Recent fabrications and applications of cardiac patch in myocardial infarction treatment

Myocardial infarction caused by coronary artery obstruction results in the loss of heart muscle, which is subsequently replaced by scar tissue with limited therapeutic options. Cardiac patch based therapy has emerged as a promising strategy for the treatment of severe myocardial infarction. They are designed to be attached to the surface of the heart, that is, they are meant to “patch” the injured heart, thus restoring the damaged or failing myocardium locally through mechanical and regeneration support. The engineered three‐dimension cardiac patch is composed of biomaterial scaffolds with/without cells or inducible factors, which is typically manufactured by decellularize extracellular matrix, electrospinning, three‐dimension printing, hydrogels, or by stacking cell sheets. In this review, we outline the latest advances of the construction methods and beneficial effects of cardiac patches used in animal and clinical treatment of cardiovascular diseases, especially in myocardial infarction. We discuss the cell souring of cell‐based patches, the types of biomolecules for cell‐free patches, and their engineering methods. In addition, the method of implanting the cardiac patch to the injured heart is also described. Finally, we briefly outlook the future challenges and probable solutions in this area.


Hydrogels
Cell sheets Genes 3D Bioprinting F I G U R E 1 Schematic illustration of the most common composites and techniques employed for fabricating cardiac patches. dECM, decellularized extracellular matrix. MSCs, mesenchymal stem cells. SMs, skeletal myoblasts. iPSCs, induced pluripotent stem cells. GFs, growth factors. AAV, adeno associated virus. EVs, extracellular vesicles heart failure. 2 Current therapies for MI, such as bypass grafts, balloon angioplasty, stents, and drug therapies, focus only on palliative care and fail to address the underlying problem of cardiomyocyte loss and replacement in early cardiomyopathy. 3 In this regard, the only definitive treatment is a heart transplant, which in turn is limited by the inadequate supply of matching hearts for the ever-increasing number of patients in need. Therefore, it is necessary to develop an effective therapy to repair the damaged myocardium and prevent end-stage heart failure.
Applying a temporary, local patch to the surface of the infarcted ventricle has been explored, namely the cardiac patch, exhibiting obvious superiority in recovering the function of damaged hearts. 4 The cardiac patch has two major features, i.e. the abilities to provide appropriate mechanical support and stimulate remodeling pathways through focused cellular or biomolecule delivery. 5 In other words, the main mechanism of tissue repair induced by the cardiac patch is not only the mechanical support provided by its scaffold properties, but also the biological function changes realized by the scaffold and its transported stem cells or biological components. 6 A variety of cardiac patches with advanced physical, chemical, and biological properties have been manufactured by cardiac tissue regenerative engineering, 7 like decellularize extracellular matrix, 8 electrospinning, 9 3D bioprinting, 10 hydrogels, or by stacking cell sheets Figure (1). 11 Here, we present an overview on the current fabrication methods and function of cardiac patches, focusing on the advances in both animal and clinical studies, primarily in the treatment of MI. Advantages and limitations, and mechanisms of prompting cardiac recovery of various patches are also discussed. Finally, we analyze and outline the major challenges and prospects about implanting cardiac patches for the treatment of MI.

CELL-BASED PATCHES
Cardiomyocytes (CMs) are terminally differentiated cells that lack the potential to regenerate, thus limiting their ability to restore cardiac function after cardiovascular diseases. 2,12 To repair or restore the myocardium, cells from many different sources have been investigated to both replace lost cardiomyocytes in the native heart and to activate cardioprotective events via paracrine signaling following their implantation. 13 The Transplanted cells can stimulate a variety of regenerative processes (e.g., angiogenesis, activation of endogenous progenitor cells, decreased apoptosis of cardiomyocytes, and reduction of fibrosis), and offer cells to assist tissue repair. 13a Taking into consideration the availability of cell sources, the technical possibility of isolating and differentiating cells, and ethical issues, skeletal myoblasts (SMs), mesenchymal stem cells (MSCs), and human induced pluripotent stem cells (hiPSCs) have been extensively used in the cardiac patch fabrication to treat MI at preclinical and clinical studies.

Skeletal myoblasts
Skeletal myoblasts are the first cells to be used in cardiac regeneration because of their abundance and contractile properties, which could help to recover the lost contractility caused by MI. Skeletal myoblasts, precursor cells of myofibers, have several advantages, including autologous availability, high proliferation, resistance to ischemia, and absence of tumorigenicity because of a myogenic lineage restriction. 14 When SMs are used as therapeutic cell source for cardiac patch engineering, they are mainly be attached to the heart surface in the form of cell sheets to treat MI. The beneficial effects of autologous SM cell sheets transplantation were confirmed in series of clinical studies by Yoshiki Sawa, suggesteing that, for the "no option" ischemic cardiomyopathy patients, SM-sheet autograft is a safe, feasible, and effective method, which is mainly based on angiogenesis induced by cell secreted growth factors (GFs), 15 for example, hepatocyte growth factor (HGF), stromal cell derived factor (SDF-1), and vascular endothelial growth factor (VEGF). 16 In a 5-year study, they evaluated the recovery of cardiac function after SM-sheet transplantation in 23 patients with end-stage ischemic cardiomyopathy and measured long-term outcomes after applying this treatment. Sixteen patients (69.6%) were defined as "responders" with a mean increase in left ventricular ejection fraction of 4.9% and an overall 5-year survival rate of 95%. 15d SMs have also been used with scaffolds to promote cell survival and angiogenesis. After being embedded in polyurethane (PU)-based scaffolds and modified with cytokines or GF genes, such as VEGF-A, HGF, SDF-1, or Akt, these patches resulted in increased angiogenesis and reduced infarct size after implantation. 17 These results suggest that the use of SMs, either in the form of cell sheets or engineered scaffolds, may be a promising approach for further clinical use.

