Overview of hydrogel-based strategies for application ...

Biomed. Mater. 10 (2015) 034005

doi:10.1088/1748-6041/10/3/034005

SPECIAL SECTION TOPICAL REVIEW

received

30 January 2015

Overview of hydrogel-based strategies for application in cardiac tissue regeneration

revised

15 April 2015 accepted for publication

23 April 2015 published

4 June 2015

Xuetao Sun1 and Sara S Nunes1,2,3 1

University Health Network, Toronto General Research Institute, 101 College St., Toronto, ON M5G 1L7, Canada Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, ON M5S 3G9E, Canada 3 Heart & Stroke/Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON M5G 1L7, Canada 2

E-mail: [email protected] Keywords: hydrogels, tissue regeneration, cardiac, stem cells, cell therapy

Abstract Cardiovascular diseases remain the leading cause of death globally. Since the adult heart lacks the capacity to regenerate, loss of myocardium following myocardial infarction is irreversible and ultimately leads to failure to maintain cardiac function. In order to repopulate the areas of cell loss in the damaged hearts for restoration of cardiac function, cell transplantation/replacement has been extensively investigated. Recently, biomaterials have emerged as an approach to improve delivery and viability of cells for the regeneration of the damaged heart. Here we review the most common approaches in hydrogel-based cardiac tissue regeneration with particular focus on the implementation of hydrogels to improve cell delivery.

1. Introduction The heart works like a pump to transport blood and supply nutrition and oxygen for all the cells of the body. Its pumping motion is generated through the synchronized contraction of heart muscle cells, or cardiomyocytes. Adult cardiomyocytes are terminally differentiated and display very low proliferation rates [1], which renders an extremely limited cardiac regenerative capacity. During myocardial infarction (MI), commonly known as heart attack, vessels that feed the heart muscle get blocked and ischemia ensues [2]. This can lead to the massive death of cardiomyocytes [2]. The remaining cardiomyocytes compensate for functional loss by growing in size rather than number [3]. Other cardiac cell types, mainly fibroblasts, form the non-functional scar in the place left by the cardiomyocytes lost due to injury or disease [2]. Such compensatory response eventually develops into an inability to provide adequate blood supply to meet metabolic needs of the body, a process known as heart failure [3]. Cardiovascular disease is the leading cause of death globally [4]. The morbidity and mortality of cardiovascular disease has been associated with long hospital stays and high health spending. Current treatments for heart failure include pharmacological interventions or mechanical assist devices [5, 6]. New potential therapies based on hydrogel delivery to the infarction site © 2015 IOP Publishing Ltd

have been the focus of pre-clinical studies for cardiac regeneration. Some of the most common approaches are discussed in section 2.1 of this review. Yet, current treatments available in the clinic and hydrogel delivery do not significantly help to regenerate cardiomyocytes. Full cardiac functional restoration can be achieved through heart transplantation [7]. However, this solution is highly invasive and limited by the availability of donor organs that does not meet the demand [9]. Thus cell-based cardiac tissue regeneration has been intensively pursued to overcome the aforementioned limitations and to serve as an alternative therapy for MI. Cell transplantation represents a potential method to repopulate irreversibly damaged heart tissue with stem/progenitor cells and/or differentiated cardiomyocytes. A wide variety of cell types, including bone marrow–derived cells, mesenchymal stem cells and cardiac stem cells have been investigated [10–14]. Some were also tested in clinical trials [15–22]. However, cell transplantation has failed to achieve substantial improvement in cardiac function. This is possibly due to the poor retention and survival of transplanted cells. To address these issues, biomaterials-based cell therapies have been pursued as means to provide deliverable, viable and sufficient cell numbers into an injured, diseased or aged heart as an attempt to circumvent cell death post transplantation. Given their versatility, allowing for either direct injection or transplantation as a heart patch, multiple hydrogels have been used

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in pre-clinical animal studies to improve cell delivery and retention, including alginate, chitosan, collagen, decellularized tissues, fibrin, hyaluronic acid (HA), keratin, Matrigel, and synthetic polymers composed of polyethylene glycol (PEG) or poly(N-isoproylacrylaminde) (PNIPAAm). We will focus on biodegradable and most commonly used hydrogels in bioengineering approaches for cardiac regeneration in this review.

