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 fumes. Do not expose DCM to plastic materials.</li>
	<li>Place the glass tube into a lab rotator until the solution becomes clear. The solution may mix overnight.</li>
	<li>If intending to embed bioactive materials such as peptides or drugs into the scaffold, create a separate solution and store at the 4 &#176;C until ready to use.
	<ol>
		<li>Prepare the fluorescent dye solution (50 &#956;g/mL) using 20 mL of PCL solution.</li>
	</ol>
	</li>
</ol>
2. Set Up the Electrospinning Apparatus (Figure 1A)
<ol>
	<li>Add the PCL solution to a 20 mL syringe with a 21 gauge blunt needle attached. Ensure that there is no air in the syringe and associated tubing.</li>
	<li>Place a rotating steel drum with the ground collector 12 cm from the needle tip.</li>
	<li>Using alligator clips, connect the Direct Current (DC) High Voltage power supply to the needle and ensure that the collector is grounded. 
	CAUTION: Always turn off the power supply before handling any connected materials.</li>
</ol>
3. Electrospinning
<ol>
	<li>For the 20 mL of PCL solution, set the parameters of the syringe pump as follows: diameter = 20.27 mm, flow rate = 0.5 mL/h. Check if the droplets are forming at the tip of the needle.</li>
	<li>If the incorporation of bioactive molecules is desired, set up the apparatus to allow for co-axial electrospinning (Figure 1B). Create a customized coaxial nozzle using hypodermic needles. 
	NOTE: Such nozzles are also available commercially. Note that a solution of dye is prepared to simulate the adding such molecules.
	<ol>
		<li>Prepare a 1% solution of the fluorescent dye Coumarin 6 in water. Add 3 mL of the 1% dye to a small syringe. Connect the syringe to the same coaxial nozzle as the PCL solution. Once again, ensure that there are no air bubbles.</li>
		<li>For this 3 mL solution, set the parameters of the syringe pump as follows: diameter = 9.49 mm, flow rate = 0.02 mL/h. Check if the droplets are forming at the tip of the needle.</li>
	</ol>
	</li>
	<li>Apply an electric potential of 20 kV between the spinneret (22-gauge needle) and a ground collector located 20 cm 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 ~1 mm.</li>
</ol>
4. Generation of PCL Nanofiber Mats with Arrayed Holes.
<ol>
	<li>Fabricate PCL nanofiber mats.
	<ol>
		<li>Dissolve PCL beads in a solvent mixture consisting of DCM and DMF with a ratio of 4:1(v/v) at a concentration of 10% (PCL) (w/v). Pump the PCL solution at a flow rate of 0.7 mL/h using a syringe pump while a potential of 20 kV is applied between the spinneret (a 22-gage needle) and a grounded collector located 12 cm apart from the spinneret.</li>
		<li>Collect the nanofiber membrane using a rotating drum with a rotating speed larger than 500 rpm.</li>
	</ol>
	</li>
	<li>Immerse the PCL nanofiber mats in liquid N2 for 5 min (i.e., become stiff). Keep the PCL nanofiber mats in liquid N2 and punch PCL nanofiber mats with a 0.5 mm-diameter punch.</li>
</ol>
5. Expansion of 2D Nanofiber Mats with/without Arrayed Holes via Subcritical CO2 Liquid (Figure 2).
<ol>
	<li>Place the PCL nanofiber mats into liquid nitrogen for 5 min and cut into 1 cm x 1 cm squares using sharp surgical scissors while submerged in liquid nitrogen to avoid deformation of the edges.</li>
	<li>Place the cut mat in a 30 mL centrifuge tube with ~1 g of dry ice. Tightly cap the lid and allow for the dry ice to change into liquid CO2.</li>
	<li>Once liquid has formed in the tube, quickly release the pressure by opening the cap. 
	CAUTION: Use proper thermal protective gear when working with liquid nitrogen and dry ice. Do not open the pressurized tube towards the face. The centrifuge tube should not be used repeatedly.</li>
	<li>Remove and observe the puffed scaffold from the tube. Place the scaffold in a new centrifuge tube with dry ice and repeat until the desired thickness is achieved. Sterilize the expanded nanofiber scaffolds in ethylene oxide prior to incubation with cells.</li>
</ol>
6. Characterization of Expanded Nanofiber Scaffolds
<ol>
	<li>Characterize the morphology and structure of the expanded nanofiber scaffolds using scanning electron microscopy (SEM).
	<ol>
		<li>Place the samples with double-sided conductive tape to the metallic stud and coat with platinum for 40 s using a sputter coater at 40 mA.</li>
		<li>Examine the fibers using SEM according to previous studies9. Collect the images at an accelerating voltage of 15 kV.</li>
	</ol>
	</li>
	<li>Characterize in vitro release profiles and bioactivity of released peptides.
	<ol>
		<li>Weight 10 mg of nanofiber membranes before and after the expansion in CO2.</li>
		<li>Immerse the samples in PBS buffer and collect 10 &#956;L of supernatant at different time points (0-28 days).</li>
		<li>Use Enzyme-Linked Immuno Sorbent Assay (ELISA) kit to quantify the LL-37 peptide concentration in the collected supernatant.</li>
	</ol>
	</li>
	<li>Examine cellular infiltration in vivo and host response.
	<ol>
		<li>Put the 9-week old Sprague-Dawley (SD) rats in an anesthesia chamber, connect to isoflurane vapor and anesthetize the rats. Transfer the rats to an operating table after the rats become complete anesthesia without feelings. Continuously anesthetize the rats using an isoflurane-nose cone with vaporizer during the surgery. Shave the hair of rat&#8217;s backs by an animal&#8217;s shaver and sterilize it with iodine and alcohol skin scrub. Create subcutaneous pockets via 1.5 cm incisions at supraspinal sites on the dorsum using a scalpel.</li>
		<li>Insert one expanded nanofiber scaffold (1.5 mm-thick) into the subcutaneous pocket using tweezers for each incision. Close the incision using a stapler.</li>
		<li>At 1, 2, and 4 weeks, euthanize the rats with 95% CO2. Gently dissect the explant and the surrounding tissue using surgical scissors. Prior to histological analysis, immerse the tissue in formalin for at least 3 days, and then embed with paraffin. Section the tissue with a microtome, then perform hematoxylin and eosin (H&#38;E), Masson&#8217;s trichrome staining.</li>
	</ol>
	</li>
</ol>

<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&quot;)</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&quot;)</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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

6.9K Views.  University of Nebraska Medical Center. 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.

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

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 ...

Bioengineering

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 ...

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.

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 ...

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 ...

Biology

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

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

Summary

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Chapters in this video

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Self-reporting Scaffolds for 3-Dimensional Cell Culture

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

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

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

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

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

Melt Electrospinning Writing of Three-dimensional Poly(ε-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

Interlinked Macroporous 3D Scaffolds from Microgel Rods