Heart Valve Tissue Engineering Research Articles

Anisotropicity and flexibility in trilayered microfibrous substrates promote heart valve leaflet tissue engineering

Heart valve leaflet substrates with native trilayer and anisotropic structures are crucial for successful heart valve tissue engineering. In this study, we used the electrospinning technique to produce trilayer microfibrous leaflet substrates using two biocompatible and biodegradable polymers—poly (L-lactic acid) (PLLA) and polycaprolactone (PCL), separately. Different polymer concentrations for each layer were applied to bring a high degree of mechanical and structural anisotropy to the substrates. PCL leaflet substrates exhibited lower unidirectional tensile properties than PLLA leaflet substrates. However, the PLLA substrates exhibited a lower flexural modulus than the PCL substrates. These substrates were seeded with porcine valvular interstitial cells (PVICs) and cultured for one month in static conditions. Both substrates exhibited cellular adhesion and proliferation, resulting in the production of tissue-engineered constructs. The PLLA tissue-engineered constructs had more cellular growth than the PCL tissue-engineered constructs. The PLLA substrates showed higher hydrophilicity, lower crystallinity, and more significant anisotropy than PCL substrates, which may have enhanced their interactions with PVICs. Analysis of gene expression showed higher α-smooth muscle actin and collagen type 1 expression in PLLA tissue-engineered constructs than in PCL tissue-engineered constructs. The differences in anisotropic and flexural properties may have accounted for the different cellular behaviors in these two individual polymer substrates.

CREATING THE IDEAL ARTIFICIAL HEART VALVE

BackgroundEmerging evidence from laboratory and clinical investigations suggests that xenogenic tissue heart valves generate an immune response that has been implicated in the eventual failure of these valve constructs. Several attempts have been made to tissue engineer functional heart valve replacements, however, there remain significant barriers to clinical translation. Our novel process for creating a widely available, immunocompatible, shelf-stable xenogenic tissue heart valve aims to address these barriers. The aim of this study was to characterize the in-vitro biomechanical and hemodynamic characteristics of our novel tissue engineered heart valve.Methods and ResultsTwo 23-mm commercially available glutaraldehyde-fixed, stented, xenogenic tissue heart valves were obtained and decellularized with subsequent enzymatic digestion of antigenic epitopes. Human mesenchymal stem cells were used for the recellularization process. Following recellularization, the tissue engineered heart valves were treated for shelf-stability and underwent biomechanical and hemodynamic testing using a pulse duplicator. One 23-mm commercially available glutaraldehyde-fixed, stented, xenogenic tissue heart valve was used as a control for baseline valve performance. All valves tested met the ISO 5840-3:2021 acceptance criteria for cardiovascular implants. Average pulsatile flow effective orifice area was 2.18 cm2 compared to 2.31 cm2 in the control valve. Transvalvular regurgitation fraction was less than 5% during forward flow conditions at 2, 3.5, 5 and 7 L for all valves tested. Visual inspection of the valves confirmed the structural integrity of the leaflets was preserved, with no evidence of tears, prolapse or perforation. Immunofluorescent nuclear staining confirmed the presence of cells on the valve surface before and after hemodynamic testing.Conclusion BackgroundEmerging evidence from laboratory and clinical investigations suggests that xenogenic tissue heart valves generate an immune response that has been implicated in the eventual failure of these valve constructs. Several attempts have been made to tissue engineer functional heart valve replacements, however, there remain significant barriers to clinical translation. Our novel process for creating a widely available, immunocompatible, shelf-stable xenogenic tissue heart valve aims to address these barriers. The aim of this study was to characterize the in-vitro biomechanical and hemodynamic characteristics of our novel tissue engineered heart valve. Emerging evidence from laboratory and clinical investigations suggests that xenogenic tissue heart valves generate an immune response that has been implicated in the eventual failure of these valve constructs. Several attempts have been made to tissue engineer functional heart valve replacements, however, there remain significant barriers to clinical translation. Our novel process for creating a widely available, immunocompatible, shelf-stable xenogenic tissue heart valve aims to address these barriers. The aim of this study was to characterize the in-vitro biomechanical and hemodynamic characteristics of our novel tissue engineered heart valve. Methods and ResultsTwo 23-mm commercially available glutaraldehyde-fixed, stented, xenogenic tissue heart valves were obtained and decellularized with subsequent enzymatic digestion of antigenic epitopes. Human mesenchymal stem cells were used for the recellularization process. Following recellularization, the tissue engineered heart valves were treated for shelf-stability and underwent biomechanical and hemodynamic testing using a pulse duplicator. One 23-mm commercially available glutaraldehyde-fixed, stented, xenogenic tissue heart valve was used as a control for baseline valve performance. All valves tested met the ISO 5840-3:2021 acceptance criteria for cardiovascular implants. Average pulsatile flow effective orifice area was 2.18 cm2 compared to 2.31 cm2 in the control valve. Transvalvular regurgitation fraction was less than 5% during forward flow conditions at 2, 3.5, 5 and 7 L for all valves tested. Visual inspection of the valves confirmed the structural integrity of the leaflets was preserved, with no evidence of tears, prolapse or perforation. Immunofluorescent nuclear staining confirmed the presence of cells on the valve surface before and after hemodynamic testing. Two 23-mm commercially available glutaraldehyde-fixed, stented, xenogenic tissue heart valves were obtained and decellularized with subsequent enzymatic digestion of antigenic epitopes. Human mesenchymal stem cells were used for the recellularization process. Following recellularization, the tissue engineered heart valves were treated for shelf-stability and underwent biomechanical and hemodynamic testing using a pulse duplicator. One 23-mm commercially available glutaraldehyde-fixed, stented, xenogenic tissue heart valve was used as a control for baseline valve performance. All valves tested met the ISO 5840-3:2021 acceptance criteria for cardiovascular implants. Average pulsatile flow effective orifice area was 2.18 cm2 compared to 2.31 cm2 in the control valve. Transvalvular regurgitation fraction was less than 5% during forward flow conditions at 2, 3.5, 5 and 7 L for all valves tested. Visual inspection of the valves confirmed the structural integrity of the leaflets was preserved, with no evidence of tears, prolapse or perforation. Immunofluorescent nuclear staining confirmed the presence of cells on the valve surface before and after hemodynamic testing. Conclusion

