Production and characterization of elastomeric cardiac tissue-like patches for Myocardial Tissue Engineering

Highlights

• The PLA/PEG/Collagen nanofiber patches were produced successfully by using electrospinning method.

• The high speed of the collector affected the nanofiber orientation by changing from random to aligned structures.

• The high amount of PEG had a toxic effect on the cardiomyocyte cell line (H9C2).

• Aligned nanofiber patches showed superior tensile strength value compared to the random nanofiber.

• The effect of aligned structure on cardiomyocyte cell behavior observed efficiently.

Abstract

Cardiovascular disease remains the leading cause of death. Damaged heart muscle is the etiology of heart failure. Heart failure is the most frequent cause of hospital and emergency room admissions. As a differentiated organ, current therapeutics and techniques can not repair or replace the damaged myocardial tissue. Myocardial tissue engineering is one of the promising treatment modalities for repairing damaged heart tissue in patients with heart failure. In this work, random Polylactic acid (PLA), Polylactic acid/Polyethylene glycol (PLA/PEG) and random and aligned Polylactic acid/Polyethylene glycol/Collagen (PLA/PEG/COL) nanofiber patches were successfully produced by the electrospinning technique. In vitro cytotoxic test (MTT), morphological (SEM), molecular interactions between the components (FT-IR), thermal analysis (DSC), tensile strength and physical analysis were carried out after production. The resulting nanofiber patches exhibited beadless and smooth structures. When the fiber diameters were examined, it was observed that the collagen doped random nanofiber patches had the lowest fiber diameter value (755 nm). Mechanical characterization results showed that aligned nanofiber patches had maximum tensile strength (5.90 MPa) values compared to PLA, PLA/PEG, and PLA/PEG/COL (random). In vitro degradation test reported that aligned patch had the highest degradation ratio. The produced patches displayed good alignment with tissue on cardiomyocyte cell morphology studies. In conclusion, newly produced patches have noticeable potential as a tissue-like cardiac patch for regeneration efforts after myocardial infarction.

Introduction

The heart is the vital organ for circulation and is composed of a complex interplay of myocardial cells with scaffolding tissue and a network of blood vessels. Arteries provide oxygen-rich blood to the cells. Heart tissue and myocardial cells require large amounts of oxygen which are provided by coronary arteries. If the coronary artery suddenly becomes occluded, the blood flow to that part of the heart is completely interrupted resulting in tissue necrosis. This condition is defined as a myocardial infarction (MI) and is the leading cause of morbidity and mortality in the developed world. Unlike other organs such as the skin or liver, the cardiac muscle's ability to regenerate itself is very limited [1]. Therefore, any damage in the heart tissue is permanent. The conversion of a healthy myocardium into non-contractile fibrous scar tissue after infarction reduces the effectiveness of cardiac contraction. Patients who survive MI frequently develop heart failure, which is defined as the clinical state resulting from the inadequacy of the heart to pump enough blood to the body's metabolic requirements. In the United States, nearly 5 million people live with heart failure. There are multiple approaches to cardiac regeneration in clinical and preclinical studies, including heart transplant, stem cell therapy, hydrogels injection, and cardiac tissue engineering. The rapid development of tissue engineering has provided new methods for the treatment of tissue damage caused by an MI. The great achievements in stem cell technology make it possible that unlimited numbers of stem cell-derived cardiac cells could be provided for regeneration purposes [2,3]. Another novel method is the production of scaffold or patches, which support cell growth and formation of an integrated tissue or organ microenvironment [4,5]. Several parameters are considered crucial when using the patch method. These are material selection, mechanical properties, biocompatibility, surface characteristics, degradation rate, and cell seeding conditions. Nanofiber patch can be produced by a variety of methods such as self-assembly nanofibers, emulsion freeze-drying, gas foaming, solvent casting/particulate leaching, computer-aided design technology, thermally induced phase separation, and electrospinning [6].

Electrospinning is one of the good choices to develop successful cardiovascular tissue regeneration systems. This method is common in Myocardial Tissue Engineering (MTE) and has been suggested for various biomaterials. Electrospinning is a simple, versatile fiber or nanofiber production method which uses electric force to draw charged threads of polymer solutions. In order to fabricate composite fiber with electrospinning, biomaterials like PLA, PEG, and Collagen are effective materials. Electrospun patch made of synthetic and natural polymers has been used for cardiac tissue engineering in several studies [[7], [8], [9]]. The biomaterials to be used for the patch for myocardium regeneration need to be compatible with the natural tissue and cells, also need to support cell adhesion, differentiation, and proliferation. Yet, the product should not compromise the elasticity of the organ. The common natural polymers used in cardiac tissue engineering include collagen, fibrinogen, chitosan, and gelatin. Natural polymers such as collagen, fibrin, and polysaccharides have shown their potential for leading to efficient cell differentiation and enhanced interaction with cardiac cells [9]. However, most natural polymers have poor mechanical properties. Researchers have investigated the combination of collagen and synthetic polymeric materials.

Synthetic biocompatible polymers such as PLA is a solution when higher mechanical properties are required. Both materials would contribute to the patch with equal importance. The synthetic polymer would provide suitable mechanical support, whereas the natural collagen polymer would confer to the cells a more in vivo-like environment. The elastic properties of successful patch materials should be similar to those of the natural heart. The effect of these fibers that can withstand this tension during cardiac contraction and relaxation movement occurs during each heartbeat and also allows cells to attach to the structure [10,11]. One of the preferred elastic polymers in tissue engineering is polyethylene glycol (PEG). The majority of the extracellular matrix (ECM) is collagen, which has to turn into an appropriate choice of material in cardiac tissue engineering due to its advantages such as it can provide mechanical support for the ischemic heart; improve stem cell attachment and engraftment; develop the delivery of bioactive agents for myocardial repair [12].

In this study, the combination of biomaterials such as Collagen, PEG and PLA were used to produce nanofiber patch with therapeutic strategies using electrospinning technique which allows the production of aligned and random nanofibers by changing the rotating speed of the collector and working distance. It is known that the aligned nanofibers had enhanced mechanical properties and superior cell orientation, migration, and differentiation [13]. To provide a particular direction; mechanical, electrical, and magnetic forces have been utilized in applications. In this presented study, the electrical field was used for aligning the nanofiber patches to observe the cardiomyocyte cell direction effect on the regeneration of MI.