Keratin-containing nanofibers for cell growth and tissue scaffolds

7 October 2015
Duygu Yüksel Deniz and Memet Vezir Kahraman
A polymer solution that includes human hair is used in a UV-electrospinning technique to fabricate non-toxic fibers with diameters of less 350nm.

Nanofibers—with diameters on the nanometer scale—are commonly used in medicine, the textile industry, biology, chemistry, aerospace technology, and in tissue engineering. There is an ever-increasing number of studies that involve nanofibers, and their application in the developing field of nanotechnology is also expanding. As such, there is a continually growing demand for new nanofiber materials. For instance, there is a need within the field of tissue engineering for cross-linked materials. This is because such materials can provide a porous surface on which cells can grow, live, and easily multiply. They also enable the exchange of necessary materials from cell membranes.

Nanofibers can be fabricated using various methods, such as self-assembly, phase separation, electrospinning, or template-assisted growth.1 The electrospinning approach, however, is more simple, efficient, and cheaper than the other methods. A typical electrospinning setup consists of several instruments, including an adjustable and regulated high-voltage power supply, a syringe pump, and a collector unit. UV-reactive electrospinning is a technique that combines an electrospinning setup with a UV lamp. Cross-linked materials can be produced with the UV version of the electrospinning approach.

In our work we aim to help meet the needs of the tissue engineering field. We have thus developed a technique to prepare nanofiber scaffolds that contain human hair keratin.2 Keratins—a group of cysteine-rich structural proteins—are the major structural fibrous proteins found in hair, feathers, wool, nails, and horns.3 Proteins such as keratin tend to act as an extracellular matrix, which helps cells form a tissue scaffold. In our technique, we synthesized and modified hydroxyapatite—a common material in bones—with the use of 4-vinylbenzene boronic acid (4-VBBA). Through this process, we incorporate a vinyl bond into the structure of the material and we are therefore able to use UV light in our electrospinning system. We used poly(vinyl alcohol)—PVA—as a carrying polymer solution for the system, which we prepared by dissolving PVA powder in distilled water. To prevent the PVA-based nanofibers from dissolving in a cell culture medium, we modified the PVA by adding 3-trimethoxysilylpropylmethacrylate and tetraethylorthosilicate to the PVA solution (in a 2:1 ratio).4

We conducted our synthesis of hydroxyapatite (HAP) and 4-VBBA-HAP according to previously published methods.5, 6 In addition, we obtained human hair samples from a local barber. To remove any naturally present residues, we cleaned the untreated hair in a flask with water. We then added a sodium hydroxide solution to the flask and the mixture was heated until the hair had dissolved. We also prepared the electrospinning solutions by adding the 4-VBBA-HAP to 50ml of the PVA solution. We added different amounts of the keratin solution to the electrospinning solution so that we obtained four formulations (see Table 1). We then stirred these mixtures at 50°C before starting the electrospinning process. After the mixtures had cooled, we added a photoinitiator.

Solution formulations used to fabricate the nanofiber scaffolds. The solutions are formed by adding different amounts of the keratin solution to the electrospinning solution. The electrospinning solution is a mixture of poly(vinyl alcohol)—PVA—and a synthesis of 4-vinylbenzene boronic acid and hydroxyapatite (4-VBBA-HAP). Tetraethyl orthosilicate (TEOS) and 3-trimethoxysilylpropylmethacrylate (MEMO) are also included in the formulations.

Formulation4-VBBA-HAP [g]PVA [g]TEOS [g]MEMO [g]Keratin [g]Photoinitiator [g]
F153.51.7510.06
F20.553.51.750.50.06
F30.553.51.7510.06
F40.553.51.7520.06

Our electrospinning setup is shown in Figure 1. In our experiments, we applied a positive high-voltage source through a wire to the tip of a syringe needle. The electrospinning solution (i.e., PVA) was contained within a syringe that was controlled by the syringe pump. The electrospinning parameters we employed were a voltage of 16kV, a distance of 10cm, and a flow rate of 0.5ml/h. We collected our fabricated nanofibers on aluminum foils and then used a scanning electron microscope (SEM) to investigate the morphological properties of the samples (see Figure 2). The images we obtained show that our nanofibers are bead-free, homogeneous, have a cross-linked structure, and diameters of less than 350nm.