Macrophage-extracellular matrix interactions: Perspectives for tissue engineered heart valve remodeling.

In situ heart valve tissue engineering approaches have been proposed as promising strategies to overcome the limitations of current heart valve replacements. Tissue engineered heart valves (TEHVs) generated from in vitro grown tissue engineered matrices (TEMs) aim at mimicking the microenvironmental cues from the extracellular matrix (ECM) to favor integration and remodeling of the implant. A key role of the ECM is to provide mechanical support to and attract host cells into the construct. Additionally, each ECM component plays a critical role in regulating cell adhesion, growth, migration, and differentiation potential. Importantly, the immune response to the implanted TEHV is also modulated biophysically via macrophage-ECM protein interactions. Therefore, the aim of this review is to summarize what is currently known about the interactions and signaling networks occurring between ECM proteins and macrophages, and how these interactions may impact the long-term in situ remodeling outcomes of TEMs. First, we provide an overview of in situ tissue engineering approaches and their clinical relevance, followed by a discussion on the fundamentals of the remodeling cascades. We then focus on the role of circulation-derived and resident tissue macrophages, with particular emphasis on the ramifications that ECM proteins and peptides may have in regulating the host immune response. Finally, the relevance of these findings for heart valve tissue engineering applications is discussed.

Towards technically controlled bioreactor maturation of tissue-engineered heart valves.

