**4. Injectable gels**

#### **4.1. Rationale**

As previously stated, cell cardiomyoplasty presents problems in terms of cell attachment and survival. Cells usually reside in a determined microenvironment which regulates their fate and function. The surrounding ECM with its chemical and biophysical cues is a key element, so the lack of cell-ECM interaction limits their survival [73]. To try to overcome the problems of cells supply, alternative approaches are considered in current studies. The use of natural or synthetic materials in an injectable format, alone or together with cells (figure 1 b), has been investigated to limit remodeling and improve both cell attachment and survival upon implantation in the heart. Ideally, they should be tailored to be amenable to delivery with minimally invasive catheter based procedures [69]. The injectable materials have to cure or self-assemble rapidly (without the need or the release of toxic components) once delivered in the site of interest. As injected, they adopt the shape of the cavity, and may increase the stiffness and thickness of the ventricular wall [74]. Simulations showed that the injection of noncontractile materials with proper mechanical properties can contribute to limit the stress the ventricular wall withstands, thus helping to limit the remodeling [75].

These materials can help to keep the cells in the site of interest, provide them a 3D environment and also protect them from the hostile environment represented by the cytokines and hypoxic conditions, reactive oxygen species, etc., consequence of the infarcted condition of the site [41]. The injected gels can provide a cell friendly environment that will prevent anoikis [69]; they can also include adhesion motifs and then actively contribute to cell attachment. Moreover, they can be used as a controlled release system providing in a sustained way drugs or growth factors to improve cells survival, integration and proliferation [32]. And in the case of bioactive materials, their degradation products may provide additional chemicals that stimulate cells.

Among others, the ideal injectable material should be biodegradable, have a low immuno‐ genity, be no cytotoxic, non-adhesive and have antithrombogenic properties, adequate mechanical properties, provide stiffness to the scar but at the same time being compliant with the heart beating and transmit properly the mechanical stimuli to the cells, induce angiogenesis or at least not disturb the angiogenic activity after incorporation, be capable of delivering cells and or bioactive molecules [76]. Next, some of the materials investigated for their potential use as injectable ones are described.

#### **4.2.** *In situ* **gelling biomaterials employed**

#### *4.2.1. Natural materials*

#### *Fibrin*

A different approach based on the same idea of providing cell-cell connections and ECM to the implanted cells to improve their retention and survival is to implant them as spherical cellbodies. Human amniotic fluid stem cells (hAFSC) cultured in a methylcellulose hydrogel to form cell aggregates were implanted in immunosuppressed rats as cell-bodies, and cell retention and engraftment were enhanced as compared with disaggregated cells. This enhancement led to functional improvement and limited the progression of heart failure [72].

As previously stated, cell cardiomyoplasty presents problems in terms of cell attachment and survival. Cells usually reside in a determined microenvironment which regulates their fate and function. The surrounding ECM with its chemical and biophysical cues is a key element, so the lack of cell-ECM interaction limits their survival [73]. To try to overcome the problems of cells supply, alternative approaches are considered in current studies. The use of natural or synthetic materials in an injectable format, alone or together with cells (figure 1 b), has been investigated to limit remodeling and improve both cell attachment and survival upon implantation in the heart. Ideally, they should be tailored to be amenable to delivery with minimally invasive catheter based procedures [69]. The injectable materials have to cure or self-assemble rapidly (without the need or the release of toxic components) once delivered in the site of interest. As injected, they adopt the shape of the cavity, and may increase the stiffness and thickness of the ventricular wall [74]. Simulations showed that the injection of noncontractile materials with proper mechanical properties can contribute to limit the stress the

These materials can help to keep the cells in the site of interest, provide them a 3D environment and also protect them from the hostile environment represented by the cytokines and hypoxic conditions, reactive oxygen species, etc., consequence of the infarcted condition of the site [41]. The injected gels can provide a cell friendly environment that will prevent anoikis [69]; they can also include adhesion motifs and then actively contribute to cell attachment. Moreover, they can be used as a controlled release system providing in a sustained way drugs or growth factors to improve cells survival, integration and proliferation [32]. And in the case of bioactive materials, their degradation products may provide additional chemicals that stimulate cells.