2.  Hydrogels in cell-based therapies Hydrogels are water-insoluble polymers which are composed of cross-linked, water-soluble precursors [23]. These can be either natural or synthetic polymers [23]. They are in liquid phase in vitro and can be polymerized into semi-solid gels after delivery to the target site under physiological conditions [24]. These biomaterials are viscoelastic and can be modified chemically and/or physically [25]. With large capacity to absorb fluids, hydrogels can become swollen but are still able to maintain their shape [24]. These properties make them highly attractive for use in cardiac tissue regeneration [26]. The gelation can be controlled spatially and/or temporally through delivery methods and/or by an environmental trigger (e.g. temperature, UV and pH), according to the physicochemical properties of the selected hydrogel [23, 24] (figure 1). Hydrogel-based cell therapies have utilized two main methods: (1) injectable hydrogels that either promote endogenous repair and regeneration, or serve as delivery vehicles for cells and other bioactive molecules and (2) pre-formed in vitro cell-seeded scaffolds for epicardial attachment (patches). Here, our goal is to provide a snapshot of the field, focusing on the most commonly used hydrogels types and combined strategies for translational applications, rather than to extensively cover current and past research; already covered elsewhere [25–29]. 2.1.  Hydrogel therapy Conventional treatment for MI and cell delivery to the damaged heart tissue may improve short-term function and remodeling, but fail to stop the progression into later stage adverse remodeling. Consequently, functional deterioration and heart failure is inevitable. Therefore in order to attain long-term cardiac functional restoration post-MI, current strategies have aimed at introducing viable cardiac cells and/or tissues to regenerate the heart [30]. One way to achieve this goal is to encourage the involvement of endogenous cells, such as prompting cardiomyocyte proliferation and migration or activating existing stem/progenitor cells from the surrounding remaining functional tissue post-MI [10, 31–34]. The use of hydrogels on their own, without addition of exogenous cells, has been shown to improve cardiac function in animal studies. Fibrin, a biodegradable protein involved in the coagulation cascade, is one of the earliest biomaterials used as hydrogels in studies of 2

cardiac repair [35]. In a rat ischemia reperfusion model of MI, fibrin injection into the scar zone was shown to improve cardiac function, reduce scar area and increase arteriole density in the infarct area [36, 37]. This demonstrates the ability of fibrin gels to recruit host cells, such as endothelial cells, to the MI area. The mechanism of this effect might be due to the molecular properties of fibrin [38]. Fibrin contains binding domains for angiogenic factors, such as basic fibroblast growth factor (bFGF) [39]. Fibrin glue also contains an arginine-glycineasparagine (RGD) motif that binds αvβ3 integrin expressed on the surface of endothelial cells [38]. Moreover, the fibrin fragment E, a degradation product of fibrin, has been shown to stimulate neovascularization in vitro [40]. However, such benefits from fibrin may be limited to short term [41]. Significant increases in neovasculature formation due to fibrin injection were later confirmed by a comparative study of fibrin, collagen type I, and Matrigel (an ECM mixture isolated from mouse sarcoma [26]), which showed that all three materials enhanced neovascularization in the infarcted rat heart [42]. Histological data indicated that compared to a control PBS group, only collagen injections significantly increased the degree of myofibroblast infiltration whereas Matrigel and fibrin groups exhibited trends for increased myofibroblast migration [42]. Other biomaterials including alginate, collagen, decellularized tissues, hyaluronic acid (HA), Matrigel, polyethylene glycol (PEG) or poly(N-isopropylacrylamide) (PNIPAAm) [7, 27] have also been used in studies of cardiac regeneration in animal models (table 1). However, the specific mechanism of action of hydrogels in the preclinical treatment of MI remains unknown. It has been speculated that the hydrogels improve cardiac function by providing mechanical support to the heart wall [26, 43–45]. However, injection of a non-degradable and bio-inert PEG hydrogel was insufficient to prevent left ventricular (LV) dilation post MI despite an increase in infarct wall thickness [46]. In another study, synthetic terpolymers alone also increased infarct wall thickness without improving angiogenesis or cardiac function [47]. These studies suggest that structural support of the heart wall on its own may not be the sole mechanism of action of hydrogels and suggest that hydrogels may also provide essential cues for the ischemic tissue to form a niche for cellular infiltration and for the recruitment of endogenous cells (e.g. aforementioned collagen-injection-increased myofibroblast density in infarcted rat heart) [27, 28, 42, 44] or the increased survival of delivered cells (see section 2.3). Ou et al [48] demonstrated that intracardiac injection of Matrigel immediately after permanent ligation of the left anterior descending (LAD) artery significantly increased LV function as well as LV wall thickness, vascularization and recruitment of host c-kit+ (expressed on cardiac progenitor cells, endothelial progenitor cells and bone marrow derived cells, including haematopoietic stem/progenitor cells and mesenchy-