Bioreactors are important tools for the pre-conditioning of tissue-engineered heart valves. The current state of the art mostly provides for timed, physical and biochemical stimulation in the bioreactor systems according to standard protocols (SOP). However, this does not meet tothe individual biological variability of living tissue-engineered constructs. To achieve this, it is necessary to implement (i)sensory systems that detect the actual status ofthe implant and (ii) controllable bioreactor systems that allow patient-individualized pre-conditioning. During the maturation process, a pulsatile transvalvular flow of culturemedium is generated within the bioreactor. For the improvement of this conditioning procedure, the relationship between the mechanical and biochemical stimuli and the corresponding tissue response has to be analyzed by performing reproducible and comparable experiments. Inthis work, a technological framework for maturation experiments of tissue-engineered heart valves in a pulsating bioreactor isintroduced. The aim is the development of a bioreactor system that allows for continuous control and documentation of the conditioning process to increase reproducibility and comparability of experiments. This includes hardware components, a communication structure and software including online user communication and supervision. Preliminary experiments were performed with a tissue-engineered heart valve to evaluate the function ofthe new system. The results of the experiment proof theadequacy of the setup. Consequently, the concept is an important step for further research towards controlled maturation of tissue-engineered heart valves. The integration of molecular and histological sensor systems will bethenext important step towards a fully automated, self-controlled preconditioning system.

Recellularized bovine pericardium with autologous mesenchymal stem cells reduces immune activation.

Current bioprosthetic heart valve replacement options are limited by structural valvular deterioration (SVD) due to an immune response to the xenogenic scaffold. Autologous mesenchymal stem cell (MSC) recellularization is a method of concealing xenogenic scaffolds, preventing recipient immune recognition of xenogenic tissue heart valves, and potentially leading to reduction in SVD incidence. The purpose of this study is to examine the effects of autologous MSC recellularized tissue on the immune response of human whole blood to bovine pericardium (BP). We hypothesized that autologous MSC recellularization of BP will result in reduced pro-inflammatory cytokine production equivalent to autologous human pericardium. Bone marrow, human pericardium, and whole blood were collected from adult patients undergoing elective cardiac surgery. Decellularized BP underwent recellularization with autologous MSCs, followed by co-incubation with autologous whole blood. Immunohistochemical, microscopic, and quantitative immune analysis approaches were used. We demonstrated that native BP, exposed to human whole blood, results in significant TNF-α and IL1β production. When decellularized BP is recellularized with autologous MSCs and exposed to whole blood, there is a significant reduction in TNF-α and IL1β production. Importantly, recellularized BP exposed to whole blood had similar production of TNF-α and IL1β when compared to autologous human pericardium exposed to human whole blood. Our results suggest that preventing initial immune activation with autologous MSC recellularization may be an effective approach to decrease the recipient immune response, preventing recipient immune recognition of xenogeneic tissue engineered heart valves, and potentially leading to reduction in SVD incidence.

Computer Model-Driven Design in Cardiovascular Regenerative Medicine

Continuing advances in genomics, molecular and cellular mechanobiology and immunobiology, including transcriptomics and proteomics, and biomechanics increasingly reveal the complexity underlying native tissue and organ structure and function. Identifying methods to repair, regenerate, or replace vital tissues and organs remains one of the greatest challenges of modern biomedical engineering, one that deserves our very best effort. Notwithstanding the continuing need for improving standard methods of investigation, including cell, organoid, and tissue culture, biomaterials development and fabrication, animal models, and clinical research, it is increasingly evident that modern computational methods should play increasingly greater roles in advancing the basic science, bioengineering, and clinical application of regenerative medicine. This brief review focuses on the development and application of computational models of tissue and organ mechanobiology and mechanics for purposes of designing tissue engineered constructs and understanding their development in vitro and in situ. Although the basic approaches are general, for illustrative purposes we describe two recent examples from cardiovascular medicine—tissue engineered heart valves (TEHVs) and tissue engineered vascular grafts (TEVGs)—to highlight current methods of approach as well as continuing needs.

Biofabrication of Sodium Alginate Hydrogel Scaffolds for Heart Valve Tissue Engineering.

