**1. Introduction**

Cardiovascular diseases (CVD) are the leading cause of mortality worldwide [1,2]. In 2017, about 17.8 million deaths globally were attributed to CVD and in the U.S. alone, CVD, which include heart disease and stroke, were among the top ten causes of death, accounting for 74% of total deaths [3]. Coronary heart disease causes the majority of deaths in CVD, with myocardial infarction (MI) often leading to heart failure. Tissue damage at the site of infarction triggers local inflammation that attracts neutrophils and monocytes to clear the area of cell debris and produce reactive oxygen species. Migration of monocytes with reparative functions induces the formation of new vasculature and collagen production and eventually, leads to tissue repair and fibrotic tissue formation [4–6]. One biomedical approach for improving cardiac tissue regeneration is the delivery of therapeutic growth factors and cytokines [7]. Growth factors and cytokines have attracted the attention of researchers and clinicians due to their angiogenic and antiapoptotic properties, as well as their ability to increase cell proliferation and mobilize endogenous cell migration [8]. Various factors and cytokines, including but not limited to, tumor necrosis factorα (TNFα) and interleukin-8 (IL-8), are also upregulated in MI and participate in triggering inflammatory cascade. Therefore, regulation of pro- and anti-inflammatory mediator functions can be used to ameliorate inflammation and to facilitate cardiac tissue regeneration [9]. However, there are some challenges associated with growth factors/cytokines. For example, the systemic administration of growth factors/cytokines is not e fficient due to a short in vivo half-life and poor bioavailability at the target sites. This, in turn, requires repeated injections, resulting in more side e ffects and greater treatment costs [10,11]. Moreover, simultaneous and rapid di ffusion can lead to formation of immature and unstable blood vessels in the case of therapy with angiogenic growth factors [12].

Biomaterials o ffer a controlled and sustained release of bound growth factors and cytokines, which makes them a promising tool for overcoming the aforementioned challenges [13,14]. Biomaterials of natural, synthetic or hybrid origins were developed. They demonstrated therapeutic benefits when used either alone or when loaded with agents such as growth factors, cytokines or stem cells [15]. The use of biomaterials alone exerts positive e ffects on cardiac tissue regeneration, possibly via mimicking the extracellular matrix (ECM) and providing direct mechanical support. Some biomaterials also help to increase electrical conductance in a fibrotic scar region, which is important for normal functioning of the heart [16,17].

The endogenous regenerative capacity of cardiac tissue is limited: adult cardiomyocyte proliferation, cardiac stem cell activation, and bone marrow progenitor cell migration are not e fficient enough to regenerate fully functional cardiac tissue. Post-MI repair often involves tissue replacement with non-functional fibrotic scarring, which can later lead to heart failure. For these reasons, stem cell therapy is considered a promising approach in MI treatment, being particularly beneficial for reducing the infarcted area and promoting cardiac function recovery [18]. Di fferent stem cell sources such as mesenchymal stem cells (MSCs), cardiac stem cells (CSCs), induced pluripotent stem cells (iPSCs), and others are now recognized for their potential use in cardiac tissue regeneration [19]. Stem cell benefits in MI treatment include di fferentiation capacity, stimulation of resident CSCs, reduction in inflammation, and ability to provide structural support by connective tissue formation and fibroblast di fferentiation [20]. Release of cytokines and growth factors by stem cells allows for immunomodulation, angiogenesis, and stimulation of adjacent cells via paracrine mechanisms [21,22]. However, harsh conditions at the infarction site present a significant burden for stem cell survival. These conditions include, but are not limited to, hypoxia, fibrogenesis, low blood supply, and inflammation [23]. Therefore, biomaterials can serve as a stem cell delivery system that increases the living potency of the cells after transplantation and enhance the exerted e ffects. This review will focus on recent findings on the use of biomaterials as drug delivery systems for growth factors, cytokines, and stem cells for improving cardiac tissue regeneration in small animal models of MI.