Among others, the ideal injectable material should be biodegradable, have a low immuno‐ genity, be no cytotoxic, non-adhesive and have antithrombogenic properties, adequate mechanical properties, provide stiffness to the scar but at the same time being compliant with the heart beating and transmit properly the mechanical stimuli to the cells, induce angiogenesis or at least not disturb the angiogenic activity after incorporation, be capable of delivering cells and or bioactive molecules [76]. Next, some of the materials investigated for their potential use

ventricular wall withstands, thus helping to limit the remodeling [75].

**4. Injectable gels**

284 Regenerative Medicine and Tissue Engineering

as injectable ones are described.

**4.1. Rationale**

Fibrin is a natural biopolymer that forms the natural provisory matrix for wound healing. It is FDA approved for many applications and there are different preparations commercially available, but it can also be obtained from autologous origin [77]; it is biocompatible, not toxic, or inflammatory [78]. Besides, some of the degradation products of fibrin have interesting properties, like improving healing promotion or a protective effect against myocardial reperfusion injury [79]. Fibrin contains arginine-glycine-asparagine (RGD), which are known cell adhesion motifs [77]; it is cytoprotective for anoxia and provides a favorable microenvir‐ onment for cardiomyogenic differentiation of marrow-derived cardiac stem cells [77]. It can also be used as a controlled release system [80]. In sum, fibrin as a gel is a potential candidate to enhance cell adhesion and survival. To obtain the fibrin, fibrinogen monomers in saline solution are mixed with thrombin and they polymerize forming a 3D net by mechanisms similar to normal clotting *in vivo* [81]. The properties of the network can be tailored by modifying the polymerization process.

A concern about translating the fibrin glue for cardiac tissue engineering into the clinic is the risk of inducing intravascular thrombosis [79]. The concentrations of fibrin amenable to delivery through current percutaneous catheters have been studied, demonstrating the feasibility of using fibrin in a non-invasive injectable application [81]. The injection of fibrin alone was proved to preserve left ventricular geometry and cardiac function in a rat acute MI model [82]. But it has also been combined with many types of cells. As an example, it was employed to deliver bone-marrow derived mesenchymal stem cells, which enhanced cell retention and prevented their redistribution in other organs, improving the beneficial effects of the treatment [81]. Injection of fibrin combined with myoblasts [82], bone marrow stem cells [83] or with autologous endothelial cells [84], improved the results obtained with cells alone.

#### *Chitosan*

Chitosan (CHT) is a natural cationic polysaccharide, obtained from the deacetylation of chitin of the mollusks, crustaceans and insects. It is soluble in acidic aqueous solution but after neutralization forms a gel-like precipitate [85]. CHT exhibits numerous positive biological and physicochemical properties: biocompatibility, non immunogenicity, and can be conjugated with various molecules thanks to the amino groups on the polysaccharide backbone [86]. A thermally responsive chitosan-based polymer was capable of scavenging the reactive oxygen species produced by the ischemic conditions and recruit key chemokines for stem cell homing such as SDF-1. As a cell delivery system with adipose-derived mesenchymal stem cells, this material was capable of improving the microenvironment for the cells when injected in the infarcted myocardium of rats, improving their survival and engraftment [87]. Chitosan mixed with collagen has been conjugated with QHREDGS (peptide thought to mediate attachment and survival responses of cardiomyocytes) in the format of a thermoresponsive hydrogel to improve maturation and metabolic activity of cardiomyocytes [86]. Alginate-chitosan nano‐ particles have been loaded with placental growth factor (PlGF) to increase the left-ventricular function and vascular density in rats [88].

#### *Matrigel*

Matrigel is a commercial ECM proteins mixture that undergoes a temperature mediated solgel transition, and is obtained from the ECM of mouse sarcoma cells [27]; its clinical application is limited precisely by the source from which it is obtained. It has been implanted alone and in combination with mouse ESC [89] or neonatal cardiomyocytes [90] into a mice model of infarcted myocardium. The gel prevented worsening of the cardiac function, but animals receiving both Matrigel and cells maintained more wall thickness and preserved better cardiac function in terms of fractional shortening and regional contractility [91].

