Biomimetic Approaches in Cardiac Tissue Engineering: Replicating the Native Heart Microenvironment.

3D bioprinted functional and contractile cardiac tissue constructs

Once injured, cardiac muscle does not regenerate. Massive and irreversible loss of cardiomyocytes due to myocardial infarction remains the main cause of heart failure. Cardiac tissue engineering has a potential to reestablish the structure and function of injured myocardium, by (1) injection of cardiogenic cells, (2) transplantation of functional cardiac tissue constructs, or (3) mobilization of endogenous repair cells. Irrespective of the therapeutic strategy, the regeneration depends on both the biological potential of the repair cells and the environment (matrix, signals) to which the cells are subjected. In general, the biological potential of the cells – the actual "tissue engineers" – needs to be mobilized by providing highly controllable "biomimetic" environments designed to enhance cell survival, differentiation, and electromechanical coupling. For cardiac regeneration, some of the key requirements include the establishment of cardiac tissue matrix, electromechanical cell coupling, robust contractile function, and functional vascularization. Engineered tissue constructs can also serve as high-fidelity models to study cardiac development and disease. We review the potential and challenges of cardiac tissue engineering to provide for the development of therapies for heart regeneration.

Biomaterializing the promise of cardiac tissue engineering

It has become increasingly apparent that cardiac tissue engineering applications can be employed to repair heart injuries. The primary purpose of the present research is to examine the use of biomaterials and bioengineering methods to create functioning cardiac tissue. Techniques for tissue maturation, scaffold design, cell sources, and biomaterial selection are important subjects. The proper growth of functioning heart tissue is dependent upon these factors. The assessment emphasizes further research and development areas and discusses the field’s challenges and potential solutions. In conclusion, this article highlights the significant advancements in cardiac tissue engineering. Regardless of its challenges, the medical discipline of cardiology has left tremendous prospects for heart repair in future generations. In this significant progress in this critically important area of regenerative medicine, biomaterials, bioengineering research, and innovation are necessary.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, with around 17.9 million deaths annually. In Europe, heart disease causes 3.6 million deaths each year, contributing to a significant healthcare burden. The main cause of heart failure is myocardial infarction (MI), where blockages in coronary vessels lead to the death of cardiac cells and loss of muscle function. Current treatments are mostly palliative, with heart transplantation being the only cure, but it is limited by donor organ availability. This thesis aims to address heart failure challenges by developing improved in vitro models and engineered tissues for therapy discovery and reparative interventions. Traditional models, such as 2D cultures, fail to replicate native cardiac physiology. Induced pluripotent stem cells (iPSCs) have revolutionized cardiac research by producing cardiomyocytes (iPSC-CMs), but these cells are immature, limiting their clinical applicability. To enhance disease modeling, Chapter 2 presents a protocol to induce rapid maturation of iPSC-CMs, improving their function. Chapter 3 discusses high-throughput solutions for cardiac spheroids, while Chapter 4 introduces the VoluHeart, a bi-chambered heart model created with volumetric bioprinting that replicates post-injury cardiac remodeling. Chapter 5 highlights the importance of integrating circadian rhythms into cardiac models to improve physiological relevance. Advances in stem cell technology and bioengineering have enabled the creation of models that better simulate human physiology and pathology. Chapter 6 stresses the importance of replicating the 3D structure of cardiac tissue, while Chapter 7 introduces pre-vascularized engineered heart tissue using a melt-electrowritten (MEW) mesh and hydrogels, offering improved functional survival. Annex 2 demonstrates the development of a 1-centimeter-thick cardiac tissue construct with precise tissue alignment, promoting natural-like contraction and enhancing potential for clinical applications. The thesis also addresses the need for long-term storage of implantable cardiac tissues, reviewing preservation methods in Chapters 8 and 9. These include hypothermic and cryogenic storage techniques, which are essential for transporting cardiac constructs between research facilities and enabling off-the-shelf tissue availability for reparative interventions. Overall, the thesis proposes a comprehensive strategy combining stem cell technology and advanced bioengineering to develop more accurate cardiac models and novel therapies for heart failure, ultimately advancing the potential for functional cardiac tissue repair.

