Research Advances of Injectable Functional Hydrogel Materials in the Treatment of Myocardial Infarction

Myocardial infarction (MI) has become one of the serious diseases threatening human life and health. However, traditional treatment methods for MI have some limitations, such as irreversible myocardial necrosis and cardiac dysfunction. Fortunately, recent endeavors have shown that hydrogel materials can effectively prevent negative remodeling of the heart and improve the heart function and long-term prognosis of patients with MI due to their good biocompatibility, mechanical properties, and electrical conductivity. Therefore, this review aims to summarize the research progress of injectable hydrogel in the treatment of MI in recent years and to introduce the rational design of injectable hydrogels in myocardial repair. Finally, the potential challenges and perspectives of injectable hydrogel in this field will be discussed, in order to provide theoretical guidance for the development of new and effective treatment strategies for MI.


Introduction
Cardiovascular diseases are the main cause of human death, while myocardial infarction (MI) is the main cause of the high incidence of ischemic heart disease [1][2][3]. MI is defined as sudden ischemic death of myocardial tissue, which is myocardial necrosis caused by acute and persistent ischemia and hypoxia of coronary arteries. Although percutaneous coronary intervention improves early survival, the patient population at risk of heart failure expands [4,5]. Epidemiological studies have shown that the increased incidence of heart failure after infarction has paralleled the decline in acute mortality in recent decades, which will significantly increase the high socio-economic burden and affect the quality of life of patients who survive acute myocardial infarction [6]. There are three main treatments for MI: drug therapy, left ventricular assist device implantation, and heart transplantation. Although the mortality of MI has decreased, the risk of donor heart transplantation and trauma is limited [7]. Furthermore, the ventricular remodeling is negative, which leads to the progressive decline of the cardiac function of myocardial infarction survivors, and there are still some limitations such as irreversible myocardial necrosis and cardiac function. Therefore, it is an important subject of cardiovascular disease research to develop new effective strategies for treating MI.
Nowadays, cardiac tissue reconstruction, prevention of ventricular remodeling and scar tissue formation, and reduction of heart failure and heart rupture have become the focus of new effective strategies [8,9], including the development and application of a variety of biomaterials. Currently, biomaterials developed to promote ischemic myocardial repair include patches, hydrogels, scaffolds, and particles [10][11][12]. It is worth mentioning that hydrogel has the following advantages. (1) It has good biocompatibility and low immunogenicity, and its three-dimensional cross-linked spatial structure might replace damaged myocardium to bear ventricular stress. Injectable hydrogels can be modified to further improve their biological properties, such as mechanical properties and electrical conductivity, enabling them to mimic native myocardium. (2) It can deliver all kinds of active cells, growth factors, drugs, and genes to specific local areas of heart tissue. (3) It can be administered through minimally invasive surgery, which can reduce surgical trauma. (4) It shows the advantages of regeneration and degradation [13][14][15][16][17][18].
Based on the above information, this review aims to focus on the role of hydrogel materials especially injectable hydrogels in the treatment of MI and to explore the therapeutic mechanism of injectable hydrogel combined with stem cells, cytokines, drugs, and genes. Last but not least, the existing problems of injectable hydrogel and the perspectives of using hydrogel formulations in the treatment of MI will also be discussed.

Biological Mechanism of Myocardial Infarction
Coronary atherosclerosis will result in a narrowing of the lumen and insufficient blood supply. When collateral circulation is not fully established and blood supply is sharply reduced, myocardial ischemia lasting more than 1 h will lead to MI [19]. After MI, with the decrease of ATP (adenosine triphosphate) level in the endocardium, ischemic heart or susceptible myocardial cells can no longer maintain their structural integrity [20]. In this case, ultrastructural changes occur, resulting in irreversible damage, which eventually leads to apoptosis or necrosis of myocardial cells. In the healing stage, MI has gone through the stages of inflammation, proliferation, and healing [21]. As shown in Figure 1, in the inflammatory phase, the generation of ROS (reactive oxygen species) activates the complement system, stimulates the expression of P-selectin, and activates the NF-κB (nuclear transcription factor-κB) system to upregulate the synthesis of cytokines and chemokines [22]. Releases pro-inflammatory mediators IL-1 (interleukin-1), (TNF)-α (tumor necrosis factor-α), and IL-6 (interleukin-6), which further upregulates the expression of CXC and CC chemokines, thereby promoting leukocyte chemotaxis, triggering an inflammatory cascade, while further helping to clear necrotic cells and cellular matrix debris [23,24]. Subsequently, negative regulation of TLR (toll-like receptor) signaling, etc., induces and releases anti-inflammatory molecules thereby transitioning to the proliferative phase. Such as TGF-β (transforming growth factor-β), IL-10 (interleukin-10), aldosterone's broad lipidderived mediators, and VEGF (vascular endothelial growth factor), etc., thereby activating anti-inflammatory pathways [25]. Cardiac fibroblasts are transformed into myofibroblasts, and the ECM (extracellular matrix) gradually deposits proteins, during which angiogenesis in the infarcted area provides oxygen and nutrients to the metabolically active mesenchymal cells in the healing infarcted area [26,27]. As the infarcted area heals, a new myocardial matrix is gradually established, scarring is formed, the ventricle dilates to maintain cardiac output and cardiac repair transitions to maturity. Healing of the infarct is intertwined with the geometric remodeling of the chamber, with dramatic changes in ventricular geometry, dilation of the chamber, and thinning of the infarcted portion, leading to ventricular remodeling [28].
The pathological process after MI is a dynamic development process as shown in Figure 2, and the related cardiac tissue cells and factors have biological roles in different stages. Therefore, according to the pathological mechanism of different stages, it requires a rational design of hydrogel materials in the treatment process, in order to conform to the pathological process of MI. In the inflammatory stage, it is a feasible way to improve the curative effect of the hydrogel by inhibiting myocardial apoptosis, promoting degradation or removal of infarct matrix fragments, and reducing the over-expression of pro-inflammatory factors. In the proliferation stage, a hydrogel strategy should be designed to promote the maturation and angiogenesis of fibroblasts, which might be beneficial to the healing of infarction. In the mature stage, functional hydrogels could be designed to prevent excessive ventricular dilatation and to reduce fibrosis or scar formation, in order to effectively prevent complications, such as heart failure. The pathological process after MI is a dynamic development process as shown in Figure 2, and the related cardiac tissue cells and factors have biological roles in different stages. Therefore, according to the pathological mechanism of different stages, it requires a rational design of hydrogel materials in the treatment process, in order to conform to the pathological process of MI. In the inflammatory stage, it is a feasible way to improve the curative effect of the hydrogel by inhibiting myocardial apoptosis, promoting degradation or removal of infarct matrix fragments, and reducing the over-expression of proinflammatory factors. In the proliferation stage, a hydrogel strategy should be designed to promote the maturation and angiogenesis of fibroblasts, which might be beneficial to the healing of infarction. In the mature stage, functional hydrogels could be designed to prevent excessive ventricular dilatation and to reduce fibrosis or scar formation, in order to effectively prevent complications, such as heart failure.