The UV-electrospinning experimental setup used in the fabrication of nanofiber scaffolds. A positive high-voltage source is applied through a wire to the tip of the syringe needle. The syringe is controlled by a pump and contains the electrospinning polymer solution. Light is generated from a UV lamp. The final fabricated nanofibers are collected on aluminum foils.


Scanning electron microscope (SEM) image of the nanofiber scaffold formulation F3 (see Table 1), obtained before the cell culturing process. Magnification: ×10,000.

After we completed the electrospinning process, we seeded sarcoma osteogenic (SAOS) and endothelium (ECV 304) cells (obtained from the American Type Culture Collection). We also used an MTT colorimetric assay to determine the cytotoxicity of our nanofibers (see Figure 3).7,8 Our measurements show that the viability of the nanofibers is very close to that of the control group. These results indicate that both the SAOS- and ECV-304-seeded nanofiber scaffolds are non-toxic. We also obtained SEM images of the nanofibers (see Figure 4) after we had conducted the MTT test, which was done after cell growth. These images show that the nanofibers are still present and the main cross-linked structure remains even after the cell seeding process. Although the morphology of the nanofibers is the same, their diameters have increased and the nanofibers have become swollen because of the cell culture medium. This structure has thus allowed cells to grow and to begin tissue formation. We clearly see—Figure 4(a) and (b)—that the seeded cells have multiplied and formed a suitable tissue scaffold. Furthermore, we observe the interaction between cells and the nanofibers—Figure 4(c) and (d)—which indicates that our fabricated nanofiber scaffolds are non-toxic.


Results from an MTT colorimetric assay used to determine the cytotoxicity of the nanofibers in a control sample and four different formulations (see Table 1). Results from tests that involved both (a) endothelium (ECV 304) and (b) sarcoma osteogenic (SAOS) cells are shown.


SEM images of the fabricated nanofibers in the four formulations (see Table 1) after cell culture. (A) F1 fibers with SAOS-seeded cells (magnification: ×5,000). (B) F2 fibers with ECV-304-seeded cells (magnification: ×5,000). (C) F3 fibers with ECV-304-seeded cells (magnification: ×5,000). (D) F4 fibers with ECV-304-seeded cells (magnification: ×10,000).

We have used a UV-electrospinning technique to produce keratin-containing nanofibers that have diameters of less than 350nm. Results from our MTT assay results, and from SEM images of our samples, are compatible. They show that the fabricated nanofibers are non-toxic and suitable for cell growth. The SEM images also show that both SAOS and ECV 304 cells had multiplied and formed a tissue scaffold for the growth of cells. In our future work, we plan to conduct MTT analyses with different cell types. In addition, we will investigate how tissue formation and viability can be used to improve tissue growth.


Authors

Duygu Yüksel Deniz
Marmara University

Duygu Yüksel Deniz has a PhD in organic chemistry. Her research areas are nanofibers, biomaterials, electrospinning, UV-curable materials, and polyurethanes.

Memet Vezir Kahraman
Marmara University

Memet Vezir Kahraman is a professor of organic chemistry. His research interests include synthetic organic–inorganic hybrid coatings, UV-curable materials, thiol-ene polymerization systems, enzyme immobilization, nanofibers, polyurethanes, electrospinning, biopolymers, membranes, organic chemistry, and polyesters.


References

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  2. D. Y. Deniz, M. V. Kahraman and S. E. Kuruca, UV-reactive electrospinning of keratin/4-vinyl benzene boronic acid--hydroxyapatite/poly(vinyl alcohol) composite nanofibers, Polym. Compos., 2015.

  3. J. Li, Y. Li, L. Li, A. F. T. Mak, F. Ko and L. Qin, Preparation and biodegradation of electrospun PLLA/keratin nonwoven fibrous membrane, Polym. Degradation Stability 94, pp. 1800-1807, 2009.

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  8. E. Çakmakç, A. Güngor, N. Kayaman-Apohan, S. E. Kuruca, M. B. Çetin and K. A. Dar, Cell growth on in situ photo-cross-linked electrospun acrylated cellulose acetate butyrate, J. Biomater. Sci. Polym. Ed. 23, pp. 887-899, 2012.

DOI:  10.2417/spepro.006120



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