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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., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The number of people affected by heart disease such as coronary artery disease and myocardial infarction increases at an alarming rate each year. Currently, the methods to treat these diseases are restricted to lifestyle change, pharmaceuticals, and eventually heart transplant if the condition is severe enough. While these treatment options are the standard for caring for patients who suffer from heart disease, limited regenerative ability of the heart restricts the effectiveness of treatment and may lead to other heart-related health problems in the future. Because of the increasing need for more effective therapeutic technologies for treating diseased heart tissue, cardiac patches are now a large focus for researchers. The cardiac patches are designed to be integrated into the patients' natural tissue to introduce mechanical support and healing to the damaged areas. As a promising alternative, synthetic biodegradable polymer based biomaterials can be easily manipulated to customize material properties, as well as possess certain desired characteristics for cardiac patch use. This comprehensive review summarizes recent works on synthetic biodegradable cardiac patches implanted into infarcted animal models. In addition, this review describes the basic requirements that should be met for cardiac patch development, and discusses the inspirations to designing new biomaterials and technologies for cardiac patches., (© 2020 Wiley Periodicals, Inc.)

Mussels are well-known for their extraordinary capacity to adhere onto different surfaces in various hydrophillic conditions. Their unique adhesion ability under water or in wet conditions has generated considerable interest towards developing mussel inspired polymeric systems that can mimic the chemical mechanisms used by mussels for their adhesive properties. Catechols like 3,4-dihydroxy phenylalanine (DOPA) and their biochemical interactions have been largely implicated in mussels' strong adhesion to various substrates and have been the centerpoint of research and development efforts towards creating superior tissue adhesives for surgical and tissue engineering applications. In this article, we review bioadhesion and adhesives from an engineering standpoint, specifically the requirements of a good tissue glue, the relevance that DOPA and other catechols have in tissue adhesion, current trends in mussel-inspired bioadhesives, strategies to develop mussel-inspired tissue glues, and perspectives for future development of these materials.

Myocardial infarction (MI) causes massive heart muscle death and remains a leading cause of death in the world. Cardiac tissue engineering aims to replace the infarcted tissues with functional engineered heart muscles or revitalize the infarcted heart by delivering cells, bioactive factors, and/or biomaterials. One major challenge of cardiac tissue engineering and regeneration is the establishment of functional perfusion and structure to achieve timely angiogenesis and effective vascularization, which are essential to the survival of thick implants and the integration of repaired tissue with host heart. In this paper, we review four major approaches to promoting angiogenesis and vascularization in cardiac tissue engineering and regeneration: delivery of pro-angiogenic factors/molecules, direct cell implantation/cell sheet grafting, fabrication of prevascularized cardiac constructs, and the use of bioreactors to promote angiogenesis and vascularization. We further provide a detailed review and discussion on the early perfusion design in nature-derived biomaterials, synthetic biodegradable polymers, tissue-derived acellular scaffolds/whole hearts, and hydrogel derived from extracellular matrix. A better understanding of the current approaches and their advantages, limitations, and hurdles could be useful for developing better materials for future clinical applications.

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.

There are compelling reasons to develop a tissue-engineered mitral valve, but this endeavor has not received the same attention as tissue engineering strategies for the semilunar valves. Challenges in regenerating a mitral valve include recapitulating the complex heterogeneity in terms of anatomy (differently sized leaflets, numerous chordae), extracellular matrix composition, biomechanical behavior, valvular interstitial cell and endothelial cell phenotypes, and interior vasculature and innervation. It will also be essential to restore the functional relationships between the native mitral valve and left ventricle. A growing amount of information relevant to tissue engineering a mitral valve has been recently collected through investigations of cell mechanobiology and collagen organization. It is hoped that the development of tissue-engineered mitral valves can build on knowledge derived from engineering semilunar valves, but the mitral valve will present its own unique challenges as investigators move toward a first-generation prototype.

