Authors would like to thank National Science Foundation through Engineering Research Center for Revolutionizing Metallic Biomaterials (ERC-0812348) and Nanotechnology Undergraduate Education (EEC 1242139) for funding support.

All protocol follows the guidelines of the North Carolina A&#38;T State University Office of Research Compliance and Ethics.
1. Chemical Preparation for Keratin Extraction 4
<ol>
	<li>To prepare 1,000 ml of 2% wt/vol peracetic acid solution (PAS), under a fume hood add 20 ml of peracetic acid to 980 ml of Deionized (DI) water.</li>
	<li>To prepare 1,000 ml of 100 mM Tris base solution (TBS), add 12.2 g of Tris Base to 1,000 ml of DI water and stir until completely dissolved.</li>
	<li>Prepare diluted hydrochloric acid solution (DHAS) in a fume hood by pouring 4 ml of concentrated hydrochloric acid into 30 ml of DI water.</li>
	<li>Procure approximately 20 g of human hair clippings that have not been chemically treated or altered. Hair can be any length.</li>
	<li>Wash the hair thoroughly, by hand, with warm water and soap. Use deionized (DI) water for the final rinse.</li>
	<li>Put the hair into two 600 ml beakers and place in an 80 &#186;C oven for 1 hr.</li>
	<li>Drain the liquid from the bottom of the beaker and place back in the oven until the hair is completely dry.</li>
	<li>Divide dried hair evenly between the 600 ml beakers, placing 10 g of hair into each beaker. Do not allow the hair to fill more than 500 ml.</li>
	<li>Pour 500 ml of PAS into each beaker making sure to cover all of the hair. Cover the beakers with parafilm and store for 12 hr.</li>
</ol>
2. Preparation of Keratin Extract Solution
<ol>
	<li>Separate the hair from the PAS using a 500 &#181;m sieve. Collect the waste PAS in a separate container. Rinse the hair thoroughly with DI water to remove any leftover PAS.</li>
	<li>Place the rinsed hair in a 500 ml flask. Pour 400 ml of TBS into the flask, making sure that the hair is covered. Place flask in a shaking bath set to 38 &#186;C, 65 rpm for 1 hr.</li>
	<li>After 1 hr, remove from shaking bath and pour the liquid, approximately 400 ml of keratin extraction solution (KES), into a 1,000 ml beaker.</li>
	<li>Pour 400 ml of DI water into the flask containing the hair ensuring that the hair is covered and place back in the shaking bath for 1 hr. Remove the flask from the shaking bath and pour the remaining 400 ml of the KES into the 1,000 ml beaker with the previously collected KES. The hair is no longer needed and can be dumped out of the flask and discarded in the nearest trash receptacle.</li>
</ol>
3. Concentration of Keratin Extract Solution
<ol>
	<li>Fill rotodistiller bath to the top line with distilled water and set to 90 &#186;C. Set rotation speed to 200 rpm. Turn on the coolant chiller-pump and set to -10 &#186;C.</li>
	<li>Using a pH meter to monitor the pH of the KES, use a pipette to slowly add 1 mM DHAS to the KES until reaching a pH 7.0. Stir slowly as the solution is added.</li>
	<li>Pour the neutralized KES into the 500 ml round-bottom flask until about quarter full. Run the distiller according to manufacturer&#39;s protocol for 1.25 hr.
	<ol>
		<li>Repeat step 3.3 until all of the KES has been distilled. Ensure that the flask is connected tightly.</li>
	</ol>
	</li>
</ol>
4. Dialysis of Keratin Extract Solution
<ol>
	<li>Pour KES solution equally into 12 separate 14 ml conical tubes. Centrifuge the tubes at 1,050 x g for 10 min.</li>
	<li>Pour the centrifuged KES into a clean beaker, making sure to pour away from the collected debris. Repeat until all of the KES has been centrifuged.</li>
	<li>Fill a 2,000 ml graduated cylinder with DI water.</li>
	<li>Cut dialysis tubing cellulose membrane to 24 inches, and clip one end of the tubing to hold it shut.</li>
	<li>Dip the length of the tube in the cylinder of DI water to make it more flexible and easier to open.</li>
	<li>Open the non-clipped end of the dialysis tubing cellulose membrane and slowly pour 60 ml of centrifuged KES solution into the tubing. Use another clip to close this end of the tubing.</li>
	<li>Put the dialysis tube in the 2,000 ml cylinder of DI water and allow to sit for 24 h. Change the DI water in the cylinder every 3 to 4 hr.</li>
	<li>After the 24 hr period, empty the dialyzed KES solution into capped jars, being sure to leave space at the top and place in the freezer at -20&#176; C for 24 hr.</li>
</ol>
5. Lyophilization of Keratin Extract Solution
<ol>
	<li>Set the lyophilizer to -86 &#186;C.</li>
	<li>Remove the jars containing frozen KES from the freezer, take the caps off of the jars, and place them into freeze dryer ampoules. Place the seals on the ampoules and turn the knob to create a vacuum pressure of 0.133 mBar. Lyophilize the samples for approximately 48 hr, until all of the moisture is gone.</li>
</ol>
6. Preparation of Electrospinning Solutions (10 wt % Keratin Solution)
<ol>
	<li>Remove the Keratin Powder from the lyophilizer and weigh it.</li>
	<li>Add DI water to the Keratin powder to create a 10% weight/weight Keratin solution.</li>
</ol>
7. Preparation of 10% wt PCL Solution
<ol>
	<li>Obtain PCL (&#949;-caprolactone polymer,Mn 70 - 90 kDa), and trifluoroethanol (TFE). Prepare a 10% by weight PCL in TFE by stirring O/N&#160;and obtain a homogeneous mixture.</li>
</ol>
8. Preparation of Keratin /PCL Solution
<ol>
	<li>Obtain the 10 wt % PCL solution previously prepared and 10 wt % keratin solution. Add keratin into the PCL solution drop wise in order to create 10 ml PCL/keratin solutions of 90:10, 80:20, 70:30, and 60:40 ratios.</li>
	<li>Use a vortexer to obtain a homogeneous mixture of PCL/Keratin solution before electrospinning.</li>