Mesenchymal stem cells
In preclinical and clinical studies, MSCs are the most commonly used cells to treat MI. 18 Characterized by pluripotency, self-renewal ability, and low immunogenicity, 19 they are capable of promoting endogenous myocardial and vascular regeneration, and involved in immunomodulatory and remodeling process. 18,20 These pluripotent stem cells can be readily obtained from a variety of tissues and organs, 21 with bone marrow (BM) and adipose tissue (AT) being the most common sources. 22 The MSC-based cardiac patch, effectively implanted, can promote cardiac recovery at the injured site in animal models of MI. In cardiac patch therapy, BM-MSCs have been embedded in decellurized extracellular matrix (dECM) hydrogel, 23 fibrin gels, 24 poly (e-caprolactone) (PCL) scaffolds, 25 or PCL/gelatin nanofibers 26 followed by being implanted onto the injured epicardium. BM-MSCs in these patches migrate to the scar tissue, significantly reducing scar size, increasing the number of blood vessels in the area around the infarct, and improving diastolic properties. To improve the efficacy of cell therapy, pre-vascularization is essential to meet the urgent need for nutritional and oxygen supply after transplantation and to successfully integrate the engineered 3D structure with the surrounding host tissue. 27 For instance, MSC sheets were pre-vascularized by co-culture with endothelial cells (ECs). By this means, the MSC successfully formed a microvascular network while maintaining the multidirectional differentiation ability. 28 Apart from BM, MSCs derived from other tissues, such as AT, 29 and umbilical cord (UC), 30 have also been shown to attenuate left ventricular (LV) remodeling and improve cardiac function when evaluated in MI animals. MSCs are an attractive cell source due to their immunoprivileged nature, as they lack major histone compatibility complex class II markers. However, a recent study conducted by Guo et al suggested that after hUC-MSCs being transplanted epicardially, immunological rejection occurred within 11 days, so the hUC-MSC sheet intervention for MI is highly possible within 11 days after transplantation. They also demonstrated that at the initial stage of AMI (days 0-3), the host heart was immersed in inflammatory cells that infiltrated the cell sheet, thus anti-inflammation was the main therapeutic effect of hUC-MSC sheets, probably via paracrine secretions and direct cell-cell contact. After the acute inflammation (days 4-11), the hUC-MSCs that survived kept producing the extracellular matrix and provided mechanical support to the host heart. 30 Although no studies have yet been conducted to compare different sources of MSCs in terms of their effectiveness in cardiac patch therapy, Hoeeg's compared MSCs obtained from human BM, UC blood, and AT in the treatment of non-ischemic dilated cardiomyopathy (NIDCM). All MSC subtypes exhibited comparable features, with improved cardiac function and anti-fibrotic, angiogenic, and immunomodulatory mechanisms. It is reasonable to suggest that the mode of action is similar regardless of cell origin. 31 Compared with autologous MSC therapy, allogeneic MSC showed an increase in left ventricular ejection fractions (LVEF), elevated all-cause rehospitalization rates at 12 months, and decreased incidence of adverse cardiovascular events. 32 Moreover, a sub-analysis by Florea et al demonstrated that the effects of MSC therapy on cardiac function and clinical outcomes were comparable in male and female patients. 33 These results show that autologous or allogeneic MSCs are both suitable for cardiac patch fabrication, and that different organ sources play an equal role in clinical trials to treat cardiovascular diseases.

iPSC-derived cardiomyocytes
The discovery of iPSCs by Yamanaka et al in 2007 opened up many new therapeutic possibilities in the field of regenerative medicine, especially for cardiac regeneration. 34 Cardiomyocytes differentiated from iPSCs contain cardiac microstructures such as sarcomere, myosin heavy chain, myosin light chain, and mitochondria. 35 Ethical problems are avoided because they can be produced from the patient's own somatic cells through overexpression of Oct4, Sox2, KLF4, and Myc. 35 However, the pluripotency and limitless proliferative ability of iPSCs can also lead to tumor formation; they are usually differentiated into CMs, smooth muscle cells (SMCs), and ECs followed by being assembled into engineered tissues. Ishida et al demonstrated that the iPSC-CM sheet was more effective than SM sheets and MSC sheets in improving the regional function in porcine models of MI. 36 iPSC-CM sheets secrete angiogenic cytokines, such as HGF and VEGF, which induce angiogenesis in damaged organs when transplanted into animal models. 37 In addition, the mechanism of functional recovery of iPSC-CM sheet transplantation also includes: (1) iPSC-CMs survived and showed synchronous contraction with the host heart; (2) the transplanted iPSC-CMs developed into sarcomere in vivo and integrated with the transplanted heart; and (3) a direct mechanical contribution was made to cardiac function. 38 Enhancement of viability and survival of transplanted hiPSC-CMs based cardiac patches may improve the clinical effectiveness for heart failure. A research team led by Yoshiki Sawa has received approval to use the hiPSC-CM sheet to treat patients with heart failure. Although promising, the method has to use only ∼100 μm of thin cell layers, since no built-in microvascular system is available, which may affect the survival of the graft. 39 Increased blood supply to the cells or homing of the cells may promote their survival. Existing approaches include cell sheet implanted on the omentum, 40 co-culture with ECs and SMAs, 41 and ECM components, such as lamin 21, 42 added to the scaffold. All of these methods have improved the survival of the transplanted hiPSC-CMs and displayed beneficial effects in animal models of MI.
Maturation of iPSC-CMs is another concern while being translated to clinical studies, which is related to the therapeutic effects and arrhythmogenicity risks. Most iPSC-CMs resemble immature CMs in morphology and function rather than adult CMs, which may hinder their applications. By culturing hiPSC-CMs on low-thickness oriented nanofibers made of PLGA, the cardiac tissue like constructs were formed with improved maturity, which showed better cardiac outcomes than acellular nanofiber scaffolds as controls when placed on the epicardium of MI rats. 43 Another method was co-culture of hiPSC-CMs with MSCs, which secreted soluble factors and exosomes to enhance the function and maturation of CMs. Cell sheets made from a mixture of these two cells showed better survival rate and protective effects when transplanted on rat MI models. 44

