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 these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (ε-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'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.
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.
All protocol follows the guidelines of the North Carolina A&T State University Office of Research Compliance and Ethics.
1. Chemical Preparation for Keratin Extraction 4
2. Preparation of Keratin Extract Solution
3. Concentration of Keratin Extract Solution
4. Dialysis of Keratin Extract Solution
5. Lyophilization of Keratin Extract Solution
6. Preparation of Electrospinning Solutions (10 wt % Keratin Solution)
7. Preparation of 10% wt PCL Solution
8. Preparation of Keratin /PCL Solution
9. Production of Electrospun PCL/keratin Fiber
10. Mechanical Analysis of PCL/Keratin Nanofibers
11. Surface Morphology and Structural Characterization
12. Study of Cell-fiber Interaction
13. Degradation of Nanofiber Matrix
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 and environmental conditions9. Generally, scaffolds are desired to have moduli close to that of the target tissue to avoid any stress shielding effects and to maintain sufficient strength during in vivo and/or in vitro cell growth10. Tensile Tester was used to measure the Young's modulus and breaking strength of the fiber. Young modulus (MPa) of PCL/keratin at ratios of 100:00, 90:10, 80:20, and 70:30 was found to be 10 ± 2, 8 ± 1, 5 ± 1.5, and 4.5 ± 1.6, respectively4. Similarly, breaking strength (MPa) of PCL/keratin at ratios of 100:00, 90:10, 80:20, and 70:30 was found to be 3 ± 1.2, 2 ± 0.5, 1 ± 0.2, and 1 ± 0.3 respectively4 as seen in Table 1. Figure 4 displays the graphical trend of variation of Young's modulus versus PCL.keratin ratios. The trendline in the graph aids in further understanding the breaking elongation rate.
Structural and Morphological Characterization
In Figure 5, FTIR transmittance spectra for PCL/keratin composite nanofibers show bands at 2,950 cm-1, 1,050 cm-1, and 1,240 cm-1 due to asymmetrical stretching vibrations of CH2, C-O and C-O-C groups, respectively. The carbonyl absorption peak at 1720 cm-1 that is characteristic of PCL is also visible4,11. Absorption bands at (3,286 cm-1), (3,056 – 3,075 cm-1), (1,600 – 1,700 cm-1), (1,480 – 1,580 cm-1), and (1,220 – 1,300 cm-1) are indicative of keratin proteins. The bands have been denoted as amide A, B, I, II, and III, respectively.
The bands and peaks present in the FTIR spectra change with the increased keratin concentrations in the composite. Their appearance indicates the presence and structural conformation of keratin chains, however, the interactions between the peaks and bands did cause some difficulty. For example, the amide I band present at 1,600 – 1,700 cm-1 is typically used to study keratin, however, the band is slightly disfigured by the PCL peak. Fortunately, the amide II band will suffice to prove keratin presence, keratin chain conformation, and bonding interactions between the PCL and keratin functional groups.
Cell-Fiber Interaction
Using Scanning Electron Microscopy the cell-fiber interaction was studied. Figure 6 shows the SEM images of the 3T3 fibroblast cells that were cultured on the nanofiber samples for 24 hr. The adhesion of the cells to the nanofiber samples is visible and shows that the cell filopodia tend to follow the alignment of the nanofibers when they are similar in diameter. Almar Blue (AB) assay was performed to quantify the viability of 3T3 cells in the fibers. AB is the chemical resazurin. This non-fluorescent dye enters the living cells, mitochondrial reductases reduces resazurin to resorrufin which is pink and fluorescent. There was not significant difference in the toxicity levels of different ratios of PCL/keratin.
Figure 1. Pictures of the Keratin Extraction Process. (A) Cleaned human hair before extraction; (B) Hair after keratin extraction; (C) Keratin extract solution; (D) Lyophilized keratin powder. Please click here to view a larger version of this figure.
Figure 2. Digital Camera Pictures of Electrospun Fibers. Images of as synthesized fibers of CL/keratin with different ratios collected on aluminum foil. Please click here to view a larger version of this figure.
Figure 3. SEM Images of PCL/keratin Nanofibers. (A-C) images of the nanofibers spun from solutions with PCL/keratin ratios of 70:30, 80:20, and 90:10, respectively. The insets show higher magnification images of each corresponding SEM image. SEM images (D-F) and (G-I) represent the fibers shown in images (A-C) after 1- and 7-week degradation tests, respectively. The scale bar in the insets represents 500 nm (see scale bar in image C). Please click here to view a larger version of this figure.
Figure 4. Graph of Modulus versus PCL/Keratin Ratios. A graph of keratin concentration versus Young's Modulus shows the graphical trend of variation of the Young's Modulus. The trendline aids in understanding breaking elongation rate. Please click here to view a larger version of this figure.
Figure 5. FTIR Spectra of PCL/keratin Nanofibers with Different Ratios. The spectrum confirms the bonding of PCL to keratin. The major peak measured at 1,722 cm-1 agrees with standard basic measurements of PCL absorption band and is visible in all spectra. Please click here to view a larger version of this figure.