Design of Functional Injectable Hydrogels
Hydrogels are useful for drug delivery and tissue engineering [29][30][31][32][33][34][35][36][37]. An injectable hydrogel, made of hydrophilic polymers, can be injected in situ into a specific area of myocardial tissue in the form of a solution, with the ability to increase the thickness of the ventricular wall and reshape the left ventricular geometry [38,39]. A sol-gel phase transition occurs upon stimulation, which provides mechanical support for myocardial tissue

Design of Functional Injectable Hydrogels
Hydrogels are useful for drug delivery and tissue engineering [29][30][31][32][33][34][35][36][37]. An injectable hydrogel, made of hydrophilic polymers, can be injected in situ into a specific area of myocardial tissue in the form of a solution, with the ability to increase the thickness of the ventricular wall and reshape the left ventricular geometry [38,39]. A sol-gel phase transition occurs upon stimulation, which provides mechanical support for myocardial tissue and promotes myocardial tissue repair [40,41]. As a typical example, a previous report has shown that the synthesized high modulus hydrogel can improve the mechanical properties of the hydrogel by changing the substitution amount of methacrylic acid in the macromolecular polymer of hyaluronic acid methacrylate [42]. More importantly, it could reduce the pressure on the ventricular wall due to poor ventricular remodeling and show smaller infarct expansion and left ventricular dilation. Furthermore, pig myocardial stroma cells after processing could be effectively maintained within in vivo and in vitro myocardial hydrogel matrix at the temperature of 37 • C [43]. This bioactive hydrogel design could achieve in situ injection in the myocardium of a rat, while it could simulate the natural extracellular environment of the myocardium. In this case, endothelial cells and smooth muscle cells could in vivo and in vitro migrate to the myocardial matrix [44]. As a result, this bionic ECM hydrogel, which significantly improves cardiac function, has good safety and therapeutic effectiveness [45]. For the other case, injection of synthetic recombinant human collagen type I (RHCI) matrix at the late proliferative stage after MI has successfully restored cardiac mechanical properties and reduced scar size [46], which maintained distal wall thickness and prevented heart enlargement. This hydrogel design provides a bionic platform for safe and effective endogenous tissue repair. Experimental evidence has shown that cardiac function was restored by promoting healing, cardiomyocyte survival, and reducing pathological remodeling of myocardium [47]. At present, hydrogel with alginate as the main component has been applied in the clinical treatment of MI. More interestingly, its therapeutic potential of inhibiting the injury process after MI has been proved in phase I and Phase II clinical trials, while the safety of implantation has also been effectively verified [48][49][50]. The above studies confirmed the preliminary feasibility and the potentials of injectable hydrogel as implantable scaffolds for cardiac tissue engineering [51].
Although injectable hydrogel can provide physical stability to infarcted myocardium, structural enhancement and improved biocompatibility might not be good enough to maintain cardiac functions over a long time [50]. Therefore, nowadays, more experimental design ideas are focused on expanding the structure and properties of hydrogel materials [13,52]. A number of in-depth studies were conducted on the mechanism of repairing MI with injectable hydrogel, so as to achieve better repair effects on infarcted myocardium [53]. As a typical example, intra-myocardial injection of chitosan hydrogel was experimentally illustrated to control infarction narrow scope, to retain wall thickness, and to improve heart function. In detail, this chitosan hydrogel could be beneficial for the removal of ROS and the recruitment of chemokines, with the ability to improve the MI microenvironment, to promote the implantation, survival, or homing of stem cells in the ischemic heart, and to accelerate myocardial repair [54]. However, the regulation of chitosan hydrogel on the MI microenvironment is mainly achieved by scavenging ROS, and its effect is relatively limited. Meanwhile, the mechanism of how chitosan hydrogel improves the MI microenvironment, promotes stem cell implantation and survival, and promotes neovascularization and cardiac repair is still unclear [54,55]. Therefore, it is necessary to further explore the possibility in repair function by chitosan hydrogels on myocardium. For this purpose, a thermosensitive CSCl-RoY (chitosan chloride-RoY) hydrogel, connected with RoY peptide to CSCl chain through amide bonds, was designed. RoY is a 12 amino acid synthetic peptide (YPHIDSLGHWRR) that specifically binds to the GRP78 (78-kD glucose-regulated protein) receptor, which is mainly expressed on the membrane surface of vascular endothelial cells during hypoxia [56]. Studies have shown that the RoY peptide-modified hydrogels can promote angiogenesis in the hypoxia state after MI and improve cardiac function [54,57]. Compared with CSCl hydrogel only, CSCl-RoY hydrogel can promote the survival, proliferation, migration, and tubular formation of human umbilical vein endothelial cells under hypoxia. In this study, the mechanism was investigated. Under hypoxia conditions, RoY ligand in CSCl-RoY hydrogel interacts with the GRP78 (glucose-regulated protein78) receptor on the surface of human umbilical vein endothelial cells, inducing the endogenization of GRP78 receptor. In this case, the expression upregulation of p-AKT (phosphorylated protein kinase B) and p-ERK1/2 (phosphorylated extracellular signal-regulated kinase 1/2) was detected, and the signaling pathway of cell survival/proliferation was activated (Figure 3a). In addition, starting from the myocardial conductance pathway, the scar after MI blocks the transmission of electrical pulse and delays local contraction, leading to ventricular dysfunction. Furthermore, in previous studies, conductive PPy (polypyrrole) was coupled to chitosan skeleton to prepare a conductive biomaterials [58]. After intramuscular injection, electrical pulse propagation of scar tissue in the body was promoted and synchronous ventricular contraction was reconstructed. Its principles are shown in Figure 3b,c based on the similar design ideas mentioned above. In short, when injected with hydrogel alone, we divided the function of hydrogel in the treatment of myocardial infarction into the following categories: (1) to reduce oxidative stress; (2) to promote angiogenesis; (3) to alleviate ischemia and hypoxia in the infarct area; (4) to inhibit the malignant remodeling caused by metalloproteinases; and (5) to increase the electrical conductivity of myocardium.   [58]. (c) Intra-myocardial injection of PPy: CHI following cardiac injury improved electrical impulse propagation of scar tissue in vivo [58]. (d) The chemical structures of peptides and their hydrogelation by co-assembly. (Reprinted with permission from Ref. [59]. 2022, Elsevier). (e) The process to prepare the ROS-scavenging/conductive composite hydrogel. (Reprinted with permission from Ref. [59]. 2022, Elsevier). (f) The illustration of the ROS-scavenging/conductive composite hydrogel to repair myocardial infarction. (Reprinted with permission from Ref. [59]. 2022, Elsevier).

To Reduce Oxidative Stress
ROS is a highly reactive oxygen derivative. As a byproduct of aerobic cell metabolism, ROS acts as a second messenger of signal transduction in regulating calcium processing mechanisms and contractile proteins [60,61]. After MI, excessive ROS acts on protein kinases including calmodulin-dependent protein kinase II, resulting in myocardial hypertrophy [60]. Secondly, when the level of ROS is high, oxidative stress might lead to apoptosis or necrosis of myocardial cells. In this case, the reduced nicotinamide adenine  [57]. 2015, American Chemical Society). (b) Generation of a polypyrrolechitosan hydrogel [58]. (c) Intra-myocardial injection of PPy: CHI following cardiac injury improved electrical impulse propagation of scar tissue in vivo [58]. (d) The chemical structures of peptides and their hydrogelation by co-assembly. (Reprinted with permission from Ref. [59]. 2022, Elsevier). (e) The process to prepare the ROS-scavenging/conductive composite hydrogel. (Reprinted with permission from Ref. [59]. 2022, Elsevier). (f) The illustration of the ROS-scavenging/conductive composite hydrogel to repair myocardial infarction. (Reprinted with permission from Ref. [59]. 2022, Elsevier).