ACELLUAR PATCHES
Mounting evidence suggests that, instead of generation of new heart tissue, the cardioprotective effects of cell therapy may be largely due to the secreted paracrine factors that enhance endogenous repair pathways. These factors include proteins, RNA, exosomes, etc. 45 Several researches attempted to use these derivatives to build acellular cardiac patches. Table 1 compares cell-based patches with acellular patches used in cardiac therapy and regeneration in preclinical and clinical studies.

Growth factors
GFs, such as VEGF, fibroblast growth factor (FGF), and HGF, induce neovascularization to improve tissue blood supply; meanwhile, platelet derived growth factor, insulinlike growth factor, and others inhibit myocardial apoptosis, thereby alleviating ischemic injury. 13b However, their short biological half-life, low specificity and the need for repeated injections to maintain therapeutic efficacy have hindered progress in clinical applications. 46 In order to protect GFs (mainly proteins) during delivery and targeting to the diseased tissue, the GF-encapsulated cardiac patch has attracted great attention. Rodness et al combined two biomaterial-based approaches to deliver VEGF to rat hearts after MI: microspheres in the patch were used for sustained release of VEGF, and the structure of patch was for mechanical support. It was demonstrated that VEGF patch had better effect on cardiac vascular growth, tissue repair, and cardiac function repair than in the control group by surgical implantation of VEGF patch in the infarction area after heart injury. 47 A large number of pro-angiogenic bioactive factors, collectively referred to as "cocktail" factors secreted by stem cells, can promote endogenous repair of ischemic damaged tissues. 48   polylactic-co-glycolicacid (PLGA) microparticles, followed by being embedded in the dECM scaffold by vacuum ( Figure 2). When transplanted into rat models of acute MI, it reduced scarring, promoted angiogenesis and increased LVEF. In addition, the artificial heart patch was biocompatible and safe in porcine models of acute MI, resulting in smaller infarct size, less myocardial fibrosis, and greater improvement in ejection fraction compared to untreated pigs. 49

Gene delivery
The efficiency of gene delivery remains a major obstacle of clinical trials of cardiac gene therapy. 50 The most efficient manner for gene transfer is using adenoviral vectors or adeno associated virus (AAV)-based vectors for its specific tropism of these vectors for postmitotic cells in vivo, notably cardiomyocytes. 51 In addition to gene transfer efficiency, another factor affecting overall efficacy of cardiac gene therapy is the route of administration. Gu et al developed an elastic epicardial patch to control the release of AAV into the heart. AAV was incorporated in elastomer polyester polyurethane urea (PEUU) and polyester ether polyurethane urea (PEEUU) core-sheath fiber scaffolds to test long-term gene delivery improvements in cardiac function after MI. The patch was labeled with green fluorescent protein to assess transfection rates. Most transfection occurred at week 1 and was still observed at week 8. After 12 weeks, large amounts of connective tissue integrated with the host and muscle bundles appeared, indicating improved heart regeneration. 52 In general, the extended release behavior, prolonged transgenic expression, and elastic mechanical properties of AAV scaffolds make them a competitive option for cardiac tissue engineering that requires gene delivery and appropriate mechanical support.

