Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.
The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.
Nanofibers are a popular utility for tissue engineering because of their ability to mimic the extracellular matrix in its structure and relative size1. However, some native tissue interfaces, such as the tendon-to-bone insertion site, contain collagen fibers, which exhibit a variable organizational structure that increases in alignment towards the tendon and decreases at the bone site2-5. So, for effective tissue regeneration there is a need to fabricate a scaffold that could effectively mimic this structural gradient.
Previously, there has been research conducted on gradual changes in fiber composition, specifically, mineral content6. However, recreating the structural component of connective tissues remains largely unexplored. An earlier study examined morphological gradients by studying the effect of surface silica particle density on the proliferation of rat calvarial osteoblasts and found an inverse relationship between silica particle density and cell proliferation7. But the morphological changes that mediated cell proliferation in previous work were mostly related to surface roughness lacking the capability in mimicking fiber organizational changes7,8. One recent study attempted to fabricate a scaffold that mimicked the unique collagen fiber orientations by using a novel collector for electrospinning9. While this study succeeded in producing a scaffold with both aligned and random fibers, it failed to mimic the gradual changes exhibited in the native tissues. Also, in producing separate components, with an immediate change from aligned to random orientation, the biomechanical properties of this scaffold decreased significantly. No previous work has been able to produce applicable nanofiber scaffolds with continuous gradations in fiber orientations from aligned and random. Our recent study has shown successful recreation of nanofiber scaffolds with gradations in fiber organization that can potentially mimic the native collagen organization at tendon-to-bone insertion10. This work aims to present the protocols used for the production of nanofiber scaffolds with a structure that closely resembles that of fiber organization in the native tendon-to-bone tissue interface.
Gradient nanofiber structures have potentially far-reaching applications across a variety of fields. We focused on the applications to tissue engineering of the tendon-to-bone insertion site by combining our scaffolds with adipose-derived stem cells (ADSCs) which are already utilized for tissue regeneration on various substrates11-14. In addition, ADSCs are very similar in nature to bone marrow stem cells in terms of multipotency and their resource is abundant which can be harvested using a simple liposuction procedure15,16. Seeding these cells to gradated nanofiber scaffolds further enhances their tissue engineering applications by allowing for the controlled distribution of the cells that can potentially differentiate into various tissues. In addition to seeding stem cells, nanofibers can be encapsulated with signaling molecules for regulation of cellular response. Coupling nanoencapsulation with the organizational gradient of these scaffolds allows for the study of cellular behavior or possible implant designs and coatings. Encapsulation of functional molecules like bone morphogenetic protein 2 (BMP2), which has been shown to induce osteoblast differentiation15,16, could further enhance the tissue engineering applications of these scaffolds10.
1. Preparation of the Solution
2. Apparatus Preparation
3. Electrospinning
4. Fiber Characterization
5. Seeding Stem Cells.
Using this protocol, a fiber mat with an organizational gradient was formed. Figure 3 shows the SEM images taken at various locations on the nanofiber scaffold. Qualitatively, it can be determined that there is a progression from the uniaxially aligned fibers at 0 mm (Figure 3A) to a random fiber assortment at 6 mm (Figure 3D). The FFT gives a quantitative value to the fiber alignment, specifics on the quantitative processes are detailed here19. Fibers at 0 mm exhibit an FFT that indicates fiber alignment, and at 6 mm the FFT pattern signifies a random orientation. There is a clear progression in the SEM images (Figure 3) from an aligned fiber organization to an increasingly random fiber deposition (Figure 3B–C).
ADSCs underwent morphological changes based on their location in the nanofiber scaffold. Figure 4 shows images taken with the fluorescent microscope (Zeiss) at 3 days (Figure 4A–D) and 7 days (Figure 4E–H). The distribution of the stem cell angle was quantitatively assessed by a customized MATLAB program and analyzed using the Kolmogorov-Smirnov test at various distances. Figure 4I shows the distribution of cell angle at different locations. At 0 mm, or the region of aligned fibers, 70% of the cells appeared within 20° of the axis of nanofiber fabrication. In contrast, the ADSCs seeded on the random portions of the fiber scaffolds lacked this organizational structure, with only 20% of the cells appearing within 20°. Finally, the formation of the chemical gradient using Coumarin 6 – loaded PCL fibers was studied using fluorescence microscopy. The chemical gradient was qualitatively confirmed using the microscopy image (Figure 5A). The image confirms the increasing chemical concentration across the scaffold, which is exhibited by the steadily increasing intensity of the fluorescent image. The graph of the fluorescent intensity (Image J) (Figure 5B) confirms the gradient of the chemical concentration by exhibiting a linear growth across the scaffold.
Figure 1: Shows the schematic of experimental setup for the preparation of the uniaxially aligned fiber substrate.
Figure 2: (A) Shows placement of the second syringe pump for the fabrication of the gradient scaffold. (B) Placement of the mask above the collector. This figure has been reprinted from [10] Macromol. Biosci., 12, Xie, J., Ma, B., Michael, P. L. & Shuler, F. D. Fabrication of Nanofiber Scaffolds With Gradations in Fiber Organization and Their Potential Applications. 1336–1341, Copyright 2012, with permission from Wiley-VCH.