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Figure 1.  Biomaterial approaches for the preclinical treatment of MI. Natural polymers (such as collagen or fibrin) and synthetic polymers (such as PEG or PNIPPAm) have been used for the cardiac regeneration. Preformed scaffolds or injectable hydrogels have been used for the transplantation in the infarct hearts alone or in combination with bioactive molecules (such as growth factors and angiogenic factors), different cell types or a combination of cells and molecules. In its injectable form, hydrogels are injected in liquid form and gels (polymerizes) in situ. Cell sheets can be generated in vitro for subsequent cardiac implantation.

Table 1.  Summary of the characteristics of hydrogels frequently used for cardiac regeneration in animal models of myocardial infarction described in this review. Category

Name

Pros

Cons

Gel mechanism

Application in animal models of MI

Natural

Collagen

Biocompatible, biodegradable

Slow gelation

Thermal

[42, 96]

Fibrin

Biocompatible, biodegradable, availability

Slow gelation, low mechanical strength, fast degradation in vivo

Peptide-self assembly

[36, 37, 41, 42, 82]

Matrigel

Biocompatible, resemble to native ECM, faster vascularization

Potentially tumorigenic

Thermal

[42, 48, 87, 99, 100]

Hyaluronic acid (HA)

Biocompatible biodegradable

Modifications needed for crosslink

Redox inhibitor

[54]

Alginate

Non-thrombogenic, relatively bio-inert

Modifications needed for cell binding

Ionic

[41, 51, 52, 94]

PEG

Bio-inert, biocompatible

Low cell adhesion, non-injectable, nondegradable

Peptide-self assembly

[46]

PNIPAAm

Extensibility

Slower degradation

Thermal

[92, 93]

Synthetic

mal stem cells) and CD34+ (expressed on hematopoietic stem cells) stem cells [48]. A hydrogel generated from the ECM of pig’s heart ventricle and delivered percutaneously via transendocardial injection to treat rat MI [49] decreased the loss of endogenous cardiomyocytes in the infarct area and preserved cardiac function [49]. This natural injectable ECM-derived hydrogel has produced positive outcomes in both small (rat) and large (pig) animal models of cardiac repair [44, 49, 50]. Furthermore, hemocompatibility studies with human blood indicated that exposure to 3

the pig’s ECM hydrogel at relevant concentrations does not affect clotting times or platelet activation, providing evidence to support moving this hydrogel to clinical studies [50]. Alginate, a naturally occurring linear polysaccharide found in brown seaweed algae [25], has been widely used as a hydrogel for tissue engineering. The injection of alginate has shown positive results in rat and porcine models MI [51, 52]. One advantage of the use of alginates versus other hydrogels described above is that they are non-thrombogenic and thus more suitable for cardiac

