**Table 1.** *Cont*.

#### **3. Biomaterials Loaded with Stem Cells for Cardiac Tissue Regeneration**

Stem cells possess self-regenerating, differentiating, and immunomodulating properties, as well as release trophic factors. Therefore, they have been considered to be promising tools for cardiac tissue regeneration [20,77,78]. Many reports have demonstrated the therapeutic potential of various stem cell types, such as bone marrow-derived stem cells (BMSCs), ADSCs, cardiac-derived stem cells/cardiac progenitor cells (CPCs), and others, on myocardial tissue regeneration [79–83]. Moreover, stem cells have shown their therapeutic efficiency in several clinical trials [84]. Treatment with MSCs can improve left ventricle remodeling and function through decreasing scar size, promoting angiogenesis, and improving contractility [85,86]. Stem cells mediate cardioprotection by lowering the number of apoptotic myocytes at the site of injection. The mechanism responsible for protection includes insulin-like growth factor 1 (IGF-1)-mediated activation of stress-signaling and inflammatory response pathways and the suppression of cardiac transcription factor, nuclear factor kappa B [20]. Stem cells also support neoangiogenesis in post-MI tissue through positive regulation of VEGF, angiopoietin-1 (Ang-1), epidermal growth factor (EGF), and PDGF. Cell survival and proliferation is regulated by the AKT signaling pathway [87]. Despite the beneficial effects of stem cells on post-MI tissue regeneration, limitations such as low engraftment and survival rates in a harsh microenvironment compromise the clinical translatability of this approach [88]. Poor engraftment of transplanted cells is linked to mechanical loss during injection, loss of viability during long-lasting pre-conditioning, hypoxia, nutritional deficiencies, and low cell proliferation rate in vivo [88]. Therefore, various approaches are now being examined to increase engraftment and enhance the survival and stability of stem cells. One such approach is the use of biomaterials. Table 2 summarizes the use of biomaterials loaded with stem cells for cardiac tissue regeneration.

Several stem cell delivery systems are now utilized, including direct needle injection, nanogels, polymers, and inorganic nanomaterials [89–91]. Needle injection is the preferred method in clinics as it is less invasive. However, it has low cell retention, with less than 5% of transplanted cells reaching and remaining in cardiac tissue [92]. Recently, Park and colleagues proposed a new e fficient direct MSC injection method to treat MI. MSCs were used in favor of other stem cell types based on their efficiency in reducing apoptosis and inflammation, as well as their ability to enhance vascularization and cardioprotection. In their study, they applied electrostatic interactions between bioengineered cationic mussel adhesive protein (MAP) and anionic HA. The resulting MAP/HA coacervate, named the adhesive protein-based immiscible condensed liquid system (APICLS), was successfully loaded with MSCs. APICLS was shown to be an innovative platform to treat MI, where stem cells demonstrated higher viability and retention and therefore, recovered infarcted tissue more e ffectively [92]. Another promising biomaterial for in vivo stem cell delivery is a collagen-based hydrogel transglutaminase cross-linked gelatin (Col-Tgel). The Col-Tgel-ADSCs system was shown to greatly improve MI treatment by enhancing engraftment of stem cells. ADSCs, which are the MSCs derived from adipose tissue, are shown to have several advantages over the bone marrow-derived MSCs. These include a more attractive cost and yield, a less invasive method for isolation, and a higher rate of cell growth [93]. In the study by Blocki and colleagues, injectable microcapsules made of agarose and ECM components were developed to enhance the survival of bone marrow-derived MSCs after their transplantation to rats with acute MI. The design was safe and e fficient as evidenced by the absence of fibrotic response and persistence of the cells in the infarcted myocardium for four weeks after injection. In contrast, when these cells were injected without microcapsules, i.e., as cell suspension, they were detectable in post-MI hearts for only two days following transplantation [94]. Gallagher and colleagues showed that delivering MSCs using an arginylglycylaspartic acid (RGD)-modified HA hydrogel improves MSCs survival in the ischemic area. This e ffect was achieved due to HA being a natural ECM component and RGD being a tripeptide sequence that promoted MSC attachment to the hydrogel [95]. Another study successfully improved post-MI heart recovery in rats by delivering iPSCs in erythropoietin-linked hydrogel. The hydrogel was administered by injection into the myocardium [96]. Moreover, Cai and colleagues developed a novel designer self-assembling peptide (DSAP) consisting of the existing synthetic SAP and angiopoetin-1-derived pro-survival peptide QHREDGS in order to improve engraftment and retention of MSCs. This system significantly improved the survival of rat MSCs when they were injected into rats with MI [97]. Enhanced cell survival could be attributed to the presence of the QHREDGS peptide in the SAP. This peptide is an integrin-binding motif of Ang-1, a growth factor that stimulates endothelial cell survival, migration, and di fferentiation [98]. It was shown that QHREDGS peptide could mediate the same e ffects on its own, without being a part of Ang-1, when it is incorporated into various biomaterials such as hydrogels, for example [97]. However, the exact mechanism by which it promotes cell survival is still to be elucidated.

