Fabrication of nanochitosan incorporated polypyrrole/alginate conducting scaffold for neural tissue engineering

The utilization of conductive polymers for fabrication of neural scaffolds have attracted much interest because of providing a microenvironment which can imitate nerve tissues. In this study, polypyrrole (PPy)–alginate (Alg) composites were prepared using different percentages of alginate and pyrrole by oxidative polymerization method using FeCl3 as an oxidant and electrical conductivity of composites were measured by four probe method. In addition, chitosan-based nanoparticles were synthesized by ionic gelation method and after characterization merged into PPy–Alg composite in order to fabricate a conductive, hydrophilic, processable and stable scaffold. Physiochemical characterization of nanochitosan/PPy–Alg scaffold such as electrical conductivity, porosity, swelling and degradation was investigated. Moreover, cytotoxicity and proliferation were examined by culturing OLN-93 neural and human dermal fibroblasts cells on the Nanochitosan/PPy–Alg scaffold. Due to the high conductivity, the film with ratio 2:10 (PPy–Alg) was recognized more suitable for fabrication of the final scaffold. Results from FT-IR and SEM, evaluation of porosity, swelling and degradation, as well as viability and proliferation of OLN-93 neural and fibroblast cells confirmed cytocompatiblity of the Nanochitosan/PPy–Alg scaffold. Based on the features of the constructed scaffold, Nanochitosan/PPy–Alg scaffold can be a proper candidate for neural tissue engineering.


Scientific Reports
| (2020) 10:22012 | https://doi.org/10.1038/s41598-020-78650-2 www.nature.com/scientificreports/ based conductive polymers. Biocompatibility and biodegradation property of conductive polymers is generally achieved by modification them with suitable natural biodegradable polymers such as alginate 12,16,18,19 . Sodium alginate (SA) is a natural biocompatible hydrophilic polysaccharide. It is non-toxic and non-immunogenic polymer, which resemble the extracellular matrix of the body. SA is biodegradable linear carbohydrate biopolymers derived from brown algae. It has been shown that alginate-based scaffolds improve cell adhesion and cell proliferation which result in repairing some organs including skin, nerve and liver 9,20 . Alginate-based scaffolds were also used as an appropriate substrate for construction of conductive scaffolds based on pyrrole monomers using electrostatic interactions between carboxylate moieties of alginate and positively charged PPy 16 . Several studies have designed alginate-pyrrole based scaffolds or hydrogels with electrical properties for neural tissue engineering [21][22][23] .
Chitosan as a natural polysaccharide and its improved derivatives have frequently been used in tissue engineering and regenerative medicine 24 . It has been shown that chitosan incorporated alginate-pyrrole scaffold provides an interactive substrate between the seeded cells and external electric field 18 . In recent years, many studies investigated the roles and biological functions of nanoparticles in the scaffold 25 . Nanoparticles possess exclusive properties such as large surface-to-volume ratio and high surface reactivity which make them as suitable substrate for adhesion and proliferation of various cells. Chitosan nanoparticles are a natural biomaterial with many advantages such as biodegradability, proper biocompatibility and being a controlled-release carrier for growth factor delivery 24,26 . Also, the use of the nanochitosan in the scaffold increases the specific surface area and surface energy which makes the scaffold more hydrophilic. The hydrophilic surface of scaffold is favorable for attachment of the cells to the scaffolds 27,28 . Chitosan nanoparticles can be crosslinked by interactions of negatively charged sodium tripolyphosphate (TPP) with positively functional groups of Chitosan 26 .
Since previous studies have reported that the amount of conductive polymers can affect the electrical properties of conductive scaffold, we hypothesized here that the application of different percentages of alginate and pyrrole affects characteristics of the electrical conductive scaffolds. Therefore, the first aim of this study was to evaluate the effects of different percentages of alginate and pyrrole on the electrical conductivity properties of PPy-Alg composites in order to find the optimal concentration. As second goal to improve the hydrophilicity of the PPy-Alg composite, nanochitosan was synthesized and characterized and then integrated with PPy-Alg polymers blend, in order to construct a novel polypyrrole based conducting scaffold with proper physiochemical characterizations for neural tissue engineering.

Materials and methods
Preparation of polypyrrole-alginate composites. Pyrrole (0.1 M, sigma) solution was made using HCl (1 N) and was added drop by drop to alginate solution (3%, sigma) with volume ratios 1: 10 and 2: 10 and stirred for 30 min at 90 °C. Then, ferric chloride (FeCl 3 , 0.2 M) was added slowly to this solution till its color changed to black within 5 h stirring 18 . The polymer mixtures were isolated from the reaction mixture using a dialysis membrane with cut off 12-14 kDa (D9402, Sigma) for 5 days to eliminate non-reactive and oxidant substances. The polymer was put in freeze dryer to dry out for 24 h (Fig. 1).

