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Electrospun PVA/keratin nanofibrous scaffold and its application in neural repair.

Introduction

In recent years damage to the peripheral nervous system has become a very serious but a common health issue. It accounts for more than $150 billion health care cost each year [1]. Which is caused primarily from trauma, motor vehicle accidents, sports injury, work and home accidents, tumour resection, diabetes, infection, toxicity, carpal tunnel syndrome, systemic or congenital diseases, complication of surgery, repetitive compression and various other reasons [2]. Injury to PNS may range from major functional losses to mild injury such as sensory or motor deficits. Regardless of severity of the injury it interferes with an individual's work performances and daily activities. Besides functional impairment these nerve injuries often result in pain, dysphasia (itching and burning), and cold intolerance [3]. Approximately 5 % to 10 % of all open wounds are complicated by peripheral nerve trauma which results in nerve gaps. In many mild injuries nerves often recover its normal function but in case of severe trauma nerve repair and regeneration creates unique clinical challenges and opportunities. In case of surgical repair the main goal is to direct regenerating sensory, motor and autonomic axons to the distal end. Though there are many modern surgical techniques available functional restoration remains incomplete [4]. Hence, it is essential to develop novel strategies and grafting options to overcome these nerve injuries.

Surgical treatments for peripheral nerve injury are not very satisfactory as it is associated with various clinical complications such as graft vs. host diseases, donor mobility and formation of neuromas. Research that are presently going on to improve the design strategies to overcome the surgical outcome involves the development of biopolymers and synthetic polymers as primary scaffold or luminal fillers as secondary scaffold with the tailored mechanical, physical and chemical properties. So an alternative to this autologous nerve graft is nerve conduits or nerve guidance tube. Biodegradable nerve guides for regenerated axon is preferred since no foreign body will be left in the host hence there is no need of second surgery so it involves less complications [5]. These nerve guide tube provides a barrier between the growing axon and surrounding environment, the barrier may help to limit unwanted scar tissue formations. In this approach peripheral nerve repair occurs through a channel or conduit, which implements chemical and physical growth and guidance cues to direct the repair process [6]. In recent years a lot of research has been carried out with different synthetic and natural polymers. Most of the work has been carried out with the synthetic polymers among which Poly L-lactic acid (PLLA), Poly glycolic acid (PGA), Poly [alpha]-caprolactone (PCL), Poly (L-lactide-co-glycolide) (PlGa), Polyvinyl alcohol (PVA) hydrogel has drawn the special attention of the researchers [7]. Besides these synthetic polymers various natural polymers have also been considered as the potential material for nerve grafting which includes chitogen, chitin, cellulose, silkfibrion, albumin, elastin, fibrinogen, and gelatin. As natural and synthetic polymer both has disadvantages, polymer blends got more attention of the researchers, work has been carried out with either blend of synthetic polymer for example PLGA and PLLA blend, Polyethylene oxide (PEO) and PVA blend or blend of synthetic and natural polymer such as PEG and chitosan, PLGA and collagen, PGA and chitosan, Polyurethane and collagen, PCL and gelatin [8].

In past few years various scaffold fabrication method has been investigated in detail which includes self assembly, phase separation and electrospinning. In this study electrospinning technique is used for the fabrication of nanofibrous scaffold. It is a wet spinning process that uses high electric voltage to draw fibers from a liquid polymer solution or melt. The process of fiber formation from the liquid is entirely physical, either by loss of solvent or freezing of a melt. Electrospinning is preferred over other fabrication methods as it can fabricate polymeric nanofibrous of various diameter ranges from nano to micrometers with tailored mechanical, physical and chemical properties with enhanced and suitable functionality for specific applications [9].

The current study mainly focuses on preparation of PVA/ keratin blended nanofibrous scaffolds. PVA hydrogel was selected for this purpose due to its high biocompatibility, high capacity to absorb water, mechanical resistance, elasticity, mobility and suturability. Keratin was chosen because of its biodegradability, biocompatibility and its integrin-binding domains and the RGD motif which provides the site for cell attachment. Keratin enhances the activity and gene expression of Schwann cells by a chemotactic mechanism, increases their attachment and proliferation, and up-regulate expression of important genes which significantly improves the electrophysiological activities [10]. The PVA and Keratin blended nanofibrous scaffold was prepared and characterized using various analytical instruments and the neural cell response towards the fabricated scaffold was evaluated with glial cell line.

Materials and Methods

Chicken feathers were collected from poultry firm. PVA with 90 % hydrolyzed, (Mn = 125 000 g/mol) were purchased from Sigma Aldrich. 25 % aqueous solution of Gluteraldehyde (GTA) was purchased from SigmaAldrich.