Figure 6. SEM Images Showing the Morphology of 3T3 Fibroblast Cells Seeded on PCL/keratin Nanofiber Membrane. Images A, B and C represent the PCL/keratin with ratios of 90/10, 80/20, and 70/30, respectively. Images (A'), (B'), and (C') are higher magnification images of (A), (B), and (C), respectively. The darker areas on the images indicate the location of each fibroblast cells attached on top of and throughout the nanofiber topography. Please click here to view a larger version of this figure.
Ratios | Young's Modulus (Mpa) | Breaking Strength (Mpa) |
100/0 | 10 ± 2 | 3 ± 1.2 |
90/10 | 8 ± 1 | 2 ± 0.5 |
80/20 | 5 ± 1.5 | 1 ± 0.2 |
70/30 | 4.5 ± 1.6 | 1 ± 0.3 |
Table 1. Mechanical Properties of PCL/Keratin Fibers
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.
The keratin extraction is the limiting step of this procedure. The yield of keratin powder is very low, 0.7 – 2%. 20g of human hair yielded 0.14 – 0.4 g keratin. Another critical step in producing the electrospun fibers is formulating a solution that is suitable for electrospinning. Keratin was easily dispersed in DI water, however, upon electrospinning, the keratin/water solution did not result in fiber formation. To provide the necessary molecular interactions to create nanofibers a copolymer was introduced to the solution. PCL dissolved in TFE, was able to interact more strongly with keratin through hydrogen bonding. The TFE was largely responsible for stability of the copolymer complexes because of its electronegativity and acidic behavior.
Mixing the keratin solution with the PCL solution presented new challenges due to that fact that PCL is known to be hydrophobic while the keratin is known to be hydrophilic. As we increased the ratio of keratin it was difficult to obtain the homogeneous mixture. This issue was resolved by adding keratin solution drop wise to the PCL solution, and vortexing it manually for 30 min.
SEM images show excellent surface morphology that is ideal for cell growth and proliferation. The comparative FTIR results demonstrate good miscibility between PCL and keratin in the electrospun fibers. This may be because of intermolecular hydrogen bonding between PCL and keratin. Another factor is the speed with which electrospun fibers solidify preventing PCL aggregation in the mixture. The structural and mechanical integrity is sustained by the molecular interactions between the PCL and keratin, making the material suitable for regenerative medicine applications. These electrospun fibers were found to have young moduli close that of the native tissue. Mechanically strong fiber is able to support cell adhesion and proliferation. The PCL/keratin nanofibers exhibited good uniformity, structural integrity, suitable mechanical properties, and cellular compatibility. Young's modulus (MPa) for PCL/keratin at ratios of 100:00, 90:10, 80:20, and 70:30 was found to be 10 ± 2, 8 ± 1, 5 ± 1.5, and 4.5 ± 1.6, respectively. The moduli decreased as the ratio of keratin added increased. Cell adhesion and proliferation on the PCL/keratin fibers confirms that the fibers are not toxic and provide support for cells growth. The filopodia growth along the nanofibers indicates favorable interaction between the fibroblasts and the PCL/keratin fibers.
Electrospinning technique was successfully used to synthesize the PCL/Keratin based nanofibers. This technique, unlike other existing methods, has proven to be reliable and cost-effective and can potentially be used in large-scale nanofiber production. From this study, we conclude that PCL/Keratin based composite nanofibrous scaffolds have the potential to be used for biomedical applications and mimic natural ECM for tissue engineering applications.
The authors have nothing to disclose.
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.
Human Hair | N/A | N/A | Obtained from Local Barber Shop in Greensboro |
Peracetic acid | Sigma Aldrich | N/A | |
PCL (e-caprolactone polymer) | Sigma Aldrich | 502-44-3 | Mn 70-90 kDa |
Trifluoroethanol (TFE) | Sigma Aldrich | 75-89-8 | |
Tris Base (TrizmaTM Base Powder) | Sigma Aldrich | N/A | > 99.9% crystalline |
Hydrochloric Acid | Fischer Scientific | A144C-212 Lot 093601 | Waltham, MA |
Kwik-Sil | World Precision Instruments | N/A | Sarasota, FL |
Cellulose membrane | Sigma Aldrich | N/A | 12-14 kDa molecular cutoff |
optical microscope | Olympus BX51M | BX51M | Japan |
scanning electron microscope | Hitachi SU8000 | SU8000 | Japan |
Table-Top Shimadzu machine | North America Analytical and Measuring Instruments AGS-X series | AGS-X Series | Columbia, MD |
Fourier transform infrared spectroscopy | Bruker Tensor 2 Instrument | N/A | Billerica, MA |
Microcal Origin software | N/A | N/A | Northampton, MA |
X-ray diffraction (XRD) | Bruker AXS D8 Advance X-ray Diffractometer | N/A | Madison, WI |
Fibroblast 3T3 cell | American Tissue Type Culture Collection | N/A | Manassas, VA |
Dulbecco’s modified Eagle’s medium (DMEM | Invitrogen | N/A | Grand Island, NY |
Spectra max Gemini XPS microplate reader | Molecular Devices | N/A | Sunnyvale, CA |
Student- Newman-Keuls post hoc test | SigmaPlot 12 software | N/A | N/A |