This work was supported by grants from the National Institute of General Medical Science (NIGMS) at the NIH (2P20 GM103480-06 and 1R01GM123081 to J.X.), the Otis Glebe Medical Research Foundation, NE LB606, and startup funds from the University of Nebraska Medical Center.

All in vivo procedures outlined below were approved by the IACUC Committee at the University of Nebraska Medical Center.
1. Prepare the Solutions for Standard Electrospinning
<ol>
	<li>In a 20 mL glass tube, dissolve 2 g of poly(&#949;-caprolactone) (PCL, Mw= 80 kDa) in a solvent mixture of dichloromethane (DCM) and N,N-dimethylformamide (DMF) with a 4:1 ration (v/v) at a concentration of 10% (w/v). 
	CAUTION: Handle DCM and DMF in a well-ventilated hood to avoid exposure to fu...

<ol>
	<li>Chen, S., 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=Recent+advances+in+electrospun+nanofibers+for+wound+healing.">Recent advances in electrospun nanofibers for wound healing.</a> Nanomedicine. 12 (11), 1335-1352 (2017).</li><li>Khandalavala, K., Jiang, J., Shuler, F. D., Xie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+Nanofiber+Scaffolds+with+Gradations+in+Fiber+Organization.">Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization.</a> Journal of Visualized Experiments. (98), e52626 (2015).</li><li>Xie, J., Li, X., 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=Put+electrospun+nanofibers+to+work+for+biomedical+research.">Put electrospun nanofibers to work for biomedical research.</a> Macromolecular Rapid Communication. 29 (22), 1775-1792 (2008).</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=Nanofiber+membranes+with+controllable+microwells+and+structural+cues+and+their+use+in+forming+cell+microarrays+and+neuronal+networks.">Nanofiber membranes with controllable microwells and structural cues and their use in forming cell microarrays and neuronal networks.</a> Small. 7 (3), 293-297 (2011).</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=Radially+aligned,+electrospun+nanofibers+as+dural+substitutes+for+wound+closure+and+tissue+regeneration+applications.">Radially aligned, electrospun nanofibers as dural substitutes for wound closure and tissue regeneration applications.</a> ACS. 4 (9), 5027-5036 (2010).</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="Aligned-to-random"+nanofiber+scaffolds+for+mimicking+the+structure+of+the+tendon-to-bone+insertion+site.">"Aligned-to-random" nanofiber scaffolds for mimicking the structure of the tendon-to-bone insertion site.</a> Nanoscale. 2 (6), 923-926 (2010).</li><li>Jiang, 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=Expanded+3D+Nanofiber+Scaffolds:+Cell+Penetration.">Expanded 3D Nanofiber Scaffolds: Cell Penetration.</a> Neovascularization, and Host Response. Advanced Healthcare Materials. 5 (23), 2993-3003 (2016).</li><li>Jiang, 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=Expanding+Two-Dimensional+Electrospun+Nanofiber+Membranes+in+the+Third+Dimension+by+a+Modified+Gas-Foaming+Technique.">Expanding Two-Dimensional Electrospun Nanofiber Membranes in the Third Dimension by a Modified Gas-Foaming Technique.</a> ACS Biomaterials Science &amp; Engineering. 10 (1), 991-1001 (2015).</li><li>Jiang, 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=CO2-expanded+nanofiber+scaffolds+maintain+activity+of+encapsulated+bioactive+materials+and+promote+cellular+infiltration+and+positive+host+response.">CO2-expanded nanofiber scaffolds maintain activity of encapsulated bioactive materials and promote cellular infiltration and positive host response.</a> Acta Biomaterialia. 68, 237-248 (2018).</li><li>Chen, S., 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=Nanofiber-based+sutures+induce+endogenous+antimicrobial+peptide.">Nanofiber-based sutures induce endogenous antimicrobial peptide.</a> Nanomedicine. 12 (10), 2597-2609 (2017).</li><li>Dhand, 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=Bio-inspired+crosslinking+and+matrix-drug+interactions+for+advanced+wound+dressings+with+long-term+antimicrobial+activity.">Bio-inspired crosslinking and matrix-drug interactions for advanced wound dressings with long-term antimicrobial activity.</a> Biomaterials. 138, 153-168 (2017).</li><li>Jiang, 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=Local+sustained+delivery+of+25-hydroxyvitamin+D3+for+production+of+antimicrobial+peptides.">Local sustained delivery of 25-hydroxyvitamin D3 for production of antimicrobial peptides.</a> Pharmaceutical Research. 32 (9), 2851-2862 (2015).</li><li>Jiang, 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=1&#945;,+25-dihydroxyvitamin+D3-eluting+nanofibrous+dressings+induce+endogenous+antimicrobial+peptide+expression.">1&#945;, 25-dihydroxyvitamin D3-eluting nanofibrous dressings induce endogenous antimicrobial peptide expression.</a> Nanomedicine (Lond). 13 (12), 1417-1432 (2018).</li><li>Ma, B., Xie, J., Jiang, J., Shuler, F. D., Bartlett, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+nanofiber+scaffolds+for+orthopedic+tissue+repair+and+regeneration.">Rational design of nanofiber scaffolds for orthopedic tissue repair and regeneration.</a> Nanomedicine. 8 (9), 1459-1481 (2013).</li></ol>

Authors declare that there is no conflict of interest.