Gold nanorod-incorporated gelatin-based conductive hydrogels for engineering cardiac tissue constructs

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

Myocardial infarction (MI) is the leading cause of death worldwide. Recent advances in stem cell research hold great potential for heart tissue regeneration through stem cell-based therapy. While multiple cell types have been transplanted into MI heart in preclinical studies or clinical trials, reduction of scar tissue and restoration of cardiac function have been modest. Several challenges hamper the development and application of stem cell-based therapy for heart regeneration. Application of cardiac progenitor cells (CPCs) and cardiac tissue engineering for cell therapy has shown great promise to repair damaged heart tissue. This review presents an overview of the current applications of embryonic CPCs and the development of cardiac tissue engineering in regeneration of functional cardiac tissue and reduction of side effects for heart regeneration. We aim to highlight the benefits of the cell therapy by application of CPCs and cardiac tissue engineering during heart regeneration.

Harnessing native blueprints for designing bioinks to bioprint functional cardiac tissue.

Progress in cardiac tissue engineering and regeneration: Implications of gelatin-based hybrid scaffolds

Although the current medical treatments have been successful in reducing the coronary heart disease related mortality rate, however there still exist various challenges in cardiac tissue engineering due to the hierarchical property of native myocardium and branched structure of blood vessels. The regeneration of progenitor cells, replacement infarcted cardiac tissues and the tissue-engineered constructs are the demand of the current era. In this contrast, 3D printing of scaffold for cardiac tissue engineering plays a crucial role. 3D printing techniques are used to generate patient-specific foundation that mimics the milieu of biological tissue. Various studies are under investigation to regenerate the cardiac tissues. Lack of regeneration capacity is a major hunt in cardiac tissue engineering. This review will provide an overview of 3D bioprinting techniques for cardiovascular tissue engineering. First, it summarises the background about bioprinting (working principles) followed by various bioink materials for cardiac tissue engineering. Recent approaches for the 3D printing of cardiac scaffolds for the regeneration of cardiac tissues are discussed.

To counteract DNA damage, cells have evolved several mechanisms collectively termed as the DNA damage response (DDR). A key member of DDR signaling in mammalian cells is ATR (Ataxia Telangiectasia and Rad3-related protein), a PIKK kinase that is activated by single-strand DNA lesions in response to replication stress. Severe ATR deficiency owing to hypomorphic germline mutations in ATR causes the Seckel syndrome, a neurodevelopmental and premature aging disorder. Besides its role in the DDR, ATR is activated by a variety of stimuli, including topological, osmotic and mechanical stress and contributes in the maintenance of proper nuclear integrity and membrane tension. Notably, ATR-defective cells exhibit intrinsic nuclear softness and fragility, raising the possibility that cells and tissues constantly undergoing mechanical strain, such as the heart muscle, might be particularly affected by ATR loss. To address this point, we analyzed cardiac muscle tissue from a mouse model of the human ATR Seckel syndrome (ATRS). By functional echocardiography and immunohistochemistry analysis, we showed that ATR Seckel cardiac tissue presents major morphological defects, particularly affecting cardiomyocytes’ nuclei, accompanied by a reduced cardiac functionality. Accumulation of nuclear and DNA damage due to lack of ATR contributes to the mis-localization of cardiomyocyte self-DNA from the nucleus to the cytoplasm and to the consequent activation of inflammatory processes through the STING pathway. In the current study, we show that lack of ATR strongly impacts cardiac tissue structure and function and contributes to development of cardiac inflammation; the insights gained from understanding the interplay between ATR signaling, DNA damage response and inflammation will help in the diagnosis of cardiac diseases in Seckel Syndrome and in the development of therapies to improve one of the pathological consequences of this disorder.