Every year, thousands of aortic valve replacements must take place due to valve diseases. Tissue-engineered heart valves represent promising valve substitutes with remodeling, regeneration, and growth capabilities. However, the accurate reproduction of the complex three-dimensional (3D) anatomy of the aortic valve remains a challenge for current biofabrication methods. We present a novel technique for rapid fabrication of native-like tricuspid aortic valve scaffolds made of an alginate-based hydrogel. Using this technique, a sodium alginate hydrogel formulation is injected into a mold produced using a custom-made sugar glass 3D printer. The mold is then dissolved using a custom-made dissolving module, revealing the aortic valve scaffold. To assess the reproducibility of the technique, three scaffolds were thoroughly compared. CT (computed tomography) scans showed that the scaffolds respect the complex native geometry with minimal variations. The scaffolds were then tested in a cardiac bioreactor specially designed to reproduce physiological flow and pressure (aortic and ventricular) conditions. The flow and pressure profiles were similar to the physiological ones for the three valve scaffolds, with small variabilities. These early results establish the functional repeatability of this new biofabrication method and suggest its application for rapid fabrication of ready-to-use cell-seeded sodium alginate scaffolds for heart valve tissue engineering.

Endothelial progenitor cells overexpressing Grb2-associated binder 1 for in vitro-constructed tissue-engineered heart valves.

Aim Endothelial progenitor cells (EPCs) play important roles in heart valve replacement surgery. Up-regulation of Grb2‑associated binder 1 (Gab1) promotes hepatocyte growth factor (HGF) - induced endothelial progenitor cell proliferation and migration. This study aimed to investigate the effects of up-regulation of Gab1 in hepatocyte growth factor-induced EPCs in tissue-engineered heart valves (TEHV).Material and methods Fresh porcine aortic valves were placed in 1 % Triton X-100 and trypsin buffer for decellularization. EPCs in the control group were cultured normally, whereas those in the experimental group were both HGF stimulated and transfected with adenovirus containing the Gab1 gene. Cells in the two groups were seeded onto the decellularized valve scaffolds and cultured for 3 or 7 days. TEHV were analyzed by HE and AB-PAS staining.Results By day 3, the experimental group had formed confluent endothelial monolayers on top of the decellularized valves, on the basis of by HE staining and AB-PAS staining. One week later, the control group showed a imperfect endothelial layer.Conclusion HGF-induced EPCs overexpressing Gab1 can endothelialize the decellularized matrix and create functional TEHV, which may then be preconditioned in a bioreactor before clinical implantation.

Surface Modification of Decellularized Heart Valve by the POSS-PEG Hybrid Hydrogel to Prepare a Composite Scaffold Material with Anticalcification Potential.

Tissue-engineered heart valves (TEHVs) are the most promising replacement for heart valve transplantation. Decellularized heart valve (DHV) is one of the most common scaffold materials for TEHVs. In actual clinical applications, the most widely used method for treating DHV is cross-linking it with glutaraldehyde, but this method could cause serious problems such as calcification. In this study, we introduced polyhedral oligomeric silsesquioxane (POSS) nanoparticles into a poly(ethylene glycol) (PEG) hydrogel to prepare a POSS-PEG hybrid hydrogel, and then coated them on the surface of DHV to prepare the composite scaffold. The chemical structures, microscopic morphologies, cell compatibilities, blood compatibilities, and anticalcification properties were further investigated. Experimental results showed that the composite scaffold had good blood compatibility and excellent cell compatibility and could promote cell adhesion and proliferation. In vivo and in vitro anticalcification experiments showed that the introduction of POSS nanoparticles could reduce the degree of calcification significantly and the composite scaffold had obvious anticalcification ability. The DHV surface-coated with the POSS-PEG hybrid hydrogel is an alternative scaffold material with anticalcification potential for an artificial heart valve, which provides an idea for the preparation of TEHVs.

A Review of the Role of Transthoracic and Transesophageal Echocardiography, Computed Tomography, and Magnetic Resonance Imaging in Cardioembolic Stroke.