Transforming traditional 2D electrospun nanofiber mats into expanded 3D scaffolds via CO2 depressurization was investigated. Traditional 2D nanofiber mats are successfully expanded via subcritical CO2 fluid. The critical steps are to fabricate 2D nanofiber mats under an optimized condition and cut the mats without deforming the edges (e.g., using sharp surgical scissors). This CO2-expanded nanofiber scaffolds have many benefits over traditional 2D mats including...

The concept of developing a synthetic scaffold that can be implanted into patients to aid in tissue repair and regeneration is one that has permeated the regenerative medicine field for decades. The ideal synthetic scaffold serves to induce cell migration from surrounding healthy tissue, provides an architecture for cell seeding, adhesion, signaling, proliferation, and differentiation, supports vascularization, allows for adequate oxygenation and nutrition delivery, and promotes host immune activity to ensure success after implantation1. Additionally, it can be used as a carrier for embedding antimicrobial molecules to assist in wound healing1,3,6,7,8,9. The ability to control the temporal release of these biologic molecules from the synthetic scaffold is another desirable attribute that is considered when engineering scaffolds1.
Electrospinning has been a well-utilized technique for producing nanofiber scaffolds1,2,3,4,5,6. Previous attempts to create a nanofiber scaffold such as the one discussed here have been done to varying degrees of success. However, traditional nanofiber scaffolds have limited abilities to achieve these goals. Traditional nanofiber scaffolds have been mostly two-dimensional mats1,3. These nonexpanded scaffolds are densely packed with small pore sizes; this limits cell infiltration, migration, and differentiation as it does not promote an environment similar enough to those found in vivo1,7,8,9. For this reason, newer techniques of 3D electrospun nanofiber scaffold preparation have been established to amend the inherent flaws that come with 2D nanofiber mats. These techniques result in 3D scaffolds; however, they have limited applicability due to the production methods requiring aqueous solutions and freeze-drying procedures. This processing results in the random distribution of the nanofibers without restricted organization, proper thickness, and/or desired porosity to provide the adequate nanotopographic cues that are necessary for cell migration and proliferation. These factors result in the previous 3D electrospun nanofiber scaffolds that lack adequate mimicry of living tissues1,7,8,9.
More recent attempts at developing an expanded, 3D scaffold with better biomimicry of extracellular matrix (ECM) have been performed using an aqueous sodium borohydride (NaBH4) solution treatment and predesigned molds to aid in better control of the shape of the resulting scaffold7,8. However, this method is not ideal as it requires the use of aqueous solutions, chemical reactions, and freeze-drying that may interfere with polymers and any encapsulated biomolecules that are water-soluble. The additives used may also cause side effects during tissue regeneration8,9. The CO2 expansion method outlined in this article greatly reduces processing time, eliminates the need for aqueous solutions, and preserves the amount and functionality of biologically active molecules to a greater extent than the previously established methods9.
In previous studies, antibiotics, silver, 1&#945;,25 dihydroxyvitamin D3, and antimicrobial peptide LL-37 were loaded into the nanofiber scaffolds individually and in combination to investigate the potential of these scaffolds to release agents to further aid in wound healing9,10,12,13. For the purpose of demonstrating this method of nanofiber scaffold expansion, Coumarine 6, a fluorescent dye, will be loaded into the scaffold to demonstrate the potential of embedding the scaffold with various desired compounds. This method of expanded nanofiber scaffold fabrication in conjunction with encapsulated bioactive molecules holds great potential in tissue regeneration, wound healing, the creation of 3D tissue models, and the topical delivery of drugs.

<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-199-1</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>Rotating Steel Drum</td>
			<td>customized</td>
			<td></td>
			<td>This serves as a collector during electrospinning.</td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Fisher Scientific</td>
			<td>14-831-200</td>
			<td>Coaxial spinning requires two single syringe pumps.</td>
		</tr>
		<tr>
			<td>Revolver</td>
			<td>Lab Net International</td>
			<td>H5600</td>
			<td>Adjustable lab rotator for mixing solutions</td>
		</tr>
		<tr>
			<td>Hypodermic Needle (27G x 1 1/2&#34;)</td>
			<td>EXCELINT International Co</td>
			<td>26426</td>
			<td>This is part of the example customized coaxial nozzel shown.</td>
		</tr>
		<tr>
			<td>Hypodermic Needle (21G x 1 1/2&#34;)</td>
			<td>EXCELINT International Co</td>
			<td>26416</td>
			<td>This is part of the example customized coaxial nozzel shown.</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>
		<tr>
			<td>LL 37 ELISA Kit</td>
			<td>Hycult Biotech</td>
			<td>HK321-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The efficacy of expanding traditional 2D electrospun nanofiber mats into 3D scaffolds via depressurization of subcritical CO2 fluid was demonstrated in different capacities: the thickness of the scaffolds increased from 1 mm when untreated to 2.5 mm and 19.2 mm with one and two CO2 treatments, respectively (Figure 3A-C). The porosity-a characteristic of the architecture critical for cell seeding-also increased in a manne...