#### *Hair keratin*

Keratin materials can be obtained from hair, importantly from autologous source. More than 30 growth factors are involved in hair morphogenesis, and the residual of them remains in the keratin, what can be beneficial for cardiac repair. Lyophilized keratin powders have the ability to self-assemble upon addition of water, and form gels. Keratin has been implanted onto infarcted rat hearts, and native cardiomyocytes as well as endothelial cells were able to infiltrate the keratin gel, promoting angiogenesis without inducing inflammation; after 2 months animals exhibited preservation of cardiac function and limited ventricular remodeling [92]. These improvements were attributed to the biomaterial's contribution to the mechanical support to the ventricular wall and the presence of cell binding motifs in it.

#### *Alginate*

Alginate is a linear block co-polymer of (1-4)-linked β-D-mannuronate and α-L-guluronate residues obtained from seaweed. It is a negatively charged polysaccharide that gels by the presence of calcium ions and is non-thrombogenic [4]. The properties of this material can be tuned either by changing the concentration of the solutions or by controlling the molecular weight. Greater concentrations will increase mechanical strength but also will increase the solution viscosity and the degradation time of the gel [27].

Alginate has been used as an injectable material in recent and old infarcts in rats, and it was observed that its injection augmented the scar thickness and limited systolic and diastolic dysfunction [93]. It has also been proposed as a controlled delivery system: based on the different binding affinity of alginate to insulin-like growth factor-1 IGF-1 and hepatocyte growth factor HGF, a dual delivery system of these factors was developed [94]. The hydrogel beads protected the proteins from degradation maintaining their bioactivity and increasing the therapeutic effect of the system.

Alginate sustains very low protein adsorption and it does not support mammalian cells attachment [95], but it can be combined with adhesion motifs to improve its attachment properties. Its conjugation with RGD increased the arteriole density in a rodent model of chronic ischemic cardiomyopathy [96]. However, the combination of alginate with RGD and tyrosine–isoleucine– glycine–serine–arginine (YIGSR) reduced the therapeutic effects of the hydrogel in terms of scar thickness, left ventricular dilation and function [97]. Another modification of alginate has been the addition of the electrical conducting polymer polypyrrole [98], which increases arteriogenesis and promotes myofibroblasts infiltration.

#### *Hyaluronic acid*

improve maturation and metabolic activity of cardiomyocytes [86]. Alginate-chitosan nano‐ particles have been loaded with placental growth factor (PlGF) to increase the left-ventricular

Matrigel is a commercial ECM proteins mixture that undergoes a temperature mediated solgel transition, and is obtained from the ECM of mouse sarcoma cells [27]; its clinical application is limited precisely by the source from which it is obtained. It has been implanted alone and in combination with mouse ESC [89] or neonatal cardiomyocytes [90] into a mice model of infarcted myocardium. The gel prevented worsening of the cardiac function, but animals receiving both Matrigel and cells maintained more wall thickness and preserved better cardiac

Keratin materials can be obtained from hair, importantly from autologous source. More than 30 growth factors are involved in hair morphogenesis, and the residual of them remains in the keratin, what can be beneficial for cardiac repair. Lyophilized keratin powders have the ability to self-assemble upon addition of water, and form gels. Keratin has been implanted onto infarcted rat hearts, and native cardiomyocytes as well as endothelial cells were able to infiltrate the keratin gel, promoting angiogenesis without inducing inflammation; after 2 months animals exhibited preservation of cardiac function and limited ventricular remodeling [92]. These improvements were attributed to the biomaterial's contribution to the mechanical

Alginate is a linear block co-polymer of (1-4)-linked β-D-mannuronate and α-L-guluronate residues obtained from seaweed. It is a negatively charged polysaccharide that gels by the

tuned either by changing the concentration of the solutions or by controlling the molecular weight. Greater concentrations will increase mechanical strength but also will increase the

Alginate has been used as an injectable material in recent and old infarcts in rats, and it was observed that its injection augmented the scar thickness and limited systolic and diastolic dysfunction [93]. It has also been proposed as a controlled delivery system: based on the different binding affinity of alginate to insulin-like growth factor-1 IGF-1 and hepatocyte growth factor HGF, a dual delivery system of these factors was developed [94]. The hydrogel beads protected the proteins from degradation maintaining their bioactivity and increasing