To Reduce Oxidative Stress
ROS is a highly reactive oxygen derivative. As a byproduct of aerobic cell metabolism, ROS acts as a second messenger of signal transduction in regulating calcium processing mechanisms and contractile proteins [60,61]. After MI, excessive ROS acts on protein kinases including calmodulin-dependent protein kinase II, resulting in myocardial hypertrophy [60]. Secondly, when the level of ROS is high, oxidative stress might lead to apoptosis or necrosis of myocardial cells. In this case, the reduced nicotinamide adenine dinucleotide phosphate oxidase containing NOX2 (nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases 2) is involved in the process of myocardial remodeling and contraction dysfunction after MI [62]. Based on the idea of reducing the damage caused by oxidative stress, in a large animal model of MI, researchers have demonstrated that injection of a temperature-responsive NIPAAm (N-isopropylacrylamide)-mPEGMA (polyethylene glycol methyl methacrylate) copolymer hydrogel with ROS scavenging properties can reduce ROS, increase wall thickness, and improve MI peripheral function [63]. Hydrogels with the function of scavenging ROS can effectively protect the myocardium from oxidative damage in vivo, such as significantly reducing cell membrane peroxidation and apoptosis, inhibiting inflammation, preserving cardiac function, and promoting left ventricular angiogenesis [64]. ROS increases dramatically after ischemia-reperfusion after MI [65], resulting in channel activity and Ca 2+ increased conductance [66]. In this case, intracellular Ca 2+ increased stimulation of Ca 2+ dependent on biochemical pathways, which leads to the development of myocardial hypertrophy. Additionally, intracellular Ca 2+ elevated levels also lead to an increase in mitochondrial Ca 2+ uptake and mitochondrial ROS levels [61,67], which is positive feedback and plays a key role in the progression of cardiac hypertrophy [66,68]. Furthermore, Ratcliffe et al. used nanoparticles mediated dual transmission of antioxidant curcumin and anti-L-type Ca 2+ channel peptide, which reduced mitochondrial metabolic activity by scavenging ROS and disrupting cytoskeletal communication between LTCC (L-type Ca 2+ channels) and mitochondria [69]. Curcumin and peptide could synergistically reduce ischemic reperfusion damage through different mechanisms, and this combination therapy will be more effective than treatment alone.

To Promote Angiogenesis in Infarct Area
One of the methods to treat ischemic tissue after MI is to enhance microvascular perfusion by delivering pro-angiogenic signals. VEGF is one of the most effective proangiogenic factors [70]. For example, Stuppd et al. developed a small peptide hydrogel that mimics VEGF for the treatment of MI in vivo [71]. The hydrogel displayed a peptide that mimics VEGF on the surface of nanofibers, which showed enhanced signal and biological activity by activating specific vascular endothelial growth factor receptors. It is found that VEGF could promote angiogenesis better than endothelial cells. In another case, citrate polymer hydrogel synthesized by citric acid and polyethylene glycol diol was injected into the left ventricle of myocardial infarction rats [72]. The citrate degradation by-products promoted angiogenesis and cardiac protection, by activating the PI3K-Akt-mTOR (phosphatidylinositol 3 -kinase-Akt-mammalian target of rapamycin) pathway and manipulating the citric acid cycle. With the effective release of MYDGF (myeloid-derived growth factor), this self-degradable hydrogel with an angiogenic effect might have great potential as medical biomaterials.

To Alleviate Ischemia and Hypoxia in Infarct Area
In addition to oxidative stress, hypoxia is one of the inflammatory characteristics of the tissue microenvironment after severe ischemic myocardial infarction [73,74]. Extremely low oxygen content in the infarct area is the main cause of massive myocardial cell death. At present, it is necessary to develop a method that can continuously deliver oxygen to the MI area [75], and a number of studies have prepared a method that can remove ROS and generate O 2 with injectable hydrogel for the treatment of MI [76]. For example, a multi-functional hydrogel under the condition of hypoxia and inflammation can not only remove ROS, but also continue to produce oxygen, with the ability to suppress apoptosis, to increase the proportion of M1/M2 macrophages, to promote angiogenesis, to reduce the effect of the infarction area, thus to regulate MI or adverse tissue microenvironment, and to improve heart function.

To Inhibit Malignant Remodeling Caused by Metalloproteinases
After MI, MMPs (matrix metalloproteinases) are activated, and the imbalance between MMPs and TIMPs (tissue inhibitor of matrix metalloproteinases) leads to left ventricular remodeling after MI [77,78]. Under normal physiological conditions, endogenous MMPs inhibition is achieved by the synthesis and release of TIMPs [79], and the relative TIMP level might not increase in the early period after MI, resulting in an imbalance between endogenous proteolytic activity and inhibition [77]. Therefore, a number of studies used hyaluronic acid hydrogel to locally deliver recombinant rTIMP-3 (recombinant tissue inhibitor of metalloproteinases-3) to promote the proliferation of myofibroblasts in the infarction area and effectively block the adverse remodeling after MI [80]. Containing MMPs shearable peptide-crosslinked HA(hyaluronic acid) based hydrogels that are responsive to MMPs [81], rTIMP-3 achieved controllable release and significantly attenuated key indicators of ventricular remodeling, such as left ventricular dilation and thinning of the infarct area at 28 d after MI.

To Increase Electrical Conductivity of Myocardium
Injectable hydrogel provides structural support for the injured hearts, while conductive injectable hydrogel enhances biological conduction, synchronizes heart contractions, and improves therapeutic effects [82,83]. Zhou et al. introduced GO (graphene oxide) nanoparticles into OPF (oligo (poly (ethylene glycol) fumarate)) hydrogels to prepare a conductive hydrogel, which not only provides mechanical support for the infarct area but also electrically connects isolated cardiomyocytes to intact tissue. In this case, the tissue with synchronize contraction could restore ventricular function and maintain cardiac function, in comparison with injections of non-conductive polymers [84]. At the same time, this study showed that OPF/GO hydrogel can upregulate Cx43 (connexin 43) and gap junctionrelated protein production, by providing mechanical support and electrical connection between healthy myocardium and cardiomyocytes in scar and by activating Wnt signaling pathways. Furthermore, homogeneous double network hydrogels [85], composed of a rigid/hydrophobic/conductive network of chemically crosslinked PTAA (polythiophen-3acetic acid) and a flexible/hydrophilic/biocompatibility network of crosslinked MAAG (methacrylate aminated gelatin), could be prepared to tunable properties. In detail, this double network hydrogel can adjust the swelling and mechanical and electrical properties by tuning the ratio of the PTAA network and MAAG network. More importantly, this double network hydrogel can promote the survival and proliferation of BADSCs (brown adipose-derived stem cells) and improve the efficiency of cardiac differentiation of BADSCs. The expression of Cx43 was up-regulated and electrical stimulation could further ameliorate the effect. This hydrogel can be used as an ideal scaffold for cardiac tissue engineering with good mechanical properties. More importantly, hydrogels with excellent electrical conductivity and anti-fatigue properties have strong benefits for cardiac repair [86,87]. The hydrogel obtained by combining the above different functions could also greatly increase the therapeutic efficiency of myocardial infarction [51,88]. The oxidant TEMPOL was integrated into the peptide, and an injectable ROS scavenging/conducting composite hydrogel was constructed through the specific binding between the conducting PPy and the multicomponent co-assembled peptide. As shown in Figure 3d-f, this therapeutic strategy, which combines increasing myocardial conductance and scavenging ROS, can significantly promote cardiac repair [59].