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applications. Two currently ongoing phase II trials are now in place to assess safety and effectiveness of alginate for the treatment of heart failure and MI (NCT01226563 and NCT01311791). One alginate, called IK-5001, was approved for PRESERVATION 1 (NCT01226563) trials and is now recruiting patients to evaluate intracoronary injection of IK-5001 into the blocked artery with the goal of preventing ventricular remodelling. Another injectable alginate subjected to clinical study is AlgisylLVR™. A pilot study showed that the alginate decreased ventricular volumes and increased ejection fraction and wall thickness [53]. The approved phase II trial AUGMENT-HF (NCT01311791) will evaluate if intramyocardial injections of the alginate hydrogel will improve heart function, increase wall thickness, reduce wall stress and restore ventricular geometry. 2.2.  Hydrogels and bioactive molecules A variety of cytokines, growth factors, and other bioactive molecules could also help to improve cardiac regeneration. However, studies have shown that the beneficial effects from exogenously delivered bioactive molecules are limited by their short half-lives in vivo [29]. In order to increase the availability and the halflife of bioactive molecules, researchers started taking advantage of the biochemical characteristics and binding affinities as well as the ability to modify the different hydrogels to increase their affinity to different biomolecules to deliver bioactive molecules. For example, an improved method was developed using a hydrogel delivery system that sustains the release of a bioactive endothelial progenitor cell chemokine, stromal cell–derived factor-1α (SDF-1α), in a rat model of MI [54]. A synthetic analog SDF-1 (engineered stromal cell– derived factor analog (ESA)) encapsulated in hyaluronic acid (HA) hydrogel induced the homing of endothelial progenitor cells in vitro. In vivo delivery of this hydrogel showed detectable presence of ESA in the rat heart for more than 3 weeks compared to saline control. ESA-HA delivery improved LV function (ventricular geometry, ejection fraction, cardiac output, and contractility) and displayed increased vascularity [54]. Fibrinogen, the precursor to fibrin, was used to make a PEG–fibrinogen hydrogel, which on its own could mechanically support the failing LV and improve cell transplantation [55, 56]. PEG–fibrinogen hydrogel was also able to store and release VEGF in a sustained and controlled fashion, significantly improving arteriogenesis and cardiac function after MI in a rat model [56]. Thus, hydrogels represent a potentially minimally invasive approach for cardiac repair [27]. More studies are required to determine the suitability of these hydrogels for application in different cardiomyopathies and the mechanism of action of each hydrogel therapy. 2.3.  Hydrogels and cells 2.3.1.  Bone marrow-derived stem cells (BMSC)  Bone marrow-derived mononuclear cells (BMMNCs) are a collection of bone marrow cells characterized with 4