</ol>
9. Production of Electrospun PCL/keratin Fiber
<ol>
	<li>Place approximately 8 ml of the PCL/keratin solution in a 10&#160;ml disposable syringe fitted with a 0.5-mm diameter plastic tube. Place the syringe in a syringe pump where the flow rate is set to be 2.5 ml/hr.</li>
	<li>Apply voltage to the tip of the needle connected to the tube (positioned ~23 cm from the fiber collecting drum, and ~30 degree from the horizontal). Apply a 19 - 22 kV voltage to the tube and rotate the collector drum at around 200 rpm.
	<ol>
		<li>Wrap the collector drum with aluminum foil before running the sample. Ensure that the aluminum foil is stable by applying the labeling tape on either end. 
		Note: The fibers will form on the aluminum foil (See Figure 2), store the fiber covered foil at RT.</li>
	</ol>
	</li>
</ol>
10. Mechanical Analysis of PCL/Keratin Nanofibers
<ol>
	<li>Cut the sample into 60 mm by 8 mm. Make sure to record the thickness by using digital micrometer. For each composition prepared, use five samples.</li>
	<li>Attach the 60 x 8 mm sample in the tensile tester. Use a custom designed specimen holder to attach the sample. Sandwich the sample between specimen holders which is made from an index card using double-sided tape. Apply the 10 N load cell with a displacement rate of 10 mm/min. 
	Note: The specimen holder was designed to be 62 mm by 40 mm with an inner window of 50 mm by 38 mm.</li>
	<li>Record extension and load values through software according to software protocol.</li>
	<li>Calculate stress by dividing the force by area of the sample tested. Next, calculate strain by dividing the change in length by initial length.</li>
	<li>Generate the stress-strain curve by plotting stress in y-axis for corresponding strain value in x-axis.</li>
	<li>Calculate the Young&#39;s modulus from the linear region where slope equals to modulus of elasticity. Determine tensile strength from the stress strain plot by taking 0.2% offset in strain and drawing a parallel line to the linear region curve.</li>
	<li>Perform the statistical analysis of the mechanical data obtained in triplicate previously using one-way analysis of variance using analysis software8. p values &#60;0.05 were considered statistically significant.</li>
</ol>
11. Surface Morphology and Structural Characterization
<ol>
	<li>Use scanning electron microscope (SEM) with parameters of accelerating voltage of 15 kV and current of 5 &#181;A to observe the morphology of the electrospun fibers.
	<ol>
		<li>Prior to imaging with SEM, cut a 2 cm2 section of the fiber and attach it to a SEM stage using copper tape. Place the stage inside the sputter coater and coat with gold at 15 mA for 1 min and 30 sec to create a gold layer approximately 11 nm thick. Load the sample into the chamber of the SEM. Observe the fiber samples.</li>
	</ol>
	</li>
	<li>Use Fourier transform infrared spectroscopy (FTIR) to characterize the chemical bonding between the PCL and keratin. Place the 2 cm2 section of the electrospun nanofiber membrane in a magnetic holder and purge the system with dry air before testing. Obtain spectra for 200 scans and analyze the spectra using standard software according to software protocol.</li>
	<li>Perform spectrum analysis using standard software and use triplicates of each ratio of nanofiber according to the manufacturer&#39;s protocol.</li>
</ol>
12. Study of Cell-fiber Interaction
<ol>
	<li>Cut fibers into 16 mm2 samples. Use a biocompatible, silicone-based glue to fix each sample to coverslips with a 12 mm diameter.</li>
	<li>Glue one end of the fiber sample to the back of the cover slip, wrap the sample around the coverslip, then glue the free end of the sample to the back of the cover slip as well, leaving the top of the slip covered with only the fiber sample.</li>
	<li>Place the fiber samples in a 24-well plate. Sterilize the samples by immersing in 80% ethanol for 1 hr.</li>
	<li>Rinse the ethanol from the samples using DI water. Then, pipette 2 ml of basal medium onto the samples, making sure to cover the entire sample. Remove the medium after 5 min.</li>
	<li>Use fibroblast 3T3 cells to seed the fiber samples. Suspend the cells in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with antibiotics and 10% fetal bovine serum (FBS) so that there are approximately 62,000 cells/cm2&#160;per 1 ml of DMEM.</li>
	<li>Drop 1 ml of the 3T3 cell-containing DMEM solution into each well plate ensuring that each fiber sample is covered with approximately 62,000 cells/cm2. Incubate the well-plates for 24 hr at 37 &#176;C and 5% CO2. Note: Use 5% CO2 because that level can usually maintain the pH of the cell media in physiological range for days.</li>
</ol>
13. Degradation of Nanofiber Matrix
<ol>
	<li>Cut dried PCL/keratin nanofiber membranes into square approximately 30 mm x 30 mm.</li>
	<li>Sterilize the 900 mm2 samples with 80% alcohol for a 10 min incubation period and wash thoroughly with DI water. Incubate the samples in 15 ml of PBS, pH 7.5, at 37 &#186;C. Replace the buffer every 3 days.</li>
	<li>Take the membranes out of the solution at specified intervals, 1 week and 7 weeks, and rinse with DI water.</li>
	<li>Place the membrane samples into freeze dryer ampoules. Place the seals on the ampoules and turn the knob to create a vacuum. Lyophilize for 24 hr, until completely dry.</li>
	<li>Attach the dried sample it to a SEM stage using copper tape. Place the stage inside the sputter coater and coat with gold at 15 mA for 1 min and 30 sec.</li>
	<li>Load the sample into the chamber of the SEM. Observe the fiber samples at an accelerating voltage of 1.5 kV and 5 &#181;A current.</li>
</ol>