Hydrogel as a Carrier for Transplanted Cells
At present, a number of studies have shown that stem cell transplantation technology can be used to repair myocardial injury and improve cardiac function, including ECSs (endogenous cardiac stem cells) [89], HSCs (hematopoietic stem cells) [90], ESCs (embryonic stem cells) [91], MSCs (mesenchymal stem cells) [92], and IPSCs(induced pluripotent stem cells) [93,94], etc. However, the treatment effect is poor due to the lack of adhesion of the transplanted cells. The mechanical loss caused by the beating of the heart, and the poor microenvironment in the infarct area, lead to the low retention rate and survival rate of the transplanted cells [89,95,96]. As an injectable polymeric formulation, hydrogel can provide a three-dimensional growth environment for cells, improve the survival rate of transplanted cells, and significantly improve cardiac function [97]. For example, Christman et al. demonstrated that fibrin hydrogel increased the survival rate of skeletal muscle myoblast transplantation [98], reduced infarct size, and increased blood flow to the ischemic myocardium. In a study of self-assembling peptide NFs (nanofibers) loaded with BMNCs (bone marrow mononuclear cells) [99], NFs injections improved cell retention and improved cardiac function at 28 d after myocardial infarction. Furthermore, these two formulations played a synergistic role in improving cardiac function. BMNCs injections significantly increased systolic function after myocardial infarction but not diastolic function, while combined injection of BMNCs and NFs improved contractility and relaxation function. It is worth mentioning that the performances of hydrogel transplanted stem cells in the treatment of myocardial infarction relied on its formulations. For example, by using hyaluronic acid hydrogel encapsulating BMNCs in the treatment of rat myocardial infarction [100], hyaluronic acid hydrogels have been experimentally proved to prevent mechanical loss and loss of nest apoptosis cells. However, cell apoptosis caused by myocardial ischemia hypoxia was not disturbed. Currently, it is still a challenge to promote the long-term survival of transplanted stem cells in hydrogel. Furthermore, it became more difficult for stem cell survival, upon the appearance of coronary myocardial infarction area of ischemia hypoxia, insufficient blood supply and subsequent inflammation, and myocardial substrate degradation, after the occurrence of myocardial infarction. In this case, it might also be valuable to explore the optimal design of cell encapsulated hydrogel designs, by considering the strategy to improve the transplanted cells survival environment, to increase the protective effect of stem cells, and to achieve the optimization of stem cell function.

To Scavenge Reactive Oxygen Species
The pathological process of myocardial infarction, especially the initial inflammation of myocardial infarction, is closely related to ROS [101]. In detail, cardiomyocytes and fibroblasts could be induced to produce a series of ROS, in case of local myocardial ischemia and hypoxia after coronary artery infarction, reperfusion injury caused by vascular recalculation, or intra-muscular injection of hydrogels. These ROS are mainly O 2 − , H 2 O 2 , HO 2 ·, ·OH, which could cause serious damage to myocardial cells and exogenous transplanted stem cells [102,103]. To prevent this undesired effect, a thermosensitive CSCl-GSH (chitosan chloride glutathione) hydrogel was designed to effectively scavenge superoxide anion [104], hydroxyl free radical, or DPPH free radical, and to inhibit oxidative stress injury or apoptosis of cardiomyocytes. In 2017, Wang et al. prepared a series of fullerenol/alginate saline gels with antioxidant activity [105], which can prevent the adverse signal transduction in cells after myocardial infarction and reduce the oxidative stress damage caused by ROS after myocardial infarction. Furthermore, the experimental results showed that fullerenol/sodium alginate hydrogel can inhibit JNK (c-Jun N-terminal kinase) signaling pathway, activate ERK (extracellular regulated protein kinases) and p38 (P38 MAPK) signaling pathways, reduce cell apoptosis, and improve the survival or proliferation of BADSCs. As shown in Figure 4a, MSCs were encapsulated in a hydrogel consisting of ROS-scavenging HBPAK (hyperbranched polymers), O 2 -generating CAT (catalase), and HA-MA (methacrylate hyaluronic acid). MSCs can maintain good viability, inhibit the activation of ROS-induced JNK/p38 apoptosis signaling pathway, and greatly improve the survival rate under oxidative stress conditions [106]. More importantly, the apoptosis level of mesenchymal stem cells also decreased in a hypoxia environment. As a result, mesenchymal stem cells were able to survive in this anti-ROS and anti-hypoxia composite, thereby enhancing the viability of the infarcted heart and promoting angiogenesis (as shown in Figure 4b-h). At present, some hydrogel materials with good electrical conductivity have a good effect in combination therapy with loaded stem cells [107]. While promoting myocardial conductance, they can also play a role in scavenging ROS to promote stem cell survival. A modified graphene oxide nanomaterial, which is both an electroactive component and can greatly scavenge ROS [108]. When this material was incorporated into silk protein hydrogels, binding to stem cells improved cardiac repair efficiency.  Injection of a bionic matrix into the myocardial infarction area not only significantly improves cardiac function, but also has certain safety and can regulate the physiological activity of stem cells [51,109]. For example, the synthesis of IGF-1C (insulin-like growth

To Simulate Extracellular Matrix
Injection of a bionic matrix into the myocardial infarction area not only significantly improves cardiac function, but also has certain safety and can regulate the physiological activity of stem cells [51,109]. For example, the synthesis of IGF-1C (insulin-like growth factor C) structure domain polypeptide chitosan hydrogel was conducted and was proved to simulate the microenvironment for stem cell survival [110]. This hydrogel provided good cell adhesion, migration, and proliferation of the microenvironment, thereby enhancing the activity of stem cells. Yao et al. demonstrated that such hydrogel could promote the proliferation, anti-apoptosis, and angiogenesis of human PMSCs (placenta-derived mesenchymal stem cells) in vitro [111]. Combined intramyocardial injection with PMSCs could improve the survival of PMSCs in the myocardial microenvironment. Meanwhile, a combined injection could promote angiogenesis in the infarcted area and reduce inflammation, prevent undesired infarct tissue fibrosis, and improve cardiac function. In addition, Yoon et al. used self-assembled biodegradable PA (peptide amphiphilic) nanomaterials to improve the therapeutic effect of PSCs (Pluripotent Stem Cells) derived CMs (cardiomyocytes) [112] and introduced a fibrin-derived cell adhesion ligand (RGDS) into PA to promote cell adhesion and survival. The incorporation of NMP-2 (nuclear matrix protein 2) biodegradable sequences enables the scaffold to be gradually degraded and replaced by the natural ECM produced by surrounding cells. This design mimicked the physical and biochemical complexity of natural ECM. In another case, folic acid peptide hydrogels were proved to mimic the natural ECM, with no immunogenicity and cell toxicity [113]. Compared with the traditional peptide hydrogel, this composite hydrogel had better gelling properties and biocompatibility and could provide a comfortable environment in vivo for IPSCs. It significantly improved the retention and survival of IPSCs cells in vivo, increased the number of IPSCs cells with the ability to differentiate into cardiomyocytes, and amplified the effect of IPSCs on angiogenesis through differentiation and paracrine.

To Improve Retention of Hydrogel-Loaded Cells in the Infarcted Area
It has been studied that when loaded with cells, hydrogels are less satisfactory in modulating the therapeutic effect of delivered stem cells and have a lower ability to support the adhesion and proliferation of encapsulated cells [114]. To solve this issue, Wang et al. modified pNIPAAM (Poly (N-isopropylacrylamide)) hydrogels with SWCNTs (singlewalled carbon nanotubes) [115]. PNIPAAm/SWCNTs hydrogels showed high bioactivity on BASCs (bone marrow mesenchymal stem cells), by promoting BASCs cell adhesion and proliferation, in comparison with PNIPAAm hydrogels alone. An injectable natural polymer hydrogel with electrical conductivity as a cell delivery vehicle not only has excellent biocompatibility and cell delivery ability for UCMSCs (umbilical cord mesenchymal stem cells), but more importantly, can promote the growth of UCMSCs, proliferation, and differentiation, thereby improving damaged myocardial tissue and rebuilding myocardial function [116]. The addition of chemokines to the hydrogel, such as SDF-1α (Stromal cell-derived factor-1 alpha), which promotes homing of stem cells in the infarcted area, also could increase their retention [117]. In addition, injection of ECM hydrogels encapsulating MSCs (mesenchymal stem cells) by means of IPC (intrapericardial cavity) injection can significantly solve the problem of low cell retention. The results showed that MSCs had a higher cardiac retention rate than IM injection when delivered by the IPC route, and the heart repair capacity of the infarcted rats was enhanced [118].