Extraction of keratin from chicken feathers

Chicken feathers collected from the poultry shops were cleaned and washed with shampoo and kept for drying in hot air oven. The dried chicken feathers were again washed with ethanol followed by drying in hot air oven. 0.1 M [Na.sub.2]S and 5 % NaOH solution was prepared and the dried chicken feathers were dissolved in it. This solution was kept in hot air oven for 2 hours at 60[degrees]C. After 2 hours the solution was filtered with Wattman filter paper and the filtered solution was kept for dialysis. A clear keratin solution was obtained after 2-3 days of dialysis. The concentration of keratin in the crude solution was found to be 6.2 mg/ml. The molecular weight was determined by using SDS-PAGE and it was found to be in the range of 30-40 KDa and 45-65 KDa. The structure of the extracted keratin from chicken feathers was determined by using Circular Dichorism Spectropolarimeter (JASCO J -715, USA).

Electrospinning

Electrospinning was carried out with an electrospinning instrument (ESPIN-NANO MODEL V2, INDIA). PVA and Keratin blends concentration were optimized after various trials and optimized at the ratio 80:20, the polymer solution was placed in a 5 ml plastic syringe fitted to a needle with tip diameter of 0.4 mm and a high voltage of 20 kv was applied using a high voltage power supply. A rotational collector drum was placed at 10 cm distance from the tip of the needle which helped to obtain the nanofibrous mat. The flow rate of polymer in the syringe was maintained at 1 ml/h. The sample was collected, vacuum dried and stored in desiccators for further trials. Pristine PVA was also prepared for comparison purpose. The nanofibrous scaffolds were crosslinked using GTA as the procedure cited in the literature [11].

Characterizations of samples

The electrospun samples were characterized using SEM (Vega3 testscan) for morphological studies , FT-IR (FT-IR spectrometer, Bruker, Germany), DSC (NETZSCH DSC 200F3), TGA (TGA Q50, Bruker, Germany) and XRD (Powder X-Ray Diffractometer, Bruker, Germany) for surface characterization .The tensile strength of the samples was tested using a universal testing machine (Instron 3345, USA).

Swelling studies

Nanofibrous scaffolds with equal weight and same dimensions were taken and kept inside closed plastic bottles containing 1 ml of phosphate buffer solution (PBS; pH 7.4). The scaffolds were incubated for various periods of time at 37[degrees]C. The amount of fluid uptake was measured by taking the wet weight after carefully wiping of excess water using a filter paper. The swelling value is calculated by using the following equation.

S = Ww - Wd / Wd x 100

Where [W.sub.d] - Dried weight

[W.sub.w] - wet weight

Degradation studies

The degradation study of the scaffolds was carried out in-vitro by incubating equal volume samples in PBS at pH 37[degrees]C for various periods of time. After each degradation periods the samples were dried in vacuum oven at room temperature for overnight. The degradation index (Di) was calculated by the following equation.

Di= Wo - Wt / Wo x 100

Where [W.sub.t-] Weight of the sample after degradation period.

[W.sub.0] - Initial weight

Protein adsorption studies

The adsorption of Bovine Serum Albumin (BSA) onto the nanofibrous scaffold was also performed. The samples of equal weight was kept in PBS 0.5 M for 24 h and samples were equilibrated with it for 2, 4 and 6 h. After respective periods of incubation samples were taken out and absorption of the BSA was measured at 272 nm in UV Spectrophotometer (Hitachi, Japan).

Cell culture study

Glial cells were cultured at 37[degrees]C and 5 % C[O.sub.2] in 24 well tissue culture plates under sterile conditions, medium was changed every third day. The nanofibrous scaffold was washed and prewetted with PBS. Samples were sterilized with 100 % ethanol, and then kept in UV light chamber for 24 h. Glial cells were seeded at a density of 5-103 cells/500 ml per well. Sterilized samples were placed on cover slips and glial cells were transferred into it. The nanofibrous scaffolds were taken out 3 days after cell seeding. Then fixed in 2 % gluteraldehyde for 2 h and post fixed with 1 % osmium tetroxide and 1 % tannic acid for 30 min and dehydrated with 50-100 % v/v ethanol. Subsequently samples were treated with Hexamethyldisilazane for 30 min two times and kept for air drying. Finally, the samples were coated with gold using sputtering and observe under Scanning Electron Microscope (SEM) to analyse the attachment, proliferation of the glial cells in the nanofibrous scaffolds and other morphological changes. The cell viability assay was carried out by 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT assay). MTT assay is as well known in-vitro analysis method for cytotoxicity. It was carried out to determine survival of glial cells on the nanofibrous scaffold. In this assay live cells reduces the tetrazolium dye to its insoluble Formazan giving a purple coloure, whereas the dead cells does not show any reaction. Cells were trypsinized, centrifuged, and resuspended in the culture medium, 6 mg/ml of synthesized scaffolds were added to each 24 well tissue culture plates. After 7 days of cell seeding in 24 well plates, cells were washed with PBS and incubated with 20 % of MTT reagent containing serum free medium, after 24 h of incubation at 37[degrees]C in 5 % C[O.sub.2], aliquots were transferred into a 96-well plate. The absorbance by each well was measured at 492 nm using UV spectrometer and the cell viability was measured after 24 h and 72 h.

