Characterization of dielectrophoresis-aligned nanofibrous silk fibroin–chitosan scaffold and its interactions with endothelial cells for tissue engineering applications

Abstract

Aligned three-dimensional nanofibrous silk fibroin–chitosan (eSFCS) scaffolds were fabricated using dielectrophoresis (DEP) by investigating the effects of alternating current frequency, the presence of ions, the SF:CS ratio and the post-DEP freezing temperature. Scaffolds were characterized with polarized light microscopy to analyze SF polymer chain alignment, atomic force microscopy (AFM) to measure the apparent elastic modulus, and scanning electron microscopy and AFM to analyze scaffold topography. The interaction of human umbilical vein endothelial cells (HUVECs) with eSFCS scaffolds was assessed using immunostaining to assess cell patterning and AFM to measure the apparent elastic modulus of the cells. The eSFCS (50:50) samples prepared at 10 MHz with NaCl had the highest percentage of aligned area as compared to other conditions. As DEP frequency increased from 100 kHz to 10 MHz, fibril sizes decreased significantly. eSFCS (50:50) scaffolds fabricated at 10 MHz in the presence of 5 mM NaCl had a fibril size of 77.96 ± 4.69 nm and an apparent elastic modulus of 39.9 ± 22.4 kPa. HUVECs on eSFCS scaffolds formed aligned and branched capillary-like vascular structures. The elastic modulus of HUVEC cultured on eSFCS was 6.36 ± 2.37 kPa. DEP is a potential tool for fabrication of SFCS scaffolds with aligned nanofibrous structures that can guide vasculature in tissue engineering and repair.

Introduction

Natural polymers have been successfully used as scaffold materials for tissue engineering [1]. In particular, Bombyx mori silk fibroin (SF) has been investigated for surgical implantation owing to its biocompatibility, relatively low thrombogenicity, low inflammatory response, degradation kinetics, high tensile strength with flexibility, and permeability to oxygen and water [2], [3], [4]. Another polymer used as a scaffold is the naturally occurring polysaccharide chitosan (CS), a partially deacetylated product of chitin. CS, which has been applied clinically as hemostatic wound dressing [5], is generally inert in vivo, has favorable degradation kinetics and mimics the glycosaminoglycan component of the extracellular matrix (ECM). Researchers have explored blending SF and CS to develop three-dimensional (3-D) SFCS scaffolds that mimic the in vivo extracellular matrix [6], [7], [8]. In addition to their excellent biocompatibility, SFCS scaffolds have biological, structural and mechanical properties that can be adjusted to meet specific clinical needs. The first generation of SFCS scaffolds have produced promising outcomes both in vitro and in vivo in repairing abdominal wall defects, healing skin wounds and regenerating bone and tracheal cartilage [9], [10], [11], [12], [13].

In vitro studies have shown that nanofibrous structures affect cellular morphology and various cellular activities, including cell attachment, proliferation and differentiation [14]. In particular, recent studies have suggested that aligned nanostructures enhance endothelial cell capillary networks in vitro, which fulfills an important need for neovascularization in tissue engineering [15], [16]. The first generation of SFCS scaffolds were smooth sheet-like structures with microfibrillar extensions that lacked the nanofibrous architecture found in the native ECM. Various methods have been used to fabricate nanofibrous scaffolds, including electrospinning, phase separation and self-assembly [1], [17], [18].

Our laboratory previously investigated the use of dielectrophoresis (DEP) to create nanofibrous structures in SFCS scaffolds [25] by manipulating current frequency and applied voltage to generate a non-uniform electric field on a microfabricated gold electrode. The electric field resulted in movement of particles in solution on the electrode surface due to polarization effects [26]. In the presence of a field gradient, an alternating current (AC) electric field induces positive DEP force (toward the high field intensity region) or negative DEP force (toward the low field intensity region). Recent work by our group and others has shown that dielectrophoresis (DEP) is a promising technique for fabricating nanofibrous scaffolds.

DEP is a non-destructive electrokinetic mechanism with great potential for manipulation of micro- or nanoparticles such as DNA, proteins, nanotubes and nanoparticles in aqueous solutions [19], [20], [21], [22], [23], [24]. Allowing scaling for massively parallel electronic manipulation of bioparticles, DEP has become an important technique in the field of microfluidics for separating DNA, viruses and bacterial spores. Recent studies have shown that DEP can be used to align actin filaments into nanofibers in vitro [19], [24]. The previous study’s model of SF fibrils self-assembly in a 3-D SFCS scaffold using DEP was based on exposing rod-shaped particles in solution to an inhomogeneous alternating electric field, generating a time-averaged, translational DEP force due to induced dipolar effects. Small-radius (<100 nm) molecules experience DEP attraction to electrode tips even at high frequencies. Molecular assembly into solid fibers of sufficiently large radius results in a sharp decrease in crossover frequency and negative DEP. The threshold radius for which the crossover frequency drops off rapidly is determined by the suspension medium conditions. The model showed that it should be possible to concentrate and orient small-radius molecules in solution by using strong attractive DEP forces at the electrode tips and repel larger-radius fibers toward low-field regions between the electrodes in the bay region. The proposed mechanism of fiber assembly is orientation of molecules in 3-D via repulsion from two-dimensional (2-D) electrode planes due to positive DEP in high-field regions at localized electrode tips and movement away from electrode tip surface structures due to negative DEP. In addition to experimentally applying DEP to a SFCS solution to fabricate nanofibrous SFCS scaffolds and aligned structures, we studied interactions of endothelial with stem cells on these scaffolds [25].

Although our previous work provided proof of concept for using DEP to create aligned nanofibrous SFCS scaffolds, little is known about the effects of system parameters such as voltage, AC frequency and solution ionic concentration on the DEP-processed SFCS scaffolds (eSFCS). In the present study, we investigated the effects of AC frequency, sodium chloride (NaCl) presence, SF:CS ratio, and post-DEP freezing temperature on scaffold properties. We used polarized light microscopy (PLM) to analyze SF polymer chain alignment within the SFCS scaffolds and scanning electron microscopy (SEM) and atomic force microscopy (AFM) to analyze the topography of the scaffolds. The interaction of human umbilical vein endothelial cells (HUVECs) with the eSFCS scaffolds was studied using AFM and immunostaining to determine the cell mechanical properties and patterning on the eSFCS scaffolds, respectively.