To Alleviate Ischemia and Hypoxia for Nutrient Provision
After MI, the infarct area is severely ischemic and hypoxic, and the cell survival rate is poor under hypoxic conditions. In this case, establishing a continuous oxygen delivery system in the infarct area might greatly improve the survival rate of transplanted stem cells and cardiomyocytes [75,119]. An oxygen release system has been prepared for in situ oxygen release in myocardial infarction [120], which consisted of hydrogen peroxide (H 2 O 2 ) released microspheres, catalase, and an injectable thermosensitive hydrogel. The design was utilized to improve the retention of microspheres and stem cells in cardiac tissue during the myocardial injection. Microspheres are based on polylactic PLGA (poly (lactideco-glycolide)), PVP (poly (2-vinlypyrridione)) and H 2 O 2 . The breakage of microspheres by catalase might produce oxygen, which lasted for at least two weeks, in a similar manner to myocardial infarction (1% O 2 ). In this case, the introduction of oxygen release in hydrogel significantly enhanced cell survival, with no cell death observation after 7 d culture. Cells even grew after 7 d and realized cardiac differentiation. In addition to the release of oxygen by hydrogen peroxide, in situ oxygen production was also studied by using an oxygen-producing hydrogel composed of CaO 2 (peroxide calcium) loaded with GelMA (gelatin methacryloyl), which could produce oxygen under hypoxic conditions (1% O 2 ) over 5 d and was sufficient to relieve the metabolic stress of the cardiac side population cells coated in this composite hydrogel [121]. More importantly, this composite hydrogel design significantly improved cell survival, compared to GelMA-only formulation.
Improving oxygen partial pressure is another way to increase oxygen content in the infarct area. Guan et al. developed a family of PFC (perfluorocarbon)-coupled hydrogels that could increase oxygen partial pressure to improve cell survival under hypoxic conditions, and these hydrogels have high oxygen preservation and fast gelation properties [122]. Fast gelation enables rapid solidification of the hydrogel, which could effectively fix cells in tissues and increase cell retention rate. The experimental results showed that under hypoxic conditions (1% O 2) , the partial pressure of oxygen of the hydrogel without PFC (pO 2 = 28.1 ± 2.9 mm Hg) was significantly lower than that of DPBS (Dulbecco's Phosphate-Buffered Saline) (pO 2 = 35.0 ± 3.1 mm Hg), while the partial pressure of oxygen was significantly greater than that of DPBS after adding 10% PFC to the hydrogel (pO 2 = 75.8 ± 8.2). MSCs were encapsulated in PFC hydrogels and cultured under 1% O 2 for 14 days. The cells survived and proliferated, while the cells in the control group hydrogels died massively. Hypoxic myocardial infarction areas can also be widely used.

To Enhance the Biological Activity of Stem Cells
The addition of active factors into the cell growth environment could enhance the biological function of the original stem cells. For example, by using biotin-streptomycin affinity, IGF-1 (insulin-like growth factor 1) could be linked to the self-assembly peptide. CPCs (cardiac progenitor cells) have an IGF-1-IGF-1 receptor system, which can promote their survival and growth [123]. Combined intracardial injection of CPCs and this self-organized polypeptide nanofiber hydrogel showed that the volume of regenerated cardiomyocytes increased and the infarct size decreased [124,125]. HE-cad-Fc (Human E-cadherin fusion protein) can effectively promote the paracrine activity of MSCs (esenchymal stem cells). PLGA (poly (lactic-coglycolic acid)) microparticles modified with hE-cad-Fc were combined with hMSCs (human mesenchymal stem cells) to construct functionalized MSC aggregates. Encapsulating them into injectable hydrogels could effectively improve the MI microenvironment compared to the direct encapsulation of MSCs by hydrogels [126]. On the other hand, adenovirus could be utilized to transfect MSCs by genetic modification. HGF (hepatocyte growth factor) could also be beneficial to MSCs [127], with significant apoptosis reduction and MSCs growth promotion. Furthermore, cytokines could also promote the differentiation of stem cells. Chan et al. transplanted FGF-10 (fibroblast growth factor-10) with self-assembled polypeptide nanofibers in combination with ESCs or IPSCs into the infarct area of mice [128]. The experimental results showed that ESCs and IPSCs cells could differentiate into cardiac cells. For the other case, a nitric oxide release controlled hydrogel, which is catalyzed by β galactosidase, was found to promote the secretion of angiogenic cytokines by AD-MSCS (adipose-derived β-mesenchymal stem cells) [129]. Thus, by promoting angiogenesis and AD-MSCS survival, this hydrogel improves cardiac function after MI by enhancing AD-MSCS implantation and paracrine effect. However, the field is still in its infancy and needs to study the role of more cytokines in stem cell differentiation.

Application of Exosomes
The paracrine effect of stem cells plays an important role in promoting the survival and angiogenesis of cardiomyocytes in the infarcted area, while the paracrine effect is mainly mediated by EV (extracellular vesicles) [130], which could be exosomes as the most abundant sub-vesicles. Compared with stem cells, stem cell-derived exosomes not only have the same beneficial functions but also have the ability to prevent the trigger of immune responses and to reduce the heterologous risk of implantation. In this case, exosomes might serve as another effective strategy for the treatment of ischemic cardiovascular disease [131,132]. For example, exosomes with high miR-675 (micro ribonucleic acid-675) content were obtained by transfecting exosome-derived mother cells with miR-675 molecules, in which miR-675 inhibited senescence and damage-induced phenotypes by targeting the TGF-β1-Smad2/3 (transforming growth factor β1-small mothers against decapentaplegic 2/3) signaling pathway [133]. The exosomes were encapsulated with a functional polypeptide hydrogel, while its myocardial injection inhibited inflammatory response and fibrosis levels, significantly improving cardiac function [134]. Since the immune system plays a central role in both the inflammatory and repair phases after MI [135], it has been found that DCs (dendritic cells) derived DEXs (exosomes) are involved in antigen presentation, immune activation, and inhibition. It is worth mentioning that DEXs can improve cardiac function through CD4 + T cells [136]. DEXs were combined with alginate hydrogel [137]. After myocardial injection, the hydrogel had better effects on immune regulation, anti-apoptosis, promotion of angiogenesis, and increase in infarct wall thickness [138]. This report also explored the mechanism of DEXs in improving cardiac function. DEXs have been found to activate regulatory Tregs (Regulatory T cells), which further regulates macrophage polarization and enables macrophages to metastasize to M2-type cells. This immunotherapy will be a novel strategy for treating myocardial infarction [138]. In addition, the exosomes were combined with a conductive hydrogel that matches the conductivity of the native myocardium to obtain the conductive Gel@Exo system. It can significantly improve the interaction between cells, promote cell proliferation and angiogenesis, and have a significant therapeutic effect on MI [139].