Extracellular vesicles
The discovery of extracellular vesicles, especially exosomes, opens up new prospects for the application of cellfree therapy in cardiac regeneration. 53 Exosomes (Exos) or extracellular vesicles (EVs) retain the characteristics of their parent cells, facilitating cell-to-cell communication through the transport of bioactive molecules (proteins and RNA) between cells, and have the ability to reduce inflammation, apoptosis and fibrosis, and stimulate angiogenesis. 54 Studies comparing the effects of MSC-derived EVs with MSCs demonstrated that EV therapy was more effective in limiting cardiac fibrosis. 55 EVs are typically administered by systemic or intramyocardial injection, or delivered by vascular stents in a recent study. 56 However, even when injected into myocardium, EVs diffuse rapidly around the infarct area. 57 Liu et al designed a hydrogel patch exhibiting slow release of iPSC-CM derived EVs to the injured myocardium. After implantation in rat MI models for 4 weeks, hydrogel-delivered EVs restored normal physiological activities of myocardial cells, reduced the infarct size, and achieved a sustained therapeutic effect. 58 Similarly, Hamada used a Poly(glycerol-co-sebacate) acrylate ethylene glycol (PGSAg-EG) polymer based adhesive cardiac patch enabling the controlled delivery of EV into the cardiac tissue. The PGSA-g-EG polymer was demonstrated to be biocompatible in a rat model of MI and was able to release bioactive EVs for at least 14 days. 59 In summary, biodegradable and high-porosity hydrogels enable EVs to be continuously released in the treatment of ischemic heart tissue by placing EV-loaded hydrogels directly on or around the target. In this way, only a small number of EVs are needed to be effective. In contrast, EVs must be given intravenously in large quantities to cope with the poor systemic bioavailability of EVs. The development of cardiac patch engineering has helped EV therapy play a more significant role in the treatment of cardiovascular diseases.

STRATEGIES FOR SCAFFLOD FABRICATING
The engineered cardiac patch scaffold is designed to mimic natural ECM. The scaffold can provide mechanical support for tissue replacement/regeneration, offer an environment conducive to cell migration, integration, proliferation, and differentiation to achieve the desired therapeutic effect, as well as accommodate a permeable structure that allows for efficient nutrient and waste transfer. 60 The chemical compositions, physio-mechanical properties (such as stiffness) and 3D architecture of the scaffold determine the interaction of cells with microenvironment and the drug release. 61 Scaffolds can be made from different natural or synthetic biological materials or their composites. Natural materials (e.g., alginate, collagen, and fibrin) induce biological stimulation, but require elaborate reproducibility and purification processes. In contrast, synthetic materials (e.g., PU, PLGA), although usually easy to synthesize with stable structure, multiple sources, and low preparation cost, require functionalized modifications to provide the desired biological signal or adhesion to endogenous and/or transplanted cells. 7 These two types of materials, even with different origins, are complementary in the field of heart regeneration, and both are in extensive use. While a universal or perfect method has not been created, highly functional and complex cardiac patches made of interwoven fibers have been engineered with diverse designs and techniques, as listed in Table 2. 9,62

Decellularized extracellular matrix
dECM is a promising biomaterial for the repair of cardiovascular tissue. dECM most effectively retains many components found in natural tissues, such as proteins, glycosaminoglycans, and proteoglycans, providing an ideal support for the regeneration and repair of damaged myocardium. 67 Decellulization is the process of removing cells from tissues or organs using detergents, enzymes, and/or salts, while preserving the composition, structure, mechanism, and biological activity of the ECM. 68 dECM can be used in a variety of forms, ranging from solid scaffolds that maintain the natural vascular structure to gelatinous scaffolds that need an extra step to decompose the ECM structure. The latter is more flexible than the former one and can be manufactured with the help of tissue engineering, 63 which will be discussed in detail later.
As sourcing of both tissue (myocardium, pericardium, or other sources such as small intestinal submucosa [SIS] or omentum) and species (human, murine, porcine) varies, many research results suggest that the sourcing is not as important as the method of decellularization and functional modification in treating MI, although cardiac specific dECM is regarded as the most effective source in the heart repair. 63 The porcine heart ECM is more accessible and easier to process than the human heart, with components similar to human cardiac ECM, making it more likely to be applied in clinical translation in the future. 69 dECM-based patches restore heart function by acting directly or by combing with cells and/or other biomaterials, such as GFs, stem cells, or polymers (e.g., chitosan, silk, gels). Implantation of dECM patches alone, without the addition of biological agents, stimulates regeneration of cardiomyocytes even after scar tissue has been formed. 70 At 7 days after transplantation, a large number of host cells into the dECM and different types of blood vessels were formed. 71 When cells were embedded in dECM, like hiPSC-CMs, they exhibited increased electrophysiological activity and maturation compared to cell aggregates. After implantation in the infarct area, the cardiac patch reduced the infarct area and improved the ejection fraction (Figure 3). 72 Another study used slices of porcine heart dECM to encase human CSC secreted factors. This off-the-shelf acellular artificial cardiac patch maintained its efficacy after long-term cryopreservation (Figure 2). 49 As for clinical translation, the SIS-based solid dECM patch, CorMatrix, has been commercially used for carotid artery repair, pericardial reconstruction, and cardiac tissue repair, most often for cardiac closure. Mewhort et al demonstrated that CorMatrix can be successfully used to treat MI in rats and pigs. 73