unilobulated or rounded nuclei and lacking granules in the cytoplasm [57]. This circulating, heterogeneous cell population has been widely investigated in preclinical animal studies, and has shown a positive effect on cardiac function after MI, mainly through multiple paracrine effects [58]. Chen et al showed that intramyocardial injection of BMMNCs (1   ×   106) in hyaluronic acid (HA) hydrogel was beneficial to the ischemic heart in a rat model of MI [59]. Then, they carried forward the combined treatment in a mini pig model of MI [60]. Although injection of HA or BMMNCs alone slightly elevated LV ejection fraction, the combined HA-BMMNC (1   ×   108) injection showed increased BMMNC retention (figure 2(A)) as well as a significant improvement in LV ejection fraction, contractility, infarct size, and neovascularization [60]. In a rabbit model of MI, injection of synthetic Dex-PCL-HEMA/PNIPAAm hydrogel-encapsulated BMMNCs (1   ×   107) increased cell engraftment (48 h post injection) and improved LV function compared with other groups. Histological analysis showed that hydrogel-BMMNCs enhanced neovascular formation and prevented scar expansion compared with the other groups [61]. These studies highlight the usage of hydrogels in combination with BMMNCs as an effective approach to enhance structural and functional recovery for injured heart. However, conflicting clinical results have been reported using these cells for intracoronary therapy in patients with acute myocardial infarction (AMI) [15, 16, 18, 22, 62–64]. Intracoronary BMMNC administration after AMI showed moderate improvement on LV function (LV ejection fraction) at short-term follow-up (up to 18 months) in TOPCARE-AMI [15, 65], REPAIR-AMI [16, 66], BOOST [17, 62, 67] and FINCEL [18]. 5-year follow-up exhibited benefits in the TOPCARE-AMI trial [65], and event-free survival in cell-treated patients compared to the placebo group in the REPAIR-AMI trial [66], in contrast to no beneficial effect in the BOOST trial [67]. Having said that, other trials have failed to demonstrate beneficial effects (LVEF) of intracoronary BMMNC administration in patients with MI [22, 68, 69]. A recent meta-analysis of the published trials suggested that intracoronary BMC treatment could lead to a moderate, but significant improvement of LVEF and reduction of LVESV at 6 months that sustained at 12 months follow-up, without a clear significant effect on LVEDV, or infarct size [63]. A larger meta-analysis (including 50 studies enrolling 2625 patients, majority using BMMNCs) suggested that these benefits persisted during long-term follow-up [64]. BMC transplantation also reduced the incidence of death, recurrent MI, and stent thrombosis in patients with ischemic heart disease [64]. However, these meta-analyses were limited by the inherited degree of heterogeneity among the selected trials and a potential bias to include publication trials with a positive outcome [64, 66]. Nevertheless, these clinical studies were not powered to assess clinical outcome [63,

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Figure 2.  Hydrogels improve cell engraftment. (A) Representative images demonstrating HA hydrogel improved transplanted BMMNC retention in pig heart post-MI. BMMNC pre-labeled with DiI before injection are shown in red. Nuclei are blue. HA, hyaluronic acid. Scale bar, 50 μm. Reproduced with permission from [60], copyright APS. (B) Matrigel improved hESC-CM survival in infarcted rat heart. hESC-CM injected into infarcted hearts of nude rats with Matrigel (Cells + Matrigel), or Matrigel combined with PSC (Cells + PSC). Sections were stained with an antibody to β-myosin heavy chain (red chromagen) as well as a humanspecific pancentromeric in situ hybridization probe (huCent, brown DAB deposit) to identify total and specifically human cardiac graft cells. The human cardiomyocytes (arrows) were significantly more abundant in the Cells + PSC group than in Cells + Matrigel group. Counterstain, fast green scale bar, 50 μm. Reprinted by permission from Macmillan Publishers Ltd: [87], copyright 2007.

66]. In order to assess the BMMNCs transplantation on clinical outcome, a large EU-funded Phase III clinical outcome study, the BAMI trial (NCT01569178), is currently recruiting. 2.3.2.  Mesenchymal stem cells (MSC)  MSCs have been deemed a promising cell type for stem-cell therapy for cardiac repair/regeneration and received enormous attention. They can be obtained from various tissues, including bone marrow and adipose, and have been used extensively due to immune privilege (for allogeneic applications) and regenerative capacity. Both autologous and allogeneic MSCs can be obtained and expanded with relative ease. MSCs have been reported to preserve tissue integrity and function after ischemia. This can happen through their direct contribution to the formation of vasculatures [12]. However, most reports ascribed preservation 5

of heart function to paracrine mechanisms (such as the secretion of vascular endothelial growth factor (VEGF) [70, 71], bFGF [71], stromal cell-derived factor 1(SDF-1) [72] and secreted frizzled related protein 2 (Sfrp2) [73]) [74, 75], through which MSCs promote angiogenesis, reduce cardiomyocyte apoptosis, dampen inflammation, potentially recruit stem/progenitor cells, and prevent adverse remodeling [73, 76–81]. Martens et al utilized adherent MSCs at passage 4 obtained from the culture of BMMNCs and display an adhered, mesenchymal cell behaviour/characteristics as opposed to the circulating, less differentiated BMMNCs described in section 2.3.1 [82]. They demonstrated that percutaneous delivery of these human bone marrow-derived MSC (BMMSC) (5   ×   105) using fibrin polymerized in situ led to an increased cell retention and survival in a nude rat model of MI [82]. However, heart functional analysis was not performed. In a