Hydrogels as Factor/Protein, Drug, Gene Release Carriers
Upon the combination of hydrogel with functional factor/protein, drug, and gene for myocardial infarction treatment, it mainly plays a role in local delivery and controllable release. Injectable hydrogel forms a stable solid scaffold in the infarct area after coagulation, and the wrapped factor/protein, drug, and gene are continuously released to avoid their rapid metabolism [140][141][142]. Injectable hydrogel systems can out-performance traditional drug delivery methods, due to their advantages such as low invasiveness, high efficiency, and sustained local delivery [41,50].

Loading with Single Active Factor/Protein
The treatment of myocardial infarction by stem cells is mainly due to the paracrine role of stem cells secreting various bioactive factors in the myocardium [143], such as VEGF, PDGF (platelet-derived growth factor), SDF-1 (stromal cell-derived factor-1) [144,145], etc. The introduction of bioactive factors is one of the approaches to treat myocardial infarction. The continuous release of hydrogel had a slow-release effect on factors and proteins in myocardial infarction. For example, FGFs (fibroblast growth factors), a family of growth factors initially identified to promote fibroblast growth, promote cardiac repair through pro-angiogenesis, anti-apoptotic, and survival mechanisms [146]. As a bioactive factor deposit, ROS-sensitive cross-linked polyvinyl alcohol hydrogels were prepared, with the ability to respond to the microenvironment where excessive ROS were generated after myocardial ischemia-reperfusion injury or myocardial infarction [147]. This hydrogel was experimentally proved to show bFGF (basic fibroblast growth factor) delivery ability and experienced fast degradation in a ROS-rich environment for myocardial repair. This design achieved enhanced retention of BFGF in the pericardium and controllable bFGF release into the heart muscle in an "on-demand" manner.
A number of proteins and peptides can also play a certain role in promoting cardiac function recovery [148,149]. For example, angiopoietin-1 (Ang1) binds to integrin and activates the presurvival pathway to promote cardiomyocyte survival, while short sequence QHREDGS peptide is the binding site of Ang1 and integrin to support cardiomyocyte adhesion and survival [150]. Therefore, researchers tried to fix the pro-survival peptide QHREDGS on the chitosan collagen hydrogel as an integrin-binding ligand, and interacted with the integrin of cardiomyocytes [151]. Experimental results showed that this design significantly improved the shape and function of the heart, including a 53% improvement in scar thickness, a 34% reduction in the scar area, a 35% improvement in shortened fraction, and a 62% improvement in ejection fraction.

Multiple Active Factor/Protein Combination Therapy
Microangiogenesis is one of the effective methods to inhibit ventricular remodeling and improve cardiac function after myocardial infarction. However, vascular regeneration is a complex process requiring the participation of many factors, and a single factor might lead to vascular leakage and immaturity [152]. VEGF can promote the proliferation and migration of endothelial cells, so as to promote the generation of new blood vessels in the infarction area, and to inhibit myocardial fibrosis in late myocardial infarction [153]. In the late stage of angiogenesis, PDGF-BB (platelet-derived growth factor-BB) could stimulate the smooth muscle cells to the newly formed blood vessels, and promote the perfection of the maturity, and function of blood vessels [154]. It has been proved that alginate hydrogel combined with VEGF and PDGF-BB could induce angiogenesis and function improvement, in comparison with single factor treatment only after myocardial infarction [155]. By designing VEGF embedding fibrin gel and PDGF embedding heparin-based coagulant, the system provides a rapid release of VEGF, while the slow and sustained release of PDGF upon single injection [156]. In comparison with the single factor approach, this composite drug delivery system is effective in improving cardiac function, ventricular wall thickness, angiogenesis, myocardial survival, and reducing fibrosis or inflammation in the infarct area. Therefore, the matching of active factors with different properties and the controlled release of different components in different stages will make them play a synergistic role in myocardial infarction and greatly increase the therapeutic efficiency [157].
In addition to combination therapy between cytokines, they can also work together with bioactive proteins. For example, BMP9 (bone morphogenetic protein 9) can effectively slow down the process of myocardial fibrosis by inhibiting the action of TGF-β1 (transforming growth factor β1) [158]. By taking these advantages, SF (silk fibroin) protein microspheres were used as the carrier of BMP9. These microspheres were added into the alginate containing VEGF and crosslinked with calcium gluconate to form a composite hydrogel [159]. This composite hydrogel can rapidly release VEGF to promote angiogenesis in the early MI and continuously release BMP9 to inhibit fibrosis formation in the long MI. In this case, the biological activity of proteins could be maintained to fit the timings of each stage of myocardial infarction, which effectively accelerated angiogenesis in MI mouse models, inhibited fibrosis formation, and enhanced cardiac function (Figure 5a-c).  [160]. 2018, American Chemical Society). Level of significance (***-p < 0.001, NS-not significant).

Material Wrapping Factor/Protein with Improved Release Performance
Nanomaterials and microspheres with bioactive factors wrapping or adsorption could be mixed into hydrogel to prolong the diffusion time and treatment effectiveness, to improve the myocardial repair rate, and to produce more favorable effects on tissue repair. For example, SF protein microspheres were prepared by a microfluidic device. IGF-1 was physically adsorbed onto SF microspheres. These microspheres were loaded into alginate brine gel to enhance the sustained release of IGF-1 [161]. Compared with the SFfree microsphere system, the composite hydrogel can release IGF-1 relatively slowly and continuously. Furthermore, intramuscular injection of complex hydrogels reduced infarct size and improved cardiac function after 28 d. To address the release limitation problem, a dual-function MI-responsive on-demand growth factor release system was developed to promote angiogenesis and inhibit cardiac remodeling by targeting MMP (matrix metalloproteinase)-2/9 upregulated after MI. By combining collagen amine with GSH (glutathione) sulfhydryl group, GSH-modified collagen hydrogels were prepared [162]. The recombinant protein GST-TIMP-bFGF was synthesized by the fusion of bFGF with GST (glutathione s-transferase) and MMP-2/9 shearable peptide PLGLAG (TIMP). The specific binding of GST and GSH significantly increased the loading capacity of GST-TIMP-bFGF in collagen-GSH hydrogel. During myocardial infarction, the TIMP peptide wrapped between GST and bFGF responds to MMP and was released on demand, which promoted angiogenesis and reduced cardiac remodeling through synchronous control of binding  [160]. 2018, American Chemical Society). Level of significance (***-p < 0.001, NS-not significant).

Material Wrapping Factor/Protein with Improved Release Performance
Nanomaterials and microspheres with bioactive factors wrapping or adsorption could be mixed into hydrogel to prolong the diffusion time and treatment effectiveness, to improve the myocardial repair rate, and to produce more favorable effects on tissue repair. For example, SF protein microspheres were prepared by a microfluidic device. IGF-1 was physically adsorbed onto SF microspheres. These microspheres were loaded into alginate brine gel to enhance the sustained release of IGF-1 [161]. Compared with the SF-free microsphere system, the composite hydrogel can release IGF-1 relatively slowly and continuously. Furthermore, intramuscular injection of complex hydrogels reduced infarct size and improved cardiac function after 28 d. To address the release limitation problem, a dual-function MI-responsive on-demand growth factor release system was developed to promote angiogenesis and inhibit cardiac remodeling by targeting MMP (matrix metalloproteinase)-2/9 upregulated after MI. By combining collagen amine with GSH (glutathione) sulfhydryl group, GSH-modified collagen hydrogels were prepared [162]. The recombinant protein GST-TIMP-bFGF was synthesized by the fusion of bFGF with GST (glutathione s-transferase) and MMP-2/9 shearable peptide PLGLAG (TIMP). The specific binding of GST and GSH significantly increased the loading capacity of GST-TIMP-bFGF in collagen-GSH hydrogel. During myocardial infarction, the TIMP peptide wrapped between GST and bFGF responds to MMP and was released on demand, which promoted angiogenesis and reduced cardiac remodeling through synchronous control of binding and responsive release, indicating a promising treatment strategy for ischemic heart disease.

