| Summary: | Cardiovascular disease (CVD) is the most leading cause of mortality in the USA and costs $300 billion per year. Heart attacks (or myocardial infarction: MI) is currently one of the most frequent types of CVD in the world. In recent years, diverse technologies, including cell transplantation, injectable biomaterials, and cardiac patches, have been developed to rebuild the function of infarcted myocardium. Among these, cardiac patches can adequately and simultaneously meet the biochemical, electrical, and mechanical demands of the native heart tissue to promote regeneration following MI. Such patches, as successful ex vivo models, can precisely replace scar tissue and improve cardiac function by supporting and thickening the damaged myocardium. From a biomaterial perspective, the engineered cardiac patches for the treatment of damaged heart tissues after MI are normally produced by seeding heart cells within 3D porous biomaterial scaffolds that mimic the extracellular matrix (ECM) of the native tissue and organs. These biomaterials, which are usually made of either biological polymers (collagen, gelatin, alginate, etc.) or synthetic polymers (i.e., PLGA, PLA, PGS), help cells to organize into functioning tissues. But poor conductivity of these materials limits the ability of these scaffolds to contract strongly as a unit. Therefore, the proper function of engineered cardiac tissues requires mimicking the anisotropic structure of the native myocardium, which can be achieved using a series of biophysical and topographical features such as the incorporation of conductive additives. In this research, we aim to integrate nanotechnology, advanced biomaterials, and biology to engineer conductive nanostructured cardiac patches that can overcome some of the key limitations that can impede heart repair after MI. We first engineered a gelatin-based (gelatin or gelatin methacryloyl) nanofibrous scaffold. A facile and cost-effective home-made electrospinning technique was carried out to electrospun highly biocompatible and biodegradable gelatin-based polymers. Since there was a major drawback to using porous acellular constructs as engineered myocardial scaffolds, we then incorporated conductive additives (including graphene nanoribbons (GNR), graphene oxide (GO), reduced graphene oxide (rGO), functionalized carbon nanotubes (CNTs), and bio- ionic liquid (Bio-IL)) to the matrices to fabricate a conductive composite scaffold. This is because the porous constructs have limited intercellular connection and electrical signal propagation due to isolating pore walls. The effects of conductive additives on physicochemical properties (such as chemical bonds, morphology, in vitro swelling and degradation, wettability, protein adsorption, mechanical properties, etc.), and electrical conductivity were evaluated. Our results revealed that, for instance, 0.1%(w/v) GNR incorporated into gelatin matrices considered as a threshold in which the maximum conductivity was obtained, which was also in the range of the conductivity of native myocardium (0.005 (transverse) ~ 0.16 (longitudinal) S/m). Therefore, incorporation of GNR could bridge the electrically resistant pore walls of the gelatin matrices, to support and facilitate the internal electrical interactions between adjacent cardiomyocytes (CMs). We then investigated the ability of the nanofibrous patches for the support, adhesion, growth, and proliferation of cardiac cells onto different patches using Live/Dead, Actin/DAPI, PrestoBlue, immunostaining against connexin 43 (Cx43) and sarcomeric alpha-actinin (SαA) cardiac biomarkers, and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Finally, we evaluated the beating characteristics of the CMs seeded on different patches. Our observations indicated that the engineered electroconductive patches could mimic the native characteristics of the myocardium and provide the beating of cardiac cells without any electrical or mechanical stimulations. As is well-known, following MI, the epicardium declines and is replaced by the myocardial expression, which has a limited capacity for innate regeneration and does not promote regeneration of the adult mammalian heart. It has been recently suggested that the epicardium of the heart (as a source of myogenic progenitors) not only plays a vital role in myocardial growth but also preserves the function of the adult myocardium after MI. Therefore, there is a need for the delivery of the epicardial-secreted factors (such as paracrine, cardiogenic, and angiogenic factors, as well as proteins) that can stimulate cell cycle entry and division of pre-existing CMs after MI, leading to improvement of cardiac function and survival. Towards this, in the next step, Follistatin-Related Protein 1 (FSTL1), which is a therapeutically effective epicardial-secreted protein, was incorporated into the optimal conductive patches obtained from the previous stage, with the aim of promoting the myocardial regeneration. The results proposed that our epicardial patch may be able to reconstitute the role of the epicardium in the regeneration of heart after MI, since they provided both proper biomechanical (stiffness) and electrophysiological support, enhanced the formation of new blood vessels (angiogenesis), as well as promoted the regeneration of myocardium due to the epicardial-secreted factors inside the patch. Our proposed strategy of integration of conductive additives and epicardial factors within 3D scaffolds may improve the therapeutic value of current cardiac patches and will open new avenues for engineering cardiac tissues. Viral or bacterial infections that lead to inflammation of heart muscle tissue (i.e., Myocarditis, which can affect the electrophysiological properties of the heart), although rare, its always been a challenge. In this regard, biodegradable and biocompatible materials with simultaneous electroconductivity and antibacterial effects have rarely been reported. Herein, we further investigated the antibacterial activity of some of the engineered graphene-incorporated patches and compared their properties with scaffolds incorporated with Chitosan as a well-known antibacterial agent. In vitro Antibacterial assays such as colony counting and bacterial adhesion were performed using multidrug-resistant Escherichia coli (MDR E. coli) and Methicillin-resistant Staphylococcus aureus (MRSA). To gain a better mechanistic understanding of the scaffold's bactericidal activity, we studied the protein adsorption capacity and analyzed the reactive oxygen species (ROS) levels. Taken all together, the patches containing graphene-based materials showed enhanced antibacterial activity compared to pristine gelatin patches. Therefore, these nanofibrous scaffolds can provide a desirable microenvironment not only as an electroconductive and antibacterial patch for bacterial-induced inflammation in cardiac tissue, but for any other tissue regenerative application that is more prone to infections, i.e., wound healing and skin tissue regeneration. We expect that our nanoengineered electroconductive and antibacterial cardiac patches may improve the therapeutic value of current cardiac patches and will open new avenues for heart muscle regeneration.--Author's abstract
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