Synthesis of nanochitosan.
Nanochitosan were synthesized as we described in our previous study with some modifications 27 . Briefly, chitosan (0.9 mg/ml, Medium molecular weight chitosan, Mw = 50,000-190,000, Sigma) was dissolved in 5 ml acetic acid 0.5% and was stirred for 24 h until a homogeneous solution is obtained. Then, NaOH (2 M) is added until its pH was adjusted to 5.5. Two milliliters TPP solution (0.25 mg/ml, Sigma) was added drop wise to the chitosan solution with rate (0.2 ml/min) and stirred for 1 h to form nanochitosan. In order to measure size and polydispersity index (PdI) of synthesized nanochitosan and their morphology, dynamic light scattering (DLS) and SEM were carried out, respectively.
Fabrication of nanochitosan/polypyrrole-alginate scaffold. Initially, 3gr of nanochitosan powder was dispersed in 100 ml acetic acid (1%) and was stirred for 30 min. one gram powdered PPy-Alg (2:10) was www.nature.com/scientificreports/ added to this mixture and again stirred for 5 h. Finally, the samples were put in freeze dryer to dry out for 24 h and then cross-linked with glutaraldehyde (0.25%) for 12 h. The scaffolds were further washed with PBS for 24 h and again dried with a freeze dryer.

Characterization
Characterization of PPy-Alg composites. The chemical structure of PPy-Alg composites was evaluated by FTIR spectrum, which was registered on a Thermo Nicolet Nexus 470 Fourier transform infrared spectrometer in the domain of 650-4000 cm −1 . In order to measure the electrical conductivity, PPy-Alg composites with volume ratios of 1:10 and 2: 10 were casted separately to form film. Electrical conductivity of the produced PPy-Alg films was measured by four-probe technique (4point probe measurement, 4PP-R2K). The conductivity was calculated using the following formula (Eqs. 1 and 2) 29 : where resistance is the amount of resistance measured by the device, V is measured voltage, I is applied current and t is the thickness of the sample.
Characterization of nanochitosan/PPy-Alg Scaffold. Electrical conductivity measurement. The amount of electrical conductivity of the produced nanochitosan/PPy-Alg scaffold was evaluated by four-probe technique. The Eqs. (1) and (2) (as mentioned above) were used to calculate the conductivity.
Scanning electron microscopy (SEM). The porous structure and morphology of scaffold and nanochitosan were analyzed through SEM instrument (Jeol, Tokyo, Japan), as described previously 30 .
Water contact angle test. The surface wettability of PPy-Alg composite and nanochitosan/PPy-Alg scaffold was investigated by measuring contact angle. This was done by placing a drop of water on the composite and scaffold surface and the contact angle was evaluated by OCA PLUS 15 device.
Porosity evaluation. The liquid displacement method was carried out to measure porosity of the nanochitosan/ PPy-Alg scaffold. Pre-weighed scaffold was submerged in a determined volume of absolute ethanol for 48 h. Then, the ethanol saturated scaffold was removed and was weighed again. Each experiment was carried out 3 times and the average of the porosity of scaffold was obtained. The porosity was measured using the following formula (Eq. 3) 30 : V1, the initial weight of the scaffold; V2, total weight of ethanol and immersed scaffold; V3, the weight of ethanol after removing scaffold.
Swelling evaluation. The water uptake ability of the nanochitosan/PPy-Alg scaffold was evaluated by immersing scaffold in PBS (pH = 7.4) for different time periods (1, 7, 14, 21 days) at 37 °C. After the mentioned time intervals, scaffolds were rinsed with deionized water and the surface water was removed using filter paper and samples were weighed (Wet weight). Experiments were done in triplicate. The swelling ratio was calculated using the following formula (Eq. 4) 31 : Degradation measurement. The scaffold was weighed (WI) and then it was immersed in PBS (1x, pH = 7.4) and incubated at 37 °C for different time intervals (1, 7, 14, 21 days). After completion of a predetermined time period, the scaffold rinsed with distilled water, dried at 37 °C for 24 h in an oven and weighed again (Wt). Degradation (%) was determined using the following formula (Eq. 5):

