REVIEW ARTICLE

Application of conductive hydrogels in cardiac tissue engineering

Xiaoyi Ren¹, Ziyun Jiang¹ and Mingliang Tang^1,2,*

¹Institute for Cardiovascular Science & Department of Cardiovascular Surgery of the First Affiliated Hospital, Medical College, Soochow University, Suzhou, 215000, China; ²Co-innovation Center of Neuroregeneration, Nantong University, Nantong 226001, China

Abstract

The fatality rate of myocardial infarction ranks first in cardiovascular disease, which is myocardial necrosis caused by persistent ischemia and hypoxia caused by coronary artery occlusion. Myocardial infarction leads to the irreversible loss of a large number of cardiomyocytes. Exogenous cardiomyocyte supplement is an ideal method for the treatment of myocardial infarction. However, myocardial infarction leads to the loss of electrical conductivity of myocardial tissue at the infarcted site, and it is difficult for exogenous cardiomyocytes to integrate effectively. So, it is necessary to reshape the microenvironment of the infarcted site and restore its electrical conductivity. The construction of heart tissue engineering combined with biomaterials, cells and bioactive molecules is a hot topic in recent years. Conductive hydrogels, as an ideal scaffold material can promote the maturation of cardiomyocytes in vitro, give effective mechanical support to the infarcted site, improve the electrical conductivity of infarcted tissue, help exogenous cardiomyocytes integrate in vivo and restore heart function gradually. In this paper, we review the natural substrate materials used to make conductive hydrogels, the emerging trend of conductive materials and the applications of conductive hydrogels in heart tissue engineering.

Keywords: myocardial infarction; heart tissue engineering; conductive hydrogels

 

 

Cardiovascular disease is one of the most fatal diseases in the world, and its prevalence rate is still on the rise. Myocardial infarction (MI) caused by coronary artery occlusion is the main cause of morbidity and death (1). The hypoxic environment formed after myocardial infarction will cause a large number of cardiomyocytes damage or necrosis, scar fibrotic tissue to replace contractile muscle tissue, and adult cardiomyocytes have limited ability to repair themselves. Once apoptosis occurs, it may form permanent trauma, which will eventually lead to heart failure (2^, 3). The existing clinical treatment methods such as coronary artery bypass grafting and cardiac stent can only delay the deterioration of the disease, but can’t restore the infarcted myocardium (4). For terminally sick patients, heart transplantation is the only suitable treatment, but the shortage of donors, high costs and subsequent immune rejection treatment put constraints on this strategy (5).

In recent years, cardiac tissue engineering has been considered as an effective method to restore cardiac function after MI. Cardiac tissue engineering uses biomaterials, appropriate cells and bioactive molecules to construct cardiac scaffolds to simulate myocardial extracellular matrix, thus forming functional cardiac tissue and repairing damaged myocardium (6). The high water content and porosity of hydrogels, as one of the ideal cardiac scaffold materials, enable biological and chemical factors to be transported and have adjustable mechanical properties (7). However, most natural hydrogels lack electrical conductivity, and repairing infarcted myocardium requires the establishment of synchronous electrical signals between scar tissue and healthy myocardial tissue to evoke synchronous contraction of the whole heart. Therefore, it is necessary to develop hydrogels with high conductivity and toughness (8). The addition of conductive materials including carbon nanotubes, graphene, gold nanoparticles and conductive polymers to the non-conductive natural hydrogel matrix can greatly improve the electrical conductivity (Fig. 1). In this review, we will introduce different types of natural hydrogels, the emerging trends of conductive materials and the application of conductive hydrogels in heart tissue engineering.

Fig. 1. Application of conductive hydrogels in heart tissue engineering.

Natural hydrogels

Natural hydrogels commonly used in heart tissue engineering include alginate, chitosan, acellular tissue and decellularized extracellular matrix (dECM), collagen, gelatin, hyaluronic acid, etc. Natural hydrogels have good biocompatibility and biodegradability, supporting cell growth and reproduction (9). Especially, these polymers are designed as heart patches or injectable hydrogels to encapsulate different cells or bioactive molecules, and then implanted into the site of myocardial infarction to alleviate further deterioration of the MI area.

