This work was supported partially from startup funds from University of Nebraska Medical Center and National Institute of Health (grant number 1R15 AR063901-01).

1. Preparation of the Solution
<ol>
	<li>Prepare a solution of Poly (&#949;-caprolactone) (PCL) (Mw= 80,000 g/mol) at an approximate concentration of 100 mg/ml. Dissolve PCL in a mixture of dichloromethane (DCM) and N, N-dimethlyformamide (DMF) at a ratio of 4:1 (v/v) with a concentration of 10% (w/v).</li>
	<li>Place the solution in a 20 ml glass tube for mixing. Place glass tube into ultrasonic cleaner for 30 minutes, or until solution is translucent.</li>
</ol>
2. Apparatus Preparation
<ol>
	<li>Add the prepared PCL solution into a 5 ml syringe with a 21 gauge blunt needle attached.</li>
	<li>Place syringe pump in a vertical electrospinning position, according to Figure 1.</li>
	<li>Use a stainless steel-gap collector with an open space of 2 cm x 5 cm for the aligned fiber substrate. Place the collector 12 cm from the needle tip.</li>
	<li>Connect the Direct Current (DC) High Voltage power supply to the needle and ground the collector. Ensure that the collector is individually grounded with no contact to the other lab equipment.</li>
</ol>
3. Electrospinning
<ol>
	<li>Set syringe pump to 1.50 ml/hr, until the droplets forming at the needle tip are immediately replaced when removed. Then, set the flow rate to 0.50 ml/hr.</li>
	<li>Turn the voltage operator to 12 kV.</li>
	<li>Electrospin until the uniaxially aligned fibers completely cover the gap on the collector.</li>
	<li>Apply glue to the edges of a small glass plate and transfer the fibers from the gap collector to the glass plate. Place the glass plate on the top of a piece of aluminum foil that has the same size as the glass plate and is grounded.</li>
	<li>Position the second syringe collector according to Figure 3.</li>
	<li>Attach the plastic mask to the second syringe pump and position it 2 mm above the collector.</li>
	<li>Add Coumarin 6 at 1% (w/w) to the PCL solution&#8212;if nanoencapsulation is desired&#8212; and mix until the solution is translucent.</li>
	<li>Load the PCL or PCL/Coumarin 6 solution into a 5 ml needle with a blunt 21 gauge needle. Set vertical pump to 0.50 ml/hr and horizontal to pull at 9 ml/hr or at an approximate speed of 1 mm/min.</li>
	<li>Electrospin until the mask has moved almost completely off the collector, but with the edge area still under the mask.</li>
</ol>
4. Fiber Characterization
<ol>
	<li>Place samples with double-sided conductive tape to the metallic stud and coat with platinum for 40 sec using a sputter coater at 40 mA.
	<ol>
		<li>Examine fibers through scanning electron microscope (SEM) according to our previous studies17,18.</li>
		<li>Collect images at an accelerating voltage of 15 kV.</li>
	</ol>
	</li>
	<li>Perform fast Fourier transform (FFT) analysis on a separate fiber sample to measure fiber alignment. The detailed information on measuring fiber alignment by FFT can refer to previous studies19,20.</li>
</ol>
5. Seeding Stem Cells.
<ol>
	<li>Sterilize the fiber samples by soaking in a 70% ethanol solution for 2 hr. Then wash the fibers with distilled water to remove any contaminants.</li>
	<li>Obtain human ADSCs and culture cells in a 25 cm2 flask at 37 &#176;C in an atmosphere of 95% air/5% CO2. Change the cell culture medium every other day10.</li>
	<li>Trypsinize the cells and count the number of cells. Specifically, remove all culture medium from the cell culture flask and wash the cells with phosphate buffered saline (PBS) twice. Then add 1 ml of a 0.25% trypsin-EDTA solution (pre-warm in water bath to 37 &#176;C) to cover the cell monolayer and incubate the flask in the cell culture incubator for 2-3 minutes. Add 4 ml culture medium and wash out all the cells from the surface by pipetting the medium all over the surface. Centrifuge the cell suspension and re-disperse the cell pellet in the culture medium. Count cells via hemacytometer. Seed around 1 &#215; 104 cells to the nanofiber scaffold placed in a 35 mm -culture dish and incubate for 3 and 7 days.</li>