Hydrogels for Direct Loading of Drugs
Curcumin, as an effective component of Chinese traditional medicine curcumin, has a significant cardiac protective effect on reperfusion injury, inhibiting ventricular remodeling caused by pressure load or myocardial infarction and improving cardiac function. However, curcumin has poor water solubility and low bioavailability [163]. Hydrogel-loaded curcumin will significantly improve its targeting activity and promote the sustained release. For example, a curcumin carrier hydrogel reduced cardiomyocytes apoptosis and hypoxiareoxygenation-injury-induced ROS formation by maintaining Cx43 phosphorylation [164]. This design also promoted autophagy, reduced mitochondrial damage, and activated the JAK2/STAT3 (Janus kinase 2/signal transducer and activator of transcription) pathway. Furthermore, in situ injection of curcumin and NO into hydrogel will exert a synergistic effect to promote angiogenesis [165]. As another example, natural ingredient colchicine has an anti-inflammatory effect, while its systemic toxicity and narrow therapeutic window restricted its biomedical application [166,167]. In order to solve this issue, a polymeric hydrogel loaded with colchicine was designed and showed its ability to effectively relieve heart inflammation [168], to inhibit myocardial apoptosis and fibrosis, to improve cardiac function and structure, and to increase the survival rate of mice, without severe systemic toxicity.

Nanomaterials Packaging for Drug Loading
TIIA (Tashinone IIA), one of the main active components of Salvia miltiorrhiza Bge, has been widely used in the treatment of cardiovascular diseases in China. It could significantly improve the cardiac function of infarcted myocardium [169], while its poor solubility, short half-life, and low drug loading of loading materials limit its application [170,171]. However, TIIA can form TIIA nanoparticles through the hydrophobic self-assembly method. In this case, the researcher deposited PDA (pyridoxylamine) layer in situ on TIIA nanoparticles to prepare core-shell TIIA@PDA nanoparticles, which had a high drug loading capacity. On the other hand, in order to restore cardiac function after myocardial infarction [172], hyperbranched ROS-sensitive macromer HB-PBAE (hyperbranched-Poly(β-amino esters)) with polyacrylate terminal group was synthesized by dynamic controlled Michael addition method. HB-PBAE was crosslinked with HA-SH (thio-hyaluronic acid) to form in situ hydrogel. Chemical crosslinking of thiocyanate and quinone groups on PDA was performed. In this case, TIIA@PDA NPs (nanoparticles) could be captured. Injection of ROS-sensitive hydrogels enhanced by TIIA@PDA NPs into infarcted hearts reversed the harmful microenvironment and inhibited the expression of inflammatory factors, such as IL-1β (interleukin 1β), IL-6 (interleukin 6), and TNF-α (tumor necrosis factor-α), thereby restoring myocardial function.
In addition to realizing high drug load and local sustained release, nanomaterials also have application in dual drug delivery systems [173], which can realize independent release of different drugs [174]. For example, tPA (tissue plasminogen activator) is commonly used for the treatment of large and small vessel myocardial infarction. The small molecule ROCK (Rho-associated kinase) inhibitor Y-27632 could eliminate the pathological process of fibrosis [175]. A dual drug delivery system was designed using a fibrin-specific poly (N-isopropylacrylamide) nanohydrogel, composed of a core-shell colloidal hydrogel structure. The architecture consists of a highly crosslinked nanogel core and loosely crosslinked shell to control the load and release of two different MI therapies. tPA would be mainly divided into relatively loose crosslinking shells, and diffusion of Y-27632 took place through crosslinked gel core layer. This design is the pioneer report of tPA release to solve primary myocardial infarction, occlusion, and fibrin deposition issues. Furthermore, the persistently released Y-27632 could block fibrosis pathogenesis and progress of key cellular responses to prevent heart scarring [160], It also addressed the need to rebuild blood flow and inhibit myocardial fibrosis after I/R injury (Figure 5d). Inspired by Ferrero's core-shell structure, another hydrophobic DMOG (dimethyloxy glycine) NPs as core and a water-soluble drug EGCG (epigallocatechin-3-gallate) encapsulated by dopamine were synthesized. EGCG was within a simulated nano-carrier composed of thick PDA shells formed by self-polymerization through π-π interaction, which had the triadic ability to load two different drugs [176]. In this case, an injectable hydrogel was obtained with the ability to eliminate ROS and acted as a cross-linking agent to HA-SH. Ferrero-like NPs and HA-SH could rapidly form hydrogels with stable mechanical properties, strong ROS capture ability, and programmed release of EGCG and DMOG. After four weeks of myocardial infraction in rats, ejection fraction (EF) increased by 23.7%, infarct area decreased by 21.1%, and fibrosis area decreased by 24.4%.

Multifactorial Combination Therapy
Hydrogel-loaded multi-drug cocktail therapy for myocardial infarction is one of the ways of multi-factor combined therapy in the treatment of myocardial infarction. For example, hyaluronic acid hydrogel was designed for myocardial joint injection, with the loading of platelet-rich plasma, allopurinol, ascorbic acid, and ibuprofen. This multifactor combination strategy could effectively thicken the infarcted myocardium, improve the host cell vitality, and realize attenuation of myocardial remodeling or dysfunction after myocardial infarction in pig animal models [177]. Combined infusion control might also have synergistic effects. For example, it might potentially improve the function of damaged myocardium while alleviating adverse cardiac remodeling after acute myocardial infarction. As a typical case, AST NPs (astragaloside IV nanoparticles) were prepared by a hydrophobic self-assembly method, and the hyperbranched polymers PEGDA-PBA (polyethylene glycol diacrylate 4-vinylbenzene boronic acid) and HA-SH formed in situ hydrogel to adsorb AST NPs for drug targeting and sustained release [178]. In the meanwhile, the hydrogel was doped with GNRs (gold nanorods) to provide electrical stimulation, which significantly improved myocardial infarction-induced cardiac dysfunction and cardiac remodeling by stimulating angiogenesis, promoting cell-cell signal transduction, and inhibiting apoptosis.