#### **2. Biomaterials Loaded with Growth Factors and Cytokines for Cardiac Tissue Regeneration**

The use of biomaterials is now rapidly evolving as a new approach for MI treatment [24,25]. They are composed of a plethora of various polymers and can be used as a drug delivery system in the field of regenerative medicine [26]. The most common types are polymeric micro and nanospheres, nanoparticles (NPs), nanofibrous structures, coacervates, hydrogels, cryogels, and sca ffolds. They di ffer in their size and assembling materials, as well as in their morphology, i.e., sheet versus vesicle-like structures [27–33]. Hydrogels, in particular, are widely investigated in the area of CVD. Hydrogel is largely composed of water and a cross-linked polymer and physically resembles tissue [34]. Hydrogels made of cardiac ECM, alginate, hyaluronic acid (HA), natural biomaterials (collagen, fibrin, and heparin), synthetic polymers, and microparticles have been studied pre-clinically for cardiac repair [35]. The e ffects of hydrogel administration include direct mechanical strengthening [36], enhanced angiogenesis and regeneration of myocardial tissue, reduced apoptosis and scar size, and improved cardiac function recovery [37]. Moreover, multiple studies showed that the use of biomaterials alone favorably a ffects various cells in the post-MI environment such as macrophages, cardiomyocytes, fibroblasts, and endothelial cells [38]. Recently, hydrogels made of ECM-based biomaterials have drawn attention because of their ability to mimic native ECM and minimize immunogenicity [39]. McLaughlin and colleagues treated mice at the end of the proliferative phase of wound healing with the injectable biomaterial, which contained human recombinant collagen I and III, one of the main proteins in the ECM of heart tissue. The treatment reduced inflammation, polarized macrophages towards M2 phenotype, increased capillary density at the border zone, and improved cardiac function [40]. The application of the self-assembling peptide (SAP) cell-free hydrogel also significantly improved the functionality of the heart post-MI through increased angiogenesis and reduced scar formation [41]. The beneficial e ffects of biomaterials are shown to depend on the time of therapy administration. In the study by Blackburn and colleagues, 3h post-MI application of collagen-based hydrogel in a murine model reduced cell apoptosis as well as increased capillary density and as a result, improved left ventricular ejection fraction. The authors also reported that biomaterial therapy is ine ffective after 14 days post-MI [37]. The mechanisms of the exerted e ffects of biomaterials are possibly mediated by modifying the inflammatory immune response. It was demonstrated that hydrogel treatment also reduced the number of macrophages and TNFα production in cardiac tissue. The in vitro culture of macrophages on biomaterials demonstrated a decrease in pro-inflammatory cytokines and an increase in anti-inflammatory cytokines [37].

Fibrosis, and its consequent non-functional scar formation, is considered to be a major problem following MI, leading to left ventricle remodeling and heart failure. Several biomaterials were designed to improve conduction of electrical signals in the scar region. For example, pyrrole was grafted onto a chitosan biomaterial to produce a conductive polypyrrole (PPy)-chitosan hydrogel. In vivo experiments used a coronary artery ligation rat model of acute MI to show reduced QRS complex on an electrocardiogram and improved transverse conduction velocity in PPy-chitosan group. It was demonstrated that both chitosan alone and PPy-chitosan were e ffective in preserving heart function, but PPy-chitosan further improved the indices, suggesting better maintenance of heart function as compared to a non-conductive biomaterial [16]. Cui and colleagues tested PPy-chitosan in a cryoablation injury rat model and reported a significant improvement in longitudinal conduction velocity in comparison to the chitosan only group. Electromyography was used to assess the conductivity of scar tissue ex vivo, which showed a significant 300–350% increase in electrical signals in the myocardial scar tissue in the group treated with PPy-chitosan [42].

Extensive research has been performed to study the importance of growth factors, cytokines, and di fferent components of ECM in the treatment of MI [43,44]. It was shown that transforming growth factor-β(TGF-β) stimulates both Smad3-dependent and independent activation of macrophages, with the involvement of Smad3 in phagocytosis activation, secretion of vascular endothelial growth factor (VEGF) and TGF-β1, and protection against adverse cardiac tissue remodeling [45]. IL-10 is also important because its deficiency increases necrosis and neutrophil migration, with an enlargement in infarct size. Moreover, IL-10 deficiency impairs the ability of endothelial progenitor cells to suppress cell apoptosis, reduce scar size, increase neovascularization, and improve left ventricle remodeling, which is mediated by upregulation of integrin-linked kinase [46]. In contrast, treatment with IL-10 suppresses inflammation, polarizes macrophages towards M2 phenotype, activates fibroblasts, and improves left ventricle remodeling [47]. Another important growth factor is VEGF, which can be released from cardiac macrophages to simulate angiogenesis and heart muscle repair by regulating endothelial cell proliferation, migration, and apoptosis [43,48]. Furthermore, VEGF-A, fibroblast growth factor (FGF), and stromal cell-derived factor-1 (SDF-1) can stimulate neovascularization [49]. IL-4 is also a key cytokine because IL-4 administration di fferentiates macrophages, which are derived from Ly6Chigh monocytes, into a M2 phenotype [50]. However, application of growth factors and cytokines in clinical practice is hindered by their short half-lives, decreased stability, and deactivation by enzymes [27]. For example, the half-life of VEGF is approximately thirty-four minutes in plasma [51]. Therefore, biomaterials can serve as promising tools for the protection, delivery, and sustained release of growth factors and cytokines [52]. Table 1 summarizes the use of biomaterials loaded with growth factors and cytokines for cardiac tissue regeneration.