Figure 3: SEM images of the PCL gradated nanofiber scaffold at 0 mm (A), 2 mm (B), 4 mm (C), and 6 mm (D). The secondary images are Fourier fast transfer patterns (FFT). Pattern at (A) is that of aligned fibers, (D) suggests random fiber deposition. This figure has been reprinted from [10] Macromol. Biosci., 12, Xie, J., Ma, B., Michael, P. L. & Shuler, F. D. Fabrication of Nanofiber Scaffolds With Gradations in Fiber Organization and Their Potential Applications. 1336–1341, Copyright 2012, with permission from Wiley-VCH.
Figure 4: Fluorescence microscopy images showing ADSCs after incubation for 3 days (A–D) and 7 days (E–H). Images exhibit the various morphologies of ADSCs in different locations of the gradated scaffold. (I): The distribution of cell angles at different locations of scaffolds. Cells were much more concentrated between 20° of the axis of nanofiber alignment on aligned fibers (0 mm). This figure has been reprinted from [10] Macromol. Biosci., 12, Xie, J., Ma, B., Michael, P. L. & Shuler, F. D. Fabrication of Nanofiber Scaffolds With Gradations in Fiber Organization and Their Potential Applications. 1336–1341, Copyright 2012, with permission from Wiley-VCH.
Figure 5: (A) Fluorescence microscopy image of Coumarin 6-encapsulated fibers. (B) The graph exhibits the fluorescent intensity across the scaffold. The linear increase signifies a gradual change in the chemical concentration through the scaffold. This figure has been reprinted from [10] Macromol. Biosci., 12, Xie, J., Ma, B., Michael, P. L. & Shuler, F. D. Fabrication of Nanofiber Scaffolds With Gradations in Fiber Organization and Their Potential Applications. 1336–1341, Copyright 2012, with permission from Wiley-VCH.
The most critical part of the protocol is generation of the gradient scaffold. It is imperative that the mask covering the collector moves at a constant velocity so there is a gradual change within the fiber scaffold. The correct preparation of PCL solution is also important to ensure electrospinning success. Checking the fiber morphology prior to electrospinning is recommendable, especially after the encapsulation of Coumarin-6, which may require a higher voltage to electrospin correctly.
Furthermore, the protocol allows for fabrication of discrete gradations in fiber organization. However, this protocol yields variable thicknesses across the scaffold, which could potentially limit its mechanical applications. The area with increased random fiber deposition will be thicker than the aligned region. This protocol also does not extend to the recreation of the chemical composition at tendon-to-bone insertion site. Construction of mineral gradients is needed to successfully recreate the native mineral contents at the insertion sites. Fabrication of fiber scaffolds with gradients in mineral composition and organizational structure will be further explored to better replicate the complete native environment in a tendon-to-bone interfacial tissue.
As of now, this is the first protocol for fabrication of an electrospun nanofiber scaffold with an organizational gradient. Previous studies have fabricated scaffolds with both random and aligned fibers, but only as two separate sections, with an immediate change between the two organizations9. Gradient structures should allow for tailoring cell orientation and their extracellular matrix deposition and thus contribute to graded mechanical properties throughout the tissue constructs. Native tendon-to-bone insertion sites have reduced stiffness by decreasing the fiber alignment on one side2, which can be replicated with our scaffold. Additionally the organizational gradient offers increased biomimicry of the extracellular matrix with the new ability to replicate the native fiber orientations. This structure offers more biomedical applications by the formation of chemical gradients through nanoencapsulation. New chemical gradients facilitate areas of study in cellular behavior, high throughput screening, and implant or implant coating design10. Additionally bone morphogenetic protein2 (BMP2), which is resembled by a dye molecule Coumarin-6 because of its ease of detection using fluorescence microscopy, could be encapsulated to further promote cellular differentiation at the end made of random nanofibers10. Encapsulation within the fiber gradient provides a method for control of its chemical concentration, as BMPs are only needed at the site of bone tissues.
The authors have nothing to disclose.
This work was supported partially from startup funds from University of Nebraska Medical Center and National Institute of Health (grant number 1R15 AR063901-01).
Polycaprolactone | Sigma-Aldrich | 440744 | |
N,N-Dimethlyformamide | Fisher Chemical | D-119-1 | |
Dichloromethane | Fisher Chemical | AC61093-1000 | |
Coumarin 6 | Sigma-Aldrich | 546283 | |
Adipose Derived Stem Cells | Cellular engineering Technologies | HMSC.AD-100 | |
Fetal Bovine Serum | Life Technologies | 26140-111 | |
Fluorescein Diacetate | Sigma-Aldrich | F7378 | |
Ethanol | Sigma-Aldrich | E7023 | |
Trypsin-EDTA | Invitrogen | 25300-054 | |
α-Modified Eagle's Medium | Invitrogen | a10490-01 | |
Acetone | Fisher Scientific | s25120a | |
Phosphate Buffered Saline | Invitrogen | 10010023 | |
Glass Slides | VWR international, LLC | 101412-842 | |
Syringe Pump | Fisher Scientific | 14-831-200 | Single syringe |
Ultrasonic Cleaner | Branson | 1510 | |
High Voltage DC Power Supply | Gamma High Voltage Research | ES30 | |
Scanning Electron Microscope | FEI | Nova 2300 | |
Fluorescence Microscope | Zeiss | Axio Imager 2 |