Tissue Engineering Strategies for Cardiac Regeneration

3D bioprinted functional and contractile cardiac tissue constructs

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.

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.

Biomaterials in cardiac tissue engineering: Ten years of research survey

Cardiovascular diseases, including heart failure, pose significant challenges in medical practice, necessitating innovative approaches for cardiac repair and regeneration. Cardiac tissue engineering has emerged as a promising solution, aiming to develop functional and physiologically relevant cardiac tissue constructs. Replicating the native heart microenvironment, with its complex and dynamic milieu necessary for cardiac tissue growth and function, is crucial in tissue engineering. Biomimetic strategies that closely mimic the natural heart microenvironment have gained significant interest due to their potential to enhance synthetic cardiac tissue functionality and therapeutic applicability. Biomimetic approaches focus on mimicking biochemical cues, mechanical stimuli, coordinated electrical signaling, and cell-cell/cell-matrix interactions of cardiac tissue. By combining bioactive ligands, controlled delivery systems, appropriate biomaterial characteristics, electrical signals, and strategies to enhance cell interactions, biomimetic approaches provide a more physiologically relevant environment for tissue growth. The replication of the native cardiac microenvironment enables precise regulation of cellular responses, tissue remodeling, and the development of functional cardiac tissue constructs. Challenges and future directions include refining complex biochemical signaling networks, paracrine signaling, synchronized electrical networks, and cell-cell/cell-matrix interactions. Advancements in biomimetic approaches hold great promise for cardiovascular regenerative medicine, offering potential therapeutic strategies and revolutionizing cardiac disease modeling. These approaches contribute to the development of more effective treatments, personalized medicine, and improved patient outcomes. Ongoing research and innovation in biomimetic approaches have the potential to revolutionize regenerative medicine and cardiac disease modeling by replicating the native heart microenvironment, advancing functional cardiac tissue engineering, and improving patient outcomes.

Biomaterializing the promise of cardiac tissue engineering

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

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.

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

Over the past decades, huge scientific efforts have been put into cardiac regeneration strategies. Although several strategies have been accepted for use in clinical practice, none has demonstrated great success in regenerating the cardiac tissue. Therefore, there are still significant challenges in repairing or regenerating cardiac tissue, which serves a predominantly biomechanical function. Furthermore, it is now evident that the mechanobiological interaction between cells and their mechanical environment is essential for tissue function and regeneration. Current cardiac regenerative strategies (i.e. cardiac cell therapy and tissue engineering) are based on administrating new healthy contractile cells within (or without) a healthy scaffold into the diseased cardiac environment. However, these strategies have widely omitted to restore the cellular mechanical environment present after cardiac injury. Therefore, the future cardiac regeneration strategies need to address the challenges of and questions on the role of biomechanics and mechanobiology in cardiac regeneration. This includes measurement and characterisation of multiscale mechanical properties of healthy and diseased cardiac tissue; development of in vitro and in silico models to understand the role of biomechanical factors in tissue regeneration; and translation of this information into the design of novel tissue-engineered strategies. In this chapter, we want to introduce the reader to the importance of mechanical considerations in the design of effective cardiac regeneration strategies.

Chapter 21 - Bioprinting of Cardiac Tissues

Cardiac tissue engineering aims to create functional tissue constructs that can reestablish the structure and function of injured myocardium. Although bioreactors have facilitated the engineering of cardiac patches of clinically relevant size in vitro, a major drawback remains the transportation of the engineered tissues from a production facility to a medical operation facility while maintaining tissue viability and preventing contamination. Furthermore, after implantation, most of the cells are endangered by hypoxic conditions that exist before vascular flow is established. We developed a portable device that provides the perfusion and electrical stimulation necessary to engineer cardiac tissue in vitro, and to transport it to the site where it will be implantated. The micropump-powered perfusion apparatus may additionally function as an extracorporeal active pumping system providing nutrients and oxygen supply to the graft post-implantation. Such a system, through perfusion of oxygenated media and bioactive molecules (e.g. growth factors), could transiently support the tissue construct until it connects to the host vasculature and heart muscle, after which it could be taken away or let biodegrade.