Pro-survival peptides were also used in the study by Lee and colleagues. In particular, they utilized collagen–dendrimer biomaterial crosslinked with pro-survival peptide analogues, namely, bone morphogenetic protein-2 peptide analogue, erythropoietin peptide analogue, and FGF2 peptide analogue, to augmen<sup>t</sup> the survival of CPCs in the MI model of mice [99]. CPCs that were transplanted with pro-survival factors enriched the collagen matrix and showed significantly greater long-term survival and engraftment compared to cells without the matrix. The authors described the molecular mechanism of enhanced cellular survival. Thus, the pro-survival matrix caused an increase in the expression of genes involved in the MAPK and phosphatidylinositol-3-OH kinase-protein kinase B (PI3K-AKT) pathways, while inhibiting pro-apoptotic pathways. In another study, silica-coated magnetic nanoparticles (MNPs) and an external magne<sup>t</sup> were utilized to enhance the survival of transplanted cells [100]. Embryonic cardiomyocytes, embryonic stem cell-derived cardiomyocytes, and BMSCs were incorporated into MNPs. Afterwards, the cell-MNP delivery system was intramyocardially injected into a murine model of MI, and a magne<sup>t</sup> was placed close to the chest of the animals to force the cells into the infarcted tissue. The treatment had drastically enhanced cell engraftment by 7-fold and 3.4-fold, two and eight weeks after application, respectively. The increased engraftment of the transplanted cells was due to a decrease in the loss of cells via the injection channel, which increased their proliferation and reduced apoptosis. The graphene oxide/alginate microgels

constructed for cell delivery also demonstrated a favorable approach to promote MI recovery of the left ventricle during transplantation of MSCs [101].

