Electrospun Hyaluronan-Gelatin Nanofibrous Matrix for Nerve Tissue Engineering

Schwann cells play a critical role in the repair of the peripheral nerve. The goal of this study was to fabricate electrospun gelatin (Gel) and hyaluronan-gelatin (HA-Gel) composite nanofibers to provide a suitable growth environment for Schwann cells. The fiber diameters of Gel, 0.5 HA-Gel, 1 HA-Gel, and 1.5 HA-Gel were 130 ± 30 nm, 294 ± 87 nm, 362 ± 129 nm, and 224 ± 54 nm, respectively. The biological performance of Gel and HA-Gel was evaluated using an in vitro culture of RT4-D6P2T rat Schwann cells. We found that the cell attachment and proliferation rates were not significantly different on these matrices. However, the Schwann cells displayed better organized F-actin onHA-Gel than onGel.Moreover, the expression levels of several genes, including Nrg1, GFAP, and P0, were significantly higher on HA-Gel than on Gel. In addition, the levels of Nrg1 and P0 protein expression were also higher on theHA-Gel than onGel.These results indicate that the hyaluronan-gelatin composite nanofibrousmatrix could potentially be used in peripheral nerve repair.


Introduction
Regeneration of the nervous system is a major challenge.However, the peripheral nervous system has a better intrinsic ability to repair and regenerate axons after axonal damage due to the ability of Schwann cells to enhance the environment for regeneration [1].For the treatment of peripheral nerve injury, nerve autografts or direct end-to-end surgical reconnection is used to repair nerve injury.Unfortunately, direct end-to-end surgical reconnection only can repair small gaps in the nerve, because the tension introduced into the nerve cable would inhibit nerve regeneration for longer nerve gaps [2].A nerve autograft harvested from another site in the body is used in these cases and is considered the "gold standard" for repair of the longer nerve damage gaps without immunological rejection problems.However, a nerve autograft has several disadvantages, including neuroma, hypofunction at the donor nerve graft site, and nerve site mismatch.Allografts and xenografts are possible replacements for autografts.Nevertheless, the major clinical problems are the risk of immune repulsion and disease transmission [3].Thus, a nerve graft made of natural and synthetic materials is a promising alternative for promoting successful nerve regeneration.
Gelatin is obtained by the partial hydrolysis of native collagen in an alkaline or acidic environment.Gelatin exhibits excellent biocompatibility and biodegradability properties; thus, it has been widely used for nerve repair [4].Hyaluronan is a naturally biopolymer composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine.It is the major constituent of the extracellular matrix (ECM) of connective tissues and has many important biological functions.Because of its excellent biocompatibility, nonimmunogenicity, and specific biological functions, hyaluronan is used in a variety of clinical therapies, including supplementing joint fluid in arthritis and facilitating wound healing and regeneration [5,6].Furthermore, hyaluronan is known to prevent perineural scar formation, which improves peripheral nerve regeneration [7].
Tissue engineered scaffolds are another choice for implantation to facilitate neural repair.Fibrous scaffolds have become very popular in tissue engineering because they mimic the physical architecture of the ECM.The architecture

Gene
Primer sequence: sense/antisense of the ECM plays an important role in regulating cell behavior with biochemical signals and topographical cues [8,9].
Electrospinning is an easy method to fabricate ultrafine fibers with submicrometer to nanometer diameters.Electrospun fibrous matrices exhibit certain special characteristics, such as a high specific surface area, a high aspect ratio, and a high porosity surface area.Most importantly, the topological structure of electrospun matrices mimics the architecture of the ECM and could enhance cell attachment, migration, proliferation, and differentiation [10].
Schwann cells are a major component of the peripheral nerve system and play an important role in Wallerian degeneration and the subsequent axon regeneration in peripheral nerve injury [11].After Wallerian degeneration, the Schwann cells begin to proliferate and migrate to the lesion, forming longitudinally oriented cell strands called the bands of Büngner; thereafter, Schwann cells secrete a number of neurotrophic factors such as nerve growth factor and cytokines that support the survival of injured neurons [12], secrete extracellular matrix components, such as laminin and collagen type IV, for axonal growth, and produce neurite promoting factors to guide the growing axon [13,14].Thus, the aim of the present study was to investigate whether a hyaluronan-gelatin electrospun matrix has potential as a neurograft for peripheral nerve repair.

Materials and Methods
2.1.Reagents.Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and trypsin were purchased from GIBCO (Grand Island, NY, USA).Gelatin type A (Gel) was purchased from the Sigma-Aldrich Chemical Company (St. Louis, MO, USA).Sodium hyaluronan was purchased from NovaMatrix (Drammen, Norway).The RT4-D6P2T rat Schwann cells were purchased from BCRC (Bioresource Collection and Research Center, Taiwan).The other chemicals used were of reagent grade unless otherwise stated.