Keloids and hypertrophic scars result from aberrant wound healing and remain a potential complication of any surgical procedure or trauma. Investigation of aberrant wound healing has been limited to the study of growth factors, collagen precursors, and DNA synthesis in simple in vitro systems, which necessitate removal or destruction of cells or factors in the growth environment of cell cultures. Multiphoton microscopy (MPM) can use endogenous chromophores such as collagen and nicotinamide adenine dinucleotide hydrogenase to produce thin optical sections of thick living tissues without the use of dyes or stains. Endogenous second-harmonic-generation (SHG) signals in collagen can be collected to form an MPM image. To present a novel wound-healing model used to investigate keloid-derived fibroblast activity and collagen production in the same intact tissue-engineered construct over time. Artificial tissue constructs called RAFTs (produced by suspension of keloid or normal dermal fibroblasts in type I collagen gel with an overlying keratinocyte layer) were cultured at air-fluid interface. Multiphoton microscopy SHG images of collagen in the intact tissue constructs consisting of normal or keloid-derived fibroblasts were obtained. The constructs were then incised with a scalpel. Serial MPM and phase-contrast microscopy images were obtained to monitor changes in the extracellular matrix in response to wounding of the artificial skin construct over 8 days. The tissue-engineered constructs formed a bilayer resembling the dermis and epidermis of human skin. Phase-contrast microscopy revealed migration of keratinocytes into the defect created by scalpel wounding. The constructs were found to contract with time after wounding. The MPM SHG images showed collagen deposition in the tissue constructs after wounding. Tissue constructs with keloid-derived fibroblasts were found to deposit collagen at a higher rate than those with normal fibroblasts. The MPM model described herein permits serial observation of the same intact specimens without the need for fixation or cytotoxic stains. Furthermore, it demonstrates the biologic activity of RAFT artificial tissue constructs.

Poor cell engraftment rate is one of the primary factors limiting the effectiveness of cell transfer therapy for cardiac repair. Recent studies have shown that the combination of cell-based therapy and tissue engineering technology can improve stem cell engraftment and promote the therapeutic effects of the treatment for myocardial infarction. This mini-review summarizes the recent progress in cardiac tissue engineering of cardiovascular cells from differentiated human pluripotent stem cells (PSCs), highlights their therapeutic applications for the treatment of myocardial infarction, and discusses the present challenges of cardiac tissue engineering and possible future directions from a clinical perspective.

As the cornerstone of tissue engineering and regeneration medicine research, developing a cost-effective and bionic extracellular matrix (ECM) that can precisely modulate cellular behavior and form functional tissue remains challenging. An artificial ECM combining polysaccharides and fibrillar proteins to mimic the structure and composition of natural ECM provides a promising solution for cardiac tissue regeneration. In this study, we developed a bionic hydrogel scaffold by combining a quaternized β-chitin derivative (QC) and fibrin-matrigel (FM) in different ratios to mimic a natural ECM. We evaluated the stiffness of those composite hydrogels with different mixing ratios and their effects on the growth of human umbilical vein endothelial cells (HUVECs). The optimal hydrogels, QCFM1 hydrogels were further applied to load HUVECs into nude mice for in vivo angiogenesis. Besides, we encapsulated human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) into QCFM hydrogels and employed 3D bioprinting to achieve batch fabrication of human-engineered heart tissue (hEHT). Finally, the myocardial structure and electrophysiological function of hEHT were evaluated by immunofluorescence and optical mapping. Designed artificial ECM has a tunable modulus (220-1380 Pa), which determines the different cellular behavior of HUVECs when encapsulated in these. QCFM1 composite hydrogels with optimal stiffness (800 Pa) and porous architecture were finally identified, which could adapt for in vitro cell spreading and in vivo angiogenesis of HUVECs. Moreover, QCFM1 hydrogels were applied in 3D bioprinting successfully to achieve batch fabrication of both ring-shaped and patch-shaped hEHT. These QCFM1 hydrogels-based hEHTs possess organized sarcomeres and advanced function characteristics comparable to reported hEHTs. The chitin-derived hydrogels are first used for cardiac tissue engineering and achieve the batch fabrication of functionalized artificial myocardium. Specifically, these novel QCFM1 hydrogels provided a reliable and economical choice serving as ideal ECM for application in tissue engineering and regeneration medicine.