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rat MI model, human BMMSCs (hBMMSC) (1   ×   106) were encapsulated in alginate and attached to the heart with a hydrogel patch [83]. Such alginate encapsulation improved retention of hBMMSCs and facilitated paracrine effects as revealed by increased peri-infarct microvasculature and decreased scar [83]. Cardiac functional analysis showed significant improvement in the post-MI heart treated with encapsulated hBMMSCs [83]. Mathieu et al demonstrated that intramyocardial injection of self-setting silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel seeded with BMMSCs (3   ×   106) could preserve cardiac function and attenuate LV remodeling during an 8 week followup study in a rat model of MI [84]. Injection of hydrogel seeded with BMMSCs not only led to improved cardiac function up to 28 d post-MI and a mid-term prevention of cardiac function alteration at day 56, but also resulted in decreased infarct size, increased scar thickness and limited the transmural extent of MI. These suggest intramyocardial injection of BMMSC and hydrogel induced short-term recovery of ventricular function and mid-term attenuation of remodeling after MI [84]. Hybrid hydrogels can also be designed by combining both natural and synthetic materials for functional cardiac regeneration. Xu et al designed biodegradable hybrid hydrogels by using thiolated collagen (Col-SH) and multiple acrylate containing oligo (acryloyl carbonate)-bpoly(ethylene glycol)-boligo(acryloyl carbonate) (OACPEG-OAC) to retain bioactivity of collagen and regulate the degradation time of the hydrogels by controlling polymer concentration [85]. In a rat model of MI, both hybrid hydrogel and BMSC-encapsulating hybrid hydrogel treatments increased the ejection fraction at 28 d postMI. The hearts receiving the hybrid hydrogel displayed a significantly reduced infarct size and increased wall thickness. These effects were more pronounced in BMSCencapsulating hydrogel group [85]. Godier-Furnemont et al used thin sections of completely decellularized human myocardium to form cell–matrix composites by applying fibrin hydrogel with suspended bone marrow derived mesenchymal progenitor cells (BMMPCs) onto decellularized sheets of human myocardium. Then the composite was implanted on the infarct bed in a nude rat model of MI and showed increased vascular network formation in the infarct bed compared to the scaffold-only group. TGF-β-preconditioned BMMPC composite enhanced such effect. Enhanced migration of BMMPCs into ischemic myocardium was also observed [86]. 2.3.3.  Embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived cardiomyocytes  Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are capable of being induced to differentiate into functional cardiomyocytes through the orchestration of developmental stage-specific growth factor gradients in defined media [87, 88]. These cells represent a potential novel unlimited autologous cell source for use in cardiac regeneration. 6

