the hearts and all resulting scaffolds could successfully be reseeded with C2C12 myoblasts, assessing their biocompatibility for three-dimensional in vitro studies, we found striking differences concerning the microcomposition of the ECM scaffolds on a histological and biochemical level. While laminin could still be detected in all groups, other crucial ECM components, like elastin and collagen IV, were completely removed by at least one of the protocols. Further, only three protocols maintained a glycosaminoglycan content comparable to native tissue, whereas the remaining DNA content within the ECM varied highly throughout all four tested protocols. This study showed that the degree of acellularity and resulting ECM composition of decellularized myocardial scaffolds strongly differs depending on the decellularization protocol.

Fabrication of cardiac patch with decellularized porcine myocardial scaffold and bone marrow mononuclear cells

Tissue engineered cardiac grafts are a promising therapeutic mode for ventricular wall reconstruction. Recently, it has been found that acellular tissue scaffolds provide natural ultrastructural, mechanical, and compositional cues for recellularization and tissue remodeling. We thus assess the potential of decellularized porcine myocardium as a scaffold for thick cardiac patch tissue engineering. Myocardial sections with 2-mm thickness were decellularized using 0.1% sodium dodecyl sulfate and then reseeded with differentiated bone marrow mononuclear cells. We found that thorough decellularization could be achieved after 2.5 weeks of treatment. Reseeded cells were found to infiltrate and proliferate in the tissue constructs. Immunohistological staining studies showed that the reseeded cells maintained cardiomyocyte-like phenotype and possible endothelialization was found in locations close to vasculature channels, indicating angiogenesis potential. Both biaxial and uniaxial mechanical testing showed a stiffer mechanical response of the acellular myocardial scaffolds; however, tissue extensibility and tensile modulus were found to recover in the constructs along with the culture time, as expected from increased cellular content. The cardiac patch that we envision for clinical application will benefit from the natural architecture of myocardial extracellular matrix, which has the potential to promote stem cell differentiation, cardiac regeneration, and angiogenesis.

Mesenchymal Stem Cells for Myocardial Infarction

Recent studies indicate that cardiac transfer of adult stem cells can have a favorable impact on tissue perfusion and contractile performance of the infarcted heart. Several cell sources are being explored in an effort to regenerate infarcted myocardium, including hematopoietic stem cells, endothelial progenitor cells, cardiac resident stem cells, bone marrow–derived multipotent stem cells, and mesenchymal stem cells (MSCs). Each of these cell types may have its own profile of advantages, limitations, and practicability issues in specific settings. Studies comparing the regenerative capacity of distinct cell populations are scarce. Most clinical investigators have therefore chosen a pragmatic approach by using unselected bone marrow cells that contain different stem cell populations. Basic scientists, by contrast, are focusing more on specific cell populations in a quest to understand the biological foundations of cell therapy and to identify the most promising stem cells for cardiac regeneration.1 See p 214 MSCs are a rare population of self-renewing, multipotent cells present in adult bone marrow. Although MSCs represent <0.01% of all nucleated bone marrow cells, they can be readily expanded in vitro. In defined culture media, MSCs differentiate into several mesenchymal cell lineages, including cardiomyocytes.2,3 When injected into normal adult myocardium, MSCs differentiate into cardiomyocyte-like cells with sarcomeric organization.4 In an earlier study in pigs with myocardial infarction (MI), MSCs grafted into the infarcted area were shown to express muscle-specific markers and to improve regional wall motion.5 Ease of isolation, high expansion capability, and cardiomyogenic potential have led to the proposition that MSCs may be a good choice for cell-based therapies of MI.6 In a report published in this issue of Circulation , Dai et al7 have …