Cardiac tissue engineering aims at repairing damaged heart muscle and producing human cardiac tissues for application in drug toxicity studies. This book offers a comprehensive overview of the cardiac tissue engineering strategies, including presenting and discussing the various concepts in use, research directions and applications. Essential basic information on the major components in cardiac tissue engineering, namely cell sources and biomaterials, is firstly presented to the readers, followed by a detailed description of their implementation in different strategies, broadly divided to cellular and acellular ones. In cellular approaches, the biomaterials are used to increase cell retention after implantation or as scaffolds when bioengineering the cardiac patch, in vitro. In acellular approaches, the biomaterials are used as ECM replacement for damaged cardiac ECM after MI, or, in combination with growth factors, the biomaterials assume an additional function as a depot for prolong d factor activity for the effective recruitment of repairing cells. The book also presents technological innovations aimed to improve the quality of the cardiac patches, such as bioreactor applications, stimulation patterns and prevascularization. This book could be of interest not only from an educational perspective (i.e. for graduate students), but also for researchers and medical professionals, to offer them fresh views on novel and powerful treatment strategies. We hope that the reader will find a broad spectrum of ideas and possibilities described in this book both interesting and convincing. Table of Contents: Introduction / The Heart: Structure, Cardiovascular Diseases, and Regeneration / Cell Sources for Cardiac Tissue Engineering / Biomaterials: Polymers, Scaffolds, and Basic Design Criteria / Biomaterials as Vehicles for Stem Cell Delivery and Retention in the Infarct / Bioengineering of Cardiac Patches, In Vitro / Perfusion Bioreactors and Stimulation Patterns in Cardiac T ssue Engineering / Vascularization of Cardiac Patches / Acellular Biomaterials for Cardiac Repair / Biomaterial-based Controlled Delivery of Bioactive Molecules for Myocardial Regeneration

Replicating the structural and functional features of native myocardium, particularly its high-density cellular alignment and efficient electrical connectivity, is essential for engineering functional cardiac tissues. Here, novel electrohydrodynamically printed InterPore microfibrous lattices with anisotropic architectures are introduced to promote high-density cellular alignment and enhanced tissue interconnectivity. The interconnected pores in the microfibrous lattice enable dynamic, cell-mediated remodeling of fibrous hydrogels, resulting in continuous, mechanically stable tissue bundles. Compared to lattices with isolated pores, the engineered aligned cardiac tissues from neonatal rat cardiomyocytes exhibit improved electrophysiological properties and synchronous contractions. Using a multiseeding strategy, an equivalent cell seeding density of 8 × 107 cellsmL-1, facilitating the formation of multicellular, vascularized cardiac structures with maintained tissue viability and integrity, is achieved. As a demonstration, human-induced pluripotent stem cell-derived cardiac tissues are engineered with progressive maturation and functional integration over time. These findings underscore the potential of InterPore microfibrous lattices for applications in cardiac tissue engineering, drug discovery, and therapeutic development.

Cardiovascular diseases pose a significant global health challenge, driving ongoing efforts to develop effective treatments. Various biofabrication technologies utilizing numerous materials have been employed to design functional cardiac tissues. Choosing the right material is crucial to support cardiac cell growth, proliferation, tissue maturation and functionality. 3D printing enables the fabrication of structures that mimic the hierarchical organization of native cardiac tissue, further enhancing its function. Electrospinning produces nanofibrous scaffolds with a high surface area and porosity, mimicking the extracellular matrix and promoting the cell behaviors required for tissue formation. Although typically employed independently, combining these technologies can enable the fabrication of patches with properties closely resembling those of native cardiac tissues. Recent research focuses on the use of electroconductive materials, which enhance cell-to-cell communication and promote the maturation of cardiomyocytes, thereby preventing arrhythmic contractions and improving the functionality of engineered cardiac tissues. In this review, recent studies showcasing the applications of electroconductive biopolymer-based fibrous materials and hydrogels designed using 3D printing and/or electrospinning for cardiac tissue engineering are discussed. Furthermore, the review evaluates the synergistic effects of biopolymer-based materials and electrical components in 3D printed electroconductive hydrogels. It also discusses the challenges faced in fabricating these hydrogels and explores their future prospects for biomedical applications.