<ol>
	<li>Huang, Z. -. M., Zhang, Y. Z., Kotaki, M., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+polymer+nanofibers+by+electrospinning+and+their+applications+in+nanocomposites.">A review on polymer nanofibers by electrospinning and their applications in nanocomposites.</a> Compos Sci Technol. 63, (2003).</li><li>Li, W. J., Laurencin, C. T., Caterson, E. J., Tuan, R. S., Ko, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+structure:+a+novel+scaffold+for+tissue+engineering.">Electrospun nanofibrous structure: a novel scaffold for tissue engineering.</a> J Biomed Mater Res. 60, 613-621 (2002).</li><li>Liu, W., 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=Electrospun+nanofibers+for+regenerative+medicine.">Electrospun nanofibers for regenerative medicine.</a> Adv Healthc Mater. 1, 10-25 (2012).</li><li>Edwards, A., Jarvis, D., Hopkins, T., Pixley, S., Bhattarai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(-caprolactone)/keratin-based+composite+nanofibers+for+biomedical+applications.">Poly(-caprolactone)/keratin-based composite nanofibers for biomedical applications.</a> J Biomed Mater Res B. 103, 21-30 (2015).</li><li>Dowling, L. M., Crewther, W. G., Parry, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+structure+of+component+8c-1+of+alpha-keratin.+An+analysis+of+the+amino+acid+sequence.">Secondary structure of component 8c-1 of alpha-keratin. An analysis of the amino acid sequence.</a> Biochem J. 236, 705-712 (1986).</li><li>Yamauchi, K., Maniwa, M., Mori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+fibroblast+cells+on+keratin-coated+substrata.">Cultivation of fibroblast cells on keratin-coated substrata.</a> J Biomat Sci-Polymer. 9, 259-270 (1998).</li><li>Shea, L. D., Wang, D., Franceschi, R. T., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+Bone+Development+from+a+Pre-Osteoblast+Cell+Line+on+Three-Dimensional+Scaffolds.">Engineered Bone Development from a Pre-Osteoblast Cell Line on Three-Dimensional Scaffolds.</a> Tissue E. 6, 605-617 (2000).</li><li>Fortin, M. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Biological+Software.">New Biological Software.</a> Q Rev Biol. 71, 169-170 (1996).</li><li>Bhattarai, N., Edmondson, D., Veiseh, O., Matsen, F. A., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+chitosan-based+nanofibers+and+their+cellular+compatibility.">Electrospun chitosan-based nanofibers and their cellular compatibility.</a> Biomaterials. 26, 6176-6184 (2005).</li><li>Yang, S., Leong, K. F., Du, Z., Chua, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+of+scaffolds+for+use+in+tissue+engineering.+Part+I.+Traditional+factors.">The design of scaffolds for use in tissue engineering. Part I. Traditional factors.</a> Tissue E. 7, 679-689 (2001).</li><li>Bhattarai, N., 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=Natural-Synthetic+Polyblend+Nanofibers+for+Biomedical+Applications.">Natural-Synthetic Polyblend Nanofibers for Biomedical Applications.</a> Adv Mater. 21, 2792-2797 (2009).</li></ol>