The incorporation of growth factors and cytokines into engineered biomaterials, such as hydrogels and NPs, offers even more opportunities for MI therapy (Figure 1). As an example, the injection of heparan sulfate proteoglycans (HSPG), which is a major component of ECM, with basic FGF (bFGF), extended the bioavailability of the growth factor by protecting it from degradation, and improved angiogenesis and cardiac function in animals with MI [53]. Another group also used bFGF that was fused with glutathione-S-transferase (GST) and matrix metalloproteinase (MMP)-2/9 cleavable peptide TIMP, and then, incorporated the complex into a glutathione-modified collagen hydrogel. This approach allowed for the controlled release of bFGF after TIMP was cleaved by the secreted MMP-2/9 at the site of tissue infarction. The use of this type of hydrogel decreased collagen deposition, increased vascularization, and improved heart function in rats with MI [54]. The mechanism of bFGF, which is a paracrine signaling protein, is mediated through binding to FGF receptor-heparan sulfate complex and further activation of tyrosine kinase. Downstream signaling proceeds via RAS-mitogen-activated protein kinase RAS-(MAPK) and phosphatidylinositide 3-kinase (PI3K) pathways [55]. In a separate study, sustained and targeted delivery of neuregulin-1β (NRG), which is a member of epidermal growth factor that regulates cardiomyocyte development and proliferation, by a hydroxyethyl methacrylate hyaluronic acid (HEMA-HA) hydrogel, demonstrated a cardioprotective effect and significantly improved ventricular function and structure [38]. The cardioprotective effect was assessed by the amount of caspase-3 in murine hearts post-MI, which was significantly reduced in the NRG-hydrogel group in comparison to the control groups treated with phosphate-buffered saline, NRG, or hydrogel alone. Caspase-3 is a key mediator of the terminal apoptotic pathway and its downregulation is associated with reduced infarct size, decreased apoptotic index of myocytes, and enhanced heart function in an experimental model of MI [56]. Awada and colleagues demonstrated that sequential delivery of VEGF followed by platelet-derived growth factor (PDGF) using a fibrin gel/heparin coacervate delivery system improves angiogenesis and cardiac function and reduces scar formation and inflammation at two and four weeks after MI in a rat model [57]. Mechanistically, VEGF promotes angiogenesis by activating or affecting different pathways and proteins, including PI3K, VRAP, Src tyrosine kinase, MAPK, and phospholipase C [58]. Recent reports show the critical role of multiple types of tyrosine and serine/threonine phosphatases, such as Shp2 and low molecular weight protein tyrosine phosphatase, in negative/positive regulation of VEGFR-2 signaling [59]. Interestingly, although VEGF demonstrated positive effects on MI in the experimental animal models, the results were not very promising according to several clinical trials [49]. One possible reason is the short period of protein bioactivity in vivo [60].

Although natural hydrogels are widely used in experiments [61], synthetic and hybrid hydrogels are also broadly investigated [62]. Synthetic glycosaminoglycan mimetic peptide nanofiber developed by Rufaihah and colleagues promoted the formation of new blood vessels and the di fferentiation of cardiomyocytes in rats [63]. Carlini and colleagues designed synthetic cyclic SAPs that were delivered to the heart through a catheter and rapidly formed a hydrogel after cleavage by enzymes MMP-2/9 and elastase, which are endogenous to the site of infarction in a rat model of MI. In addition to their low viscosity and ability to form a gel-like structure, the novel SAPs showed hemocompatibility, biocompatibility, and non-thrombogenicity that open up the possibility for implementation in drug delivery for the treatment of MI [35]. A novel hybrid temperature-responsive poly(N-isopropylacrylamide) gelatin-based injectable hydrogel was developed for cardiac tissue engineering and it exhibited a high level of cardiomyocyte and cardiac fibroblast survival and enhanced cytoskeletal organization [64]. Moreover, myeloid-derived growth factor (Mydgf) was incorporated into an injectable citrate-based polyester hydrogel to investigate its e ffects on improving cardiac tissue repair following MI. The combination of the released Mydgf and citrate, which is an important substrate in cellular energy metabolism, reduced cell apoptosis and scar formation as well as improved angiogenesis and cardiac function [65]. In the study by Waters and colleagues, therapeutic biomolecules, such as growth factors and cytokines, secreted by human adipose-derived stem cells (ADSCs), were loaded

into laponite/gelatin hydrogel and injected into the peri-infarct region in an acute MI rat model, which resulted in increased angiogenesis and reduced fibrosis as well as a significant improvement in ejection fraction and cardiac output [66]. The hydrogel could accommodate growth factors due to laponite, which is a synthetic nanoclay composed of discoid NPs that can bind growth factors through electrostatic forces.

