Human Mesenchymal Stem Cells being Encapsulated in Alginate/reduced Geraphen Oxide Improving Infarct Expansion, and Inducing Neovasculariziation Formation in Ischemic Myocardium

Currently, one of the new therapeutic strategies is injection of hydrogel and cells to myocardial infarction (MI) patients, which has some limitations such as lack of electromechanical properties and neovascularization. In this study, we investigated the therapeutic potential of new electroactive hydrogel [Reduced graphene oxide (rGO)/Alginate (ALG)] encapsulated human bone marrow mesenchymal stem cell (BMSC) in different experimental groups. The study was done in rat model of chronic ischemic cardiomyopathy by ligating the left anterior descending coronary artery (LAD). Echocardiograms were analyzed at 4 and 8 weeks after MI induction. Experimental groups particularly (BMSC) encapsulated in rGO-ALG increased signicantly improvement of fractional shortening (FS) and ejection fraction (EF) compared to the control group. Eighth week after injection, Eosin (H&E) staining was performed. MSC-ALG-rGO demonstrated that signicantly increasing vascularization and connective tissue in comparison with control. Masson's trichrome staining indicated reduced scarring/collagen deposition and improved remodeling LV thickness in the MSC-ALG-rGo group. Anti-CD31 antibody applied to detect neovascularization in the experimental groups. Microvessel density was signicantly higher in MSC-ALG-rGO treatment than other groups (MSC-ALG and control) (p<0.01). This study demonstrates, use of rGO/ALG a bio-electroactive hydrogel appears safe for intramyocardial injection, improving (LV) function, neovascularization, adjust the electrical properties following MI.


Introduction
Myocardial infarction (MI), made by blood ow blockage of the cardiac coronary arteries, is one of the most serious diseases with a high death ratio around the world [1].MI results in wall thinning, brosis, left ventricular (LV) dilation and reduced cardiac function [2]. Due to the lack of cardiomyocyte selfgeneration ability, regeneration of the infarcted area is not possible. The progression of structural and functional weakening of the LV happens in the process of LV remodeling and lead to deteriorating of the clinical symptoms, exercised intolerance and ultimately cause death of the a icted patient [3].The 5 year mortality of heart failure (HF),as a major health problem around the world ,surpass 50% [4]. Following HF progression, the heart develops progressive LV remodeling.
Despite existing developments in drug and device treatment, the morbidity and mortality made by HF remain to increase [5]. Transplantation of Mesenchymal stem cells (MSCs) is promising to repair heart tissue after MI. Particularly, paracrine impacts of the transplanted MSCS play important roles in heart regeneration through secretion of many growth factors and immune modulatory cytokines. However, because the transplanted MSC are subjected to high shear stress caused by injection and the harsh postinfarction environment with high oxidative stress, their viability and e cacy still remains low [6].
Although cardiac cell injection has demonstrated promising improvements in cardiac function, but there are still limitations that need to be addressed before their clinical application [7].Signi cant issues associated with cell injection is the low engraftment in which most of the cells are being lost to the vasculature or moving out of the injection site [8,9]To overcome this problem, injected cells can be supported by being delivered with biomaterial matrix. It has been stated that delivery of proper material with cells or growth factors can be a more effective method to restore cardiac function compared with injecting only materials or cells [7,9,10]. In fact, injectable biopolymers allow the host body to perform as a bioreactor and recreate the injured tissue in situ [2].
Among biopolymers which have been utilized for MI treatment, alginate has been shown improvement in LV functioning animal models of MI [2,11]. Previously, it was shown that intramyocardial injection of alginate in dogs with chronic heart failure could increase LV thickness and recover LV structure and function. Apart from decreasing wall stress that underlies the bene ts of alginate therapy, increase of LV wall thickness and reduction of LV size are other mechanisms which can lead to a reduced end-diastolic length of contractile element. Besides, alginate therapy can lessen cardiomyocyte hypertrophy induced by continuous LV enlargement and consequent augmentation of mechanical stretch. Alginate hydrogel implant, also, improved LV function and stopped progressive remodeling in chronic HF dogs [11,12].This combination therapeutic approach was considered to prevent patients with advanced HF experiencing end-stage disease. Electrical integration of the insulated hydrogels with the infarct myocardium can be delayed leading to the arrhythmias. However, electroactive hydrogels could improve cell-cell electrical coupling and synchronous contraction of the cardiac cells; which is necessary for the effective integration with the host tissue [13,14].Graphene-based nanomaterials, especially graphene oxide (GO) are likely to effectively reinforce materials for the restore of ischemia in heart tissues due to their outstanding high mechanical and electrical properties. Reduced graphene oxide (rGO) as a carbon based nanomaterials shows itself anti-oxidant activity in this way sp2 carbons play a major role in radical scavenging such as ROSs leads to protect the cells from excessive oxidative stress via radical adduct formation and electron transfer [6,15].