Cardiac cell patches (Table 2) can be constructed from natural or synthetic materials, albeit natural materials are more favorable due to their biocompatibility and comparatively low cost [102]. Studies show that cardiac patches loaded with stem cells, where MSCs are preferable compared to CPCs, embryonic or iPSCs, facilitate a higher engraftment rate of transplanted cells. Moreover, cell patches also provide a positive impact on cardiomyogenesis and angiogenesis [102,103]. Wang and colleagues transplanted poly(ε-caprolactone)/gelatin patch loaded with MSCs into the epicardium of the murine model of MI. The patch reduced MI-induced damage by promoting angiogenesis, lymphangiogenesis, and cardiomyogenesis, decreasing scar size and enhancing the release of paracrine factors from stem cells. They also showed an increase in the expression of hypoxia-inducible factor 1<sup>α</sup>, TGF-β, VEGF, and SDF1 factors and a negative regulation of CXCL14. Cytokine release enhanced the recruitment of endogenous c-kit+ cells and activated the epicardium [103]. Chen and colleagues designed a novel chitosan and silk fibroin microfibrous cardiac patch that significantly improved the survival of murine adipose tissue-derived MSCs in infarcted hearts of a rat model [104]. This was achieved due to the structural resemblance of the patch to the native ECM of the heart. Thus, the patch provided a suitable environment for the retention and survival of the transplanted cells. Nevertheless, the detailed mechanism of this process is ye<sup>t</sup> to be identified. Similarly, in the study by Gaetani and colleagues, it was shown that a 3D-printed HA/gelatin cardiac patch could support long-term survival and differentiation of the CPCs when they were tested on the mouse model of MI [105]. Su and colleagues used a cardiac patch not only to provide an adhesion and retention framework for stem cells, but to also nutritionally support them [106]. Specifically, they developed a vascularized fibrin gel that could accommodate CSCs. Such a construct would help stem cells receive nutrients through biomimetic blood vessels (BMV) within the hydrogel and consequently, enrich their survival. In addition, the BMV were made of fibronectin, a constituent of the natural ECM, and hence, provided the appropriate environment for the transplanted cells. Moreover, Dong and colleagues constructed a patch made of gold NPs coated with a combination of ECM and silk proteins [107]. The patch was loaded with rat bone marrow-derived MSCs and tested in a cryoinjury model of MI in rats. The construct was found to greatly improve stem cell survival and retention as well as significantly decrease the infarct size 28 days post-infarction. The authors proposed several mechanisms to achieve beneficial effects of the patch on cell viability. Namely, the construct possesses antioxidant properties and acts as a mechanical scaffold, thus, protecting the transplanted cells from the harsh environment in the infarcted region. Gao and colleagues used an ECM scaffold to deliver human iPSC-derived cardiomyocytes, smooth muscle cells, and endothelial cells to mice with MI [108]. This treatment significantly reduced infarct size and improved cell proliferation, cardiac function, and angiogenesis. Furthermore, Tang and colleagues developed new microneedle patches loaded with cardiac stromal cells (CSCs) for post-MI tissue regeneration. Poly(vinyl alcohol)-made microneedles served as channels between myocardial tissue and regenerative factors released from CSCs. In vivo studies on a rat MI model showed that microneedle patches could promote angiogenesis, reduce fibrosis, and repair the left ventricular wall [109]. Combinatorial dual stem cell delivery is another approach to enhance the survival of transplanted stem cells. Park and colleagues used MSCs seeded on polycaprolactone patch and iPSC-derived cardiomyocytes for in vivo treatment of the rat MI model. Analysis with immunohistochemistry, gene expression, and echocardiography demonstrated significant enhancement in MI recovery. Cardiomyocytes contributed to myocardium regeneration, while growth-promoting paracrine factors from MSCs accelerated angiogenesis as well as caused iPSC-cardiomyocytes to resemble adult-like cardiomyocyte morphology [110]. Interestingly, the cardiac patch can be 3D printed for iPSC-derived cell delivery to effectively enhance post-MI treatment [111].

Another positive effect of biomaterials on stem cell therapy is the enhanced release of paracrine factors produced by the cells. Melhem and colleagues developed a microchanneled hydrogel patch that can sustain a continuous release of stem cell synthesized factors [112]. The patch was loaded with human bone marrow-derived MSCs and tested in vitro and in the murine model of MI. Patch-protected MSCs released a variety of angiogenic, anti-inflammatory, cardioprotective, antifibrotic, and antiapoptotic factors in vitro. Furthermore, the sustainable release of paracrine factors by the system was confirmed by the assessment of the VEGF release profile for one week. Over this period of time, the amount of VEGF linearly increased. The microchanneled hydrogel patch loaded with MSCs showed other benefits as well. Namely, mice treated with the patch showed significant improvement in cardiac function, which was established by echocardiographic examinations of ejection fraction and stroke volume five weeks after infarction. Importantly, the therapeutic effects of the treatment were significantly greater with MSCs, the patch without MSCs, or MSCs alone as compared to the patch without microchannels. Moreover, the effects of the patch did not depend on the number of transplanted cells, implicating that the construct could reduce the number of stem cells required for treatment. Similarly, Mayfield and colleagues showed that single cell hydrogel microencapsulation of human CSCs significantly improves the production of pro-angiogenic/cardioprotective cytokines, angiogenesis, and angiogenic cells recruitment after direct intramyocardial injection into mice with MI [113]. Less is known about biomaterial distribution after injection in vivo; Ahmadi and colleagues reported that a collagen matrix is retained mostly in the injected area with minimal distribution to non-target areas [114]. Han and colleagues utilized iron NPs that were co-cultured with rat cardiomyoblasts to boost the therapeutic efficiency of human bone marrow-derived MSCs [115]. The modified MSCs showed increased expression of various paracrine factors, namely, bFGF, HGF, VEGF, Ang-1, urokinase type plasminogen activator, placental growth factor, and monocyte chemoattractant protein-1. Moreover, pre-treated MSCs reduced infarct size, prevented fibrosis, decreased apoptosis of myocardial cells, increased angiogenesis, and improved cardiac function and the survival of rats with acute MI overall. The authors stated that improvements in the therapeutic potential of MSCs should be attributed to the increased expression of connexin 43 gap junction protein by cardiomyoblasts, which was stimulated by iron NPs. The greater expression of connexin, in turn, leads to a more efficient electrophysiologic and paracrine crosstalk between MSCs and cardiomyoblasts [116–118].