Alginate sustains very low protein adsorption and it does not support mammalian cells attachment [95], but it can be combined with adhesion motifs to improve its attachment properties. Its conjugation with RGD increased the arteriole density in a rodent model of chronic ischemic cardiomyopathy [96]. However, the combination of alginate with RGD and tyrosine–isoleucine– glycine–serine–arginine (YIGSR) reduced the therapeutic effects of the

The properties of this material can be

function in terms of fractional shortening and regional contractility [91].

support to the ventricular wall and the presence of cell binding motifs in it.

presence of calcium ions and is non-thrombogenic [4].

the therapeutic effect of the system.

solution viscosity and the degradation time of the gel [27].

function and vascular density in rats [88].

286 Regenerative Medicine and Tissue Engineering

*Matrigel*

*Hair keratin*

*Alginate*

Hyaluronic acid (HA) is a non-sulfated glycosaminoglycan prevalent in the extracellular matrix of many tissues. HA plays an important role in homeostasis, transport of nu‐ trients and also mediates the inflammation and repair processes. It is biocompatible, nonimmunogenic, biodegradable and has different biological activities depending on its molecular weight. Precisely the low molecular weight degradation products of HA stimulate angiogenesis and endothelial cell proliferation and migration [99]. It can be functionalized to improve its biological development, for example with PEG-SH4 [100]. Moreover, it is a FDA-approved material for its use in humans in certain applications like dermal and intra-articular injection.

There are already commercially available *in situ* crosslinkable HA-derived hydrogels. Differ‐ ent types of HA hydrogels have been compared with commercial fibrin, poly(vinyl alcohol) chitosan and elastin hydrogels, in terms of *in vitro* degradation rates and cytotoxicity and *in vivo* degradation, immune response and angiogenic potential [76]. Traut's grafted HA hydrogel and periodate oxidated HA hydrogel, especially the first one, demonstrated to be the most suitable for new artery formation in ischemic myocardium because they were both digested within 2 weeks with low immune response and strong angiogenesis compared with the other examined hydrogels.

HA alone does not support cell adhesion. Cardiosphere-derived cells were delivered using a thiolated hyaluronan-based hydrogel crosslinked with thiol-reactive poly(ethylene glycol) diacrylate and covalently linked or not with thiolated denatured collagen. It was observed that the retention rate achieved with the hydrogel without collagen was similar to that of cells delivered in phosphate buffer saline (PBS), either by a low physical retention or poor cell survival and adhesion of HA [101]. In the *in vivo* study in a mouse model of myocardial infarction, some functional benefits were observed though.

#### *Collagen*

Collagen supports growth and survival of cardiomyocytes *in vitro*, and is one of the main components of the ECM in the adult heart [102]. Commercial collagen alone has been im‐ planted in animal models showing improvements in ventricular cardiac function and geom‐ etry [103]. In another study in a myocardial infarction model in rats (with ischemia-reperfusion model this time) increased capillarity density and myofibroblasts infiltration after 5 weeks were reported [104].

The therapeutic potential of injectable collagen has been evaluated in combination with different cell types. Bone marrow stem cells were injected via catheter in a swine model in combination with collagen, demonstrating the feasibility of a non-invasive delivery of this system [105]. Collagen was also used as a carrier for mesenchymal stem cells (MSC) transplantation to improve the retention of the cells in the infarcted myocardium [106]. 4

weeks after implantation, rats receiving cells in saline suspension, had the implanted cells in remote organs, whereas in animals receiving the cells with collagen, were detected to a lesser extent in remote organs. However, cardiac function was improved in animals receiving cells in saline and collagen alone but not in the combined collagen MSC group. The mechanisms underlying this negative interaction (controverted in other works) are unknown, but is suggested that collagen may limit oxygen and nutrients diffusion, and compromise cell-cell interactions. In another study, collagen combined with chondroitin 6 sulfate was employed to deliver CD-133+ progenitor cells derived from peripherial blood after expansion *in vitro* [107]. It was expected that the material would improve cell adhesion and survival into ischemic hind limb athymic rats. The collagen increased two-fold the number of cells retained when implanted alone; the implanted material was vascularized and the injected cells added into vascular structures.