Alginate

Alginic acid is a natural polysaccharide from brown algae or bacteria, which is insoluble in water and organic solvents. Its sodium salt is soluble in water and can form a stable viscous solution (10, 11). The structure of alginate saline gel (AlgGel) is similar to that of extracellular matrix and it can provide mechanical support by injection or implantation into the infarct site. Sack et al. injected alginate into the left ventricle of large animal pigs, which significantly reduced poor left ventricular remodeling, compared with the untreated control group (12). Roche et al. proved that AlgGel-based heart patches allow structural self-organization of cardiomyocytes and endothelial cells (13). However, in clinical experiments, alginate degrades so fast in the body that it only has a temporary therapeutic effect.

Chitosan

Chitosan is a natural polysaccharide formed by deacetylation of chitin, which has biocompatibility and antibacterial properties (14). Its assembled hydrogels, sensitive to light and temperature, allow cells or bioactive molecules to dissolve into the solution without affecting their activity and then fix hydrogels in the injection area at nearly body temperature. Therefore, chitosan is widely used to synthesize thermosensitive chitosan-based hydrogels (15). In cardiac tissue engineering, Bejleri et al. found that injecting 24% acetylated chitosan hydrogels into the epicardium in ischemic environment can reverse harmful cardiac remodeling and inhibit the degree of fibrosis (16).

Decellularized extracellular matrix

Hydrogels made from extracellular matrix and obtained from acellular tissues or organs have drawn much attention in heart tissue engineering. Compared with other natural hydrogels, they can better simulate the extracellular matrix of the body and provide an ideal environment for repairing damaged myocardium (15). It could be shown that dECM-based hydrogels promote repair in animal models such as mice and pigs (17). Diaz et al. injected myocardial extracellular matrix hydrogels into the mouse model of chronic myocardial infarction (18). Four weeks later, it was observed that the left ventricular volume was retained and the apical wall thickened, which effectively prevented the further deterioration of the chronic MI model. Decellularized extracellular matrix can be spontaneously assembled to form gel at physiological temperature, but the degradation rate was fast and the cell retention in the target area was less (19).

Collagen

Collagen is not only the main structural protein of myocardial intima, but also the main component of extracellular matrix. The collagen hydrogels obtained by extraction or acellular method have good tissue compatibility and are beneficial to the attachment and survival of cardiomyocytes (20). Blackburn et al. confirmed that collagen hydrogels stimulated myocardial cytokine spectrum, promoted angiogenesis, and reduced fibrosis and cell death in injured heart tissue of MI mice (21). Although collagen hydrogels have been widely used in cells or drug delivery systems, their mechanical and electrical properties are weak and need to be further improved to overcome these limitations (20).

Gelatin

Gelatin as the product of partial hydrolysis of collagen, is more stable than collagen (22). Methacryloyl gelatin (GelMA) is often modified in tissue engineering. It has the characteristics of photocross-linking, is conducive to cell adhesion and growth, supports enzymatic degradation, and plays a key role in dermal wound healing, morphogenesis and tissue repair (23). Previous studies have shown that injection of GelMA carrier cells into rats can promote the formation of vascular network and tissue healing, but the UV cross-linking of GelMA may have a negative impact on the normal function of myocardial tissue, so that clinical transformation is limited (24).

Hyaluronic acid

Hyaluronic acid is also an important part of extracellular matrix and an acidic mucopolysaccharide. Viscosity gives it resistance to compression, which allows it to play a crucial role in load-bearing tissue, to be modified by a variety of functional groups and to introduce reaction points for specific cross-linking agents (20). Injectable hydrogels made from hyaluronic acid showed a significant increase in ejection fraction and short axis contraction, a decrease in infarct volume, and better angiogenesis in the treatment of MI (25).