Preparation of Hyaluronan-Gelatin Composite Nanofibrous Matrix (HA-Gel).
To allow complete evaporation of the solvent, the solvent used in this study was a mixture of formic acid and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at a volume/volume ratio of 3/7.The gelatin powder was dissolved in the solvent mixture to a concentration of 12% (w/v).Various concentrations of sodium hyaluronan (0%, 0.5%, 1%, and 1.5% w/v) were added to the gelatin solution and dissolved by vortexing until the solution was clear.For the electrospinning process, the polymer solution was placed in a plastic syringe fitted with an 18 G needle and attached to a syringe pump that provided a steady flow rate of 8.5 L/min.The electrospinning voltage of 2.3 kV/cm was applied using a high-voltage power supply (Glassman High Voltage, Inc.).

Characterization of the Electrospun Nanofibers.
The fiber morphology and diameter of the HA-Gels were determined by a field emission scanning electron microscope (SEM; model S-4800, Hitachi, Tokyo, Japan).Briefly, the electrospun matrices were sputter-coated with gold and visualized by SEM at an accelerating voltage of 5 kV.The fiber diameter was analyzed using image analysis (ImageJ software 1.42, National Institutes of Health, USA).The average fiber diameter and standard deviation were calculated from 100 random measurements.

Cell Culture on HA-Gel.
Because the HA-Gels readily dissolved in aqueous media, it was necessary to cross-link them before using them for cell culture.The electrospun matrices were cross-linked by treatment with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC).The matrix was immersed in 1% EDC solution in 95% ethanol for 2 hrs and then washed repeatedly with 0.01 M PBS to remove the residual EDC.Finally, the electrospun matrices were sterilized by exposure to ultraviolet light in a sterile hood overnight.The electrospun matrices were placed in 24-well tissue culture plates containing a suspension of RT4-D6P2T rat Schwann cells (BCRC no.60508) (5 × 10 4 cells/well) in DMEM supplemented with 10% (v/v) FBS, 100 U/mL of penicillin, and 100 g/mL of streptomycin.The cultures of cellseeded electrospun matrices were harvested at 1, 2, 3, and 4 hrs for cell attachment assays and on days 1, 3, and 6 days for cell proliferation assays.Cell viability was determined using the MTT assay.At each time point, three samples were used to measure the number of cells attached to the electrospun matrices.The experiments were performed in triplicate.

Fluorescent Staining of the Cytoskeleton.
The morphology of cells with their F-actin cytoskeleton fluorescently stained with fluorescein isothiocyanate (FITC)-conjugated phalloidin and nuclei stained with DAPI was examined.After 1 or 4 hrs of cell culture, cells were fixed with 3.7% (v/v) paraformaldehyde in PBS for 10 min, followed by 2 times washing with PBS and permeabilization with 0.1% (v/v) Triton X-100 in PBS for 5 min.After washing with PBS, the samples were blocked with 1% bovine serum albumin in PBS for 1 hr.After blocking, the samples were stained with 6.4 M FITCconjugated phalloidin for 20 min.Cell nuclei were stained

Fabrication of Hyaluronan-Gelatin Electrospun Nanofibers.
Formic acid is a good solvent for hyaluronan and gelatin.However, gelatin and hyaluronan/gelatin which dissolved in a formic acid solution cannot be fabricated into nanofibrous matrices by electrospinning.In a previous study, hexafluoropropanol was found to be a suitable solvent to dissolve hyaluronan/collagen [15].To obtain hyaluronan-gelatin nanofibers (HA-Gel), we used the cosolvent method by mixing formic acid and HFIP.
Figure 1 shows that the synthetic product was a three-dimensional nonwoven nanofibrous hyaluronan-gelatin matrix with interconnected pores.The fiber diameters were 130 ± 30 nm, 294 ± 87 nm, 362 ± 129 nm, and 224 ± 54 nm for Gel, 0.5 HA-Gel, 1 HA-Gel, and 1.5 HA-Gel, respectively.These dimensions are similar to those of the native fibrous proteins in the ECM.We found that the diameters of the fibers in the HA-Gel matrix were significantly different from those in the Gel matrix ( < 0.05, one-way ANOVA with Tukey's post hoc test) (Table 2).In addition, the diameters of the fibers gradually increased and then decreased as the concentration of hyaluronan was gradually increased.
The Gel and HA-Gel fibers cross-linked with EDC are shown in Figure 2. The Gel and HA-Gel fibers still maintained their morphology.However, the fibers appeared to be more rubbery and were fused at fiber junctions.