Mouse ESC-derived cardiomyocytes (5   ×   106) seeded on elastic poly(lactide-co-caprolactone) scaffolds subjected to cyclic stretch, followed by implantation into infarcted rat hearts showed reduced fibrotic tissue formation and upregulation of cardiac gene expression as compared with unstrained controls [89]. Matrigel was used in the first human ESC-derived cardiomyocytes (hESC-CM) for preclinical animal studies for cardiac repair in infarcted heart [87] (discussed in detail in section 2.4). The feasibility of delivery of hESC-CM (3   ×   106) together with a biodegradable hydrogel made from PEGylated-fibrinogen was revealed by studies in a rat MI model [55]. A significant increase in fractional shortening (FS) and wall thickness was observed 30 d after transplantation in [55]. A novel cell delivery system, termed cell sheet technology, was developed and could avoid some unwanted effects from conventional method of cell/hydrogel delivery, such as needle injection, leakage of transplanted cells, injury and mechanical stress caused by the needle [90]. In this method, cells were cultured in plate surfaces which contains a temperature-responsive, Ultrathin Poly(N-isopropylacrylamide) (PNIPAAm) Gel Modified Surface [91]. Given the gel characteristics, cells can be detached as a sheets by simply reducing the temperature without any enzymatic treatment [91]. Using this gel, Kawamura et al [92] generated hiPSC-CM sheets and transplanted over the myocardial infarcts in a pig model of ischemic cardiomyopathy induced by ameroid constriction of the LAD [92]. Transplantation significantly improved cardiac performance and attenuated LV remodeling [92]. hiPSC-CM could be detected 8 weeks after transplantation, but very few survived long term [92]. The method was also used to make cardiac tissue sheets derived from mouse iPSCs and implanted epicardially in the nude rat heart 2 weeks after LAD ligation [93]. Such treatment showed that the implanted cardiac tissue sheet survived 4 weeks after implantation and significantly improved cardiac function and attenuated LV remodeling compared with the sham group [93]. 2.4.  Combination strategies: hydrogels, bioactive molecules and cells Strategic attention has also been paid to the combination of cells with biomolecules, such as growth factors, or other therapeutic reagents. These more sophisticated approaches hold the potential to bring synergistic interaction desired for therapeutic purpose. For this purpose, the biomaterial can be used as carrier for the co-delivery of multiple elements and modified to maximize their efficacy. Multiple combinations of cells and growth factors have been investigated in the infarcted myocardium. In a rat model of MI, neonatal rat cardiac cells (2.5   ×   106) were seeded on an alginate patch incorporated with the pro-survival and pro-angiogenic factors: insulinlike growth factor-1 (IGF-1), SDF-1 and VEGF. The patch was prevascularized on the omentum before

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implantation on the infarcted heart [94]. Patches containing growth factors demonstrated enhanced vascularisation on the omentum, and these prevascularized patches fully integrated into the host tissue after 4 weeks and improved cardiac function (increased LV function and reduced ventricular remodelling). The effects were more pronounced in groups receiving cardiac cellular patches than in those with patches loaded with growth factors only (although no significant difference was found between these two groups) [94]. In a pig model of MI, a gelatin hydrogel sheet incorporated with bFGF, human cardiosphere derived cells (hCDCs) (2   ×   107) or combination of both were delivered to ischemic heart [95]. bFGF/gelatin improved myocardial perfusion and LVEF while hCDCs improved LVEF and reduced infarct size. Co-delivery of hCDCs and bFGF/gelatin significantly enhanced hCDC engraftment and resulted in synergistically improved ventricular function and regional wall motion and reduced infarct size. However, such synergistic effects were not found when BMMSCs was co-delivered with bFGF [95], suggesting that the transplanted cell type is important in this particular method. In a sheep infarct model, the MI was treated with polyester CorCap ventricular constraint device together with adiposederived MSC (~5   ×   107), which were encapsulated in collagen and delivered to the infarct by injection. Such combination improved systolic and diastolic function and also reduced adverse remodeling [96]. Combination strategies have also been used to delivery vascular cells (VC) derived from hESC for cardiac repair [97]. In a rat model of MI, delivery of hESC-VCs (1   ×   106) using a synthetic metalloproteinase (MMP)-responsive matrix hydrogel containing a pro-angiogenic and pro-survival factor thymosin β4 (Tβ4) to the infarcted heart formed microvascular grafts and effectively preserved contractile performance at 3 d and 6 weeks post-MI, attenuated LV dilation, and decreased infarct size as compared to the PBS-injected control [97]. These hydrogels were formed by in situ crosslinking of vinyl sulfone-functionalized branched poly(ethylene glycol) (PEG) with a peptide that contains two cysteine residues flanking a matrix MMP substrate site [97], thus can be remodeled in response to elevated MMP-levels that occur after MI [98]. Laflamme et al [87] developed a Matrigel-based transplantation strategy to improve viability of hESCCM after transplantation in the infarcted rat heart [87]. Four days after ischemia-reperfusion (I/R) injury, hESC-CMs (1   ×   107) were delivered in control media, Matrigel, or a combination of Martrigel and a pro-survival cocktail (PSC), which consisted of immunosuppressant drugs, apoptosis inhibitors and pro-survival growth factors [87]. Grafts with surviving cells were found in hearts treated with hESC-CMs/Matrigel and hESC-CMs/Matrigel/PSC but not in hESC-CM/ media (control group) (figure 2(B)) [87]. hESC-CMs/ Matrigel/PSC group showed a significantly larger graft than hESC-CMs/Matrigel, suggesting a compounded 7