Watch this Scientific Journal Video about Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds at JoVE.com

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

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 ...

This protocol reported the transformation of traditional electrospun nanofibremembranes from 2D to 3D, via depressurization of subcritical CO2 fluid which was not realized previously. This method eliminates many issues associated with previous approaches, including the use of aqueous solutions and chemical reactions, multiple step processes, loss of activity of encapsulated biological molecules, and limitations of hydrophobic polymers. Demonstrating this procedure is Shixuan Chen, a postdoc from my lab.In a 20 mililitre glass tube, dissolve two grams of PCL in a solvent mixture of dichloromethane and DMF, with a four to one ratio at a concentration of 10 percent. Place the glass tube into a lab rotator until the solution becomes clear. The solution may mix over night.To set up the Electrospinning Apparatus, first, add the PCL solution to a 20 milliliter syringe with a 21 gauge blunt needle attached. Ensure there is no air in the syringe, and dissociated tubing. Place a rotating steel drum with the ground collector 12 centimeters from the needle tip.Using alligator clips, connect the direct-current high-voltage power supply to the needle, and ensure that the collector is grounded. For the 20 milliliters of PCL solution, set the parameter of the syringe pump using a diameter of 20.27 millimeters, and a flow rate of 0.5 milliliters per hour. Check if the droplets are forming at the tip of the needle.Apply an electric potential of 20 kilovolts between the spinneret and a ground collector located 20 centimeters away from the spinneret. Collect the aligned nanofiber mats in a drum, rotating at 2, 000 RPM. Collect the PCL nanofiber mats once they reach a thickness of approximately one millimeter.Immerse the PCL nanofiber mats in liquid nitrogen for five minutes. Keep the PCL nanofiber mats in liquid nitrogen, and punch PCL nanofiber mats with a 0.5 millimeter diameter punch. Place the PCL nanofiber mats into liquid nitrogen for five minutes.Cut the mats into one centimeter by one centimeter squares using sharp surgical scissors while submerged in liquid nitrogen to avoid deformation of the edges. Place the cut mat in a 30 milliliter centrifuge tube with approximately one gram of dry ice. Tightly cap the lid, and allow for the dry ice to change into liquid carbon dioxide.Once the liquid has formed in the tube, quickly release the pressure by opening the cap. Remove and observe the puffed scaffold from the tube. Place the scaffold in a new centrifuge tube with dry ice, and repeat util the desire thickness is achieved.Sterilize the expanded nanofiber scaffolds in ethylene oxide prior to incubation with cells. The effectiveness of expanding traditional 2D electrospun nanofiber mats into 3D scaffolds via depressurization of subcritical CO2 fluid is shown on the left after the second treatment. The thickness of the scaffold increased from one millimeter when untreated to 2.5 millimeters with one CO2 treatment, to 19.2 millimeters with two CO2 treatments.The porosity of the scaffolds increased from 79.5 percent for the untreated mats, to 92.1 percent after the first treatment, to 99.0 percent after the second treatment. This is significant because the degree of cell penetration into a scaffold, and thus its efficacy to induce regeneration, is largely dependent on the porosity. SEM images reveal that the densely-packed fibular structure of untreated 2D mats were transformed into ordered, layered structures with aligned nano fibers after expansion with CO2.In Vevo studies were carried out by subcutaneous implantation of CO2-expanded nano fiber scaffolds with square-arrayed holes to rats. This allows for cellular migration and proliferation within the holes, as well as further infiltration within the nano fiber layers that were created during expansion. From week one to four post-implantation, the expanded scaffolds showed a significant increase in the number of blood vessels formed, and multinucleated giant cells when compared to a traditional nano fiber mat.Following this procedure, different molecules including growth factors, amino-modulating compounds, hemostatic agents, and anti-microtubular agents can be incorporated into the nano fiber mats and expanded in subcritical CO2 fluid. Such functionalized expanded nano fiber scaffolds could be used to explore new questions in other scientific fields, such as hemostasis, prevention and treatment of infection, immunology, and tissue regeneration and repair. Organic solvents are toxic, and should be handled in a chemical hood.Furthermore, a container that can endure the high pressure of subcritical CO2 fluid should be used for the expansion.

expansion-two-dimension-electrospun-nanofiber-mats-into-three

Research

JoVE Journal

Genetics

6.8K visualizações.  University of Nebraska Medical Center. Este protocolo relatou a transformação de nanofibremembranos eletrofibremsos tradicionais de 2D para 3D, via despressurização do fluido de CO2 subcrático que não foi realizado anteriormente. Este método elimina muitas questões associadas a abordagens anteriores, incluindo o uso de soluções aquosas e reações químicas, processos de múltiplas etapas, perda de atividade de moléculas biológicas encapsuladas e limitações de polímeros hidrofóbicos. Demonstrando este procedimento ...