Keratin Nanofibers as a Biomaterial

Zhi-Cai Xing, Jiang Yuan, Won-Pyo Chae, and Inn-Kyu Kang*

Department of Polymer Science and Engineering, Kyungpook National University

Daegu 702-701, South Korea e-mail: ikkang@knu.ac.kr

Suk-Young Kim School of Materials Science and Engineering,

Yeungnam University Gyongbuk 712-749, South Korea

Abstract—Keratin is one of the most abundant proteins which can be used in a variety of biomedical applications due to its biocompatibility and biodegradability. However, keratin is insoluble in solvents and soluble in few solvent systems, which lead to a limitation in its processability for further utilization. In this study, keratin was extracted and chemically modified by reacting sulfide side group with iodoacetic acid to enhance its solubility in organic solvent. The modified keratin (m-keratin) was then blended with poly (ethylene oxide) (PEO) in different proportions, dissolved in 2,2,2,-trifluoroethanol (TFE), and electrospun to produce nanofibrous mats. The m-keratin nanofibers were obtained by crosslinking the m-keratin/PEO nanofibrous mats with glutaraldehyde vapor (25%) and washed with distilled water for 3 times to remove PEO. The morphology, diameter distribution, biodegradation, and interaction of NIH 3T3 cells with nanofibrous mats were investigated. The results showed that the crosslinked m-keratin nanofibrous mats have a potential to be used in tissue engineering and wound dressing.

Keywords- biodegradability, NIH3T3 cells, keratin, nanofiber, poly(ethylene oxide)

I. INTRODUCTION In recent years, much attention has been focused on the

electrospinning of biopolymers such as silk fibroin [1-2], collagen [3], fibrinogen [4], gelatin [5] and elastin [6]. However, keratin has received poor attention even though it is one of the most abundant proteins, being the major component of hair, feathers, wool, nails and horns of mammals, reptiles and birds. Keratin is well known as biocompatible and biodegradable proteins [7], which can accelerate the growth of fibroblast [8]. Thus, keratin is expected to be applicable for biomedical use in a similar manner to collagen and fibroin. Unfortunately, the poor mechanical properties of regenerated keratin hinder its processability and restrict its practical applications to blending with appropriate polymers having better structural properties. In the previous work, Lee at al [9] blended keratin and fibroin and found that the films composed of silk fibroin and S-carboxymethyl kerateine showed lower blood coagulation compared to silk fibroin or keratin alone [10]. Yuan et al. [11] fabricated the poly(hydroxybutylate-co-hydroxyvalerate) (PHBV)/keratin composite nanofibrous mats and concluded the resulted keratin nanofibers contained many beads due to the broad molecular weight distribution

and low dissolvability of keratin. Aluigi et al [12] fabricated composite nanofibers consisted of keratin and poly (ethylene oxide) (PEO) using water as a solvent. As a result, regularly shaped nanofibers could be obtained at the ratio of 50/50 and polymer concentration of 7-10%. They only extracted the keratin from wool and studied the chemical, physical and rheological characteristics of the electrospun PEO/keratin mats [13].

PEO is an amphiphilic, water-soluble, and non-degradable polymer with good biocompatibility [14] and low toxicity [15]. To produce keratin nanofibers, PEO has been added to keratin solution with different ratio to improve the processability of the keratin itself because it can be electrospun without defects from solutions [12, 13, 16].

In this study, keratin was chemically modified with iodoacetic acid for enhancing its solubility in organic solvent. The modified keratins (m-keratin) were mixed with PEO at different ratio, dissolved in 2,2,2,-trifluoroethanol (TFE) and electrospun to produce m-kerain/PEO composite nanofiber mats. The m-keratin nanofibers were obtained by crosslinking the m-keratin//PEO nanofiber mats with glutaraldehyde vapor followed by removal of PEO by washing with water. The morphology of the nanofibrous mats were studied with field-emission scanning electron microscope (FE-SEM). Biological performances of the nanofibrous mats including biodegradation and cell-scaffold interaction were also studied.