Tissue engineering holds great promise for treatment of valvular diseases. Despite excellent progress in the field, current approaches do not fully take into account each patient's valve anatomical uniqueness, the presence of a middle spongiosa cushion that allows shearing of external fibrous layers (fibrosa and ventricularis), and the need for autologous valvular interstitial cells. In this study we propose a novel approach to heart valve tissue engineering based on bioreactor conditioning of mesenchymal stem cell-seeded, valve-shaped constructs assembled from layered collagenous scaffolds. Fibrous scaffolds were prepared by decellularization of porcine pericardium and spongiosa scaffolds by decellularization and elastase treatment of porcine pulmonary arteries. To create anatomically correct constructs, we created silicone molds from native porcine aortic valves, dried two identical fibrous scaffolds onto the molds, and stabilized them with penta-galloyl-glucose a reversible collagen-binding polyphenol that reduces biodegradation. The layers were fused with a protein/aldehyde scaffold bio-adhesive and neutralized to reduce cytotoxicity. Spongiosa scaffolds, seeded with human bone marrow-derived stem cells, were inserted within the valve-shaped layered scaffolds and sutured inside the original aortic root. The final product was mounted in a heart valve bioreactor and cycled in cell culture conditions. Most cells were alive after 8 days, elongated significantly, and stained positive for vimentin, similar to native human valvular interstitial cells, indicating feasibility of our approach.

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., ((c) 2010 Wiley Periodicals, Inc.)

Scaffolds for heart valve tissue engineering must function immediately after implantation but also need to tolerate cell infiltration and gradual remodeling. We hypothesized that moderately cross-linked collagen scaffolds would fulfill these requirements. To test our hypothesis, scaffolds prepared from decellularized porcine pericardium were treated with penta-galloyl glucose (PGG), a collagen-binding polyphenol, and tested for biodegradation, biaxial mechanical properties, and in vivo biocompatibility. For controls, we used un-cross-linked scaffolds and glutaraldehyde-treated scaffolds. Results confirmed complete pericardium decellularization and the ability of scaffolds to encourage fibroblast chemotaxis and to aid in creation of anatomically correct valve-shaped constructs. Glutaraldehyde cross-linking fully stabilized collagen but did not allow for tissue remodeling and calcified when implanted subdermally in rats. PGG-treated collagen was initially resistant to collagenase and then degraded gradually, indicating partial stabilization. Moreover, PGG-treated pericardium exhibited excellent biaxial mechanical properties, did not calcify in vivo, and supported infiltration by host fibroblasts and subsequent matrix remodeling. In conclusion, PGG-treated acellular pericardium is a promising scaffold for heart valve tissue engineering.

In engineered tissues we are challenged to reproduce extracellular matrix and cellular deformation coupling that occurs within native tissues, which is a meso-micro scale phenomenon that profoundly affects tissue growth and remodeling. With our ability to electrospin polymer fiber scaffolds while simultaneously electrospraying viable cells, we are provided with a unique platform to investigate cellular deformations within a three dimensional elastomeric fibrous scaffold. Scaffold specimens micro-integrated with vascular smooth muscle cells were subjected to controlled biaxial stretch with 3D cellular deformations and local fiber microarchitecture simultaneously quantified. We demonstrated that the local fiber geometry followed an affine behavior, so that it could be predicted by macro-scaffold deformations. However, local cellular deformations depended non-linearly on changes in fiber microarchitecture and ceased at large strains where the scaffold fibers completely straightened. Thus, local scaffold microstructural changes induced by macro-level applied strain dominated cellular deformations, so that monotonic increases in scaffold strain do not necessitate similar levels of cellular deformation. This result has fundamental implications when attempting to elucidate the events of de-novo tissue development and remodeling in engineered tissues, which are thought to depend substantially on cellular deformations.

