Elsevier

Acta Biomaterialia

Volume 31, February 2016, Pages 134-143
Acta Biomaterialia

Full length article
Hybrid hydrogel-aligned carbon nanotube scaffolds to enhance cardiac differentiation of embryoid bodies

https://doi.org/10.1016/j.actbio.2015.11.047Get rights and content

Abstract

Carbon nanotubes (CNTs) were aligned in gelatin methacryloyl (GelMA) hydrogels using dielectrophoresis approach. Mouse embryoid bodies (EBs) were cultured in the microwells fabricated on the aligned CNT-hydrogel scaffolds. The GelMA-dielectrophoretically aligned CNT hydrogels enhanced the cardiac differentiation of the EBs compared with the pure GelMA and GelMA-random CNT hydrogels. This result was confirmed by Troponin-T immunostaining, the expression of cardiac genes (i.e., Tnnt2, Nkx2-5, and Actc1), and beating analysis of the EBs. The effect on EB properties was significantly enhanced by applying an electrical pulse stimulation (frequency, 1 Hz; voltage, 3 V; duration, 10 ms) to the EBs for two continuous days. Taken together, the fabricated hybrid hydrogel-aligned CNT scaffolds with tunable mechanical and electrical characteristics offer an efficient and controllable platform for electrically induced differentiation and stimulation of stem cells for potential tissue regeneration and cell therapy applications.

Statement of significance

Dielectrophoresis approach was used to rapidly align carbon nanotubes (CNTs) in gelatin methacryloyl (GelMA) hydrogels resulting in hybrid GelMA-CNT hydrogels with tunable and anisotropic electrical and mechanical properties. The GelMA-aligned CNT hydrogels may be used to apply accurate and controllable electrical pulses to cell and tissue constructs and thereby regulating their behavior and function. In this work, it was demonstrated that the GelMA hydrogels containing the aligned CNTs had superior performance in cardiac differentiation of stem cells upon applying electrical stimulation in contrast with control gels. Due to broad use of electrical stimulation in tissue engineering and stem cell differentiation, it is envisioned that the GelMA-aligned CNT hydrogels would find wide applications in tissue regeneration and stem cell therapy.

Introduction

The capability of stem cells to undergo unlimited self-renewal and differentiate into multiple cell types has prompted their widespread application in tissue engineering (TE) and cell therapy fields [1]. The stem cell microenvironment has a major effect on stem cell renewal and differentiation. This microenvironment, termed the niche, was first described by Schofield [2]. The stem cell niche is composed of extracellular matrix (ECM) components, soluble factors, and supportive cells, which provide spatiotemporal signals to stem cells to direct their fate. Creating biomimetic stem cell niches in vitro is crucial for controlling stem cell behavior in therapeutic applications [3]. Therefore, the design and fabrication of biomaterials and tools to mimic various aspects of the stem cell niche to control stem cell behavior are an interesting area of research. In particular, the precise control of stem cell differentiation and fate needs to be further achieved [4].

Carbon nanotubes (CNTs) have recently attracted significant attention in biological applications ranging from bioimaging [5], drug delivery [6], biosensing [7], cancer therapy [8], and TE scaffolding [9]. This recent interest in CNTs arises from their outstanding chemical, mechanical, electrical, and optical properties [10]. Incorporating CNTs into TE scaffolds leads to enhanced scaffold flexibility, strength, and electrical conductivity. For example, adding CNTs to gelatin methacryloyl (GelMA) hydrogel scaffolds increases their Young’s modulus and electrical conductivity without significantly affecting gel porosity [11]. GelMA is a biocompatible, biodegradable, and photocrosslinkable hydrogel that is suitable for culturing different cell types and fabricating various tissues [12]. However, the relatively poor electrical conductance of this gel limits its application in the regulation of electro-active cell behaviors and electrical stimulation (ES) of cell and tissue constructs [13]. Here, hybrid GelMA-CNT hydrogels with tunable electrical and mechanical characteristics were used as scaffolds to culture and electrically regulate the cardiac differentiation of mouse embryoid bodies (EBs).

Micro- and nanoscale technologies have widely been used in biomedicine [14]. They can be used to precisely fabricate biomaterials or cellular structures that mimic the complex architecture of native biological constructs. Dielectrophoresis (DEP) is one such useful and versatile technology. DEP is based on particle polarization and manipulation in a medium by applying a non-uniform electric field [15]. For example, we recently reported the use of DEP for the rapid formation of three-dimensional (3D) EBs in GelMA hydrogel [16]. It was possible to fabricate 3D EBs of varying sizes and shapes using a high-throughput approach. The DEP method was also employed to align CNTs in GelMA gels [17], [18]. The hybrid GelMA-aligned CNT scaffolds showed better performance in the generation of functional and contractile skeletal muscle myofibers in contrast with pure GelMA and GelMA-random CNT scaffolds.

Here, DEP was utilized to fabricate hybrid GelMA-aligned CNT gels. The mechanical and electrical properties of these gels were measured and compared against pure GelMA and GelMA-randomly dispersed CNT hydrogels as the control samples. We then used the GelMA-aligned CNT hydrogels to support the cardiac differentiation of EBs in response to ES. The efficiency of the GelMA hydrogel containing the aligned CNTs and control hydrogels (i.e. pure GelMA and GelMA-randomly dispersed CNT gels) in supporting the cardiac differentiation of EBs was determined by using gene and protein expression analyses and beating activity of the differentiated cells.

Section snippets

Materials

The following materials were used: developer (MF CD-26; Shipley Far East, Japan); photoresist (S1818; Rohm and Haas, USA); SU-8 3050 and SU-8 developers (MicroChem, USA); methacrylic anhydride, gelatin type A from porcine skin, trichloro (1H,2H,2H-perfluorooctyl)silane, 3-(trimethoxysilyl)propyl methacrylate (TMSPMA), and penicillin/streptomycin (P/S) (Sigma–Aldrich Chemical, USA); multi-walled CNTs (Hodogaya Chemical, Japan); 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone

Results and discussion

Prior to the DEP procedure, carboxyl groups were chemically bonded to the CNTs to make their stable dispersion in aqueous medium. The high quality of the synthesized CNTs was demonstrated using Raman spectroscopy and microscopy images in our previous work [18]. The CNTs were then dispersed in a 10% (w/v) GelMA prepolymer solution. For the DEP experiment, an IDA-ITO was made on a glass slide using lithography and chemical etching techniques (Fig. 1A). A SU-8 microstamp (well diameter, 700 μm) was

Conclusions

DEP approach was employed to generate GelMA-aligned CNT hydrogels in a rapid and facile manner. The GelMA hydrogels containing the aligned CNTs exhibited superior performance in supporting the cardiac differentiation of EBs in contrast with the pristine GelMA and GelMA-random CNT gels, as confirmed by the protein and gene expression analyses and the beating activity of the EBs. The obtained cardiomyocytes may be useful in broad applications within regenerative medicine and cell therapy.

Disclosures

The authors declare no conflict of interest.

Acknowledgments

S.A. conceived the idea, designed the experiments, and analyzed the results. M.E. functionalized the CNTs and performed the I–V measurements. X.L. helped with the AFM measurements under the supervision of K.N. S.Y. and S.A. performed all other experiments. S.A. wrote the paper. H.S., T.M., and A.K. analyzed the results and supervised the project. All authors read the manuscript, commented on it, and approved its content. This work was supported by the World Premier International Research Center

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