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&#160;</td>
			<td >N/A</td>
			<td >N/A</td>
			<td >Obtained from Local Barber Shop in Greensboro</td>
		</tr>
		<tr >
			<td >Peracetic acid</td>
			<td >Sigma Aldrich</td>
			<td>N/A</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 >N/A</td>
			<td>&#62; 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 >N/A</td>
			<td>Sarasota, FL</td>
		</tr>
		<tr >
			<td >Cellulose membrane</td>
			<td >Sigma Aldrich</td>
			<td >N/A</td>
			<td>12-14 kDa molecular cutoff</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&#160;</td>
			<td>Columbia, MD</td>
		</tr>
		<tr >
			<td >Fourier transform infrared spectroscopy</td>
			<td >Bruker Tensor 2 Instrument&#160;</td>
			<td >N/A</td>
			<td>Billerica, MA</td>
		</tr>
		<tr >
			<td >Microcal Origin software</td>
			<td >N/A</td>
			<td >N/A</td>
			<td>Northampton, MA</td>
		</tr>
		<tr >
			<td >X-ray diffraction (XRD)</td>
			<td >Bruker AXS D8 Advance X-ray Diffractometer</td>
			<td >N/A</td>
			<td>Madison, WI</td>
		</tr>
		<tr >
			<td >Fibroblast 3T3&#160; cell</td>
			<td >American Tissue Type Culture Collection</td>
			<td >N/A</td>
			<td>Manassas, VA</td>
		</tr>
		<tr >
			<td >Dulbecco's modified Eagle's medium (DMEM</td>
			<td >Invitrogen</td>
			<td >N/A</td>
			<td>Grand Island, NY</td>
		</tr>
		<tr >
			<td >Spectra max Gemini XPS microplate reader</td>
			<td >Molecular Devices</td>
			<td >N/A</td>
			<td>Sunnyvale, CA</td>
		</tr>
		<tr >
			<td >Student- Newman-Keuls post hoc test</td>
			<td >SigmaPlot 12 software</td>
			<td >N/A</td>
			<td>N/A</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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15.3K 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

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

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 ...

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

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 of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb monoliths (MHMs) with micrometer-scale channels are expected as efficient platforms for reactions and separations because of their large surface areas. Up to now, MHMs have been prepared by a unidirectional freeze-drying (UDF) method only from very limited precursors. Herein, we report a protocol from which a series of MHMs consisting of different components can be obtained. Recently, we found that cellulose nanofibers function as a distinct structure-directing agent towards the formation of MHMs through the UDF process. By mixing the cellulose nanofibers with water soluble substances which do not yield MHMs, a variety of composite MHMs can be prepared. This significantly enriches the chemical constitution of MHMs towards versatile applications.

Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...