Previously, we demonstrated the promising in vitro results of ALG-rGO electroactive hydrogel for cardiac application. Toxicities or biocompatibilities and producing of collagen and connective tissue of GO have not been fully studied. Consequently, we employed our previously developed reduction strategy (GOcontaining gel formation followed by mild chemical reduction) to improve the anti-oxidizing ability by producing rGO with its minimal aggregation and then MSCs were encapsulated in this composite micro gels to increase the therapeutic activities. Here, we further evaluated the therapeutic outcomes of ALG injection combined with MSCs and rGO in a rat model of ischemic myocardial infarction to improve cardiac function and induce neovascularization.
Within each group, we compared echo-cardiologist parameters before and after MI induction, at 4 and 8 weeks after treatment. Compared to control, the myocardial injection of rGO-ALG-MSC signi cantly improved stroke volume, ejection fraction, fractional shortening, wall thickness and internal diameter (Table 1). The rGO-ALG-MSC group also showed changes in 30.13±7.30to 32.95±6.12 improvement in LVFS compared to the control group (11.56±7.61 to10.56±5.6) respectively 4 and 8 weeks after MI (Fig. 1C,   1D).

Discussion
One of the major problems in the cardiovascular disease is inability of the myocardium to self- Ventricular remodeling which is a consequence of mechanical stress leads to ventricular enlargement and progression to ventricular dilation and heart failure [16].The e cacy of alginate and hMSCs in preventing or reversing cardiac remodeling has been proven but the injection of hMSCs along with rGO/alginate is a novel method to improve cardiac function and reverse cardiac remodeling. Reducing active oxygen species in the ischemic region has negative affect on the adhesion of cells in the injured area. Some biomaterials which support local infarct healing while limiting myocardial remodeling, and establishing a stress gradient and increase myocyte division by the proliferation of pre-existing myocytes are several. These mechanisms can be considered to increase the wall thickness of the infarcted myocardium and improving myocardial function caused by rGO/alginate encapsulated hMSCs [16][17][18][19].
According to the results of H&E staining and immunohistochemistry, the rate of neovascularization and reduced collagen brosis area (indicating reduced scarring/collagen deposition) was far better in the rGO/alginate encapsulated hMSCs group than other groups. In a study performed on a model of MI in dogs, results in the alginate-treated group con rmed the presence of less in ammation, increased muscle repair and increased collagen production and decreased brous tissue containing tangled and irregular collagens. The Mason-Trichrome staining results in that study also con rmed the increase in collagen production and decrease in brotic in ammation in all groups treated with alginate material [11,20]. Graphene-based nanomaterials can effectively scavenge reactive oxygen species (ROS) providing antioxidant activities to the transplanted cells and MI tissues. The antioxidant activity of rGO was considered as the reason to increase the survival and therapeutic e cacy of hMSC delivered for MI treatment [3]. In addition, it seems that the presence of rGO can improve the adhesion of mesenchymal stem cells in the damaged area and consequently increase the paracrine secretion of pre-vascular factors such as VEGF and FGF2, and increase the rate of regeneration of damaged heart tissue [16,18,19].
After MI, the left ventricle gradually undergoes deformation and cardiac myo brils go to remodeling because of increased mechanical pressure due to an increase in the volume of blood remaining in the left ventricle. Alginate as physiological support prevents the deformity of the left ventricle and prevents thickness of the left ventricle change. On the other hand, due to the structural properties of this hydrogel, less in ammation occurs in the left ventricular margin after injection into the affected area [3]. In a study by Young Lee et al, investigated the effect of rGO/alginate encapsulated adipose-derived mesenchymal stem cells, the results of trichrome-mason staining showed rGO/alginate encapsulated mesenchymal stem cells had less brous tissue than the control group and alginate/GO, indicating a reduction in scar tissue and the presence of more collagen as a result, there was an improvement in the ectopic deformation of the heart [6]. It seems that rGO/hMSCs composition injected into the infarcted area, improved the cardiac repair via expression of angiogenic growth factors in addition to conductivity made by rGO besides mechanical support and electrical bonding provided by GO between isolated cardiomyocytes and native healthy myocardium [16].