#### *Gelatin*

It is a non-immunogenic partially degraded product of collagen [108]. It has been inject‐ ed as a hydrogel in rat infarcted hearts bare or loaded with basic fibroblast growth factor; adding the factor improved arteriogenesis, ventricular remodeling and function [109]. Basic fibroblast growth factor has also been delivered with gelatin microspheres [110], induc‐ ing angiogenesis and improving cardiac function. The loaded nanoparticles induced an increase in the blood flow in the infarct border (thanks to stimulated angiogenesis), and as a result left ventricular function was improved.

#### *ECM-derived materials*

A different approach is based on decellularized tissues, their digestion and injection. This type of materials has the advantage of containing a physiological proportion of the native compo‐ nents of the ECM [102] and cues for cell-matrix interactions. ECM coming from different tissues has been studied, and apparently the ECM of each tissue has its unique combination of proteins and proteoglycans. This makes of myocardial decellularized matrix, among all other tissues matrices, the best candidate for myocardial repair when it is available [111]. Decellularized porcine myocardial tissue able to self-assemble into a nanofibrous structure similar to collagen *in vitro* at 37°C and deliverable *in vivo* upon catheter injection was tested in rats. It induced endothelial cells and smooth muscle cells migration increasing the arteriole formation at 11 days post-injection [111].

Small intestinal submucosa (SIS) is a dense sheet of acellular extracellular matrix. This material is used in the clinic for accelerated wound healing. SIS supports proliferation, attachment and migration of various cell types and stimulates angiogenesis thanks to the growth factors and binding motifs embedded in the matrix. Two different types of commercial available SISderived gels have been studied as an injectable material for cardiac repair in a murine model [112]. The two materials differed in the concentration of basic fibroblast factor, obtaining best result the material richer in this factor. In another work, an emulsion of digested ECM from SIS was injected into infarcted rat hearts, improving cardiac function, increasing neovascula‐ rization and promoting cell recruitment [113].

#### *4.2.2. Synthetic materials*

weeks after implantation, rats receiving cells in saline suspension, had the implanted cells in remote organs, whereas in animals receiving the cells with collagen, were detected to a lesser extent in remote organs. However, cardiac function was improved in animals receiving cells in saline and collagen alone but not in the combined collagen MSC group. The mechanisms underlying this negative interaction (controverted in other works) are unknown, but is suggested that collagen may limit oxygen and nutrients diffusion, and compromise cell-cell interactions. In another study, collagen combined with chondroitin 6 sulfate was employed to deliver CD-133+ progenitor cells derived from peripherial blood after expansion *in vitro* [107]. It was expected that the material would improve cell adhesion and survival into ischemic hind limb athymic rats. The collagen increased two-fold the number of cells retained when implanted alone; the implanted material was vascularized

It is a non-immunogenic partially degraded product of collagen [108]. It has been inject‐ ed as a hydrogel in rat infarcted hearts bare or loaded with basic fibroblast growth factor; adding the factor improved arteriogenesis, ventricular remodeling and function [109]. Basic fibroblast growth factor has also been delivered with gelatin microspheres [110], induc‐ ing angiogenesis and improving cardiac function. The loaded nanoparticles induced an increase in the blood flow in the infarct border (thanks to stimulated angiogenesis), and

A different approach is based on decellularized tissues, their digestion and injection. This type of materials has the advantage of containing a physiological proportion of the native compo‐ nents of the ECM [102] and cues for cell-matrix interactions. ECM coming from different tissues has been studied, and apparently the ECM of each tissue has its unique combination of proteins and proteoglycans. This makes of myocardial decellularized matrix, among all other tissues matrices, the best candidate for myocardial repair when it is available [111]. Decellularized porcine myocardial tissue able to self-assemble into a nanofibrous structure similar to collagen *in vitro* at 37°C and deliverable *in vivo* upon catheter injection was tested in rats. It induced endothelial cells and smooth muscle cells migration increasing the arteriole formation at 11

