Direct-write, highly aligned chitosan-poly(ethylene oxide) nanofiber patterns for cell morphology and spreading control
© Fuh et al.; licensee Springer. 2013
Received: 4 December 2012
Accepted: 26 January 2013
Published: 22 February 2013
Near-field electrospinning has been demonstrated to be able to achieve direct-write and highly aligned chitosan nanofibers (CNF) with prescribed positioning density. Cell spreading in preferential direction could be observed on parallel-aligned nanofibers, and the CNF patterns were capable of guiding cell extension when the distances between them are 20 and 100 μm, respectively. Alignment of the cells was characterized according to their elongation and orientation using the fast Fourier transform data and binary image analysis. Parallel CNF indicates that the alignment values sequentially increased as a function of positioning density such that incrementally more aligned cells were closely related to the increasing CNF positioning density. These maskless, low-cost, and direct-write patterns can be facily fabricated and will be a promising tool to study cell-based research such as cell adhesion, spreading, and tissue architecture.
KeywordsChitosan nanofibers Near-field electrospinning Direct-write patterns
Physicochemical properties of scaffold materials are found to be critical in regulating cell behaviors and cell-material interaction in tissue engineering. For example, altering the various substances of different chemical compositions, wettability, and topography is the most common practice to control cell responses in the past decades[1, 2]. Extracellular matrix consist of nanoscaled fibrous morphology has been considered beneficial in tissue regeneration due to its bio-mimicking characteristics. On the other hand, behaviors of cells on conducting polymers such as polypyrrole (PPy) and polyaniline have demonstrated enhanced growth and differentiation of cardiac myoblasts[4, 5], neurons[6, 7], and skeletal muscle cells[8, 9] because of direct electrical stimulation or electroactivity effect. In terms of biocompatible materials, chitosan is widely adopted due to its unique properties such as being naturally nontoxic, biodegradable, and antimicrobial. It has been demonstrated as a promising scaffolding material in tissue engineering.
Electrospinning is a simple yet versatile technique for producing nanofibers. An electrically driven jet initiating from a polymeric solution through so-called Taylor cones can deposit a rich variety of polymers, composites, and ceramics with diameter ranging from tens of nanometers to few microns. Previously, chitosan solutions blended with poly(ethylene oxide) (PEO) and poly(vinyl alcohol) (PVA) have been successfully electrospun via a conventional electrospinning process. However, the chaotic nature of conventional electrospinning process will result in instability of the polymer jet and deposit nanofibers in a disordered and random fashion. Continuous near-field electrospinning (NFES) was recently developed as a favorable technology due to its precise location control for nanofiber deposition and sophisticated patterns[15, 16]. Fundamentally, when the needle-to-collector distance implemented a significant reduction from several centimeters to few millimeters, the applied bias voltage can be reduced to few hundreds of volts. A recent application of direct-write, well-aligned chitosan-poly(ethylene oxide) nanofibers deposited via near-field electrospinning was carried out to exhibit excellent deposition of aligned nanofiber patterns. Electrospun nanofiber-based scaffolding systems were found to be able to achieve good cell alignment[18, 19]. The cell interaction between the prescribed microscale patterns of nanofibers and macroscale specimen was experimentally observed with particular focus on cellular alignment and associated tissue architecture. Furthermore, microfluidic synthesis of pure chitosan microfibers without any chemical additive for bio-artificial liver chip applications was proposed, and the chemical, mechanical, and diffusion properties of pure chitosan microfibers were analyzed. Micropatterns of double-layered, multifunctional nanofiber scaffolds with dual functions of cell patterning and metabolite detection have been developed consisting of multiple layers of nanofiber scaffolds and nanofiber-incorporated poly(ethylene glycol) hydrogels. Recent micro/nano technologies have opened up emerging interests to investigate relevant biological effects. For example, new nanomaterial-based assays are developed to quantitatively assess dose effect issues and related size dependence response. Furthermore, under the action of rare earth oxide nanoparticle with respect to the nature of cytotoxin, cell proliferation and apoptosis are presented in. In this paper, NFES was utilized to achieve direct-write and highly aligned chitosan nanofiber (CNF) with prescribed positioning density. The controlled and well-aligned CNFs are used to investigate cell spreading phenomena and related issues of cellular biocompatibility. The fundamental issues of cell spreading and extension guiding in a preferential direction are experimentally performed on parallel-aligned and grid patterns for the purpose of better realization of the ability to manipulate cellular architecture.
Chitosan from crab shells with 85% deacetylation (Mw = 50 to 190 kDa) was purchased from Sigma Chemical Co (St. Louis, MO, USA). PEO (Mw = 900 kDa; Triton X-100™) was provided by Acros Co. (Geel, Belgium), and dimethylsulfoxide (DMSO) was obtained from Tedia Co. (Fairfield, OH, USA). All reagents were used as received from the manufacturer without further purification.
Preparation of stock solutions for electrospinning
Chitosan solution (5%) and 1% PEO solution were first prepared separately by dissolving chitosan in 0.5 M acetic acid, then vacuumed in an oven at 0.8 Torr to remove air bubbles. Solutions containing 0.5 wt.% of Triton X-100™ and 5 to 10 wt.% of DMSO were mixed with the chitosan/PEO solutions, and the mixtures were again stirred for 16 h and vacuumed to remove air bubbles before use.