We believe that the positive ndings in this study using rGO along with alginate and hMSCs can provide research impetus to investigate the bene ts of this composition in other models of tissue degeneration.

Methods
Rat acute myocardial infarction model.
In this experiment, 42 male Wistar rats (250-300 g) were used in ve experimental groups that were randomly arranged. Experiments performed according to the protocol approved by the Ethics Committee of Tarbiat Modares University, Tehran, Iran (IR.TMU.REC.1396.700). After induction of anesthesia by intraperitoneal injection of ketamine and xylazine, animals were tracheal intubated and inhaled anesthesia was continued with iso urane (1%) mechanically ventilated. The mechanical ventilation was done with connected to a rodent ventilator. Ventilation was controlled with room air and oxygen using a Harvard rodent ventilator (VentElite Small Animal, Harvard model 683, Holliston, USA) (tidal volume 2-3 mL, respiratory rate 65-70 per min). Acute myocardial infarction was made via occlusion of the left anterior descending (LAD) coronary artery by making a small suture undergoing 20 minutes, followed by reperfusion. After the process, the chest was closed and the animals were kept in a safe condition (12 h: 12 h light/dark controlling room temperature with suitable humidity (60 ± 5%). Moreover, su cient food and water were provided for animals for 8 weeks for further evaluation of LV functionality [2,21].
Experimental groups.
Animal models were randomly categorized to ve experimental groups (n = 7/group) (Table2). In addition, sham group (surgical operation without any treatment) and control group (receiving PBS as treatment) were considered (n = 7/group). Fractional shortening (FS) and ejection fraction (EF) as inclusion parameters were measured in this study. Preparation of hydrogels.
Brie y, a solution of 1%(w/v) Alginate Hydrogel (Sigma-Aldrich) and 102 mmol/L calcium chloride solution (CaCl2; Sigma-Aldrich) as a cross-link was prepared at room temperature and ltered through 0.22 µm nylon membrane syringe. In addition, after sterilization of rGO (Sigma-Aldrich) by UV irradiation for 20 min they were added to prepared hydrogel with a total concentration of 10 µl/ml. Intramyocardial injection of hydrogel in Rat MI model.
Twenty-eight days after LAD ligation, animals were anesthetized (iso urane 1%) and underwent thoracotomy surgery, then intubation via mouth was done and inhaled anesthesia was continued and placed on mechanical ventilation. After identi cation of the infarct zone, which was darker, compared to other LV border zones, near the anterior apx of the LV wall. Animals received an intramyocardial injection (28G insulin needle, 20µl) in ve parts of the infarct border zone. 5×106 cells/µl were applied in each group that must receive hBMSC. Immediately after injection of the cells with a biopolymer, cross-linked (CaCl2 102Mmol) was used in the same area, in separate syringes.
Four and eight weeks after injection, the LV functionality assessment was performed with transthoracic two-dimensional (2D) echocardiography using a 10-MHz linear array transducer connected to a Vivid 7 expert ultrasound system at speed of 100 mm/s (General Electric-Vingmed Ultrasound, Horten, Norway), under anesthesia of sodium thiopental (50 mg/kg, ip). Fractional shortening (FS) and left ventricular ejection fraction (LVEF) were two parameters that evaluated. For the measurement of FS%, we ought to analyze other parameters; such as left ventricular internal diameter in diastole (LVIDd), left ventricular internal diameter in systole (LVIDs), the posterior wall thickness in diastole (LVPWd) and posterior wall thickness in systole (LVPWs). FS% was calculated, based on the following formula [2,22]. A cardiologist with enough experience on small animal echocardiography, performed total echocardiographic evaluation.
Based on acquired results on echocardiography, this assessment performed on three groups of ALG, ALG-rGO and results of them were compared with the control group. Eight weeks after MI treatments, rats were sacri ced with pentobarbital overdose (200 mg/kg). Their chest was opened and the hearts were immediately transferred to paraformaldehyde 4% (Merck, Germany) for xation. The para n-embedded blocks were prepared and sectioned with a microtome (at 5µm thickness) (Roto-CUT 100 Scilab England) and the process was continued by representative slides were stained with hematoxylin and eosin (H&E) staining for evaluation of vascularization, and Masson's trichrome staining was done for collagen observation (Accustain; Sigma, St Louis, Mo).
Immunostaining for evaluating of neovascularization was done by CD31 antibody (1:100Abcam, Cambridge, MA). Images were further quanti ed using ImageJ software (1.46r version, USA) for