Cell Attachment and Proliferation.
Cellular functions, including spreading, proliferation, and differentiation, are sensitive to the composition and surface topography of the matrix.To test the effects of the hyaluronan/gelatin weight ratios of the nanofibrous matrix on cell morphology, we incubated the RT4-D6P2T rat Schwann cells on hyaluronan and gelatin matrices with different weight ratios.The morphologies of the Schwann cells that adhered to the different matrices were investigated by staining the actin cytoskeleton with FITC-phalloidin and visualizing the cells with a fluorescence microscope (shown in Figure 3) and a laser scanning confocal microscope (shown in Figure 4).We found that most of the Schwann cells cultured on the HA-Gel matrix appeared to have spread during the 4 hr attachment period.However, it was obvious that the cells cultured on the Gel matrix remained round after 4 h (Figure 3    and HA-Gel matrices were quantified using the MTT assay.There were no significant differences in attachment or proliferation among the cells cultured on 0.5 HA-Gel, 1 HA-Gel, or 1.5 HA-Gel ( > 0.05) (as shown in Figure 5).

Real-Time PCR Analysis. The expression levels of
Schwann cell-specific genes were analyzed using real-time PCR for Nrg1, S100, NGF, p75, GFAP, and P0 and 18S ribosomal RNA (as shown in Figure 6).On day 3, the Schwann cells displayed insignificantly increased gene expression levels of Nrg1, S100, NGF, p75, GFAP, and P0 when grown on 0.5 HA-Gel, 1 HA-Gel, or 1.5 HA-Gel relative to those obtained from cells grown on Gel ( > 0.05).However, on day 6, the Schwann cells showed significantly higher gene expression levels of Nrg1, GFAP, and P0 when grown on 0.5 HA-Gel, 1 HA-Gel, or 1.5 HA-Gel compared to cells grown on Gel ( < 0.05) (as shown in Figure 6).

Western Blot Analysis.
The CD44 levels expressed on day 3 and day 6 by the RT4-D6P2T rat Schwann cells grown on the different matrices were not significantly different ( > 0.05).The levels of Nrg1 and P0 were higher in the cells grown on 0.5 HA-Gel, 1 HA-Gel, or 1.5 HA-Gel than in the cells grown on Gel on day 6 ( < 0.05) (as shown in Figure 7).
However, the levels of Nrg1 and P0 on day 3 and day 6 were not significantly different among the cells grown on 0.5 HA-Gel, 1 HA-Gel, or 1.5 HA-Gel ( > 0.05).

Discussion
Sponge forms of gelatin, hyaluronan, and their composites have been widely used to fabricate scaffolds for tissue engineering [16][17][18].However, the physical structure of the sponge form of these matrices is not acceptable for the tissue  regeneration.A porous and nanofibrous biodegradable matrix that mimics a natural ECM is the optimal scaffold for tissue engineering [19].It is widely accepted that cell adhesion and most cellular activities, including spreading, migration, proliferation, gene expression, and cytoskeletal function, are sensitive to the nanotopography [20] and molecular composition of the matrix [15].Xu et al. demonstrated that there were stronger interactions between the cells and the nanofibers [21].Consequently, most researchers believe that a nanofibrous matrix may promote cell growth more effectively than a sponge substrate.
Several methods have been used to fabricate the fibrous matrix, but the electrospinning is a simple and effective fabrication technique to produce nano-/microfibers.The morphology and diameter of electrospun fibers are dependent on many processing parameters, including the molecular weight of the polymer, the concentration (or viscosity), the conductivity, the applied voltage, and the feeding rate of the polymer solution.In our previous study, we found that increasing the concentration of hyaluronan would increase the charge density on the surface of the electrospinning jet, which would induce higher electrostatic forces and result in the formation of smaller diameter fibers [15].On the other hand, the diameter of the electrospun fibers increases with increasing concentrations of a polymer solution.A combination of the aforesaid factors might explain why the mean fiber diameters increased at lower hyaluronan concentrations and decreased at higher hyaluronan concentrations.
Hyaluronan is found in all vertebrate tissues and is important for the organization of pericellular and extracellular matrices [22].Hyaluronan plays a crucial role in tissue hydration and lubrication and in the regulation of cell functions,including cell attachment, cell mitosis, cell migration, tumor development, metastasis, cell apoptosis, and inflammation [23,24].The function of hyaluronan depends upon its interaction with the cell surface receptors, including the receptor for hyaluronan-mediated motility (RHAMM), cluster determinant 44 (CD44), lymphatic vessel endothelial hyaluronan receptor (LYVE-1), hyaluronan receptor for endocytosis (HARE), liver endothelial cell clearance receptor (LEC receptor), and toll-like receptor 4 (TLR4) [25,26].Hyaluronan has been shown to influence the interaction of glial cells with axons, to reduce the formation of scar tissue and to provide a suitable environment for tissue repair [27][28][29].Wang et al. found that hyaluronan played a vital role in the biological processes involved in the deposition and remodeling of the fibrin extracellular matrix to increase the level of myelination of axons after 4 weeks in vivo [30].Sherman noted that the CD44 transmembrane glycoprotein plays a key role in Schwann cellneuron interaction.The reduction of CD44 expression Journal of Nanomaterials in vitro decreased Schwann cell-neurite adhesion and caused the apoptosis of Schwann cells [31].
The peripheral nerve repair process depends on Schwann cells, which synthesize and secrete important substances such as neuregulin-1 (Nrg1), GFAP, and P0.Nave proposed that Nrg1 is essential for every developmental stage of Schwann cells and for the myelination of axons [32].Nrg1 is an epidermal growth factor-like ligand that interacts with the tyrosine kinase ErbB receptor to regulate many aspects of neural development.Moreover, Ghatak et al. demonstrated that ErbB2 can be activated by HA through CD44-mediated mechanisms to promote the proliferation and differentiation of Schwann cells [33].
GFAP is an intermediate filament protein that functions as a scaffold for cytoskeletal assembly and maintenance [34].GFAP also plays a vital role in Schwann cell proliferation and the upregulation of Schwann cell-specific cytoskeletal constituents after nerve damage [35].Myelin protein zero (P0) is the major adhesive and structural protein of the myelin sheath of peripheral nerves [36].P0 is expressed by myelinating Schwann cells and is necessary for normal myelin structure and function [37].Some studies have demonstrated that P0 promoted the regeneration of injured axons [38].From the results of real-time Q-PCR and western blotting, we found that Nrg1, GFAP, and P0 gene expression and Nrg1 and P0 protein expression were enhanced at day 6 when Schwann cells were cultured on HA-Gel.We speculate that the HA-Gel enhanced myelination via the interaction of the surface CD44 receptors of Schwann cells with hyaluronan.

