Name: synthesis of keratin-based nanofiber for biomedical engineering
Uploaded: 2016-02-07T15:00:00
Description: Watch this Scientific Journal Video about Synthesis of Keratin-based Nanofiber for Biomedical Engineering at JoVE.com

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 ...

<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>

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

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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