These natural hydrogels have a wide range of sources, good biocompatibility and biodegradability, and can alleviate the deterioration of MI. However, because of their poor electrical conductivity, it is difficult to restore the electrical conductivity of infarcted tissue and limit the in vivo integration of exogenous cardiomyocytes. Therefore, natural hydrogels doped with conductive materials to develop ideal conductive hydrogels for the repair of damaged myocardium is the development direction of heart tissue engineering.

Conductive hydrogels

Ideal cardiac scaffold materials need to be equipped with the following properties at the same time: good biocompatibility, degradability, biomimetic mechanical and electrical properties, etc. Among them, conductivity is greatly essential to repairing myocardial tissue, because the conductivity of normal myocardium is about 1 mS/m, and non-conductive materials will hinder the transmission of electrical signals between myocardium and inhibit cardiac contraction (26). And the thickness of conductive Purkinje fibers in the heart is about 70 – 80 μm, so conductive nanomaterials have great potential in imitating the micro / nano composition and conductivity of these heart tissues (27). Therefore, doping different conductive nanomaterials into the natural hydrogels matrix to maintain the original elasticity of the hydrogels and enhance the electrical conductivity is an effective method to develop bionic conductive structures in vitro.

Electroactive polymer conductive hydrogels

Polypyrrole (PPy) is widely used in heart tissue engineering due to its simple synthesis, low production cost and good electrical conductivity. Although it can’t be directly used as a substrate for cell culture, it can regulate its electrical and biological activity by chemical modification, and prepare conductive hydrogels in soft hydrogels. He et al. synthesized a conductive polypyrrole-chitosan hydrogel (PPy-CHI) (28). In the equivalent circuit model, the resistance of PPy-CHI was 10 times lower than that of the control group. In the rat MI model, PPy-CHI reduced the resistivity of fibrotic scars and resynchronized cardiac contraction. Parchehbaf-Kashani et al. designed a conductive hydrogel based on cardiac ECM, and the doped PPy assumed the conductive function (29). The results showed that it was effective for the maturation and synchronous beating of newborn mouse cardiomyocytes in vitro. After that, a cardiac gel patch containing PPy (CG-PPy) was designed and transplanted into the MI rat model together with cardiac progenitor cells (CPCs) to effectively improve the left ventricular ejection fraction.

Polyaniline (PANI) and polythiophene (PT) are also often studied in the field of heart. The former shows biocompatibility in vitro and in vivo, and has been added to biodegradable polymers to prepare composites with mechanical, electrical and surface properties (30). Chakraborty et al. developed a new Arg-Gly-Asp (RGD) tripeptide sequence-based hydrogel and integrated it with pre-synthesized PANI in a nanosized manner to create a conductive hydrogel (31). The composite hydrogel has antibacterial properties, can bind to DNA, and promote cardiomyocyte tissue to become a spontaneous contraction system. In particular, the application of electrical stimulation will lead to the reversal of tissue contraction and has the potential to promote the growth of electrical cells. Polythiophene family members are an attractive alternative, showing better water dispersibility and high level of biocompatibility than polypyrrole and Polyaniline (32).

Carbon nanomaterial conductive hydrogels

Carbon nanomaterials, including carbon nanotubes (CNTs), graphene-based nanosheets, carbon nanoangles and carbon nanofibers, have attracted great attention in heart tissue engineering because of their excellent mechanical, topological and electrical properties (33). Carbon nanomaterials are often combined with non-conductive biomaterials due to their good electrical conductivity. Hydrogels have been a recent research hit.

Carbon nanotubes with diameters less than 1–100 nm are cylindrical hollow nanostructures with high strength, low weight and high conductivity, and their electrical activity and interconnection of cardiomyocytes exposed to CNTs are better (33). It has been successfully demonstrated that conductive hydrogels formed by CNTs encapsulated in methacrylate-based gelatin reproduce the layered anisotropic structure of myocardial tissue and produce a stronger, spontaneous and synchronized beat frequency in cardiomyocytes (34). The addition of CNTs to chitosan hydrogels also produces higher mechanical strength. Carbon nanotubes act as an electrical nano-bridge between cardiomyocytes to enhance electrical coupling and improve cardiomyocyte function.