Stroke is a major source of morbidity and mortality worldwide, accounting for the second largest cause of mortality and the third greatest cause of disability. Stroke is frequently preceded by a transient ischemic attack (TIA). The etiologies of 20–30% of ischemic strokes are unknown, and thus are termed “cryptogenic strokes”. About 25% of ischemic strokes are cardioembolic. Strokes occur at a rate of around 2% per year in individuals with heart failure with reduced ejection fraction (HFrEF), with a strong correlation between stroke risk and the degree of ventricular impairment. Furthermore, stroke risk is augmented in the absence of anticoagulation therapy. Cardioembolic strokes, when treated inadequately, have a greater predilection for recurrences than atherothrombotic strokes, both early and late in life. The role of a patent foramen ovale in strokes, specifically in “cryptogenic strokes”, is a matter of concern that deserves due attention. The use of tissue-engineered heart valves and aspirin for minimizing the risk of stroke is recommended.Transthoracic echocardiography (TTE) is advantageous for assessing heart function in the acute phase of ischemic stroke. Transesophageal echocardiography (TEE) is considered the criterion standard procedure for detecting LAA thrombi. Computed tomography (CT) scans are good imaging modalities for identifying and excluding bleeding. Magnetic resonance imaging (MRI) images are by far the most effective imaging technique available for assessing the brain parenchymal state.We conducted a thorough review of the literature on the use of imaging modalities, highlighting the important contribution of TTE, TEE, CT, and MRI in the evaluation of cardioembolic stroke.

Design of a Mechanobioreactor to Apply Anisotropic, Biaxial Strain to Large Thin Biomaterials for Tissue Engineered Heart Valve Applications.

Repair and replacement solutions for congenitally diseased heart valves capable of post-surgery growth and adaptation have remained elusive. Tissue engineered heart valves (TEHVs) offer a potential biological solution that addresses the drawbacks of existing valve replacements. Typically, TEHVs are made from thin, fibrous biomaterials that either become cell populated in vitro or in situ. Often, TEHV designs poorly mimic the anisotropic mechanical properties of healthy native valves leading to inadequate biomechanical function. Mechanical conditioning of engineered tissues with anisotropic strain application can induce extracellular matrix remodelling to alter the anisotropic mechanical properties of a construct, but implementation has been limited to small-scale set-ups. To address this limitation for TEHV applications, we designed and built a mechanobioreactor capable of modulating biaxial strain anisotropy applied to large, thin, biomaterial sheets in vitro. The bioreactor can independently control two orthogonal stretch axes to modulate applied strain anisotropy on biomaterial sheets from 13×13mm2 to 70×40 mm2. A design of experiments was performed using experimentally validated finite element (FE) models and demonstrated that biaxial strain was applied uniformly over a larger percentage of the cell seeded area for larger sheets (13×13 mm2: 58% of sheet area vs. 52×31 mm2: 86% of sheet area). Furthermore, bioreactor prototypes demonstrated that over 70% of the cell seeding area remained uniformly strained under different prescribed protocols: equibiaxial amplitudes between 5 to 40%, cyclic frequencies between 0.1 to 2.5 Hz and anisotropic strain ratios between 0:1 (constrained uniaxial) to 2:1. Lastly, proof-of-concept experiments were conducted where we applied equibiaxial (εx = εy = 8.75%) and anisotropic (εx = 12.5%, εy = 5%) strain protocols to cell-seeded, electrospun scaffolds. Cell nuclei and F-actin aligned to the vector-sum strain direction of each prescribed protocol (nuclei alignment: equibiaxial: 43.2°±1.8°, anisotropic: 17.5°±1.7°; p<0.001). The abilities of this bioreactor to prescribe different strain amplitude, frequency and strain anisotropy protocols to cell-seeded scaffolds will enable future studies into the effects of anisotropic loading protocols on mechanically conditioned TEHVs and other engineered planar connective tissues.

Polysaccharides, as biological macromolecule-based scaffolding systems in heart valve tissue engineering: a review

Polysaccharides, as biological macromolecule-based scaffolding systems in heart valve tissue engineering: a review

Natural Polymers in Heart Valve Tissue Engineering: Strategies, Advances and Challenges.

