Tunable 3D Nanofiber Architecture of Polycaprolactone by Divergence Electrospinning for Potential Tissue Engineering Applications
KeywordsDivergence electrospinning 3D nanofiber scaffold Tissue engineering Microstructure gradient
A novel 3D divergence electrospinning technique of tunable fibrous microarchitecture for tissue engineering.
Versatile capability of controlling both the microstructure and macroscopic shape of the scaffold.
Nanofiber scaffold with microstructure gradient coupled with element gradient.
The fabrication of biomimetic cell microenvironments closely resembling native tissues is critical for regenerative medicine. Recent bioinspired approaches have focused on creating biomimetic cell microenvironments that closely resemble the natural gradients of cell distribution, extracellular matrix (ECM), and tissue topology. Studies have shown that micro and nanotopography and the local environment of the ECM influence trends in cell behavior by providing biochemical and biophysical stimuli to promote cell adhesion, proliferation, morphogenesis, and motility [1, 2]. Critical physical features of the ECM include dimensionality, architecture, stiffness, ligand topography, and density . One of the important biofabrication strategies is to integrate tunable microarchitecture in heterogeneous scaffolds to closely resemble the patterned structures of native tissues [4, 5].
Electrospinning has been extensively studied as a nanofiber fabrication technique for tissue engineering. Two-dimensional (2D) mats composed of aligned or grid nanofibers can be created by adopting rotation collectors or arrayed pins. Some important electrospinning-based methods to construct three-dimensional (3D) nanofiber structures include vertically stacking layers of nanofiber membranes , incorporating nanofibers in hydrogels , rolling nanofiber mats into a tubular structure , and combining nanofibers with 3D printing . In addition, a 3D microfiber architecture can be fabricated through melt electrospinning . Our research group discovered that with tailored electrical and rheological properties of polymer solutions, a divergent electric field induced a fiber-bridging phenomenon between two separately grounded bevels, resulting in self-assembly of patterned nanofiber arrays into a functional architecture. In this study, we present a novel 3D divergence electrospinning technique of preparing tunable fibrous microarchitecture for potential musculoskeletal tissue engineering. Divergence electrospinning could control not only the microstructure of the highly aligned nanofiber scaffold, but also the macroscopic shape of the scaffold. In addition, hydrogels with element gradients were fabricated by incorporating the element-loaded nanofiber scaffolds. The scaffolds provided microtopographical cues to promote cell adhesion, proliferation, and morphogenesis. This approach enables the integration of 3D microtopographical cues and biomolecular gradients in a one-station top-down additive manufacturing process.
3 Experimental Methods
3.1 Configuration of the Divergence Electrospinning
We adopted 15% (w/v) PCL solution as the nanofiber material. The solution was prepared by dissolving PCL pellets (MW = 80,000) in N,N-dimethylformamide and chloroform (1:1) through magnetic stirring for 4 h at room temperature. All materials above were purchased from Sigma-Aldrich® (St. Louis, MO). To stain the nanofibers, 0.1 mg mL−1 DilC12(3) perchlorate (Thermo Fisher Scientific, Waltham, MA) was added to the PCL solution. We first adopted a preset collector (width = 40 mm, height = 40 mm, inclination angle = 60°) to determine the appropriate ranges of process parameters. Through the preliminary study, the electric field intensity and the pump rate were set to be 1.1 kV cm−1 and 0.375 mL h−1, respectively. Then we investigated the effects of inclination angle on the nanofiber attributes. Two levels of angles, 45° and 60°, were tested. The width and height of the scaffolds were 40 and 20 mm, respectively.
3.2 Characterization of Scaffold Microstructure
The 3D microstructures of the nanofiber scaffolds were characterized through a series of sectioning in both vertical and horizontal directions. Vertical sectioning was performed at four equidistant points across the y-axis (Fig. 1a) of the scaffold using thin glass slides attached with double-sided tapes. The distance was 5 mm for the 20 mm-wide collectors and 10 mm for the 40 mm-wide collectors. Horizontal sectioning was performed at seven equidistant points (distance = 1.1 mm) across the z-axis. The sectioned samples were observed by scanning electron microscopy (SEM, Phenom ProX, NanoScience, Alexandria, VA) and analyzed using ImageJ. The images were segmented and processed for measurement of fiber diameter, fiber density, and fiber alignment.
3.3 Changes in Scaffold Shape
Given that the nanofibers were only deposited on the grounded area, we hypothesized that the scaffold shape could be altered according to the profiles of the conductive areas on the collector bevels. To test this hypothesis, we adopted four different geometries of conductive areas: a triangle with vertical orientation, a triangle with horizontal orientation, a circle, and a circle with a concentric hole. In addition, a four-bevel collector (with a rectangular conductive area) was adopted to test whether a 3D nanofiber scaffold with a grid structure could be obtained.
