(64b) Adhesive and Electroconductive Cardiac Patches for Cardiac Tissue Regeneration Following Myocardial Infarction

Introduction:Following myocardial infarction(MI), a fibrous scar forms that cannot contribute to the normal mechanical and electrical coupling of the heart. Due to the inability of the adult myocardium to self-regenerate, significant cell death ultimately leads to cardiac failure [1]. One aim of cardiac tissue engineering is to develop biomaterials that may be delivered to the infarcted heart to improve cardiac function following MI. Recently, various microfabrication techniques have been used to develop biomimetic scaffolds for cardiac tissue engineering applications that emulate different properties of the native extracellular matrix (ECM). For instance, electrospinning is a technique that relies on an electrical field to draw charged threads of different diameters, from a polymer mixture to form scaffolds that mimic the fibrous architecture of the myocardium. Here, we propose to develop cardiac patches based on electrospun gelatin methacryloyl (GelMA) as the base material, due to its tunable mechanical properties and biodegradation rate, as well as the presence of cell binding sites that promote cell adhesion and proliferation. In addition, we covalently bond a bio-ionic liquid (Bio-IL) to electrospun GelMA polymers using UV irradiation. The presence of Bio-IL provides cardiac patches with electrical conductivity needed to propagate electrical signals across damaged heart tissue, as well as, strong adhesiveness to wet tissues. Tissue adhesion allows cardiac patches to be easily placed on the surface of the heart, eliminating the need for additional sutures. This fibrous patch has the potential to be used for heart tissue regeneration applications following MI.

Methods:GelMA was synthesized as described previously [2]. Then different concentrations of GelMA (10, 12.5, and 15% (w/v)) were dissolved in hexafluoroisopropanol (HFIP) solution and placed in a syringe. Fibrous mats of GelMA were fabricated using the electrospinning technique, with a high voltage power supply set at 20 kV. Fibrous mats were then soaked in 1.25% (w/v) of photoinitiator Irgacure 2959 in ethanol for 2 h, and placed in -80 °C. An acrylatyed choline-based Bio-IL was synthesized using the previously described method [3], and added to cold water at different ratios (0:1, 1:2, 2:1 and 1:0) and pipetted on the frozen GelMA fibers. Fibrous mats were then immediately exposed to UV light for 300 s on each side. Electrical conductivity was determined using a two-point probe electrical station. Mechanical stiffness and adhesive properties of cardiac patches were evaluated using an Instron 5542 mechanical tester. We evaluated the in vitrobiocompatibility of GelMA and GelMA/Bio-IL electrospun cardiac patches using co-culture of primary rat cardiomyocytes (CMs) and cardiac fibroblasts (CFs) at a 2:1 ratio. We also evaluated the expression of the cardiac differentiation markers sarcomeric α-actinin and Connexin 43 (Cxn43) using immunofluorescent staining [4].

Results: GelMA/Bio-IL cardiac patchesexhibited an increasing electrical conductivity with an increasing concentration of Bio-IL used during fabrication. For example, the conductivity of a 10% GelMA cardiac patch increased from 1.23 ±0.16 S ×m^-1to 3.56 ±0.47 S ×m^-1when the Bio-IL concentration was increased from 33% to 66%. The conductivity of these patches could be tuned to closely mimic that of native heart tissue, which has been reported to be 0.16 S ×m^-1(longitudinally) [5]. Mechanical tests demonstrated that the scaffolds possessed tunable elastic modulus, which was dependent on the concentrations of both GelMA and Bio-IL. The elastic modulus increased concomitantly with an increase in the concentration of the biopolymer GelMA, and for higher concentrations of Bio-IL. Tissue adhesion was also investigated to determine if fibrous patches may be placed on the surface of the heart without the need for sutures. These results demonstrated that cardiac patches fabricated with higher concentrations of Bio-IL exhibited significantly higher adhesion to heart tissues than pure GelMA patches. Patches fabricated with 100% Bio-IL exhibited the highest adhesion strength to cardiac tissue with an adhesion strength of 24.89 ±4.05 kPa. In vitrobiocompatibility tests demonstrated that CMs/CFs seeded on 66% Bio-IL/GelMA cardiac patches showed a significant over-expression of the gap junction protein Cxn43, when compared to pure GelMA controls at day 7. As a gap junction protein, Cxn43 is associated with many biological functions, such as the electrical coupling of cells. This cell-cell communication allowed for strong and synchronous muscle tissue contraction, and an increase in Cxn43 expression would lead to improved cardiac function.

Conclusions: We present here a new method to fabricate highly conductive and adhesive fibrous GelMA/Bio-IL scaffolds, with tunable mechanical and conductive properties. We demonstrated the ability for the conductivity and mechanical properties of these cardiac patches to be finely-tuned by optimizing the concentration of GelMA and Bio-IL. Further, GelMA/Bio-IL cardiac patches demonstrated excellent adhesiveness to cardiac tissues, and biocompatibility in vitro. CMs and CFs seeded on the surface of cardiac patches demonstrated good cell attachment and proliferation, and the scaffold supported the expression of gap junction proteins necessary for heart tissue contraction. This conductive GelMA/Bio-IL cardiac patch has the potential to be readily implanted to the site of MI, to restore the electromechanical coupling of the native myocardium and improve cardiac function. In addition, the tunable properties of the cardiac patch made it suitable for other tissue engineering applications and the development of flexible electronics.

References:

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4. Tandon, N., et al., Electrical stimulation systems for cardiac tissue engineering.Nat Protoc, 2009. 4(2): p. 155-73.
5. Stout, D.A., et al., Mechanisms of greater cardiomyocyte functions on conductive nanoengineered composites for cardiovascular application.Int J Nanomedicine, 2012. 7: p. 5653-69.

Acknowledgements:This work is supported by American Heart Association (AHA, 16SDG31280010), National Institutes of Health (NIH) (R01EB023052; R01HL140618).