The potential for decellularized aortic heart valves (AVs) as heart valve replacements is based on the assumption that the major cellular immunogenic components have been removed, and that the remaining extracellular matrix (ECM) should retain the necessary mechanical properties and functional design. However, decellularization processes likely alter the ECM mechanical and structural properties, potentially affecting long-term durability. In the present study, we explored the effects of an anionic detergent (sodium dodecyl sulfate (SDS)), enzymatic agent (Trypsin), and a non-ionic detergent (Triton X-100) on the mechanical and structural properties of AV leaflets (AVLs) to provide greater insight into the initial functional state of the decellularized AVL. The overall extensibility represented by the areal strain under 60 N/m increased from 68.85% for the native AV to 139.95%, 137.51%, and 177.69% for SDS, Trypsin, and Triton X-100, respectively, after decellularization. In flexure, decellularized AVLs demonstrated a profound loss of stiffness overall, and also produced a nonlinear moment-curvature relation compared to the linear response of the native AVL. Effective flexural moduli decreased from 156.0+/-24.6 kPa for the native AV to 23.5+/-5.8, 15.6+/-4.8, and 19.4+/-8.9 kPa for SDS, Trypsin, and Triton X-100 treated leaflets, respectively. While the overall leaflet fiber architecture remained relatively unchanged, decellularization resulted in substantial microscopic disruption. In conclusion, changes in mechanical and structural properties of decellularized leaflets were likely associated with disruption of the ECM, which may impact the durability of the leaflets.

Heart valve interstitial cells (VICs) appear to have a dynamic and reversible phenotype, an attribute speculated to be necessary for valve tissue remodeling during times of development and repair. Therefore, we hypothesized that the cytoskeletal (CSK) remodeling capability of the aortic and pulmonary VICs (AVICs and PVICs, respectively), which are dominated by smooth muscle alpha-actin, would exhibit unique contractile behaviors when seeded on collagen gels. Using a porcine cell source, we observed that VIC populations did not contract the gels at early time points (2 and 4 hours) as dermal fibroblasts did, but formed a central cluster of cells prior to contraction. After clustering, VICs appeared to radiate out from the center of the gels, whereas fibroblasts did not migrate but contracted the gels locally. VIC gels treated with transforming growth factor beta1 contracted the gels rapidly, revealing similar sensitivity to the cytokine. Moreover, we evaluated the initial mechanical state of the underlying CSK by comparing AVIC and PVIC stiffness with atomic force microscopy. Not only were AVICs significantly stiffer (p < 0.001) than the PVICs, but they also contracted the gels significantly more at 24 and 48 hours (p < 0.001). Taken together, these findings suggest that the AVICs are capable of inducing greater extra cellular matrix contraction, possibly manifesting in a more pronounced ability to remodel valvular tissues. Moreover, significant mechanobiological differences between AVICs and PVICs exist, and may have implications for understanding native valvular tissue remodeling. Elucidating these differences will also define important functional endpoints in the development of tissue engineering approaches for heart valve repair and replacement.

Heart valve (HV) diseases are among the leading causes of death and continue to threaten public health worldwide. The current clinical options for HV replacement include mechanical and biological prostheses. However, an ongoing problem with current HV prostheses is their failure to integrate with the host tissue and their inability grow and remodel within the body. Tissue engineered heart valves (TEHVs) are a promising solution to these problems, as they are able to grow and remodel somatically with the rest of the body. Recently, decellularized HVs have demonstrated great potential as valve replacements because they are tissue specific, but recellularization is still a challenge due to the dense HV extracellular matrix (ECM) network. In this proof‐of‐concept work, we decellularized porcine mitral valve chordae, aortic valve leaflets, and mitral valve leaflets and processed them into injectable hydrogels that could accommodate any geometry. While the three valvular ECMs contained various amounts of collagen, they displayed similar glycosaminoglycan contents. The hydrogels had similar nanofibrous structures and gelation kinetics with various compressive strengths. When encapsulated with NIH 3 T3 fibroblasts, all the hydrogels supported cell survivals up to 7 days. Decellularized HV ECM hydrogels may show promising potential HV tissue engineering applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1732–1740, 2019. [ABSTRACT FROM AUTHOR]