In the history of biomedicine and biomedical devices, heart valve manufacturing techniques have undergone a spectacular evolution. However, important limitations in the development and use of these devices are known and heart valve tissue engineering has proven to be the solution to the problems faced by mechanical and prosthetic valves. The new generation of heart valves developed by tissue engineering has the ability to repair, reshape and regenerate cardiac tissue. Achieving a sustainable and functional tissue-engineered heart valve (TEHV) requires deep understanding of the complex interactions that occur among valve cells, the extracellular matrix (ECM) and the mechanical environment. Starting from this idea, the review presents a comprehensive overview related not only to the structural components of the heart valve, such as cells sources, potential materials and scaffolds fabrication, but also to the advances in the development of heart valve replacements. The focus of the review is on the recent achievements concerning the utilization of natural polymers (polysaccharides and proteins) in TEHV; thus, their extensive presentation is provided. In addition, the technological progresses in heart valve tissue engineering (HVTE) are shown, with several inherent challenges and limitations. The available strategies to design, validate and remodel heart valves are discussed in depth by a comparative analysis of in vitro, in vivo (pre-clinical models) and in situ (clinical translation) tissue engineering studies.

Cardiovascular Tissue Engineering and Regeneration: A Plead for Further Knowledge Convergence

Cardiovascular tissue engineering and regeneration strive to provide long-term, effective solutions for a growing group of patients in need of myocardial repair, vascular (access) grafts, heart valves, and regeneration of organ microcirculation. In the past two decades, ongoing convergence of disciplines and multidisciplinary collaborations between cardiothoracic surgeons, cardiologists, bioengineers, material scientists, and cell biologists have resulted in better understanding of the problems at hand and novel regenerative approaches. As a side effect, however, the field has become strongly organized and differentiated around topical areas at risk of reinvention of technologies and repetition of approaches across the areas. A better integration of knowledge and technologies from the individual topical areas and regenerative approaches and technologies may pave the way toward faster and more effective treatments to cure the cardiovascular system. This review summarizes the evolution of research and regenerative approaches in the areas of myocardial regeneration, heart valve and vascular tissue engineering, and regeneration of microcirculations; and discusses previous and potential future integration of these individual areas and developed technologies for improved clinical impact. Finally, it provides a perspective on the further integration of research organization, knowledge implementation, and valorization as a contributor to advancing cardiovascular tissue engineering and regenerative medicine. Impact Statement Despite ongoing convergence of disciplines, research in the field of cardiovascular tissue engineering and regeneration is organized and differentiated around focal areas, including myocardial regeneration, heart valve tissue engineering, vascular tissue engineering, and engineering of microcirculations. Cross-area integration of knowledge, supported by a more holistic, overarching research approach, may lead to faster and more effective treatments and prevent the reinvention of technologies across the areas. Herein, we review the evolution of research and technologies in the individual focal areas and discuss how to enhance integration of-and collaboration between-these areas for improved scientific and clinical impact.

Engineering Efforts to Refine Compatibility and Duration of Aortic Valve Replacements: An Overview of Previous Expectations and New Promises.

The absence of pharmacological treatments to reduce or retard the progression of cardiac valve diseases makes replacement with artificial prostheses (mechanical or bio-prosthetic) essential. Given the increasing incidence of cardiac valve pathologies, there is always a more stringent need for valve replacements that offer enhanced performance and durability. Unfortunately, surgical valve replacement with mechanical or biological substitutes still leads to disadvantages over time. In fact, mechanical valves require a lifetime anticoagulation therapy that leads to a rise in thromboembolic complications, while biological valves are still manufactured with non-living tissue, consisting of aldehyde-treated xenograft material (e.g., bovine pericardium) whose integration into the host fails in the mid- to long-term due to unresolved issues regarding immune-compatibility. While various solutions to these shortcomings are currently under scrutiny, the possibility to implant fully biologically compatible valve replacements remains elusive, at least for large-scale deployment. In this regard, the failure in translation of most of the designed tissue engineered heart valves (TEHVs) to a viable clinical solution has played a major role. In this review, we present a comprehensive overview of the TEHVs developed until now, and critically analyze their strengths and limitations emerging from basic research and clinical trials. Starting from these aspects, we will also discuss strategies currently under investigation to produce valve replacements endowed with a true ability to self-repair, remodel and regenerate. We will discuss these new developments not only considering the scientific/technical framework inherent to the design of novel valve prostheses, but also economical and regulatory aspects, which may be crucial for the success of these novel designs.