Electrospinning
Electrospinning can produce continuous fibers and form nanofibrous structure, replicating the myocardium ECM with controllable aligned architectures by optimizing several parameters like electric field, rotating speed of the collector, solution concentration, flow velocity, and jet parameters such as height. 74 Except for its high surface area, another advantage of electrospinning is its versatility, which can be attributed to the diversity of polymers selected to achieve their different physical and chemical properties to meet different needs, 75 and cells can be loaded into the electrospinning solutions to form cell laden scaffolds. 76 Lin et al prepared aligned-and randomorientated nanofibrous patches with or without seeding of CMs and ECs using electrospinning techniques. CMs seeded in anisotropic cardiac patches showed better beating frequency, prevented electro uncoupling, and improved the therapeutic efficacy of cardiac repair when implanted on the heart of MI rats. 77 Others fabricated anisotropic cardiac patches using biodegradable polycarbonate polyurethane urea elastomer (PECUU) and dECM, which combined the biological activity of cell sheets and the mechanical performance of electrospun scaffolds. After being implanted into the rat MI model, the patch increased the survival of the transplanted MSC, enhanced more new blood vessels around the infarction area, and the left ventricle injection was also improved ( Figure 4A). 78 In an earlier study, the same materials were used to fabricate a bi-layered scaffold with isotropic properties, that is, a dECM rich layer (containing sparse PECUU fibers) and a PECUU microfiber layer. The polymer fibers in the ECM rich layer were anisotropic, due to the combined action of electrospinning and electrostatic spraying, while in PECUU layer were isotropic. The former provided biological activity and the latter mechanical support. This bilayered patch altered the progression of several key aspects of adaptive remodeling after MI ( Figure 4B). 79 Thus, electrospinning, with its diversity, offered a unique method for fabricating scaffolds that mimic the 3D geometry, mechanical and electrical properties (by adding polypyrrole, polyaniline, carbon nanotubes, etc.) 80 of cardiac tissue engineering.

3D Bioprinting
3D bioprinting is the process of depositing sequential layers of biological materials (biomaterial/cell/growth factor mixtures, commonly known as "bio-inks") on top of one another to form 3D structures. 81 This technique allows precise control of the geometry of cardiac patches and adjustment of characteristics such as bundle diameter and pore size. 64 The controllable complex porous network established in this way permits efficient delivery of nutrients and ensures the uniform distribution of cells in the whole patch, which is conducive to the proliferation of cells with sufficient transport of oxygen and nutrients in the patch. 82 Extrusion-based bioprinting, ink-jet 3D printing, and stereolithography-based bioprinting have been developed and increasingly used in tissue engineering applications.
The key to 3D printing lies in the selection of biological ink, which should consider both biological activity and printability. 10 Tissue-specific heart dECM hydrogel is a promising bio-ink that can repair murine models of MI either when printed alone or in combination with cells. Its limitations are soft mechanical properties and very low viscosity, which are generally improved by increasing the concentration of dECM, 83 cross-linking modifications (like vitamin B2), 84 adding low melting point materials (like GelMA 85 ) or depositing on polymer scaffolds as support materials (like PCL). 23,86 Bioprinting based on extrusion is the most commonly used technology in 3D printing because of its low cost and high speed, which can print highly viscous cell-loaded biological ink by adjusting the air pressure in the process of pneumatic deposition. Jang generated a pre-vascularized stem cell patch through spatial organization of two cells (cardiac progenitor cells/dECM and MSCs/dECM) using 3D cell printing methods. The two bio-inks can be printed alternately on the PCL support layer based on the generated code. After one layer was printed, it was exposed to UVA light for 30-60 s to initiate the vitamin-B2-induced post-crosslinking process. Angiogenesis, cell survival and tissue reconstruction were observed in the patch after transplantation. In addition, the pre-vascularized stem cell patch provided enhanced beneficial effects, including reduced cardiac remodeling and fibrosis, as well as promotion of myocardial generation and neovascularization in the damaged myocardium ( Figure 5A). 84 A recent study conducted by Huang et al designed a perfusable, multifunctional epicardial device, called PerMed, which was assembled using a biodegradable elastic patch, permeable hierarchical microchannel networks (PHMs) and a delivery system . The PHM, made of PCL and PCL/gelatin (3:1), was fabricated by 3D bioprinting consisting of multilayer interconnected branch network with many open and uniformly permeable micropores in the channel walls. With good elasticity and fatigue resistance, the PHM withstood dynamic deformation and maintained a tubular structure during long-term epicardial applications. In addition, new vessels infiltrated into the PHMs, resulting in an angiogenic effect in the infarcted zone and border zone. 87 Extrusion 3D printing cannot produce structures with the size at which individual cells interact. Gao et al used multiphoton-excited 3D printing technology to create a dECM scaffold with submicron resolution (<1 μm) and then seeded with CMs, SMAs, and ECs (2:1:1 ratio). The distribution of fibronectin in mouse was used as a template, and calcium transients and beating synchronously was generated within 1 day of cells being seeded in the scaffold. In experiments on surgically induced MI mice, animals treated with this cardiac patch significantly outperformed those treated with cell-free scaffolds in terms of cardiac function, infarct size, apoptosis, vascular and arteriole density, and cell proliferation. 88 To better simulate the structural and biological characteristics of natural cardiac tissue, a physiologically adaptive 4D heart patch was printed by beam-scanning stereolithography. GelMA and polyethylene glycol diacrylate (PEGDA) were used to prepare the cardiac anisotropic cardiac patch with myocardial fiber orientation. The smart patch provided mechanical support, physiologically adjustable structure, and a suitable matrix environment (elasticity and bioactivity) for cell implantation. Successful vascularization of the patch satisfied the continuous metabolic requirements of hiPSC-CMs and maintained its viability and function throughout the in vivo study ( Figure 5B). 85 3D bioprinting depends on the selection and modification of the bioink. Bioprintability, mechanical property, and biological property are major issues to be considered. 82 If the ink is too adhesive, then it will need high extrusion pressure, which may do damage to living cells