	<li>Stain cells using fluorescein diacetate (5 mg/ml in acetone) (FDA) then image the samples using a fluorescent microscope. Specifically, add 100 &#956;l of FDA solution to the culture dish and incubate for 30 min. Wash cells with PBS three times before fluorescent imaging. Analyze cell orientation by a customized mat-lab program based on our previous studies10.</li>
</ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fiber+orientation+at+the+tendon+to+bone+insertion+and+its+influence+on+stress+concentrations.">Collagen fiber orientation at the tendon to bone insertion and its influence on stress concentrations.</a> J. Biomech. 39 (10), 1842-1851 (2006).</li><li>Thomopoulos, S., Williams, G. R., Gimbel, J. A., Favata, M., Soslowsky, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+of+biomechanical,+structural,+and+compositional+properties+along+the+tendon+to+bone+insertion+site.">Variation of biomechanical, structural, and compositional properties along the tendon to bone insertion site.</a> J. Orthop. Res. 21 (3), 413-419 (2003).</li><li>Thomopoulos, S., Genin, G. M., Galatz, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+morphogenesis+of+the+tendon-to-bone+insertion+-+What+development+can+teach+us+about+healing.">The development and morphogenesis of the tendon-to-bone insertion - What development can teach us about healing.</a> Musculoskelet Neuronal Interact. 10 (1), 35-45 (2010).</li><li>Li, X., Xie, J., Lipner, J., Yuan, X., Thomopoulos, S., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofiber+scaffolds+with+gradations+in+mineral+content+for+mimicking+the+tendon-to-bone+insertion+site.">Nanofiber scaffolds with gradations in mineral content for mimicking the tendon-to-bone insertion site.</a> Nano Lett. 9 (7), 2763-2768 (2009).</li><li>Kunzler, T. P., Huwiler, C., Drobek, T., V&#246;r&#246;s, J., Spencer, N. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+cells+from+human+adipose+tissue:+implications+for+cell-based+therapies.">Multilineage cells from human adipose tissue: implications for cell-based therapies.</a> Tissue Eng. 7 (2), 211-228 (2001).</li><li>Xie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+differentiation+of+embryonic+stem+cells+seeded+on+electrospun+nanofibers+into+neural+lineages.">The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages.</a> Biomaterials. 30 (3), 354-362 (2009).</li><li>Xie, J., MacEwan, M. R., Li, X., Sakiyama-Elbert, S. E., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+on+nanofiber+scaffolds+with+different+orders,+structures,+and+surface+properties.">Neurite outgrowth on nanofiber scaffolds with different orders, structures, and surface properties.</a> ACS Nano. 3 (5), 1151-1159 (2009).</li><li>Ayres, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+anisotropy+in+electrospun+tissue+engineering+scaffolds:+analysis+of+fiber+alignment+by+the+fast+Fourier+transform.">Modulation of anisotropy in electrospun tissue engineering scaffolds: analysis of fiber alignment by the fast Fourier transform.</a> Biomaterials. 27 (32), 5524-5534 (2006).</li><li>Ayres, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+fiber+alignment+in+electrospun+scaffolds:+a+user&#8217;s+guide+to+the+2D+fast+Fourier+transform+approach.">Measuring fiber alignment in electrospun scaffolds: a user&#8217;s guide to the 2D fast Fourier transform approach.</a> J. Biomater. Sci. Poly. Ed. 19 (5), 603-621 (2008).</li></ol>

