Engineering the heart: Evaluation of conductive nanomaterials for improving implant integration and cardiac function

Mohammad Ali Darabi (Department of Mechanical Engineering, Faculty of Engineering, University of Manitoba, Winnipeg, Mani)
Jin Zhou (Department of Advanced Interdisciplinary Studies, Institute of Basic Medical Sciences and Tissue Eng)
Malcolm (Mengqiu) Xing (Department of Mechanical Engineering, Faculty of Engineering, University of Manitoba, Winnipeg, Mani)
Changyong Wang (Department of Advanced Interdisciplinary Studies, Institute of Basic Medical Sciences and Tissue Eng)

Introduction

Engineering cardiac tissues in vitro offers new perspectives for the therapy of myocardial infarction (MI) (1). The Engineered cardiac tissues (ECTs) exert beneficial effects on heart function after implantation; however, the therapeutic efficacy in general is restricted to inhibit further pathological deterioration of infarct myocardium without expected complete reversal of myocardial dysfunction (2). The biomaterial scaffold is the main component of engineered cardiac tissues. It has been demonstrated that carbon nanotubes (CNTs) can improve the viability and proliferation of cardiomyocytes and promote their electrophysiological maturation. Given that gelatin is a kind of biocompatible materials derived from extracellular matrix, it has been reported that CNTs-incorporated gelatin methacrylate (CNTGelMA) hydrogel thin film can be used to engineer 2D cardiac patches. Nevertheless, it remains to be elucidated that whether cardiac patches based on CNT composites can exert beneficial effects on the heart function after myocardial infarction. In this study, we hypothesized that single walled carbon nanotubes (SWNT)/gelatin composite scaffolds can be used to fabricate ECTs with strong contractile and electrical properties, promote the repair efficacy of ECTs to infarct myocardium, and enhance the integration between ECTs and host myocardium. Here we demonstrated a paradigm to construct ECTs for cardiac repair using conductive nanomaterials. SWNTs were incorporated into gelatin hydrogel scaffolds to construct three-dimensional ECTs. The functional measurements showed that SWNTs were essential to improve the performance of ECTs in inhibiting pathological deterioration of myocardium.

Materials and Methods

To prepare SWNT/gelatin scaffolds, gelatin aqueous solution and the dispersed SWNT solution was added to make the final ratio at 2 wt% of gelatin. A 25% glutaraldehyde solution (20 mL) was added to 180 ml of solution of SWNT/gelatin to give a final concentration of glutaraldehyde at 2.5%. Ventricular cardiac cells were isolated from 1- day-old neonatal Sprague-Dawley rats and seeded into SWNT/gelatin scaffolds to construct c-ECT. All c-ECTs were cultured under static conditions for 3 days. The cell constructs were cultivated in a cell incubator, under electrical stimulation. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the Chinese Academy of Military Medical Science (Beijing, China). The data distributions were checked for normality with the Shapiro-Wilk test and for equality of variances with the Levene procedure. One-way analysis of variance was used for multiple group comparisons of left ventricular function after 4 weeks’ implantation

Results

Scanning electron microscopy (SEM) observation of SWNT/ gelatin showed that the scaffolds possessed a highly microporous structure and a well-developed network in which coil SWNTs were uniformly distributed (Fig. 1a). The surface of pore walls appeared smooth without adherence of aggregated SWNTs. To assess the maturation of cardiomyocytes, we investigated the ultra-microstructure of c-ECTs under transmission electron microscopy (TEM) (Fig. 2). It is noted that most of the cardiomyocytes were densely packed with myofibrils and displayed a predominant orientation of sarcomere composed of Z bands along the longitudinal cell axis, while the cardiomyocytes showed progressively less organized sarcomeres within gelatin scaffold (Fig. 2). Intercalated discs, specialized cell-cell junctions that were responsible for mechanical and electrical coupling of myocardium, formed between adjacent cardiomyocytes. SWNTs were observed to disperse in the interspaces between cardiomyocytes and in direct contact with the cardiac cell membranes. After 21 days of culture, the distribution of SWNTs on cardiac membrane was extended and accompanied with adjacent membranes concavity and vesicles. Immunostaining showed c-ECTs-derived DiI+ cardiomyocytes developed a differentiated phenotype with abundant expression of Cx43 (Fig. 3). Besides, DiI1 implanted cardiomyocytes and SWNTs located within c-ECTs and few could be detected in the host myocardium and scar areas after 1 week of engraftment. DiI-+vWF+ (von Willebrand factor) blood vessels were detected in c-ECTs, suggesting that host’s vasculature has invaded into the c- ECTs at early stage.

Discussion and Conclusion

We have constructed 3D ECTs with good structure, phenotype and function based on conductive SWNT incorporated hydrogel scaffolds in vitro. Furthermore, we demonstrated, for the first time, that ECTs based on conductive nanomaterials could improve heart function in vivo. Notably, ECTs appeared obvious structural fusion with the infarct myocardium after implantation, which enhanced the remodeling and regeneration of the infarct myocardium. Despite the unresolved questions, our study provides a promising therapeutic perspective of conductive nanomaterials in cardiac tissue engineering/regeneration

Figure 1. SEM images showed the highly microporous structure of gelatin hydrogels and SWNT/gelatin hydrogels. Carbon nanotubes were well dispersed in gelatin hydrogels (G) with the appearance of networks connecting the pore of gelatin hydrogels.

Figure 2. TEM showed the ultra-microstructure of c-ECTs at day 8 with apparent oriented sarcomeres, Z bands, newly formed intercalated disc, and directly contact of carbon nanotubes at localized sites of cardiac membrane surface (black arrow)

Figure 3. H and E staining of the mid-ventricular 4 weeks after engraftment showed the morphology of c-ECTs attached to the infarct surface (black dotted line). Carbon nanotubes dispersed throughout the c-ECTs and migrated into the scar area (black arrow).

References

1. Fujimoto KL, Tobita K, Merryman WD, Guan J, Momoi N, Stolz DB, et al. An Elastic, Biodegradable Cardiac Patch Induces Contractile Smooth Muscle and Improves Cardiac Remodeling and Function in Subacute Myocardial Infarction. Journal of the American College of Cardiology. 2007 6/12/;49(23):2292-300. 2. Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H, et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med. 2006 04//print;12(4):459-65.

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