Endothelial Progenitor Cell-Based in vitro Pre-Endothelialization of Human Cell-Derived Biomimetic Regenerative Matrices for Next-Generation Transcatheter Heart Valves Applications.

Hemocompatibility of cardiovascular implants represents a major clinical challenge and, to date, optimal antithrombotic properties are lacking. Next-generation tissue-engineered heart valves (TEHVs) made from human-cell-derived tissue-engineered extracellular matrices (hTEMs) demonstrated their recellularization capacity in vivo and may represent promising candidates to avoid antithrombotic therapy. To further enhance their hemocompatibility, we tested hTEMs pre-endothelialization potential using human-blood-derived endothelial-colony-forming cells (ECFCs) and umbilical vein cells (control), cultured under static and dynamic orbital conditions, with either FBS or hPL. ECFCs performance was assessed via scratch assay, thereby recapitulating the surface damages occurring in transcatheter valves during crimping procedures. Our study demonstrated: feasibility to form a confluent and functional endothelium on hTEMs with expression of endothelium-specific markers; ECFCs migration and confluency restoration after crimping tests; hPL-induced formation of neo-microvessel-like structures; feasibility to pre-endothelialize hTEMs-based TEHVs and ECFCs retention on their surface after crimping. Our findings may stimulate new avenues towards next-generation pre-endothelialized implants with enhanced hemocompatibility, being beneficial for selected high-risk patients.

Tri-Layered and Gel-Like Nanofibrous Scaffolds with Anisotropic Features for Engineering Heart Valve Leaflets.

3D heterogeneous and anisotropic scaffolds that approximate native heart valve tissues are indispensable for the successful construction of tissue engineered heart valves (TEHVs). In this study, novel tri-layered and gel-like nanofibrous scaffolds, consisting of poly(lactic-co-glycolic) acid (PLGA) and poly(aspartic acid) (PASP), are fabricated by a combination of positive/negative conjugate electrospinning and bioactive hydrogel post-processing. The nanofibrous PLGA-PASP scaffolds present tri-layered structures, resulting in anisotropic mechanical properties that are comparable with native heart valve leaflets. Biological tests show that nanofibrous PLGA-PASP scaffolds with high PASP ratios significantly promote the proliferation and collagen and glycosaminoglycans (GAGs) secretions of human aortic valvular interstitial cells (HAVICs), compared to PLGA scaffolds. Importantly, the nanofibrous PLGA-PASP scaffolds are found to effectively inhibit the osteogenic differentiation of HAVICs. Two types of porcine VICs, from young and adult age groups, are further seeded onto the PLGA-PASP scaffolds. The adult VICs secrete higher amounts of collagens and GAGs and undergo a significantly higher level of osteogenic differentiation than young VICs. RNA sequencing analysis indicates that age has a pivotal effect on the VIC behaviors. This study provides important guidance and a reference for the design and development of 3D tri-layered, gel-like nanofibrous PLGA-PASP scaffolds for TEHV applications.

Spatially Heterogeneous Tubular Scaffolds for In Situ Heart Valve Tissue Engineering Using Melt Electrowriting

AbstractHeart valve tissue engineering (HVTE) aims to provide living autologous heart valve implants endowed with regenerative capabilities and life‐long durability. However, fabrication of biomimetic scaffolds capable of providing the required functionality in terms of mechanical performance and tunable porosity to enable cellular infiltration remains a major challenge. Here, the additive manufacturing of bioinspired, spatially heterogeneous, tubular scaffolds enclosing the leaflets, inter‐leaflet triangles, and their interface for in situ HVTE using melt electrowriting (MEW) is demonstrated. The innovative platform enables the digital fabrication of scaffolds with ad hoc architecture (e.g., tunable location, specific fiber pattern, and orientation) and customizable geometry via a custom‐made control software. The user‐friendly interface allows for the definition of areas of the scaffold with specific patterns to obtain properties such as tunable J‐shaped stress–stain curve and anisotropy typical of the heart valve leaflet, compliant inter‐leaflet triangles, and reinforced curvilinear boundary between them. Heterogeneous, tubular, heart valve MEW scaffolds are then embedded with a microporous elastin‐like recombinamer (ELR) hydrogel to develop a soft‐network composite favoring cell infiltration and ensuring hemocompatibility. The acute systolic hemodynamic functionality of the MEW/ELR composite satisfies the ISO 5840 requirements, under aortic and pulmonary conditions.