The authors have nothing to disclose.

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.

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.

<table><tbody><tr><td>Polycaprolactone</td><td>Sigma-Aldrich</td><td>440744</td><td></td></tr><tr><td>N,N-Dimethlyformamide</td><td>Fisher Chemical</td><td>D-119-1</td><td></td></tr><tr><td>Dichloromethane</td><td>Fisher Chemical</td><td>AC61093-1000</td><td></td></tr><tr><td>Coumarin 6</td><td>Sigma-Aldrich</td><td>546283</td><td></td></tr><tr><td>Adipose Derived Stem Cells</td><td>Cellular engineering Technologies</td><td>HMSC.AD-100</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>26140-111</td><td></td></tr><tr><td>Fluorescein Diacetate</td><td>Sigma-Aldrich</td><td>F7378</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Invitrogen</td><td>25300-054</td><td></td></tr><tr><td>&amp;alpha;-Modified Eagle&#039;s Medium</td><td>Invitrogen</td><td>a10490-01</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>s25120a</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Invitrogen</td><td>10010023</td><td></td></tr><tr><td>Glass Slides</td><td>VWR international, LLC</td><td>101412-842</td><td></td></tr><tr><td>Syringe Pump</td><td>Fisher Scientific</td><td>14-831-200</td><td>Single syringe</td></tr><tr><td>Ultrasonic Cleaner</td><td>Branson</td><td>1510</td><td></td></tr><tr><td>High Voltage DC Power Supply</td><td>Gamma High Voltage Research</td><td>ES30</td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>FEI</td><td>Nova 2300</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Zeiss</td><td>Axio Imager 2</td><td></td></tr></tbody></table>

electrospun nanofiber scaffolds with gradations in fiber organization

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.

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&#176; 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&#176;. 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.
<img alt="Figure 1" src="/files/ftp_upload/52626/52626fig1.jpg" /> 
Figure 1: Shows the schematic of experimental setup for the preparation of the uniaxially aligned fiber substrate.
<img alt="Figure 2" src="/files/ftp_upload/52626/52626fig2.jpg" /> 
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. &#38; Shuler, F. D. Fabrication of Nanofiber Scaffolds With Gradations in Fiber Organization and Their Potential Applications. 1336&#8211;1341, Copyright 2012, with permission from Wiley-VCH.
<img alt="Figure 3" src="/files/ftp_upload/52626/52626fig3.jpg" /> 
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. &#38; Shuler, F. D. Fabrication of Nanofiber Scaffolds With Gradations in Fiber Organization and Their Potential Applications. 1336&#8211;1341, Copyright 2012, with permission from Wiley-VCH.
<img alt="Figure 4" src="/files/ftp_upload/52626/52626fig4.jpg" /> 
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&#176; 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. &#38; Shuler, F. D. Fabrication of Nanofiber Scaffolds With Gradations in Fiber Organization and Their Potential Applications. 1336&#8211;1341, Copyright 2012, with permission from Wiley-VCH.
<img alt="Figure 5" src="/files/ftp_upload/52626/52626fig5.jpg" /> 
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. &#38; Shuler, F. D. Fabrication of Nanofiber Scaffolds With Gradations in Fiber Organization and Their Potential Applications. 1336&#8211;1341, Copyright 2012, with permission from Wiley-VCH.

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Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

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 ...

electrospun-nanofiber-scaffolds-with-gradations-in-fiber-organization

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.8K Views.  University of Nebraska Medical Center.  Hier presenteren we een protocol om elektrogesponnen nanovezel steigers met gradated organisatie van vezels fabriceren en hun toepassingen verkennen in het reguleren van de cel morfologie / oriëntatie. Verlopen met betrekking tot fysische en chemische eigenschappen van de nanovezel scaffolds biedt een grote verscheidenheid aan toepassingen op biomedisch gebied. 

Video: Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Neuroscience

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease control of composition, structure, and diameter of fibers. However, despite these advantages, traditional electrospun nanofiber scaffolds come with limitations including disorganized nanofiber orientation, low porosity, small pore size, and mainly two-dimensional mats. As such, there is a great need for developing a new process for fabricating electrospun nanofiber scaffolds that can overcome the above limitations. Herein, a novel and simple method is outlined. A traditional 2D nanofiber mat is transformed into a 3D scaffold with desired thickness, gap distance, porosity, and nanotopographic cues to allow for cell seeding and proliferation through the depressurization of subcritical CO2 fluid. In addition to providing a scaffold for tissue regeneration to occur, this method also provides the opportunity to encapsulate bioactive molecules such as antimicrobial peptides for local drug delivery. The CO2 expanded nanofiber scaffolds hold great potential in tissue regeneration, wound healing, 3D tissue modeling, and topical drug delivery.

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease ...

Genetics

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

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