Soluble PPy was synthesized chemically using ammonium persulfate (APS) as an oxidant and a dopant. Pyrrole of 0.3 mol and 1:50 ratio of APS and pyrrole solution were mixed with 500 ml of distilled water. The solution was spin-cast on a polystyrene Petri dish to obtain a PPy film, and the electrical conductivity was measured to be 7.25 kΩ/square using the four-point probe method.
The stock solution for electrospinning was fed into a 1-ml disposable syringe fitted with a 0.4-mm-wide needle tip, the applied electrostatic voltage was in the range of 800 to 1,000 V (AU-1592, Matsusada Precision Inc., Kusatsu, Japan), and the distance between the syringe tip and the grounded collector was 500 μm. The substrate was mounted onto a programmable XY stage (Yokogawa Inc., Tokyo, Japan), controlled by a personal computer, which allows movement of the sample during nanofiber deposition. The experiment was carried out at room temperature and atmospheric pressure.
Cell culture, adhesion, and spreading
Human embryonic kidney cells (HEK 293T) were cultured in 25-cm2 flasks in Dulbecco's modified Eagle medium containing 10% fetal bovine serum. The cell suspension was added to each nanofiber pattern in a PPy-modified polystyrene Petri dish and cultured in an incubator at 37°C with 5% CO2.
In order to seed HEK 293T cells onto the CNF, a confluent monolayer of cells was trypsinized and centrifuged at 1,000 rpm for 4 min. After supernatant removal and re-suspension in fresh culture medium, cells were transferred to a PPy-modified polystyrene Petri dish.
Quantification of HEK 293T alignment
Fast Fourier transform (FFT) analysis was used to characterize the alignment of HEK 293T as a function of the positioning density of CNF previously. Relative alignment of CNF in electrospun scaffolds can be quantitatively evaluated via FFT analysis. FFT was conducted using ImageJ software (NIH, Maryland, USA) supported by an Oval Profile plug-in. Bright-field microscopic images of cells in a grayscale 8-bit TIF format were initially cropped to 1,024 × 1,024 pixels and imported into the Oval Profile plug-in for detailed FFT analysis. Typically, the degree of alignment can be reflected by the height and overall shape of the peak. The principal angle of HEK 293T orientation can be represented by the position of the peak.
Results and discussion
Integrity of nanofibrous structure in water
Cell viability, adhesion, and spreading
HEK 293T cell was selected in the present study to assess cell viability and spreading on aligned CNF. HEK 293T cells are often used as an in vitro model to assess cytotoxicity and has been well characterized for its relevance to toxicity models in human[30, 31]. Here, HEK 293T cells are seeded onto PPy substrates with prescribed unidirectional CNF at a dense 20-μm spacing, and cell cultivation for 1 and 3 days are shown in Figure 4b,c, respectively, similar to the culture period described before[32, 33]. It is observed that cells on the aligned CNF show morphology characteristics of nanofiber-dependent orientation, i.e., a majority of the cells was dramatically influenced and elongated along the orientation of the CNF. When the CNFs were spaced more sparsely at 100 μm, cell shape and ordering were considerably less elongated, and a slight orientation is acquired as shown in Figure 4d,e. For the two different positioning densities with a controlled 20-μm and 100-μm spacing, respectively, cell spreading in preferential direction could be observed on parallel-aligned nanofibers, and the nanofiber alignment was capable of guiding cell extension, though cell orientation is noticeably less significant for the sparse 100-μm spacing. In contrast, HEK 293T cells seeded onto a nanofiber-free PPy substrate formed cells of isotropic, disordered orientation and polymorphic shapes, as shown in Figure 4f,g. Therefore, the enhancement of CNF alignment could have positive effects on cellular elongation behavior, possibly including cell spreading, as compared with nonuniformly distributed shapes of the nanofiber-free substrate[34, 35].
Figure 5a shows the schematic of the NFES CNF grid pattern at controlled 20- and 100-μm spacing, respectively. Qualitatively speaking, cell alignment revealed a relatively weak influence of the positioning density of the CNF grid patterns on cell shape and ordering. It is observed that no distinct elongated shape in cell morphology between the dense grid about 183 fibers/mm2 (Figure 5b,c), the sparse grid about 37 fibers/mm2 (Figure 55d,e), and randomly distributed mat (Figure 5f,g). However, the cells do exhibit confluence to some degree such that the dense CNF grid and randomly distributed mat seem to provide a specific contact guidance and oriented growth to the cells to result in spontaneously contracting cultures. The confluence and contracting cultures are less significant in the sparse grid. We experimentally observed that CNF with distinct patterns, such as aligned or grid configurations, could have a significant impact and control the cell spreading in a different perspective.
Relation between cell spreading and positioning density of CNF
Degree of HEK 293T alignment as judged by FFT
In this study, we utilized NFES to prepare CNF in a direct-write manner and deposit prescribed patterns of different positioning densities. The cell ordering and alignment of HEK 293T was grown on PPy substrate with CNF of different orientations and positioning densities. Our experiments showed that the presence of parallel-aligned CNF greatly influenced cell shape.
Fast Fourier transform
Poly (ethylene oxide)
Poly (vinyl alcohol)
This work was supported in part by the Taiwan National Science Council under contract no. NSC 101-2221-E-008-014.
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