II. MATERIALS AND METHODS

A. Preparation of m-keratin/PEO blend solutions Keratin was extracted and chemically modified according

to the method previously reported [11]. In brief, raw keratin (MP Biomedical Company, Germany) was first mixed with urea, sodium dodecyl sulfate (SDS), 2-mercaptoethanol and water. The mixture was stirred for 12 h at 60ºC and then filtered. Subsequently, the filtrate was dialysed against deionized water to afford a colorless solution. The dialysate (unmodified keratin solution) was allowed to react with iodoacetic acid for modification. Finally, S-(carboxymethyl) keratin was lyophilized to obtain modified keratin (m-keratin).

PEO powder with a viscosity-average molecular weight of ca. 9×105 g/mol (Sigma–Aldrich, St. Louis, MO) was dissolved in 2,2,2,-trifluoroethanol (TFE) at the room

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2010 International Conference on Nanotechnology and Biosensors IPCBEE vol.2 (2011) © (2011) IACSIT Press, Singapore

temperature for about 12 h. The concentration was adjusted at 2 wt %.

The m-keratin/PEO blend solutions were prepared by adding m-kerain to the PEO solution and stirring for 12 h at room temperature. The blend solutions of the m-keratin/PEO were adjusted at the concentration of 2 wt% and the ratio of m-keratin and PEO was changed from 50:50 to 90:10.

B. Preparation of m-keratin/PEO blend nanofibers The blend solution was delivered to a metal needle

connected to a high-voltage power supply. Upon applying a high voltage, a fluid jet was ejected from the needle. As the jet accelerated towards a grounded collector, the solvent evaporated and a charged polymer fiber was deposited on the collector in the form of a nanofibrous mat. The typical parameters for electrospinning were as follows: 9 kV (voltage), 12 cm (distance between tip and receptor), 1.0mLh−1 (feed rate), 60% (humidity) and 25ºC (temperature). For analysis of the morphology of the electrospun fibers, the samples were sputter-coated with gold, and examined using FE-SEM (Hitachi S-4300, Japan). The diameters of the electrospun nanofibres were measured at 100 different points from SEM pictures for each sample produced.

C. Preparation of m-keratin nanofibers The m-keratin/PEO nanofiber mats need to be

crosslinked to reduce their solubility in water. The electrospun m-keratin/PEO nanofibrous mats were crosslinked by treating them with glutaraldehyde vapor and saturated with a 25% glutaraldehyde aqueous solution at room temperature for 4 h. This was followed by treatment with 0.1 M glycine aqueous solution to block unreacted aldehyde groups. The crosslinked m-keratin/PEO nanofibrous mat was then washed with distilled water for three times (10 minutes each) to produce m-keratin nanofiber mats. To examine the presence of PEO in the crosslinked m-keratin/PEO mat after removal by water, fluorescein-tagged PEO (F-PEO) was synthesized by reacting hydroxyl end group of PEO with group of fluorescein isothiocyanate (FITC) [20].

D. In vitro biodegradation The m-keratin/PEO and m-keratin nanofiber mats were

cut into rectangles (20 × 20 × 0.05 mm) for in vitro degradation testing. Each specimen was placed in a test tube containing 10 ml of phosphate-buffered saline (PBS, pH 7.0, Gibco) and incubated for 12 h at 37ºC. After incubation, the samples were washed and lyophilized for 24 h. In order to measure the enzymatic degradation of nanofibrous mats, the samples were incubated in a PBS containing trypsin (10 mg/ml) at 37ºC. After incubation for a requisite time (2 h or 12 h), the samples were washed with distilled water and then lyophilized for 24 h. Morphological changes were observed with a FE-SEM.

E. Cell adhesion In order to examine the interaction of nanofiber mats

with cells (NIH 3T3), the circular nanofibrous mats were

fitted in a 24-well culture plate and subsequently immersed in a DMEM medium containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin G-streptomycin. To seed the cells, 1 ml of NIH 3T3 cell solution (3×104 cells) was added and incubated in a humidified atmosphere of 5% CO2 at 37ºC. After incubation for a 4 h, the medium solution was removed. These samples were washed twice with the PBS, and fixed by 2.5% glutaraldehyde aqueous solution for 20 min. The sample mats were then dehydrated in a graded concentration of ethanol (25, 50, 75, 90, and 100) for 10 min each. Finally, the sample mats were air dried in a fume hood overnight. Dry cellular structures were sputter-coated with gold and observed with a FE-SEM.