3.4 Hydrogel with Element Gradient
To demonstrate the potential of incorporating element gradients by divergence electrospinning, we fabricated PCL/collagen nanofiber scaffolds through the basic two-bevel configuration. The solution was prepared by mixing 10% (w/v) collagen type I powder and 10% (w/v) PCL in hexafluoroisopropanol. The electrospun scaffolds were placed in 2% sodium alginate solution and cross-linked by 2% CaCl2 solution. The nanofiber-incorporated hydrogel was freeze-dried at − 50 °C for 48 h. The cross-sections of the dried hydrogel scaffolds were examined by energy-dispersive X-ray spectroscopy (EDS) for elemental composition analysis. We selected nine equally spaced spots along the z-axis of the scaffold for EDS and calculated the nitrogen-to-carbon (N/C) ratio at each spot. The fitting curve for each scaffold was generated based on the nine points, and the gradient of N/C ratio was plotted by MATLAB.
3.5 Cell Culture
We performed a cell culture study to investigate the potential effect of the biomimetic nanofiber architecture on cell growth. Human fibroblasts (ATCC® MRC-5) were seeded on UV-sterilized electrospun scaffolds at a concentration of 2 × 105 cells mL−1 in Eagle’s Minimum Essential Medium (ATCC®, Manassas, VA) with 10% fetal bovine serum (ATCC®, Manassas, VA). The scaffold was incubated at 37 °C (CO2 = 5%) for 1 day for cell attachment. Because some cells were attached to the bottom of the well instead of the nanofiber scaffold, the scaffold with cells was transferred to another well and cultured continuously for 7 days. Alamar Blue (Thermo Fisher Scientific, Waltham, MA) tests were conducted on day 1 and 7 to quantify cell proliferation. After day 7, cells were fixed with 4% formaldehyde and stained with Phalloidin CruzFluor™ 488 Conjugate (Santa Cruz Biotechnology, Dallas, TX) and 4′,6-diamidino-2-phenylindole (Santa Cruz Biotechnology, Dallas, TX) for filamentous F-actin and nuclei, respectively. Fluorescence images were taken from both the top and side of the scaffold.
4 Results and Discussion
While 2D scaffolds have been widely used in tissue engineering applications, they fall short in investigating the factors proven to be crucial to an in vivo environment, such as cell communication in the context of ECM, mechanical cues, and nutrient transportation . Although tremendous effects have been made in creating 3D nanofiber scaffolds, including post-processing nanofiber mats [30, 31, 32], self-bundling of random nanofibers with selective polymers or conditions, and adopting ground-pin arrays , solution-based electrospinning is still widely considered as a 2D or 2.5D manufacturing process . Moreover, electrospun nanofiber mats tend to have a high fiber density, which impedes cell infiltration. Divergence electrospinning overcomes the drawbacks and enables the rapid assembly of uniaxially aligned nanofibers into centimeter-scale 3D scaffolds with a tunable gradient in fiber density. It is not clear what causes the fiber density gradient along the z-axis of the scaffold. We speculate that the electrostatic repulsion due to the residual charges on the nanofibers result in the low density at the bottom of the scaffold. Thus, the homogeneity of nanofiber distribution can be enhanced by dissipating charges on the deposited fibers. A potential strategy will be adjusting the conductivity of the nanofibers by introducing conductive polymers or salts in polymer solutions.
A novel 3D method of polymer microfiber array assembly was explored in this study as a one-station, top-down approach for integration of biomimetic microarchitecture with tissue scaffold. Our study showed the feasibility of direct electrospinning of centimeter-scale scaffolds with tunable nanofibrous structures within several minutes. The inclination angle of the collector influenced the nanofiber attributes, including diameter, density, and alignment. By altering the projection geometry on the collecting bevels, a polyhedron and a cylinder composed of aligned nanofibers were directly fabricated through divergence electrospinning. A grid nanofiber structure was also created by adopting a four-bevel configuration. In addition, hydrogels with element gradients were made by incorporating the element-loaded nanofiber scaffolds. The scaffolds provided microtopographical cues to promote cell adhesion, proliferation, and morphogenesis in 3D. In conclusion, divergence electrospinning provides a highly efficient and scalable biofabrication platform for nanofiber scaffolds with both microstructure and element gradients. This technique will promote the development of novel nanoarchitecture with modulated functionality, composite materials, and complex features in response to the dynamic physiological and mechanical environments for biomedical application. It will facilitate the development of biomimetic artificial tissues with patterned nanofiber structures, such as tendon, ligament, cartilage, and muscle.
This paper was financially supported by the Foundation of the Whitacre College of Engineering and the Office of Vice President for Research at Texas Tech University.
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