Results and Discussion

Electrospinning

The presently available treatments for peripheral nerve injury are not very satisfactory. The gold standard of treatment for peripheral small nerve gaps is the autologous nerve graft, however this treatment is associated with a variety of clinical complications. Thus, the development of a nerve guidance conduit, that matches the effectiveness of the autologous nerve graft, is required for neural repair. The use of biomaterials processed by the electrospinning technique has gained significant interest in neural tissue engineering. Electrospinning can generate fibrous meshes having fiber diameter dimensions at the nanoscale and these fibers can be nonwoven or oriented to facilitate neuritis extension via contact guidance. Electrospun scaffolds made of natural polymers, proteins and compositions enhance neural cell function, electrospun scaffold has considerable promise in repair and regeneration of damaged nerves both in-vitro and in-vivo. PVA was selected for this purpose as it is one of the most biocompatible, hydrophilic and biodegradable polymer and seems to have a great potential as nerve graft, on the other hand Keratin is also biocompatible, supports cell growth and besides that studies showed it to be a potential guiding material for axons. Fabrication of keratin nanofiber by electrospinning combines the aforementioned properties to keratin which include high surface to volume ratio and the high porosity. Keratin is ideal for adhering and proliferating cells, but they show poor mechanical properties. PVA was blended to improve the mechanical strength, further it was cross linked to obtain the desired mechanical strength and stability. Thus, the combination of protein and PVA will provide composite materials with good cell adhesion and mechanical properties. PVA and PVA/Keratin nanofibrous scaffolds were fabricated by using electospinning technology. It was found that their properties were strongly influenced by the concentration of the polymeric solution, voltage and flow rate. The fibre diameter was highly influenced by tip diameter and concentration of the solution, the fibre diameters are directly proportional to the polymer concentration. Crude keratin was added to the PVA polymer which gave a high probability of beads formation, to minimize such defects various parameters were maintained. The scaffold can be fabricated to any thickness by adjusting the collecting time. An electrically charged polymer jet is created when the applied electrical force overcomes the surface tension of the polymer solution. After the polymer jet get dries, it forms charged fibres. These residual charges exerts a repel force on the next depositing fibre by changing its orientation or pushing it aside, which leads to pores or voids between fibres. The electrospun nanofibrous scaffolds were fabricated, as shown in figure 1. Various studies were carried out and the results are discussed as follows.

Structural conformation of protein extracted from chicken feathres

The structural details of the extracted keratin proteins were obtained from the CD spectrum as shown in figure 2. The spectrum was constructed from the aqueous solution of keratin that confirms ant parallel beta sheet structure with negative minimum absorption band at 225 nm and a weak positive maximum absorption band at 190 nm The CD spectrum confirms to have a-pleated sheets twisted together with disulfide bonds.

Scanning electron microscopy (SEM)

The SEM micrographs of representative Scaffolds before and after crosslinking are given in figure 3. Both PVA and PVA/Keratin scaffold surface is open with continuous heterogeneous pores which are interconnected. The surface of the scaffolds composed of mostly thin fibres, diameter of these fibres ranges from 150 nm-360 nm. In case of PVA/Keratin nanofibrous scaffold as crude keratin was blended with PVA it showed some beads formation. After crosslinking with gluteraldehyde fibre morphology PVA scaffold has disturbed whereas the fibre diameter has increased and it showed more flattened fibres. On the other hand fibre morphology of the composite scaffold remained undisturbed but the fibre diameter has increased. Most importantly beads disappeared from the cross linked PVA/Keratin scaffold. Chemical crosslinking by gluteraldehyde immersion produced a more compressed fibrous network which appeared swollen or melted.