The authors declare that they have no competing financial interests.

Extraction of keratin from human hair was successfully achieved. The peracetic acid acted as an oxidizing agent on the human hair, allowing the keratin to be extracted by the Tris Base. The production of keratin powder was small scale due to the fact that it was only done for research purposes. This procedure has already been established in industry for large-scale production. The purpose of extracting the small-scale keratin was to control contamination, batch variability, and cost-effectiveness.
<p class="jove_cont...

Electrospinning is recognized as a prevalent method of achieving polymer nanofibers. The fibers can be produced on a nanoscale and the fiber properties are customizable 1. These developments and the characteristics of electrospun nanofibers have been especially interesting for their applications in biomedical engineering especially in tissue engineering. The electrospun nanofibers possess similarities to the extracellular matrix and thus promote cell adhesion, migration and proliferation2. Due to this similarity to the extracellular matrix (ECM), electrospun fibers can be used as materials to assist in wound dressing, drug delivery, and for engineering tissues such as liver, bone, heart, and muscle3.
A variety of different polymers of synthetic and natural origin have been used to create electrospun fibers for different biomedical engineering applications4. Recently there has been growing interest in the development of composite nanofibers by blending synthetic and natural polymers4. In these compositions the final products typically inherit the mechanical strength associated with the synthetic polymer while also adopting biological cues and properties from the natural polymer.
In this experiment, PCL and keratin are presented as the synthetic and natural polymers to be used for the synthesis of a composite nanofiber. Keratin is a natural polymer that is found in hair, wool and nails. It contains many amino acid residues; of notable interest is cysteine4,5. Ideally a naturally occurring polymer would be biorenewable, biocompatible and biodegradable. Keratin possesses all three of these characteristics while also enhancing cell proliferation and attachment to the biomaterials it has been incorporated in6.
Polycaprolactone (PCL) is a resorbable, synthetic polymer that is significant in tissue engineering4. This polymer has previously been praised for its structural and mechanical stability, however, it lacks cell affinity and exhibits a lengthy degradation rate. The hydrophobic nature of PCL is likely responsible for the lack of cell affinity7. However, PCL makes up for its limitations by being highly miscible with other polymers. A PCL/keratin composite should demonstrate the mechanical properties of PCL and incorporate the biological properties of keratin, making it an ideal choice for various biomedical applications.