Hydrogels
Hydrogels are characterized by a large network of hydrophilic polymer chains, which form 3D conformation due to the cross-linking within the network and have been widely used in MI treatment. 89 They are considered to be one of the most promising drug delivery systems with minimally invasive techniques while maintaining all the desirable characteristics. 65 The properties of hydrogels depend on the biomaterials used to make them, as these materials determine important factors such as electrical conductivity, cross-linking, density, degradation and interaction with inducible drugs (drug release and protection, cell attachment). Fibrin gel (FG) is the most convenient and common hydrogel used to generate cardiac patches, composed of component I (fibrinogen and factor XIII) and component II (thrombin and calcium chloride). 90 FG mimics the final stage of hemostasis, and commercial allogeneic fibrin sealants are in clinical operation for hemostasis. What's more, FG has been reported to enhance the therapeutic effect of MI. Fibrin can be used in a variety of approaches, including in prefabricated fibrin patches, 91 instantly produced FG-cell complex by directly dropping on the heart surface, 24 or as an adhesive glue. They can be polymerized in molds of different sizes and shapes, 92 up to 36 × 36 mm, a size suitable for a human heart. 93 Furthermore, these different sized fibrin patches maintain spatial uniformity with high conduction velocity and contractile stresses. 94 Fibrin-based composite scaffolds usually degrade within two weeks after implantation onto the heart and can be gradually replaced by ECM secreted by cardiomyocytes. 95 Addition with PEG can significantly improve the stiffness and stability of fibrin scaffolds. Furthermore, hydrogels can be modified to enhance the binding of GFs and achieve slow release. For example, an alginate hydrogel was modified to mimic the heparin/heparan sulphate binding groups by sulphating the uronic acids in the saccharide backbone to improve the binding ability of GFs. 96 Revascularized hydrogel could also be realized to improve their thickness and force generation. 97 For example, Schaefer et al developed a mechanically constrained double-layer fibrin patch, composed of the cardiomyocyte layer and the microvascular layer (ECs and the pericyte). After implantation, the patch microvessels germinated in the myocardial cell layer of the patch and combined with the host vascular system. 98 Similar to fibrin gels, PuraMatrix R (PM) is another hydrogel of this class that has also been extensively studied. Under physiological conditions, the peptide components of PM self-assemble into 3D structures exhibiting highly organized nanoscale fiber structures with an average pore size of 50-200 nm. These solutions can be spread onto the surface of the heart and then a hydrogel is formed subsequently. This controlled gelation, adhesiveness, and easy of operation of PM make it a rapid and convenient method for epicardial placement. For example, an epicardium "coating" PM-MSC complex was produced immediately during surgery in the operating room. This technology requires neither manual/costly GMP production nor transportation of cell sheets or premade patches. 99 Of note, both fibrin gel and PM have achieved more significant improvement in cardiac function with greater initial retention and survival of donor MSCs, compared to intramyocardial delivery. This enhanced efficacy is due to the upregulation of genes related to myocardial tissue repair, thereby enhancing the repair of damaged myocardium, augmenting microvascular formation, and reducing interstitial fibrosis. 100

Scaffold-free patches
Although scaffold-based patches provide a template for structural organization of the cells, they are expected to degrade over time and gradually be replaced by a new matrix after implantation onto the heart. Scaffold-free 3D structure formed by staking multiple cell monolayers is capable of delivering large numbers of cells to the damaged heart muscle without loss of transplanted cells or damage to the host heart muscle, and has demonstrated feasibility and safety in clinical studies. SMs, 11 MSCs, 101 and hiPSC-CMs are commonly used cells to establish scaffold-free cell sheets for the treatment of cardiomyopathy, which benefits from the integration of graft and host tissue or from secondary paracrine mechanisms that promote a favorable response.
Cell sheets for MI repair are produced by culturing cells on dishes coated with temperature-responsive polymer substrate, poly(N-isopropylacrylamide) (PIPAAM), 102 which releases the attached cells when the temperature drops below 32 • C and maintains the ECM and intracellular connects produced during culture. 66 These ECM and intracellular connects facilitate cells of adjacent layers forming communication rapidly, including gap junctions, causing the cardiomyocytes in the cell sheet to form electrical connections rapidly. 103 Transplantation of hiPSC-CM sheets in MI areas in porcine models improves cardiac performance through structural and electromechanical integration into the host myocardium. 36,104 Additionally, vasculogenesis in the layered cell sheets are introduced by establishing stacks consisting of CMs and ECs or their coculture in the same layer. 105 Cell sheets have unique properties to enhance cell function by stretching. When the cell sheet is detached from the temperature-responsive dish, its surface area decreases and its thickness increases due to cytoskeleton shrinkage. 106 After stretching the harvested CM sheet along one direction, the sheet was significantly longer than before. When transplantation of the stretched sheet onto the heart, CMs were observed to be arranged unidirectionally on the stretched sheet while randomly on the unstretched sheet. Two weeks after transplantation, the stretched CM retained the unidirectional properties of myocardial fiber, and its orientation intensity was higher than that of the control cell sheet after transplantation and the stretched cell sheet before transplantation. 107 Overall, the cell-sheet method has been shown to maximize the number, survival, function, and integration of transplanted cells into the heart.

APPROCHES TO PATCHING THE HEART
The effectiveness of cardiac patch engineering techniques in the treatment of cardiovascular diseases depends on in vivo implementation and evaluation of functional results in animals or humans. Unlike other delivery routes, the patch needs to be placed firmly against the beating heart for a short period of time.