Graphene-based nanosheets, including graphene (G), graphene oxide (GO) and reduced graphene oxide (rGO) nanosheets, are connected by monolayer carbon atoms in the form of hexagonal honeycomb lattices (33). Like CNTs, it has excellent electrical conductivity. Zhu et al. developed a hydrogel system, based on GelMA and oxidized dextran, and added reduced graphene oxide (35). By adjusting the amount of rGO, it could be found that the conductivity of hydrogels with concentration of 0.5 mg/mL rGO was similar to that of natural myocardium. Injecting human umbilical cord mesenchymal stem cells (UCMSCs) wrapped with conductive hydrogels into the MI site of rats can increase the expression level of cardiac troponin-I antigen (cTnI) and connexin 43, decrease the expression level of caspase-3 and improve the damaged heart tissue.

Carbon nanofibers are also often mixed with natural hydrogels. For example, Serafin et al. filled alginate and gelatin hydrogels with carbon nanofibers (CNF) to produce conductive hydrogels with uniform dispersion of CNF (36). In vitro experiments confirmed that the cell proliferation in the hydrogels was improved, compared with the control group, which may be due to its excellent mechanical properties.

Gold nanomaterial conductive hydrogels

Gold nanomaterials (AuNPs) are attractive nanomaterials in heart tissue engineering. Compared with carbon nanomaterials, gold nanomaterials are chemically inert and safer. It has been confirmed that gold nanoparticles can be used as a synthetic coupling agent for cardiomyocytes to improve electrical signal transmission between adjacent cells (33). Li et al. incorporated gold nanoparticles into methacrylate-based gelatin, and as a result, the conductive hydrogels were created, which showed greater vitality and maturity than the cardiomyocytes cultured on GelMA hydrogels. It could be proved that its electrical conductivity and mechanical properties were enhanced (37). Shilo et al. made the conductive hydrogels by mixing gold nanoparticles with acellular matrix and wrapping cardiomyocytes, which can absorb reactive oxygen species in vivo and in vitro (38). After injecting it into the mouse ischemia-reperfusion model, the scar size and inflammatory reaction can be reduced. Given that metal-based nanomaterials have been widely used in tissue engineering, gold nanomaterials have become one of the most promising materials for making conductive hydrogels because of their biocompatibility and high electrical conductivity.

Application

Promote myocardial regeneration

After myocardial infarction, a large number of cardiomyocytes will be lost, and the original viable myocardial tissue in the ischemic site will gradually be replaced by non-conductive fibrous tissue, destroying the coordinated electrical activity of the heart, causing the heartbeat to be out of sync, thus affecting cardiac contraction. Most functional cardiomyocytes in adult heart are difficult to regenerate. Given that, recently, it is hot to transfer functional cardiomyocytes to MI region. Induced pluripotent stem cells, mesenchymal stem cells and myocardial progenitor cells have been widely used in stem cell therapy (39). However, directly injected stem cells can’t fully differentiate into cardiomyocytes, most of which will die in the process of transplantation and be unable to grow in the specified location (40). In addition, the surviving cells can’t integrate well with the host myocardium, so wrapping stem cell-induced cardiomyocytes into the damaged heart in conductive hydrogels is a good strategy to promote cardiomyocyte maturation and help myocardial regeneration (2).

Conductive hydrogels based on GelMA and reduced graphene oxide have similar electrical conductivity to natural myocardium. Umbilical cord mesenchymal stem cells (UCMSCs) cultured in hydrogels network can be stably released, and the expression of CTnI is upregulated compared with stem cells cultured on tissue culture plate (35). CTnI involved in calcium-induced cardiac contraction is a unique regulatory protein in myocardial tissue. It is proved that conductive hydrogels can effectively promote the differentiation of UCMSCs, improve the survival rate of intracardiac injection of stem cells and help myocardial regeneration. Li et al. also incorporated graphene oxide into GelMA and applied electrical stimulation to promote the maturation of hydrogels central muscle cells (41). The results showed that the conductive hydrogels scaffold improved the orientation order of muscle fibers under electrical stimulation.