Biodegradable Poly-ε-Caprolactone Scaffolds with ECFCs and iMSCs for Tissue-Engineered Heart Valves.

Clinically used heart valve prostheses, despite their progress, are still associated with limitations. Biodegradable poly-ε-caprolactone (PCL) nanofiber scaffolds, as a matrix, were seeded with human endothelial colony-forming cells (ECFCs) and human induced-pluripotent stem cells-derived MSCs (iMSCs) for the generation of tissue-engineered heart valves. Cell adhesion, proliferation, and distribution, as well as the effects of coating PCL nanofibers, were analyzed by fluorescence microscopy and SEM. Mechanical properties of seeded PCL scaffolds were investigated under uniaxial loading. iPSCs were used to differentiate into iMSCs via mesoderm. The obtained iMSCs exhibited a comparable phenotype and surface marker expression to adult human MSCs and were capable of multilineage differentiation. EFCFs and MSCs showed good adhesion and distribution on PCL fibers, forming a closed cell cover. Coating of the fibers resulted in an increased cell number only at an early time point; from day 7 of colonization, there was no difference between cell numbers on coated and uncoated PCL fibers. The mechanical properties of PCL scaffolds under uniaxial loading were compared with native porcine pulmonary valve leaflets. The Young’s modulus and mean elongation at Fmax of unseeded PCL scaffolds were comparable to those of native leaflets (p = ns.). Colonization of PCL scaffolds with human ECFCs or iMSCs did not alter these properties (p = ns.). However, the native heart valves exhibited a maximum tensile stress at a force of 1.2 ± 0.5 N, whereas it was lower in the unseeded PCL scaffolds (0.6 ± 0.0 N, p < 0.05). A closed cell layer on PCL tissues did not change the values of Fmax (ECFCs: 0.6 ± 0.1 N; iMSCs: 0.7 ± 0.1 N). Here, a successful two-phase protocol, based on the timed use of differentiation factors for efficient differentiation of human iPSCs into iMSCs, was developed. Furthermore, we demonstrated the successful colonization of a biodegradable PCL nanofiber matrix with human ECFCs and iMSCs suitable for the generation of tissue-engineered heart valves. A closed cell cover was already evident after 14 days for ECFCs and 21 days for MSCs. The PCL tissue did not show major mechanical differences compared to native heart valves, which was not altered by short-term surface colonization with human cells in the absence of an extracellular matrix.

Evaluation of Pericardial Tissues from Assorted Species as a Tissue-Engineered Heart Valve Material.

Decellularized pericardial tissue is a strong candidate for a TEHV material as ECM is present to guide cellular infiltration and fixed porcine and bovine pericardial tissue have existing use in bioprosthetic heart valves. In this work, we compare the mechanical and microstructural properties of decellularized-sterilized (DS) porcine, bovine, and bison pericardial tissues with respect to use as a TEHV. H&E staining was used to verify removal of cellular content post-decellularization and to evaluate collagen fiber structure. Additionally, uniaxial and biaxial tension testing were used to compare mechanical performance and, for the latter, acquire constitutive model parameters for subsequent finite element (FE) modeling. H&E staining revealed complete removal of cellular content and good collagen fiber structure. Tensile testing showed comparable mechanical strength between the three DS pericardial tissues and considerably stronger mechanical properties compared to native tissues. Bovine and bison DS pericardial tissues showed the strongest mechanical performance in the FE models with bison demonstrating the overall best mechanical characteristics. The increased thickness of bovine and bison tissues coupled with the strong mechanical behavior and ECM structure indicates that these materials will be resistant to damage until sufficient cellular infiltration has occurred such that damaged tissue can be repaired.