Microtopographical effects of natural scaffolding on cardiomyocyte function and arrhythmogenesis

Abstract

A natural myocardial patch for heart regeneration derived from porcine urinary bladder matrix (UBM) was previously reported to outperform synthetic materials (Dacron and expanded polytetrafluoroethylene (ePTFE)) used in current surgical treatments. UBM, an extracellular matrix prepared from urinary bladder, has intricate three-dimensional architecture with two distinct sides: the luminal side with a smoother surface relief; and the abluminal side with a fine mesh of nano- and microfibers. This study tested the ability of this natural scaffold to support functional cardiomyocyte networks, and probed how the local microtopography and composition of the two sides affects cell function. Cardiomyocytes isolated from neonatal rats were seeded in vitro to form cardiac tissue onto luminal (L) or abluminal (Ab) UBM. Immunocytochemistry of contractile cardiac proteins demonstrated growth of cardiomyocyte networks with mature morphology on either side of UBM, but greater cell compactness was seen in L. Fluorescence-based imaging techniques were used to measure dynamic changes in intracellular calcium concentration upon electrical stimulation of L and Ab-grown cells. Functional differences in cardiac tissue grown on the two sides manifested themselves in faster calcium recovery (p < 0.04) and greater hysteresis (difference in response to increasing and decreasing pacing rates) for L vs Ab side (p < 0.03). These results suggest that surface differences may be leveraged to engineer the desired cardiomyocyte responses and highlight the potential of natural scaffolds for fostering heart repair.

Introduction

Cardiac tissue is terminally differentiated and therefore has limited repair and regeneration ability in the event of disease or injury. Cardiovascular disease, more specifically ischemic heart disease, remains a leading cause of death for adults throughout the world [1]. Definitive care usually requires whole heart transplantation, but the insufficiency of donor organs necessitates another approach. One alternative is a surgical technique, the Dor procedure, in which infarcted myocardium is replaced with an inert patch [2]. Typically, the patch is composed of biologically inert woven polyethylene terephthalate fibers (Dacron) [3]. Dacron is well tolerated by the body, has high tensile strength and high resistance to stretching and degradation in wet and dry forms [4]. However, Dacron’s limitations include mineralization, fibrosis, risk of infection and its inability to become a functional constituent of the heart portion it replaces [5]. These drawbacks are shared by other synthetic scaffolds, precluding full integration within the cardiac muscle. Therefore, an alternative material that is biocompatible, biodegradable and can be replaced by host-derived tissue to restore cardiac function is in demand.

A potential candidate is natural scaffolding derived from an extracellular matrix (ECM). ECM serve as natural scaffolds for all organ and tissue growth; they are composed of various structural and functional proteins, growth factors, glycosaminoglycans and cytokines [6] arranged in a three-dimensional architecture which facilitates cell maturation. The composition and topography of ECM are essential in determining specificity, cellular structure, organization and function. Finally, ECMs’ natural self-degradation allows regenerated tissue to exist on its own after maturation [5].

Naturally derived scaffolding with little to no immunological response can be prepared by careful removal of overlying tissue and cells to preserve ECM structure and composition. Various tissue types have been repaired with such natural scaffolding, including the lower urinary track, the esophagus, dura matter, blood vessels and musculotendinous tissues [5]. In fact, ECM scaffolding has been previously reported to outperform Dacron and ePTFE as a ventricular patch, and to aid in the improvement of overall heart function [7], [8], [9].

There are currently two commercially available acellular ECM scaffold materials: one is derived from porcine urinary bladder (UBM) (A-Cell, Jessup, MD), and the other porcine jejunum small intestinal submucosa (SIS) (Cook Biotech, West Lafayette, IN). SIS is rich in epithelial cells, tends to be well vascularized and is composed primarily of type I collagen [5]. UBM is extracted from the region below the smooth muscle cells along the lining of the bladder, and is composed mostly of type IV collagen, laminin and entactin [10]. Of the structural proteins, cardiomyocytes adhere most readily to collagen IV and laminin [11]. Furthermore, the growth factors within UBM include VEGF [12] and PDGF [13], which are critical for induction of angiogenesis, especially essential for highly oxygen-demanding heart tissue.

Another characteristic of UBM which may support heart tissue growth is its structure, composed of two different sides: one with a mesh of random fibers (abluminal side) and the other a flat base (luminal side). This is in contrast to Dacron’s regular woven surface (Fig. 1). UBM’s “loose” and fine fiber configuration with high porosity results in a high surface-to-volume ratio and thus potentially larger surface area for cell growth. These characteristics are suspected to play a significant role in enhancing cell adhesion and packing [14], thus promoting high cell density – essential for electrical and mechanical synchronization within cardiac tissue.

Based on these qualifications, it was hypothesized that UBM may serve as a suitable scaffolding material for engineering cardiac tissue. The authors set out to determine whether UBM could provide the environment needed for electrical stability, and whether functional response of the cardiomyocytes is sensitive to the architecture of the two UBM sides.