Small intestinal submucosa (SIS) is a dense sheet of acellular extracellular matrix. This material is used in the clinic for accelerated wound healing. SIS supports proliferation, attachment and migration of various cell types and stimulates angiogenesis thanks to the growth factors and binding motifs embedded in the matrix. Two different types of commercial available SISderived gels have been studied as an injectable material for cardiac repair in a murine model [112]. The two materials differed in the concentration of basic fibroblast factor, obtaining best result the material richer in this factor. In another work, an emulsion of digested ECM from SIS was injected into infarcted rat hearts, improving cardiac function, increasing neovascula‐

and the injected cells added into vascular structures.

288 Regenerative Medicine and Tissue Engineering

as a result left ventricular function was improved.

*Gelatin*

*ECM-derived materials*

days post-injection [111].

rization and promoting cell recruitment [113].

Synthetic materials are made in the laboratory from primary building blocks, so their proper‐ ties can be tuned to match desired characteristics. Besides, they are free from animal origin components and the risks related therewith.

#### *Thermosensitive hydrogels*

This group of materials has temperature-dependant sol to gel transition. The great advantage of this group of materials is the possibility to tune their properties for them to undergo the gelation transition around body temperature [114]. In this way they can be comfortably manipulated and injected and only when they are inside the body they will undergo the transition.

Some of the materials of this group are based on N-isopropylacrylamide (NiPAAm). It is non biodegradable, but copolymerized with degradable polymers becomes biodegradable. For instance, NiPAAm was copolymerized with acrylic acid (AAc) and hydroxyethyl methacry‐ late-poly(trimethylene carbonate) (HEMAPTMC) [115]. The ratio of each material was adjusted to obtain a hydrogel at 37ºC. It can also be degraded *in vitro* with a mass loss over 85% after 5 months. This material was injected *in vivo* in rats and proved to preserve the area of the left ventricular cavity and contractility. Tissue ingrowth, a thicker left ventricle (LV) wall and greater capillarity density were also found when compared with PBS controls. After 8 weeks, a layer of smooth muscle cells with contractile phenotype was formed next to the remaining material.

Another family of thermoresponsive hydrogels based on polycaprolactone, N-isopropylacry‐ lamide, 2-hydroxyethyl methacrylate and dimethyl-g-butyrolactone acrylate has been developed [116]. Cardiosphere derived cells (CDC) combined with the hydrogel were suitable for myocardial injection and the solutions formed solid gels within 5 s at 37ºC. Hydrogels with different mechanical properties were obtained and it was shown that they influence the fate of the CDC differentiation. Another thermoresponsive material containing biodegradable dextran chain grafted with hydrophobic poly(ε-caprolactone)-2-hydroxylethyl methacrylate (PCL-HEMA) chain and thermoresponsive poly(N isopropylacrylamide) (PNIPAAm) (Dex-PCL-HEMA/PNIPAAm) has been synthesized. It can shift from sol to gel within 30 s and is reversible within the same time frame [117]. It was injected in rabbits, 4-days post-infarction. Histological analyses one month later indicated that the material prevented the scar expansion and thinning of the wall. Left ventricular ejection fraction was increased and it attenuated left ventricular systolic and diastolic dilation.

#### *Poly (Ethylene Glycol) (PEG)*

A strategy based on non-biodegradable *in situ* crosslinkable PEG hydrogel has been developed, to provide a permanent support to limit the remodeling [118]. Its therapeutic effects were tested in rat myocardial infarction model at short and long term. Beneficial effects were observed at 4 weeks, but at long term (13 weeks) it was unable to prevent the dilation. Besides, the material injection induced some inflammatory response.

An injectable α-cyclodextrin/poly(ethylene glycol)–b-polycaprolactone-(dodecanedioic acid) polycaprolactone–poly(ethylene glycol) (MPEG–PCL–MPEG) hydrogel was used to deliver and encapsulate bone marrow stem cells into infarcted myocardium [119]. The CD/MPEG-PCL-MPEG hydrogel alone does not induce angiogenesis, but can serve as a support in the infarcted zone and contribute to inhibit the left ventricular remodeling. One month after the injection of the gel combined with cells, cell retention and survival and the density of vessels were increased when compared with cells injection alone; moreover, the gel was absorbed, ventricular dilation was limited and the ventricular ejection fraction improved.