Background: Defects in the pulmonary valve (PV) occur in a variety of forms of congenital heart diseases. Quantitative information on PV collagen fiber architecture, and particularly its response to diastolic forces, is necessary for the design and functional assessment of approaches for PV repair and replacement. This necessity is especially the case for novel tissue-engineered PV, which rely on extensive in-vivo remodeling for long-term function. Methods: Porcine PV and aortic valves (AV) were fixed under a 0 to 90 mm Hg transvalvular pressure. After dissection from the root, small-angle light-scattering measurements were conducted to quantify the collagen fiber architecture and changes with increasing applied transvalvular pressure over the entire cusp. Histomorphologic measurements were also performed to assess changes in cuspal layer thickness with pressure. Results: While the PV and AV displayed anticipated structural similarities, they also presented important functionally related differences. In the unloaded state, the AV cusp demonstrated substantial regional variations in fiber alignment, whereas the PV was surprisingly uniform. Further, the AV demonstrated substantially larger changes in collagen fiber alignment with applied transvalvular pressure compared with the PV. Overall, the AV collagen fiber network demonstrated greater ability to respond to applied transvalvular pressure. A decrease in crimp amplitude was the predominant mechanism for improvement in the degree of orientation of the collagen fibers in both valves. Conclusions: This study clarified the major similarities and differences between the PV and the AV. While underscoring how the PV can serve as an appropriate replacement of the diseased AV, the observed structural differences may also indicate limits to the ability of the PV to fully duplicate the AV. Moreover, quantitative data from this study on PV functional architecture will benefit development of tissue-engineered PV by defining the critical fiber architectural characteristics. [Copyright &y& Elsevier]

Abstract: The design and development of functional engineered tissues is dependent on multiple considerations, with biomechanics paramount for load-bearing constructs such as tissue engineered heart valves. As the cryopreserved allograft is the current standard for valve replacement in pediatric patients, identifying and quantifying essential structural–mechanical properties of the native valve leaflet is a crucial step in the engineered valve leaflet design process. Native valve leaflet properties provide an intuitive basis for assessing engineered valvular tissue performance, and can potentially be used as biomechanical endpoints for qualifying engineered leaflets prior to clinical applications. In this short review, we present three analysis techniques that have been used by our lab and others for characterizing heart valve leaflet biomechanical response and discuss the relevance of these properties as candidate endpoints for engineered leaflet tissues. The studies presented herein focused primarily on the aortic valve, which most frequently warrants repair or replacement in the general population and has been useful in our understanding of bioprosthetic heart valve mechanics. However, these analysis techniques are directly applicable for pulmonary and most engineered valve leaflets. Where data is available, initial studies applying these techniques for in vitro assessment of scaffolds and engineered valve leaflets are presented. The development of a tissue engineered heart valve for the pediatric population is conceptually appealing, since few options currently exist due to the lack of growth potential in non-viable prosthetics and size limitations. While significant challenges remain, we believe that a derivative of the current tissue engineered heart valve paradigm will ultimately yield a design suitable for clinical evaluation. The role of biomechanics in this process will be to identify and quantify the structural–mechanical endpoints essential for appropriate heart valve leaflet function, while guiding investigators prior to and during clinical evaluation. [Copyright &y& Elsevier]

Abstract: There is a major need for scaffold-based tissue engineered vascular grafts and heart valves with long-term patency and durability to be used in diabetic cardiovascular patients. We hypothesized that diabetes, by virtue of glycoxidation reactions, can directly crosslink implanted scaffolds, drastically altering their properties. In order to investigate the fate of tissue engineered scaffolds in diabetic conditions, we prepared valvular collagen scaffolds and arterial elastin scaffolds by decellularization and implanted them subdermally in diabetic rats. Both types of scaffolds exhibited significant levels of advanced glycation end products (AGEs), chemical crosslinking and stiffening -alterations which are not favorable for cardiovascular tissue engineering. Pre-implantation treatment of collagen and elastin scaffolds with penta-galloyl glucose (PGG), an antioxidant and matrix-binding polyphenol, chemically stabilized the scaffolds, reduced their enzymatic degradation, and protected them from diabetes-related complications by reduction of scaffold-bound AGE levels. PGG-treated scaffolds resisted diabetes-induced crosslinking and stiffening, were protected from calcification, and exhibited controlled remodeling in vivo, thereby supporting future use of diabetes-resistant scaffolds for cardiovascular tissue engineering in patients with diabetes. [Copyright &y& Elsevier]