FT-IR results

The FT-IR spectra of PVA, PVA/Keratin, and PVA GTA and PVA/Keratin GTA are shown in figure 4. The major functional groups present in PVA are Hydroxyl and acetate groups. All major peaks related to PVA were observed in the FT-IR spectrum. After blending with keratin all the other major groups were remain in the spectra with shifts in peaks along with additional functional groups. The major functional group of keratin are amide, amine and thiol groups. The broad band observed between 3550 and 3200 [cm.sup.-1] is associated with the OH stretch caused by the intermolecular and intramolecular hydrogen bonds. The vibrational band observed between 2840 and 3000 [cm.sup.-1] is the result of the CH stretch from alkyl groups. The peaks between 1730 and 1680 [cm.sup.-1] are due to the CO stretches from the remaining acetate groups in PVA and the peak at 1420 is due to CH-OH bond. At 3500-4000 [cm.sup.-1] the peak observed was due to N-H group, 1500-2000 [cm.sup.-1] peaks represents the amide groups, wavenumbers from 3250 to 3300 [cm.sup.-1] are the NH stretch, peaks at 1600-1700 [cm.sup.-1] are mainly the C-O stretching and the peaks at 1203 and 1024 [cm.sup.-1] are related respectively to the asymmetric and symmetric S-O stretching vibrations. These shows that after blending the two did not generate new chemical bond but there was a strong action of hydrogen bond. After crosslinking FT-IR spectra did not show much variation as the same functional group is present in gluteraldehyde, only the peaks become more sharper due to increase in absorbance.

Thermogravimetric analysis (TGA)

Thermal degradation behaviour of PVA and PVA/Keratin was examined by thermogravimetric analysis (TGA) as shown in figure 5. TGA curves cover a wide temperature range 0[degrees]C - 700[degrees]C which includes the melting points, physical transition and the degradation temperatures of homopolymers. Therefore, the higher values of weight loss in the first decomposition stage indicate the existence of a chemical degradation process resulting from bond scission (carbon-carbon bonds). The lower weight loss may have corresponded to the breaking of the ester linkages and the second stage to the degradation of the whole polymer. The PVA and PVA/Keratin scaffold showed 90 % and 85 % weight loss (first stage) at 280[degrees]C, followed by 10 % weight loss around at 380[degrees]C, complete decomposition occurs at 400[degrees]C and 480[degrees]C respectively. PVA crosslinked scaffold showed decrease in residual amount, on the other hand PVA/Keratin blended scaffold after modified with GTA showed increase in residual amount. After crosslinking PVA scaffold shows similar weight loss in the first stage but there is an increase in thermal stability at the second stage, the fibre degradation started at 500[degrees]C. It can be concluded from this analysis that after blending PVA and keratin and further cross linking with GTA shows increase in thermal denaturation temperature.

X-ray diffraction (XRD) studies

Every crystalline material shows unique diffraction pattern and their characters are represented by distance (d) between each diffraction crystal face which is related to the shape and size of the crystal cell and relative intensity (I) which is related to the species of particles and its position in the crystal cell. Pure keratin is amorphous by nature and its crystallinity is 0. PVA is semi crystalline in nature and strong absorption peak was found at 18, 21, and 23 as shown in figure 6. After blending PVA with keratin, there are diffraction peak at 17 and 19 .When compared with pure PVA scaffold the absorption peak at 23 disappeared, which indicates that crystal cell in the composite scaffold has changed. After crosslinking PVA with GTA comparatively flat peaks were found at 21, 23 and in PVA/Keratin GTA modified scaffold peaks were observed in 18 and 20. It can be concluded that the flat peaks and shifts in the peaks are due to the influence of the semi crystalline nature of PVA on keratin which indicates the strong bonding of the two at the molecular level which was further influenced by GTA and showed disappearance and shifting of peaks.

Tensile strength measurement

It is very important to find out the mechanical properties of the fabricated scaffold to know the strength and stability in adverse conditions or in the in-vivo environment. The mechanical properties of the scaffold involve the load bearing capacity, stress and young modulus which were obtained by tensile testing. Figure 7(a) shows the variation in modulus of the fabricated scaffold which emphasised on the resistance of a material to return to its elastic (recoverable) deformation state under load. It was observed that there is an increase in Young's modulus in both the fabricated scaffold PVA and PVA/keratin after crosslinking with GTA where as tensile strength of the fabricated scaffold decreased after modified it with GTA as shown in figure 7(b). Materials having higher modulus are stiffer and have better resistance to deformation. The result obtained confirms that the crosslinked scaffolds showed better mechanical properties and good potential as nerve implant.