Sutures and glues
Cardiac patches are usually be sutured or glued onto the epicardium of damaged hearts in animals or humans, 23,108 including scaffold-based tissue patches 41,109 and scaffold free patches. Although suturing allows the patch to adhere firmly to the desired site, it risks disrupting blood supply to the patch, bleeding, damage to healthy tissue, and infection. Such a trauma may even deteriorate the LV function and extend the scope of the damage. Fibrin glue is commonly used as an adjuvant to attach cell sheets 15d or dECM scaffolds 72 to the epicardium. While fibrin glue is more convenient and less invasive, it may not be sticky enough to attach a large heart patch to the surface of a beating heart. In addition, the glue may create an unwanted gap between the cardiac patch and the epicardium, thereby inhibiting the infiltration of cell secretaries into the myocardium.
Reportedly, stitching has no adverse effect on the patch compared with the gluing group.
In the case of cell sheets, although they may attach to the heart without sutures because of the deposited ECM, 110 they need to be placed and sutured when they were fabricated to be applied on a human heart, for example, 4 cm in diameter and 100-150 μm in thickness. Another case is the microneedle (MN)-based cardiac patch. Due to its special structure and polymer poly(vinyl alcohol) composition, MN can be directly attached to the epicardial surface, but as reported in the literature, the authors applied fibrin glue on the MN side of the patch to aid adhesion. 111

Photocurable adhesive polymers
In addition to fibrin and PM-3D, photocurable adhensive polymer based cardiac patches may also adhere strongly to the heart directly. For example, the PGSA-g-EG patch adhered to the heart surface due to its hydrophobic and viscous properties, allowing on-demand adhesion to biological tissues using light, and was attached to the heart surface for 1 month. 59 A choline based bio-ionic liquid (Bio-IL) was used to develop a conductive and adhesive cardiac patch. Due to the formation of ionic binding between the Bio-IL and native tissue, the engineered patch had a strong adhesion to the mouse heart muscle without suturing. Moreover, the patch exhibited mechanical and electrical properties similar to those of natural heart muscle, and provided mechanical support and restored electromechanical coupling at the infarction site to reduce cardiac remodeling and maintain normal cardiac function. 112 Other nanocomposite scaffolds, consisting of albumin electrospun fibers and gold nanorods (AuNRs), also adhered to the heart surface when irradiated with a nearinfrared laser (808 nm) without suturing. The mechanism is that AuNRs were able to absorb light and convert it to thermo energy, in situ altering the molecular structure of the fibrous scaffold, perhaps either by denaturing and cross-linking the albumin and collagen upon heating, or by melting the polymers, so that they adhered firmly and safely to the heart. 113

CONCLUSIONS AND PERSPECTIVES
Engineered cardiac patches, composed of scaffold materials and bioactive molecules, have shown progress in preclinical experiments and clinical trials in the treatment of MI. Despite the differences in construction and delivery method, these observations agree that the cardiac patch can provide adequate mechanical and regenerative support to treat MI. It mainly reduces myocardial infarction area, reverses ventricular remodeling, and enhances the ejection fraction and other cardiac functions through the processes of anti-inflammation, anti-apoptosis, promoting angiogenesis, and anti-fibrosis. However, cell sourcing and maturation, low biophysical and electronical integration, and the lack of a minimally invasive delivery approach remain critical obstacles that must be overcome for effective functional repair and clinical transformation of MI treatment.

Promising iPSC-CM based patches
The best source of cells for heart repair has long been controversial. CMs differentiated from iPSCs have been regarded as a promising source of cardiac regeneration since they have phenotypes and/or characteristics similar to those of normal CMs, making them better integrated into host cells. Currently, the most prospective method for delivering iPSC-CMs is combination with the cell sheet technology. 114 With the progress in the field of cell sheets, issues encountered are being addressed, such as the realization of large-scale culturing of hiPSC-CMs, elimination of undifferentiated iPSCs to reduce the risk of tumor formation, and improvements in myocardial tissue manufacturing techniques. Apropriate iPSC-CMs maturation is an important factor to promote clinical translaiton progress, by reducing the incidence of arrhythmia and improving the recovery outcomes. However, it is unclear to what extent iPSC-CMs matures to obtain the desired optimal results. For instance, in one study, iPSC-CMs was purified on day 20 of initiation of differentiation, and its transplantation effect was better than that on day 8 or 30. 36 Further investigation is warranted to explore the optimal stage of differentiation to maximize the implantation rate and therapeutic effect.