<table><tbody><tr><td>Human Hair&amp;nbsp;</td><td></td><td></td><td>Obtained from Local Barber Shop in Greensboro</td></tr><tr><td>Peracetic acid</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>PCL (e-caprolactone polymer)</td><td>Sigma Aldrich</td><td>502-44-3</td><td>Mn 70-90 kDa</td></tr><tr><td>Trifluoroethanol (TFE)</td><td>Sigma Aldrich</td><td>75-89-8</td><td></td></tr><tr><td>Tris Base (TrizmaTM Base Powder)</td><td>Sigma Aldrich</td><td></td><td>&gt;99.9% crystalline</td></tr><tr><td>Hydrochloric Acid</td><td>Fischer Scientific</td><td>A144C-212 Lot 093601</td><td>Waltham, MA</td></tr><tr><td>Kwik-Sil</td><td>World Precision Instruments</td><td></td><td>Sarasota, FL</td></tr><tr><td>Cellulose membrane</td><td>Sigma Aldrich</td><td></td><td>12 - 14 kDa molecular cut off</td></tr><tr><td>optical microscope</td><td>Olympus BX51M</td><td>BX51M</td><td>Japan</td></tr><tr><td>scanning electron microscope</td><td>Hitachi SU8000</td><td>SU8000</td><td>Japan</td></tr><tr><td>Table-Top Shimadzu machine</td><td>North America Analytical and Measuring Instruments AGS-X series</td><td>AGS-X Series&amp;nbsp;</td><td>Columbia, MD</td></tr><tr><td>Fourier transform infrared spectroscopy</td><td>Bruker Tensor 2 Instrument&amp;nbsp;</td><td></td><td>Billerica, MA</td></tr><tr><td>Microcal Origin software</td><td></td><td></td><td>Northampton, MA</td></tr><tr><td>X-ray diffraction (XRD)</td><td>Bruker AXS D8 Advance X-ray Diffractometer</td><td></td><td>Madison, WI</td></tr><tr><td>Fibroblast 3T3&amp;nbsp; cell</td><td>American Tissue Type Culture Collection</td><td></td><td>Manassas, VA</td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s medium (DMEM</td><td>Invitrogen</td><td></td><td>Grand Island, NY</td></tr><tr><td>Spectra max Gemini XPS microplate reader</td><td>Molecular Devices</td><td></td><td>Sunnyvale, CA</td></tr><tr><td>Student- Newman-Keuls post hoc test</td><td>SigmaPlot 12 software</td><td></td><td></td></tr></tbody></table>

synthesis of keratin-based nanofiber for biomedical engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Fiber Morphology 
SEM images of the fibers were obtained for all the fiber compositions. See Figure 3. Fiber image confirms that the fibers are randomly oriented.
Mechanical Testing 
Mechanically strong fibers are generally required for various tissue engineering applications. These fibers should retain sufficient strength and flexibility under certain stress ...

Watch this Scientific Journal Video about Synthesis of Keratin-based Nanofiber for Biomedical Engineering at JoVE.com

Synteza nanowłókien na bazie keratyny dla inżynierii biomedycznej | Bioinżynieria | Wideo

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

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15.4K Views.  North Carolina A&T State University. Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Video: Synthesis of Keratin-based Nanofiber for Biomedical Engineering

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 ...

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 ...

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 ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

Procedure for Fabricating Biofunctional Nanofibers

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, &alpha;,&omega;-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of &alpha;-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The &alpha;,&omega;-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-&isin;-amino caproic acid, by DCC coupling, resulting in the formation of &alpha;,&omega;-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, 1H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.

An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups ...

Fabricating Engineered Nanofibrillar Collagen with Directed Patterning

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...

Formation and Attachment of FN-Silk Membranes to Inserts of Cell Culture 

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.

Author Spotlight: Enhancing In Vitro Cell Culture Models with Recombinant Functionalized Spider Silk Membranes

Tissue culture inserts with plastic membranes are the golden standard in cell culture labs as permeable supports to establish cell layers and models of barrier tissues. Herein, we present a simple method to replace the plastic membrane with a more biologically relevant membrane made from a recombinant functionalized spider silk protein.

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using ...