F. Determination of cell viability A standard Live/Dead assay was used to image cell

survival, adhesion, and spatial organization. After 6 days incubation, cells were collected by centrifugation and incubated in calcein-AM (1 mM in PBS) and ethidium homodimer-1 (2.5 mg/ml PBS) solution for 15 min. Cells with compromised membranes exhibit red-fluorescence from the live-cell impermanent nucleic acid stained with ethidium homodimer-1. Cells with intact membranes are able to use nonspecific cytosolic esterases to convert nonfluorescent calcein-AM into bright green-fluorescent calcein. Cells were observed under a fluorescence microscope using a band-pass filter (Nikon Eclipse E600-POL, Japan).

Cell viability was measured after 2, 4 and 6 days of culture using a commercially available MTT assay kit (Sigma). After incubation of certain time, the medium was replaced with a (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) solution and incubated for further 4h. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, yielding purple formazan crystals. Formazan crystals were then dissolved in PBS solution. The optical density (OD) of the solvent is proportional to the mitochondrial activity of the cells on the surface. OD was measured at 570 nm using a kinetic microplate reader (EL 9 800, Bio, Tek, Instruments, Inc, Highland Park, USA). Background absorbance at 690 nm was subtracted from the measured absorbance.

G. Statistical analysis Results are displayed as mean ± standard deviation.

Statistical differences were determined by a student’s two-tailed t-test. Scheffe’s method was used for multiple comparison tests at a level of 95%.

III. RESULTS AND DISCUSSION

A. Nanofiber morphology Figure 1 shows the SEM images of nanofibers obtained

from electrospinning of the m-keratin/PEO blend solutions. The electrospinning of a 2 wt% PEO pure solution produced nanofibers without defects at the condition of a flow rate of 1ml/h and a voltage of 9 kV. However the diameter distribution of PEO nanofibers was so broad, ranging from 200 nm to 2000 nm. The diameter distribution of PEO became narrow with an increase of m-keratin content (Figure

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1b,c,d). Therefore, it could be say that the blend composition plays an important role in determining the diameter distribution of the nanofibers. In addition, the average diameter of the blend nanofibers gradually decreased from 950±50 to 400±30 nm as the m-keratin content increases (Figure 1b and Figure 1d). The viscosity of m-keratin/PEO (50/50) blend solution (224 cP) was significantly decreased down to 34 cP with the increase of m-keratin (90/10). The conductivity of m-keratin/PEO blend solution also increased with an increase of the m-keratin data not shown. In fact, it is known that lower viscosity promotes the formation of finer nanofibers [17] and higher charge density carried by jet forms smoother and finer nanofibers because the stronger whipping instability of the jet enhances the filament stretching [18,19].

B. Crosslinking of m-keratin/PEO nanofibers The m-keratin/PEO blend nanofibers can be easily

dissolved in water. Therefore, the nanofibers need to be crosslinked to reduce their solubility. The most popular crosslinking reagent used in proteins is glutaraldehyde vapor. Glycine solution was used to block residual aldehyde group after treatment of m-keratin/PEO nanofibers with glutaraldehyde vapor. Figure 2 showed the morphology of m-keratin/PEO (90/10) nanofibrous mats before and after the crosslinking with glutaraldehyde vapor for 4h. Obviously, the m-keratin/PEO nanofiber mat lost its fibrous morphology slightly after the crosslinking. Figure 3 showed the fluorescence images of the m-keratin/F-PEO(90/10) mat (Figure 3a) and the m-keratin nanofibrous mat obtained by removal of F-PEO by water. As a result, the m-keratin/F-PEO showed the image of green color due to the presence of F-PEO. However, the green color was almost disappeared after washing the crosslinked m-keratin/F-PEO nanofibrous mats with water (Figure 3b). It is revealed, from the data of fluorescence image, that m-keratin nanofiber mat could be obtained by crosslinking the m-keratin/PEO mat with glutaraldehyde vapor followed by washing with water.

Figure 1. SEM images of electrospun nanofibers with different ratio of m-keratin and PEO (a) pure PEO, (b) 50/50, (c) 70/30 and (d) 90/10.

Figure 2. SEM images of the m-keratin/PEO (90/10) nanofibrous mats before (a) and after (b) crosslinking with glutaraldehyde vapor for 4h.