Yet another advantageous effect of biomaterials on stem cell treatment is their ability to accommodate factors that could act synergistically with stem cells, thus, enhancing their therapeutic actions [119,120]. For instance, Yokoyama and colleagues tested the efficiency of statins and human ADSCs combinations incorporated into NPs [121]. The treatment was injected into the tail vein of mice with MI, and its therapeutic effects were assessed for four weeks after infarction. The statin-ADSCs encapsulating NPs significantly increased the ejection fraction and several other parameters, which reflect left ventricular function. This positive effect of statin-ADSCs combination was superior compared to the use of statins or ADSCs alone. The mechanism by which the treatment brought about these improvements is likely through stimulation of the sustained and localized release of the statins by ADSCs. This, in turn, resulted in the inhibition of local inflammation, promotion of circulating stem cell recruitment, and stimulation of their differentiation to cardiomyocytes and angiogenesis [121]. Importantly, in this study, treatment efficiency was achieved with a smaller cell number of ADSCs than has ever been reported. A conductive hydrogel was also used to deliver plasmid DNA encoding endothelial nitric oxide synthase and ADSCs by injection into the infarcted myocardium. The results again demonstrated improved cardiac function with the conductive hydrogel [17]. Yao and colleagues also combined adipose-derived MSCs with a nitric oxide (NO) releasing system [122]. They utilized a naphthalene hydrogel that could maintain a controllable release of NO. In addition to demonstrating an excellent cell survival rate, the hydrogel stimulated the synthesis of angiogenic factors VEGF and SDF-1α in the MI model of mice. In ye<sup>t</sup> another study, the therapeutic benefits of ADSCs were enhanced by NRG1 growth factor [123]. The ADSCs-NRG1 mixture was encapsulated into microparticles and injected into rats with MI. This combination improved cell survival as demonstrated by the persistence of the transplanted cells at three months after injection. Furthermore, ADSCs induced the shift of macrophages found in the infarcted myocardium from pro-inflammatory M1 to regenerative M2 phenotype. At the same time, NRG1 reduced the infarct size and stimulated

cardiomyocyte proliferation. Compared to a separate administration, the combined treatment with ADSCs-NRG1 microparticles resulted in a more pronounced regeneration of the damaged myocardium. Chung and colleagues showed that cardiac patch-supported co-transplantation of CSCs and VEGF had a synergistic e ffect on angiogenesis, cell proliferation, and the recruitment of stem cells [124]. In particular, they developed a poly(l-lactic acid) mat and loaded it with rat CSCs and VEGF. When the system was tested in the rat MI models, it had greater angiogenic and cardiomyogenic e ffects compared to either VEGF with the patch or CSCs with the patch. Thus, numerous developments have been achieved in recent years in the usage of biomaterials to deliver stem cells to the infarction site. These include coacervates, various modifications of the hydrogels, NPs, and cardiac patches. Moreover, the combination of NPs and hydrogels also promoted transplanted cell survival. Stem cells derived from di fferent sources were loaded into biomaterials alone, preconditioned or loaded in combination with bioactive molecules. Transplanted or tail vein injected stem cells delivering biomaterials have significantly enhanced recovery of the MI in small animal models and show promising results for their therapeutic applications. Small animals are commonly used in cardiovascular research due to their small size, low cost, short gestation time, and ease in maintenance and genetic manipulations [125]. However, there are limitations to their use that are responsible for their high failure rates in human clinical trials. These include a small heart size and anatomical di fferences in the coronary artery and conduction system. [126–128].


**Table 2.** Biomaterials loaded with stem cells for cardiac tissue regeneration.


**Table 2.** *Cont*.


**Table 2.** *Cont*.