PEG-based temperature-sensitive hydrogels have also been combined with growth factors or other molecules. VEGF was mixed or conjugated with the aliphatic polyester hydrogel poly(δvalerolactone)-block-poly(ethylene glycol)-block-poly(δ-valerolactone) (PVL-b-PEG-b-PVL); the sustained VEGF release during the degradation time of the hydrogel translated into an improvement of the myocardial and functional recovery, in dependence of the preparation method [120]. In another work, a metalloproteinase-responsive PEG-based hydrogel was synthesized to be a thymosin β4 (a pro-angiogenic and pro-survival factor) delivering scaffold. It was implanted combined with endothelial and smooth muscle cells derived from human embryonic stem cells (hESC) in rats [121]. The gel provides structural organization and when was loaded with cells and thymosin b4 enhanced more contractile performance than when the hydrogel was only loaded with the factor, because of their paracrine effect. Another PEG-based hydrogel, α-cyclodextrin/MPEG–PCL–MPEG, was tested as a delivery system for erythro‐ poietin (EPO) [122], a hormone that plays a protective role in the infarcted myocardium. Rats treated with this system showed limited cell apoptosis and increased neovasculature forma‐ tion; also infarct size was reduced and cardiac function improved.

PEG in the format of nanoparticles has also been studied. They can be injected intravenously, circulate in the body for long periods and bind only to desired tissues. Nanoparticles targeting the infarcted myocardium were developed based on the overexpression of angiotensin II type 1 (AT1) receptor in the infarcted heart [123]. The system was formed by a vehicle and a targeter, a ligand specific to AT1 that will make the nanoparticles bind specifically. The vehicle was 142 nm diameter PEGylated liposomes, which could carry therapeutic molecules and release them in a controlled way. This system was proved to target the infarcted heart in mice model, but not the healthy.

#### *Self Assembling Peptides (SAPs)*

SAPs are short peptides capable of forming hydrogels at physiological pH and osmolarity [124]. When the SAPs solution is placed in contact with ions or pH is changed, the charges are partially neutralized and a hydrophobic packing takes place forming beta-sheet structures, constituting fibers that build a 3D network if the concentration is high enough. Fibers shape is different depending on the nature of the employed peptides. In the particular case of the RAD16 ionic peptides family (R: arginine, A: alanine, D: aspartate) fibers thicknesses are of 5-10 nm.

Peptides can be combined with cells to encapsulate them within the peptide network [125]. RAD16-I (AcN-RADARADARADARADA-CNH2) has proved to be a useful synthetic gel capable of maintaining the cells in the site of interest, and has been used as a delivery system of different types of cells to the heart. On the contrary, when it was implanted alone limited improvements were observed in the infarct area and the remodeling process. RAD16-II (AcN-RARADADARARADADA-CNH2) peptide has been shown to create microenvironments in the infarcted myocardium that are infiltrated with endothelial and smooth muscle cells, suggesting a potential for vascularization [124]. It was also observed that combining RAD16- II with neonatal cardiomyocytes the density of endogenous *α*-sarcomeric actin positive cells increased.

As stated, SAPs gels can be modified to incorporate growth factors or drugs. The self assem‐ bling peptide RAD16-II has been used as a drug vehicle to deliver both platelet derived growth factor and fibroblast growth factor (PDGF-BB and FGF-2) [126]. The first is arteriogenic and the second is angiogenic; their combination targets endothelial cells (EC) and vascular smooth muscle cells (VSMC). Infarct size and cardiomyocyte apoptosis were considerably reduced in rats. The capillary and arterial density was recovered, and cardiac function was almost recovered. This system also induced long-lasting vessel formation. RAD16-II combined with IGF-1, a cytokine that protects and promotes cardiomyocytes growth, has also been used as a delivery system for cardiomyocytes [127]. The addition of IGF-1 acted reducing cell apoptosis and improving systolic function.