Full functionalization and integration of the patch to the host
Full functional and mechanical/electrical integration of the transplanted biomaterial/stem cell constructs with the host myocardium remain to be critical challenges. Electrical integration helps not only to restore electrical conductivity of the host heart and reduce the arrhythmogenic risks due to no or partial electrical integration, but also to monitor the electrical activity of the patched cells. Full functionalization could be realized by prevascularization through co-culture with ECs or addition of vascular growth factors. The truth is, the vessels may not develop quickly enough (about half a day to get blood from the host to the transplant), while cell apoptosis occurs in approximately 30 min when transplanted into the damaged myocardium. Moreover, vascularized cardiac patches maintained their pregraft histological structure and electrical properties, but no anterograde or retrograde conduction was observed between the patch and host cardiomyocytes, suggesting a lack of electrical integration. 92 Electrical integration is crucial for the cardiac patch, which is of help in restoring electrical conductivity of the host heart, reducing the arrhythmogenic risks, and monitoring the electrical activity of the patched cells.
The MN approach provides an idea to solve this problem, in which transplanted cells receive nutrients from the heart and release paracrine factors to repair the heart. 111,115 A good case in point is the use of an integrated system, containing MN patches, arraying carbon nanotubes (CNTs) and iPSCs, in the treatment of heart diseases. The anisotropic structure of MN patch induced directional arrangement of CMs, and its conductive elements provided a platform for intercellular interactions. The functional MN array patch adhered firmly to the heart and released the encapsulated drugs, thus increasing the functionality. Besides, the presence of aligned CNT layers both ensured synchronous contraction of CMs distributed across the patch, and allowed these cells to maintain a synergistic effect with the heart in vivo (Figure 6). 115 The integration seemed to be an ideal recovery strategy for the clinical treatment of heart diseases.
Besides, ablation of the epicardium before transplantation may enhance the integration of transplanted cells, while other studies demonstrated that MSCs in cell sheets could differentiate into host cells or fused with host cells when implanted on the epicardium. 30,101b

Minimally invasive or noninvasive delivery methods needed
Although some progress has been made in treating cardiovascular diseases with cardiac patches in animals and humans, implantation still requires an invasive surgical approach; that is, a median sternal incision or lateral thoracotomy is needed to access the wider surface area of the heart. Generally, mediastinal inflammation (1.5-2% of patients 116 ) is associated with open-heart surgery, 117 and has been reported to be more prevalent in immunosuppressive heart transplanted patients. 118 Simultaneously, open-chest surgery brings panic to patients with psychological anxiety. 119 Considering that cardiac patch transplantation may inevitably involve the use of immunosuppressive agents, in order to reduce the possibility of postoperative infection, minimally invasive surgery with small incisions must be carried out to reduce these discomforts and risks, and thus achieve better cardiac outcomes.
Several studies have demonstrated the possibility for minimally invasive delivery of cardiac patches, either by simulation in vitro or through in vivo implantation 87,120 Osada et al developed a novel endoscopic cell sheet delivery device for minimally invasive surgery, and the feasibility of endoscopic cell sheet delivery to the anterior and outer walls of the left ventricle was verified in a 3D printed simulator. The commercialization of this prototype may provide a safe, minimally invasive cardiac regeneration therapy in the future ( Figure 7A). 121 In addition, Montgomery et al developed an elastic and microfabricated scaffold, and the shape memory of the scaffold was based on the design of a micromachined grid. The scaffold material and the cardiac patch (1 cm × 1 cm) were injected through a 1 mm hole to restore the original shape without affecting the viability and function of CMs. In allogeneic rat models, this method was comparable to open-heart surgery in terms of vascularization, macrophage aggregation, and cell survival. In particular, this minimally invasive delivery of the cell based patch to the epicardium, aorta, and liver was successfully achieved in large animal models. 122 Although this result was achieved with synthetic materials, we think that other natural or synthetic polymers, such as dECM, can also simulate this effect by combining with shape memory polymers.
Garcia et al developed a method for delivering hydrogels through the pericardial cavity to the epicardium. This device used an existing anatomical structure to form a temporary compartment for gel delivery, which could bypass many of the obstacles that impede vascular or intracardiac delivery with no risks of premature gelation, embolism, arrhythmias, and off-target effects ( Figure 7B). 123 There were no clinically relevant acute or subacute adverse F I G U R E 7 Minimally invasive delivery of cardiac patches. (A) Schematic illustrating the prototype of the totally endoscopic cell sheet delivery device and its epicardial placement using 3D printed simulators. Reprinted with permission. 121 Copyright 2020, Elsevier. (B) (I) Schematic showing deliver hydrogels to the pericardial space. (II) Schematic representation of lateral (a) and frontal view (b) of the device placed over the cardiac epicardium within the pericardial space. Once in position, the device is secured in place by gentle suction ports (c, white arrows) to the epicardium as floor, and the pericardium as ceiling. Gel components are delivered through separate lumens and combined only after exiting the device (a and b, asterisk) through ports arrayed around fence (c, black arrows). Reprinted with permission. 123 Copyright 2017, Elsevier reactions in pigs in this way, and we believe that combined with the advantages of fibrin gels, this method can be a strategy for delivering fibrin gel coated cells to the heart, thus exerting their synergic beneficial role in the treatment of MI.
Although some major challenges have been made clear in creating a structurally acceptable and biocompatible cardiac patch, there are still biosafety, mechanical properties, degradation, functional involvement, cost of GMP production, storage, as well as transportation methods that need to be fully considered. At present, it is still in the process of combining different materials (scaffolds and the embedded biological substances) and developing fabrication methods of engineering cardiac patches to achieve the best therapeutic outcomes with the lowest possible side effect in MI animals. Nevertheless, with the development of cell biology, and advanced engineering materials, we hold promise that the cardiac patch may become a comprehensive and effective treatment for heart diseases, thus mitigating the burden and casualties from cardiovascular diseases.

A C K N O W L E D G M E N T S
We highly appreciate financial supports from the National Key Research and Development Program of China (2017YFA0104302, 2019YFA0210104), the National Natural Science Foundation of China (61821002, 51832001, 81971701), and the Natural Science Foundation of Jiangsu Province (BK20201352).

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.