In light of tissue engineering, development of a functional and controllable scaffold which can promote cell proliferation and differentiation is crucial. In this study, we introduce a controllable collection method of the electrospinning process for regularly-distributed and uniaxially oriented nanofiber scaffold and evaluate the effects of aligned nanofiber density on adhesion of dermal fibroblasts. The suggested spinning collector features an inclined void gap, which allows easy transfer of uniformly aligned fibers onto other surfaces. By undergoing multiple transfers, the density of the nanofibers can be quantitatively controlled. The resultant polycaprolactone (PCL) nanofibers had well-defined nanotopography in a 400–600 nm range. Human dermal fibroblasts were seeded on aligned nanofiber scaffolds of different densities achieved by varying the number of transfers. Cell morphology and actin stress fiber formation was accessed after seven days. The experimental results indicate that the contact guidance of the cells along the fiber alignment can be more activated with more than one guidance feature on a cell; that is, the high density of fiber is attained in so much that fiber spacing gets below the cell size.
The importance of nanostructured topography in view of cell biology has been recognized as one of the key factors in tissue engineering applications. Nano- or micro-architectures that can mimic natural extracellular matrix (ECM) environments have been employed in fabrication of functionalized scaffolds to enhance cellular responses, such as adhesion, migration, proliferation, and locomotion [
The electrospinning process, in particular, has advantages for fabricating cytocompatible scaffolds thanks to its high efficiency and simplicity in producing submicron to nanometer scale fibers from a wide range of biomaterials [
The potential of applying these alignment techniques to scaffold fabrication is significant in that it can provide a defined architecture to guide cellular behavior as well as nanoscaled dimensions for the biomimetic ECM for a wide range of cell lines such as fibroblasts, neuron cells, and muscle cells, and so forth [
We previously reported on the fabrication process of aligned electrospun nanofibers realized by the proposed inclined gap method [
Polycaprolactone (PCL, average
The schematic of the electrospinning method is illustrated in Figure
(a) Schematic diagram of the electrospinning setup for the inclined gap method. (b) The transfer of collected fibers onto PDMS substrate. (c) SEM image of the transferred nanofiber array.
The collector for the formation of uniaxially aligned aluminum strips (0.2 mm thickness), which were fixed horizontally and vertically at upper and lower position, respectively. This height difference between the strips formed the void space and the electrospun nanofibers were suspended along the void gap. The spinning distance from the needle tip to the upper strip was determined as 160 mm. The suspended nanofibers with uniaxial alignment were transferred onto a surface of the PDMS membrane adhered to the slide glass and both ends of the fiber array were anchored with Permount (Fisher Scientific, Pittsburgh, Pa, USA).
The resultant samples that were transferred with electrospun nanofibers were photographed using a scanning electron microscope (SEM; JSM-6300, JEOL) and an optical microscope (i-Camscope, Sometech Vision). The fiber alignment was quantified by employing the angles between the desired direction (perpendicular to the edge lines of two strips) and the longitudinal axes of the fibers. The angle distribution of each nanofiber was measured from the captured images by using ImageTool 3.0 (University of Texas Health Science Center in San Antonio, Tex, USA).
Cell studies were performed using human dermal fibroblasts (6th and 10th passage) purchased from Lonza (Basal, Switzerland). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, Basal, Switzerland) containing 10% fetal bovine serum (FBS) supplemented with penicillin-streptomycin (Sigma, St. Louis, Mo, USA). Cultures were incubated in a humidified atmosphere of 5% CO2 in air at 37°C. Prior to the cell seeding, all nanofiber scaffolds were sterilized in ethanol for 24 hours under ultraviolet irradiation, treated with O2 plasma for 2 mins (Femto Science) and coated with fibronectin at 10
After experiments, cells were washed with the warmed DPBS and fixed for 15 min in 3.7% formaldehyde. After DPBS washing, cells were permeabilized for 20 min in 2% Triton X-100 and blocked in 3% BSA (Invitrogen). Following three washes in DPBS, cells were stained for F-actin cytoskeleton with Alexa-568-phalloidin (Molecular Probes) at 1 : 50 dilution in blocking buffer. After rinsing, the nuclei of the cells were labeled with DAPI (300 nM, D1306, Molecular Probes), and then, they were finally mounted in a Vectashield (H-1000, Vector Laboratories) to minimize photobleaching. The cells were imaged using a Zeiss fluorescence microscope (Axiovert-200M, Zeiss) equipped with a CCD camera (Axiocam HSM, Zeiss) and image analysis software (Axiovision, Zeiss).