effect of Matrigel and PSC in promoting cell survival [87]. The engrafted hESC-CMs improved cardiac function after MI compared with controls receiving hESC derived non-cardiac cells or vehicle [87]. hESC-CMs/ Matrigel/PSC injected 3 weeks after initial MI also led to a successful engraftment, but failed to restore heart function or to alter adverse remodeling [99]. This strategy is now being tested in intra-myocardial delivery of hESC-CMs (1   ×   109)/Matrigel/PSC into non-human primate hearts after I/R injury [100]. Despite the low number of animals shown so far, a preliminary analysis showed hESC-CM survival and engraftment after 84 d. hESC-CM grafts were perfused by host vasculature. However, this preliminary study did not show the impact of transplanted cells on cardiac function. In addition, the animals that received treatment developed early arrhythmias [100], which can be problematic when translating to the clinic.

3.  Challenges and future studies Cardiac repair/regeneration is a multi-faceted process. Given the insufficient potential of the heart for self-regeneration after injury, replacement of the cardiomyocytes lost post MI is a promising strategy to achieve functional restoration. Therefore cellbased therapies for cardiac regeneration are aiming at delivering cells with potential to repair or regenerate to the damaged heart tissues. Though these transplanted cells reveal regenerative potential, functional gain has been small, primarily due to low cell retention and survival. This has been improved by the utilization of hydrogels, which improves retention of delivered cells and biomolecules and increase vascularization [7]. These hydrogel-based cell therapies have shown positive results for cardiac repair and regeneration in both small animal and large animal models. In hydrogel-based cardiac tissue regeneration, the hydrogels, cell source and cardiac tissue regeneration are three main aspects to be considered for the development of an effective cardiac regeneration strategy. Significant progress has been attained: (1) improved cell delivery and retention; (2) increased understanding of stem cell biology to optimize the cell source; (3) better comprehension of biological events associated with cardiac tissue repair/regeneration from molecular, cellular and physiological perspectives. Therefore continued advance in these aspects should continue to improve cell delivery strategies for biomaterials-based cardiac regeneration and will aid in the generation of new therapies to promote heart regeneration post ischemia. In order to ease the challenges for clinical translation of these approaches, the above-mentioned criteria, as well as clinical situation, should be integrated in the rational for the selection of animal model. A common concern for many approaches is the timing for cell delivery. The delivery of cells and hydrogels immediate following MI in animal models used in multiple studies

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is in favor of laboratory implementation of the procedure rather than considering the most common clinical scenery for patients where interventions will likely occur at a later time point [101]. Thus, consideration should also be given to delivery timing with regard to the stages of progress of MI [101].

4. Conclusions Significant progress has been made since the beginning of the use of hydrogels in cardiac regeneration. As described above, multiple hydrogels have been tested and the field is now moving towards more complex therapies by combining different hydrogels with bioactive molecules and cells. These studies have also given us a better understanding of the behaviour of cells when transplanted on their own and in combination with different strategies showing, for example, higher survival and retention when combination strategies are used. The advance of the studies using combination therapies for transplantation of human stem cell-derived cardiomyocytes with hydrogels and bioactive molecules into non-human primate models [100] demonstrate a tendency in the field to continue to explore these more sophisticated strategies to ameliorate cell transplantation and facilitate cardiac regeneration.

Acknowledgments The authors wish to acknowledge the following funding sources: This work was supported by a grant-in-aid from the Heart and Stroke Foundation of Canada (G-14-0006265) and by an operating grant from the Canadian Institutes of Health Research (137352) to SSN.

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