Conclusion
Schwann cells grown on hyaluronan-gelatin nanofibrous matrices showed better organized F-actin stress fibers, higher levels of Nrg1, GFAP, and P0 mRNA, and higher levels of Nrg1 and P0 protein compared to those grown on the gelatin nanofibrous matrix.These findings suggest that the hyaluronan-gelatin nanofibrous matrix could potentially be used in the repair of injured peripheral nerves.
(e) and Figure4(e)).The attachment and proliferation of the Schwann cells on the Gel

Figure 5 :Figure 6 :
Figure 5: Quantification of RT4-D6P2T Schwann cells on Gel and HA-Gel matrices.The MTT assay was used to quantify cell attachment and proliferation on Gel and HA-Gel.(a) The attachment of RT4-D6P2T Schwann cells on various matrices after culturing for up to 4 hrs.(b) The viability of RT4-D6P2T Schwann cells on various matrices after culturing for up to 6 days.The data are presented as the means ± SD,  = 4.No statistically significant differences in cell attachment or proliferation on the HA-Gel and Gel were found.

Table 1 :
Oligonucleotide primer for real-time PCR amplification.

Table 1
shows the sequences of the oligonucleotides that were used as PCR primers.Briefly, the reaction is followed by the manufacture protocol of SYBR Green PCR Master Mix Kit (Protech SA-SQGLR-V2).The real-time PCR conditions for the promoter region were 15 mins at 94 ∘ C, 1 min at 62.5 ∘ C, and 15 s at 94 ∘ C. Real-time PCR reactions were performed using an iQ5 Gradient Real-Time PCR system (Bio-Rad).The levels of RNA expression were calculated using the 2 −ΔΔCt method.2.7.Immunoblotting Analysis.Cells were seeded on substrates at 3 × 10 4 cells/cm 2 in medium.Immunoblotting to detect the Schwann cell-specific proteins neuregulin 1 (Nrg1), myelin protein zero (P0), and CD44 was performed after day 3 and day 6 of culturing.Cells were collected and lysed in lysis buffer.The supernatants were obtained by centrifugation at 4 ∘ C, 15,000 ×g for 10 min.The concentration of protein was analyzed using a Bradford Coomassie assay.The proteins (30 g/L) were fractionated by electrophoresis and electrotransferred to polyvinylidene difluoride film (PVDF).2.8.Statistical Analyses.Statistical analyses were performed using SPSS v.11 software.For each condition, the diameters of at least 100 randomly chosen fibers were measured.The fiber diameters were analyzed using a one-way ANOVA with Tukey's post hoc test.The cell viability, gene expression, and