Swelling and degradation studies

Fluid uptake is a very important parameter, it shows how efficient the fabricated material will be to absorb nutrients and growth factors when implanted in-vivo. PVA is a highly hydrophilic material, hence it is having high fluid uptake capacity. As show in figure 8(a) blending of PVA with keratin showed comparatively slow fluid uptake which may attribute to a more rigid network formed by the inter and intra polymer reactions and also to the reduction of hydrophilic groups in PVA/Keratin blend. The latter is also due to the amine groups of Keratin being more reactive to GTA than to hydroxyl groups of PVA. Even though the PVA/keratin scaffold showed slower fluid uptake rate, since the difference is very minimum the potential of PVA/ Kratin as nerve graft is not affected.

Biodegradation is one of the most important properties that a material should inherit to be used as scaffold. The process through which useful physicochemical properties of the materials are lost once the purpose is fulfilled is known as degradation. It includes the loss of polymer mass through mechanisms such as salvation and depolymerisation. Degradation study is very important as it gives an idea about the degradation rate of polymeric and protein composite scaffolds when implemented inside the body. From the degradation behaviour of PVA and PVA/Keratin shown in figure 8(b) it was clearly observed that the degradation rate of PVA/Keratin nanofibrous scaffold was much slower than PVA scaffold. This is due to higher density of chemical cross-linking between gluteraldehyde and amine groups of Keratin which leads to the slower depolymerisation. These results indicates that after blending of Keratin/PVA it gives comparatively controlled degradation rate which is very important as the damaged nerve requires specific time for its complete recovery.

Protein adsorption study

Protein adsorption study is carried out to know about behavioural response of the scaffolds towards the adsorption of various important cell adhesion molecules like fibronectin or vitronectin and neural proteins. The adsorbed protein concentration was increased with increase in incubation time from 2 to 6 h. The graph plotted for time of incubation vs. total amount of protein adsorbed as shown in the figure 9 revealed that protein adsorption capacity of the PVA/Keratin nanofibrous scaffold was much

higher than PVA nanofibrous scaffold due to increase in binding site on the material surface and the increased electrostatic interaction between the protein and material surfaces.

In-vitro study

MTT assay was used to evaluate the relative cell viability. The microscopic observation of glial cells, which were used to determine the neural cell response towards the fabricated scaffold were shown in figure 10. The cell viability of Glial cells on the nanofibrous scaffolds for a three day period is shown in figure 11. Cell viability of glial cells cultured on both PVA/Keratin (before and after crosslinking) nanofibrous scaffold is significantly enhanced as compared to PVA scaffold. Similarly, the cell viability on PVA/keratin after crosslinking is greater than that on PVA/Keratin scaffold before crosslinking. It was observed that cells which attached on the PVA/Keratin (after crosslinking) spread largely along the fibres and also the proliferations of glial cells on PVA/Keratin (after crosslinking) nanofibrous scaffold are significantly higher. The results obtained from in-vitro studies which are also evident in scanning electron micrograph as shown in figure 12, confirms that PVA/Keratin (after crosslinking) scaffold is having immense potential as nerve repair alternative.

Conclusion

The objective of this study was to fabricate an electrospun PVA/keratin blended nanofibrous scaffold that facilitated the neural cell growth which intended to be a nerve repair alternatives in case of peripheral nerve injuries. Various characterization studies were carried out during this study to optimize the required environment for nerve cell growth. These studies showed the efficacy of the fabricated nanofibrous scaffold in nerve repair. To evaluate the response of the nerve cells in-vitro study was done, glial cells were used for this purpose. The in-vitro study indicates that the fabricated scattold is immensely promising nerve repair system. The glial cells were adhered properly to the scaffold and also showed good proliferation rate. MTT assay was carried out to evaluate the cytotoxic effect and it showed good cell viability. The fabricated scaffold is also found to be highly biocompatible along with all other desired properties required for nerve repair and proves to be an appropriate alternative in case of severe peripheral nerve injuries.

References

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Babita Mahanta (1), S. Agnes Mary (2), Ahana Bhaduri (1), V.R. Giridev (2)

(1) Biomedical Engineering Division, VIT University, Vellore, Tamilnadu, India.

(2) Department of Textile Engineering, Anna University, Chennai, Tamilnadu, India

Received 7 July 2014; Accepted 5 December 2014; Available online 10 December 2014
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Title Annotation:Original Article; polyvinyl alcohol
Author:Mahanta, Babita; Mary, S. Agnes; Bhaduri, Ahana; Giridev, V.R.
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Date:Oct 1, 2014
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