After 7 days of cell culture, the stained cells were observed under a light microscope (Carl Zeiss Axiovert 200 M) with phase contrast. 60 cells from each scaffold were randomly selected and the fluorescent images of actin and nuclei were analyzed using ImageTool 3.0 to measure the extent of cell alignment. The orientation angle of cell long axis was measured with respect to the reference, which is the desired direction of fiber alignment, that is, perpendicular axis to the collector edges. In addition, cell elongation factor
In order to improve the uniformity of the aligned nanofiber array, the fiber collector was modified from a conventional design for the general gap method [
As reported previously [
Comparison between results of the inclined and the planar gap collection: photographs of the fiber arrays suspended on (a) the inclined gap collector and (b) the planar gap collector. Histograms of angle distribution of fiber alignment in the arrays transferred from (c) the inclined gap collector and (d) the planar gap collector. Homogeneous fiber arrays collected from the inclined gap by (e) single transfer and (f) 3 transfers. Inhomogeneous fiber arrays collected from the planar gap by (g) single transfer and (h) 3 transfers. Arrows indicate the inhomogeneous conditions of the fiber distributions. The scale bars are 10 mm (a, b) and 100
Although the utilized electrospinning technique has the advantage of producing the relatively large area of fiber array, repulsion caused by the residual charges on the collected fibers creates an inherent difficulty in generating controlled nanofibrous scaffold containing a sufficient quantity of fibers. As shown in Figure
SEM images of the nanofiber arrays transferred repetitively: (a) 1 transfer, (b) 3 transfers, (c) 10 transfers, and (d) 20 transfers. The scale bar on (a) is 20
While conventional electrospinning uses a simple electrically grounded collector without a gap, the collector used in our method comprises of two aluminum sheets separated by a gap. Because this collector configuration with a gap is designed such that as-spun fibers become suspended between both edges of the sheets, relatively rigid jets and fibers are preferred for stable suspension. When the initial concentration of the utilized solution is high, the whole jets and fibers throughout the spinning process exhibit more solid-like behavior. Thus, we increased the solution concentration to 20 wt% from the typical concentration of 10–12 wt%.
One concern about increasing the solution concentration for better mechanical stability is its consequence in the increased diameter of the nanofibers [
(a) Photograph of nanofibers collected on the upper strip and within the gap space. SEM images of (b) the microscale fibers deposited directly on the upper strip (the scale bar is 5
In order to test how substratum topographies generated from the transferred nanofiber scaffolds influence cell morphology, several specimens of different densities of nanofibers were prepared by varying number of transfers from 1, 3, to 10. Fibroblasts were seeded and cultured for seven days on each scaffold. As shown in Figure
Phase contrast micrographs illustrating the cell alignment effect of different fiber arrays for (a) 1, (b) 3, and (c) 10 transfers. The scale bar on (a) is 100
To visualize the interaction between the nanofibers and the cultured cells better, cells were fluorescently stained for nuclei and cytoskeletal actin with DAPI and rhodamine phalloidin, respectively. As a control, a smooth PDMS substrate without nanofibers (Figure
Immunofluorescent staining images of cell morphologies and quantitative assessments of cell alignments on (a) smooth PDMS substrate without fiber (control), (b) fiber mesh deposited randomly (random), (c) singly transferred substrate with aligned nanofibers (1 tr), and multiply transferred substrate with (d) 5 (5 trs) and (e) 20 transfers (20 trs). Red and blue correspond to actin and nucleus, respectively. The scale bar on (a) is 50
On the scaffold with a single transfer (Figure
Figure
Immunofluorescent staining images to visualize the formation of actin stress fiber on the substrate (a, b) with single transfer and (c, d) with 20 transfers. The scale bar on (a) is 30
This study presents an initial effort to apply uniaxially aligned and uniformly distributed nanofibers to scaffold-based tissue engineering in a quantitatively controlled manner and implies that a highly dense scaffold with nanofiber alignment would have functional significance. Besides as a dermal fibroblast used for wound healing strategy, the developed nanofiber scaffold could potentially contribute to other tissue engineering applications where tissue anisotropy is a critical factor (e.g., blood vessels, muscles and nerves), and thus other cell types should be tested for efficacy of the scaffolds. In addition, because of the material universality of the electrospinning process, naturally occurring polymers such as collagens can be tested as scaffold materials.
In this study, uniaxially electrospun nanofiber arrays were constructed with quantitatively controlled density. As a single transfer of the nanofibers collected from the inclined gap provided a well-aligned and regularly distributed form, the developed nanofiber array could be applied to tissue scaffold by employing multiple transfers. The fiber density within a scaffold could be controlled in proportion to the number of transfers. Diameters of the nanofibers in the scaffold were in a submicrometer range, which means that the fibers were appropriate to utilize as nanotopographic features. In regard to the contact guidance along the aligned direction of the nanofibers, favorable interactions between the fibroblasts and the developed scaffold, especially in a highly dense scaffold with the fiber spacing reduced down to less than cell size, were revealed in observation of cell morphologies. The results regarding the aspect of the fiber density control are believed to help practical scaffold design with aligned configuration. Further studies with other cell types utilizing more quantitative biochemical assays are under way to evaluate the feasibility of the developed scaffolds to tissue engineering applications.