C. In vitro biodegradation The image of the mat biodegraded was examined with

FE-SEM. Figure 4 illustrates the morphological changes of the electrospun mats during in vitro degradation. The crosslinked m-keratin nanofibrous mats lost their nanofibrous form after 2h degradation in trypsin aqueous solution, and the mat seriously degraded and remained as debris after 12h incubation. Yuan et al [11] reported on biodegradation of m-keratin/PHBV nanofibrous mats by trypsin solution. As a result, fibrous morphology almost not changed when the m-keratin/PHBV mat was incubated in trypsin agueous solution for 24 h. It is concluded that m-keratin/PEO nanofibrous mats underwent a higher biological degradation than the keratin/PHBV nanofibrous mats. This result suggests that the m-keratin mats studied in this study are suitable for further biomedical and biotechnological applications [21].

Figure 3. Fluorescence images of the crosslinked m-keratin/F-PEO mat before (a) and after (b) removal of F-PEO by water nanofibours mats.

Figure 4. SEM images of the crosslinked m-keratin nanofibrous mats incubated in trypsin solution for 2h (a) and 12h (b).

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D. Cell-scaffold interaction. To evaluate cellular behavior on electrospun fibers,

fibroblasts were seeded and cultivated on the crosslinked m-keratin nanofibers and the tissue culture polystyrene (control). As shown in Figure 5, cells were more adhered to the surface of the crosslinked m-keratin nanofiber mat, and showed much more spread morphology than the tissue culture polystyrene. The viability of NIH 3T3 cells on the surface of crosslinked m-keratin nanofibrous mat and the tissue culture polystyrene were also investigated. NIH 3T3 survival was assessed through live/dead fluorescence staining. Images taken on a fluorescence microscope indicate that the surface of crosslinked m-keratin nanofiber mat was a favorable template for cell adhesion. NIH 3T3 cells seeded on crosslinked m-keratin nanofibers displayed a high level of viability as assessed using a standard MTT assay (Figure 7). The viability of cells on the surface of crosslinked m-keratin nanofiber is significantly higher than that on the tissue culture polystyrene after incubation of 6 days, indicating that the NIH 3T3 cells seeded on the crosslinked m-keratin nanofiber surface are healthy and there are no cytotoxic effects (Figure 6). Further, fluorescence images of live cells seeded on surface of crosslinked m-keratin nanofiber showed higher degree of spreading compared to that on the tissue culture polystyrene. These results suggest that the crosslinked m-keratin nanofiber mat is a good scaffold for the adhesion and spread of NIH3T3 cells compared to tissue culture polystyrene.

Figure 5. SEM images of NIH 3T3 cells cultured for 4 h on the tissue culture polystyrene (a) and the crosslinked m-keratin nanofibrous mats (b).

Figure 6. Fluorescence images of NIH 3T3 cells cultured for 6days on the tissue culture polystyrene (a) and the crosslinked m-keratin nanofiber mat (b).

Figure 7. MTT assay, Formozan absorbance expressed as a measure of cell viability from the NIH 3T3 cells cultured on the tissue culture polystyrene and the crosslinked m-keratin/PEO nanofibrous mats (Data are expressed as means ± SD (n=6) for the specific absorbance, * p < 0.05, values are significantly different from those of the previous group).

IV. CONCLUSIONS The m-keratin/PEO blend solutions prepared in 2,2,2,-

trifluoroethanol (TFE) were electrospun to produce nanofibers. All blend solutions were electrospun successfully. Morphological investigation showed that as the m-keratin amount in the blend solution increased, the nanofibers became thinner and more homogeneous. In addition to being biocompatible and biodegradable, crosslinked m-keratin nanofibers induced an enhanced NIH 3T3 cells response. The results demonstrated that the crosslinked m-keratin nanofibers enhanced NIH 3T3 cells adhesion and proliferation as compared to the tissue culture polystyrene. The performance of the crosslinked m-keratin nanofiber warrants future work aimed at in vivo characterization and fabrication of 3-D mats.

ACKNOWLEDGMENTS This research was supported by the research grants of the

Biotechnology development project (2009-0090907) and by the grant of 2010-0011125 from Ministry of Education, Science and Technology of Korea.

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