**Figure 1.** Representative images of biomaterials. Hydrogels, scaffolds/patches, and nanoparticles loaded with growth factors/cytokines and stem cells, and their combination are shown.

Along with hydrogels, nanoscale carriers (Table 1) are extensively studied for cardiac tissue repair following MI [67]. Targeted delivery, maintenance of protein stability, presence in blood circulation for an extended time, and controlled release of loaded agents make NPs attractive carriers for cardiac tissue therapy. For the purpose of targeting MI, Nguyen and colleagues developed NPs that respond to a specific enzymatic stimulus of MMP-9 and MMP-2 enzymes, which are upregulated upon infarction. This method allows for better accumulation at the MI site and longer clearance from the system [68]. Moreover, DNA enzymes conjugated to gold NPs have been demonstrated to produce an anti-inflammatory effect and improve cardiac function in a rat model of acute MI via silencing TNF-α and downregulating pro-inflammatory mediators, such as IL-12β, IL-1β, IL-6, as well as inducible nitric oxide synthase [69]. Another group loaded liraglutide in poly(lactic-co-glycolic acid)-poly(ethylene glycol) nanoparticles (NP-liraglutide) and delivered it to the infarcted rats via intramyocardial injection to overcome the challenges posed by its short half-life [70]. As a result, the NP-liraglutide system is retained in the myocardium over four weeks, thus, enhancing heart function, attenuating adverse cardiac remodeling, stimulating angiogenesis, and suppressing cardiomyocyte apoptosis. Although NPs appear to be a promising drug delivery system, the main concerns are their toxicity and tendency to aggregate, which lead to changes in physical and chemical properties and the formation of protein corona on the surface of NPs that prevents specific targeting [67].

Hydrogels and NPs can be used separately, as previously mentioned, or in combination. For example, a sulfonated hydrogel incorporated with VEGF and IL-10 and combined with PDGF-loaded micelle NPs showed a sequential and sustained release of all three factors for 28 days in vitro and a significant increase in the formation of mature vessels in vivo on a subcutaneous injection murine model [12]. As a result, this novel system significantly promoted angiogenesis and demonstrated the potential to ameliorate inflammation for improving cardiac repair post-MI. Another study used a novel, shear-thinning biocompatible and catheter-deliverable HA-based hydrogel loaded with dimeric fragment of hepatocyte growth factor (HGFdf) and a variant of stromal cell-derived factor 1α (ESA) to demonstrate a dual stage release that decreased infarct size and improved angiogenesis

and heart function following MI [71]. ESA is a potent chemokine that attracts endothelial progenitor cells to infarcted areas and displays significant pro-angiogenic and wound healing e ffects. Moreover, hepatocyte growth factor prevents tissue fibrosis by inhibiting TGF-β production and stimulating MMP-1 to increase collagen degradation, as well as possessing pro-angiogenic and cardiomyogenic properties [72].

Another type of biomaterial is a cardiac patch that is directly applied to the myocardium. An acellular epicardial patch, developed from hydrogel, was also shown to prevent left ventricle remodeling and improve cardiac function in acute and subacute MI models in rats [73]. Wan and colleagues developed a novel cardiac patch derived from human heart valves. It is thought that the use of a human heart valve-derived sca ffold (hHVS) may be superior to other approaches in cardiac repair by providing a native myocardial ECM. An in vitro study showed increased cellular proliferation and induction of cardiomyogenic di fferentiation of cells attached to a hHVS. An in vivo experiment demonstrated that patch application of hHVS alone reduced infarct size in a murine MI model. However, c-kit+ stem cell-seeded hHVS was more e ffective [74]. Cardiac patches have also been used for growth factor delivery (Table 1). Rodness and colleagues demonstrated that VEGF-containing calcium-alginate microsphere patches increased capillary density and improved tissue regeneration and cardiac function [75]. Transplanted human cardiomyocyte patches, which contained cardiomyocytes derived from human iPSCs and NPs loaded with FGF1 and CHIR99021, an inhibitor of the enzyme glycogen synthase kinase-3, reduced infarction size and improved angiogenesis and cardiac function. The combination of these factors reduced apoptosis and increased proliferation of transplanted cardiomyocytes [76].

In summary, biomaterials including micro and nanospheres, lipid NPs, nanofibrous structures, coacervate, hydrogels, and sca ffolds appear to be a promising drug delivery system for cardiac tissue repair following MI. They can be administered alone or loaded with powerful therapeutic agents, such as growth factors and cytokines, that regulate cardiac tissue regeneration following MI. Biomaterials loaded with growth factors/cytokines have been shown to enhance angiogenesis and tissue regeneration, reduce cardiac cell death and scar size, ameliorate inflammation, and improve cardiac function (Table 1).


**Table 1.** Biomaterials loaded with growth factors and cytokines for cardiac tissue regeneration.