Vascular remodeling

The heart is an organ composed of myocardium and blood vessels, which are excessively damaged after MI, so the treatment of myocardial infarction should not only promote myocardial regeneration, but also repair damaged blood vessels and reconstruct functional vessels (42). In addition, the formation of new blood vessels can restore the blood and nutritional supply of ischemic myocardium. Xiong et al. designed a vascularized conductive elastic patch, in which porous reduced graphene oxide and PPy were added to poly (hydroxyethyl methacrylate) gel, coronary artery casting was used as a preset template, three-dimensional bionic blood vessels were formed in the conductive hydrogels patch, and rat aortic endothelial cells and cardiomyocytes were cultured in the channel (8). In vitro experiments demonstrated excellent cardiomyocyte functionalization and synchronous contraction. Compared with the simple cell injection group, the density of microvessels and arterioles in the infarcted area was the highest, which significantly reversed the malignant expansion of the left ventricle. Vascular remodeling is not only an important process of myocardial repair, but also the key application of conductive hydrogels.

Antioxidation

Antioxidation is a myocardial repair strategy for MI microenvironment. A large number of reactive oxygen species (ROS) are formed in the anoxic environment of MI region, which further accelerates cardiomyocyte injury (43). In addition, ROS can produce inflammatory cytokines through rapid stimulation signal transduction, leading to severe inflammation. Therefore, conductive hydrogels that absorb ROS are being developed. Zhan et al. constructed a conductive hydrogel capable of scavenging reactive oxygen species, by integrating antioxidant TEMPOL into the peptide, and then uniformly combining polypyrrole with the target peptide by nanoengineering (44). In vitro experiments show that this hydrogel can effectively remove ROS from cardiomyocytes. Echocardiography and histological analysis also show that this hydrogel can promote cardiac repair and reconstruction of cardiac function. The conductive hydrogel formed by the combination of gold nanoparticles and dECM showed good antioxidant capacity in vivo and in vitro, especially in the ischemia-reperfusion model, showing good cardiac repair potential (38). When the occluded blood vessels are reopened, oxygen molecules return to the ischemic tissue, resulting in an imbalance between the rate of ROS production and the ability of tissue to detoxify these molecules. If the existing oxidative stress environment is not excluded, the effect of heart patch implantation will be affected, and the treatment results will be endangered.

Summary and prospect

Diverse natural hydrogels are introduced in this paper. The hydrogels which are easy to obtain are more likely to gain popularity among researchers. Good biocompatibility, biodegradability and low immunogenicity are their advantages, while poor mechanical properties and non-conductivity are their disadvantages. Conductive materials have been widely used in tissue engineering, whether it is convenient synthesis of conductive polymers or nano-sized carbon materials, metal materials have excellent electrical conductivity. Therefore, it is a promising strategy to obtain conductive hydrogels by crosslinking / mixing natural hydrogels with excellent mechanical properties and conductive materials. However, it is worth noting that the addition of high content of conductive materials may lead to the decline of the original mechanical properties of hydrogels or cause a certain degree of cytotoxicity, so different doping ratios need to be tested in the process of making conductive hydrogels.

Conductive hydrogels have great potential in cardiac tissue engineering, like promoting myocardial regeneration, helping vascular remodeling, reducing oxidative stress and so on. First of all, the ideal conductive hydrogels are non-cytotoxic and have mechanical properties that can meet the contraction of the heart. Secondly, it has a conductivity similar to that of the myocardial tissue. Finally, it needs to show a variety of biological functions to repair the cardiac function of MI damage. All the hydrogels mentioned above merely have one or two functions, so it cannot perfectly simulate heart tissue. Apart from that, these hydrogels are basically used for short-term experiments in small animals, and there is still a long way to go, in order to achieve the real clinical transformation. Therefore, it is necessary to develop conductive hydrogels with a variety of biological functions and conduct long-term experiments in large animals to ensure that they are not potentially toxic. Although there are many ups and downs in the research, cardiac patches or injectable conductive hydrogels show great potential in cardiac tissue engineering and will eventually be used in cardiac clinical research.

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