Loading of Genes and Plasmids
With the development of gene transfer technology, introducing gene therapy for cardiovascular diseases such as heart failure has been gradually arriving at the stage of clinical trials [179,180]. It is worth mentioning that non-viral transfection vectors were of satisfying stability and biological safety [181]. In the case of using hydrogels as non-viral gene delivery vectors, it might also show good biocompatibility as a gene delivery carrier and prolonged gene expression [182,183]. As a typical case, the luciferase plasmid loaded within temperature-responsive hydrogels showed a four times increase in gene expression in mice, in comparison with naked plasmids only [184]. While upon HVEGF (human vascular endothelial growth factor) plasmid could be loaded in gel formulation within the infarction area, continuous HVEGF expression might increase capillary density and vascular formation in an effective manner.
In another case, RNAi (ribonucleic acid interference) is an effective gene silencing technology, and the local targeting RNAi therapy strategy has strong specificity and high efficiency in the treatment of heart disease [185][186][187]. Previous reports have synthesized the plasmid of ACE-shRNA (angiotensin-converting enzyme-short hairpin RNA) [188]. The DNA (deoxyribonucleic acid) sequence of rat ACE-shRNA was cloned into pgenesil-1 plasmid and mixed with Dex-PCL-HEMA/PNIPAAm (dextran-poly(e-caprolactone)-2hydroxylethylmethacrylate-poly(N-isopropylacrylamide) hydrogel [189]. The hydrogel loaded with ACE-shRNA plasmid prolongates the expression time of the gene in vivo, thus prolonging the silence time of the ACE gene and achieving greater myocardial protection. This method of silencing the ACE gene could completely block the effect of ACE after myocardial infarction compared with lowering Ang II (Human angiotension II) level with ACE inhibitor [190]. In addition, the induction of MSCs by microRNA and cardiomyocytes combined with hydrogel can improve the differentiation efficiency of MSCs into iCMs (induced cardiomyocyte-like cells) to improve the cardiac function of the MI model after transplantation [191]. In particular, miR-21-5p (microRNA-21-5p), which is highly expressed in endothelial cells, can stimulate angiogenesis by targeting anti-angiogenic genes [192]. miR-21-5p was loaded with MSN (mesoporous silica nanoparticles) with anti-inflammatory potential, and the complex was loaded with pH-responsive hydrogel to obtain Gel@MSN/miR-21-5p. In this report, on-demand delivery was achieved using pH triggering in the acidic infarct zone, and miR-21-5p was delivered by the MSN/miR-21-5p complex in the second stage. The synergistic effect of anti-inflammatory and pro-vascular effects can effectively reduce myocardial infarct size in pigs [193], restore contractility of damaged myocardium, and significantly increase capillary density in the border zone, thereby significantly improving myocardial function [194]. In short, the gene therapy with the assistance of injectable hydrogels could also be favored.

Combination Therapy
MI, resulting in massive myocardial cell death, adverse microenvironmental changes, loss of electrical communication in fibrotic scars, and inadequate blood supply to the infarcted myocardium, are three key aspects of MI treatment [195]. Lipid/plasmid DNA-eNOs (endothelial nitric oxide synthase) nanocomplexes were loaded into soft conductive hydrogels with equivalent myocardial conductivity and anti-fatigue properties. By coencapsulation with ADSCs (adipose-derived stem cells) in vitro, the expression of NO was also significantly up-regulated by examining the enhanced NOx concentration in the medium after seven days, indicating gene transfection of ADSCs [196]. After the hydrogel was injected into the infarcted heart, QRT-PCR (quantitative real time polymerse chain reaction) showed that eNOs, VEGFA, Ang-1, Cx43, and Cdh-2 mRNA gene expressions were all up-regulated. The cardiac ejection fraction (EF) was significantly increased, the QRS interval was shortened, the infarct size was reduced, the fibrosis area was reduced, and the blood vessel density was increased, indicating a significant improvement in cardiac function (as shown in Figure 6).
This conceptual study of combination therapy demonstrated a holistic approach to MI that enhances electrical communication to restore cardiac function, while ADSCs injected into hydrogels of infarcted myocardium alleviate the inflammatory environment, compensate for cell loss, and upregulate NO expression in myocardial tissue, with the ability to promote neovascularization and myocardial maturation.  [196]. 2022, Elsevier).

Conclusions and Perspective
At present, hydrogel has made breakthrough progress in the treatment of myocardial infarction. As a substitute for myocardial infarction, the material itself not only has the potential of treating myocardial infarction, but also has great development potential in effective cell encapsulation, as well as bioactive factors, proteins, drugs, or genes delivery. However, the injection volume and time of injectable hydrogel, the concentration of loading factors and drugs, the matching of degradation rate and myocardial remodeling, and the physical and biological mechanisms of hydrogel for myocardial repair need to be further clarified.
First of all, regarding the existing hydrogel treatment strategies, the injection amount of hydrogel in different treatment approaches and the injection time after MI need to be further studied, due to infarction area size differences in the clinical application. It is  [196]. 2022, Elsevier).

Conclusions and Perspective
At present, hydrogel has made breakthrough progress in the treatment of myocardial infarction. As a substitute for myocardial infarction, the material itself not only has the potential of treating myocardial infarction, but also has great development potential in effective cell encapsulation, as well as bioactive factors, proteins, drugs, or genes delivery. However, the injection volume and time of injectable hydrogel, the concentration of loading factors and drugs, the matching of degradation rate and myocardial remodeling, and the physical and biological mechanisms of hydrogel for myocardial repair need to be further clarified.
First of all, regarding the existing hydrogel treatment strategies, the injection amount of hydrogel in different treatment approaches and the injection time after MI need to be further studied, due to infarction area size differences in the clinical application. It is necessary to individualize infarction and injection amounts in order to achieve the optimal treatment effect. Furthermore, for the case of bioactive factor, drug, and gene loading, hydrogel injection time and the release of the load composition concentration should also be adapted to the corresponding pathologic stage. At present, most studies are limited to the effect of hydrogel injection at a single time on cardiac function, and there are few studies on the effect of injection time after MI. However, it has been proved that the time of hydrogel injection after myocardial infarction has a certain correlation with the efficacy [197,198], which is worth further study on this issue.
Secondly, the pathological change of myocardial infarction is a progressive process, which gradually leads to ventricular remodeling. The degradation rate of hydrogel should match that of myocardial remodeling, and the degradation rate of hydrogel should be automatically degraded with myocardial remodeling. In this case, the degraded hydrogel should have sufficient mechanical force to continue to support the myocardial tissue. Recently, the EMH (elastin simulated hydrogel) loaded with SAB-PDA (salvianolic acid and polydopamine nanoparticles) solved the problem that the mechanical strength of hydrogel structure was reduced by the continuous exercise of myocardial tissue [199]. Under the up-regulation of TGase (glutamine transaminase) in myocardial tissue after myocardial infarction, SAB-PDA/EMH with higher mechanical strength can be formed, which makes the hydrogel material have better self-healing ability. More studies are also needed on how different hydrogel materials can be degraded while maintaining their mechanical properties. In addition, it is necessary to consider that the degraded fragments of hydrogel should not cause other adverse reactions in vivo, and the massive release of cells, drugs, and factors caused by degradation may also affect the treatment efficiency or produce side effects. Therefore, we also need to establish a more complete evaluation method for the efficacy and side effects of hydrogels in the treatment of myocardial infarction [200].
Finally, MI is the physiological and pathological mechanism study upon hydrogel treatments after MI had revealed that hydrogel might reduce stress, influence the geometrical shape and thickness of the ventricular wall, and promote angiogenesis [201]. However, it is also worth mentioning that the interaction between this hydrogel formulation and the microenvironment of MI still lacks exploration [202,203].
In short, the application of hydrogel in the treatment of MI has made significant progress in clinical research [204,205]. Based on this, different treatment strategies related to hydrogel will provide a variety of treatment means and approaches to serve patients. This review summarized a number of functional hydrogels in the treatment of MI, as well as various biological therapies combining cell, factor, protein, and drug-gene therapy for MI. Injectable hydrogel as a new biological material, although there is no precise and systematic study on the efficacy of various aspects, the satisfactory progress of hydrogel formulation in the field of myocardial tissue engineering will definitely bring better therapeutic effects for patients with MI. To achieve an optimal therapeutic effect, it might need to find the optimal injection time and load component concentration in the next few decades to explore the important mechanism of hydrogel in the direction of MI. Furthermore, the intelligent release of load components based on the myocardial microenvironment and better self-healing ability of hydrogel based on the dynamic process of myocardial continuous movement and ventricular remodeling will also be the future research direction. It is one of the key points of materials science and myocardial tissue engineering research to create a "cocktail therapy" with the optimal curative effect by rationally matching the modification of hydrogel, the combination of cells, growth factors, proteins, and drugs, in order to achieve a long-term curative effect.

Conflicts of Interest:
The authors declare no conflict of interest.