Cell culture study
OLN-93 neural cell line (rat brain neural cells) and Normal human dermal fibroblasts (NHDF) were purchased from Pasteur Institute. The cells were cultured in flask (25 cm 2 ) with Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin and incubated in an incubator at 37 °C containing 5% CO 2 and 95% air. The media was exchanged every 3 days. The experimental procedures in this study were approved by Ethic Research Committee of Shahid Beheshti University of Medical Sciences under the ethical code number of IR.NIMAD.REC.1398.388. All methods were performed in accordance with the relevant guidelines and regulations of Shahid Beheshti University of Medical Sciences.  Histology study. Histological studies were carried out to investigate cell distribution on the nanochitosan/ PPy-Alg scaffold. The scaffolds were sterilized using UV, and then incubated for 24 h in DMEM. The next day, medium was completely removed and OLN-93 neural cells were cultured on the scaffold and incubated in an incubator at 37 °C containing 5% CO 2 and 95% air for 24 h. Then glutaraldehyde (2.5%) was used for fixation of the cells for 2 h and dehydrated with different concentrations (50, 70, 90, and 100% v = v) of alcohol for histological assay. Afterwards, the scaffolds seeded with the cells were embedded in paraffin by standard techniques. Then the sections of 4 µm thickness were cut with a microtome (MICRO DS, 4055) and were stained with hematoxylin and eosin (H&E) technique for light microscope investigation.
Statistical analysis. All data were presented as means ± SD. Analysis of variance (ANOVA) was used with Turkey's post-test to determine statistical significance. A p value of < 0.05 was considered to be statistically significant.

Results and discussion
Fourier transform-infrared spectroscopy. PPy-Alg composites were produced by chemical polymerization of the pyrrole monomer using FeCl 3 as an oxidant agent. Pyrrole monomers was allowed to react with alginate in acidic condition. Then, polymerization was initiated by adding FeCl 3 . It was put in freeze dryer for 24 h to form PPy-Alg Composites powder is illustrated in Fig. 1. FTIR spectrum analysis of pyrrole, alginate and PPy-Alg composites have shown in Fig. 2. The spectrum of FTIR obtained for pyrrole indicates the presence of characteristic absorption peaks of C=C (stretching of pyrrole ring) at 1550 cm −1 , C=N at 1427 cm −1 , Electrical conductivity. Measurement of the electrical conductivity of films produced from PPy-Alg composites and nanochitosan/polypyrrole-alginate scaffold has been performed by four-probe apparatus at room temperature. The calculated electrical conductivity values are shown in Table 1. It is obvious that the films conductivity increased by increasing the amount of pyrrole. The composites with ratio 2: 10 of pyrrole and alginate possess higher conductivity. It has been reported that the amount of conductive polymers can impress the electrical properties of conductive scaffolds 21 . Based on this result, polymers with 2:10 ratio of pyrrole and alginate was determined to incorporate with chitosan nanoparticles to synthesize the final scaffold. In this study, we used concentrations higher than 2 ml of 0.1 M pyrrole in our pilot studies. The films made from compositions with concentrations higher than 2 ml of PPy were fragile, so they were broken to pieces during the drying process or within evaluation of their surfaces by four-point probe. These results were consistent with previous studies in which showed more PPy concentration more fragility of film 10,21,22 . As a result, the composite ratio 2:10 was chosen which provides a suitable mechanical property and electrical conductivity.
Morphology and size of nanochitosan. Nanochitosan particles were usually synthesized by the iontropic gelation method. Nanochitosan particles were created by the electrostatic interaction between the protonated amine groups in chitosan and the polyanion sodium tripolyphosphate (TPP). The DLS results for synthesized nanochitosan particles is indicated that their distribution was in the range of 39-243 nm (PdI = 0.234). SEM image of nanochitosan particles in Fig. 3 shows that nanoparticles were spherical and clustered shape. These result demonstrated that the chitosan nanoparticles mostly distributed in nano size. It has been shown that the nano size of chitosan demonstrate better results in cell growth and nanochitosan incorporated scaffolds provide a superior support for tissue regeneration 32 . Narrow size distribution of PdI (near 0.2) demonstrates a low tendency to accumulate 33 . Morphology and porosity of nanochitosan/PPy-Alg scaffold. Designing a proper scaffold for neural tissue engineering is crucial for the successful regeneration of tissue. Therefore, electrically conductive polymers are promising substrates in manufacturing of scaffolds for neural tissue engineering 9,34 . In this study,  www.nature.com/scientificreports/ nanochitosan particles were incorporated into PPy-Alg (2:10) composite for obtaining a hydrophilic, processable and stable scaffold. The results showed that incorporation of nanochitosan into PPy-Alg composite did not change the electrical conductivity and the nanochitosan/PPy-Alg scaffold possesses electrical conductivity. SEM images showed that the nanochitosan/PPy-Alg scaffold has a clear porous structure (Fig. 4). The porous structure of nanochitosan/PPy-Alg scaffold can improve cell adhesion, enhance delivery of nutrients, mediums and soluble signaling molecules to the seeded cells, and also provide a condition for metabolic waste removal. Hence, it can be used as a scaffold for tissue engineering applications. In addition, the porosity of the prepared scaffold was evaluated based on Eq. (3). The scaffold exhibited a porosity of about 97.17 ± 0.14%. Total porosity greater than 90% is optimal for polymeric scaffolds to be used in tissue engineering and can allow the cells to migrate into the scaffolds 30 .
Scaffolds' wettability measurements. The water contact angle values of PPy-Alg composite was 96.7 which shows that PPy-Alg composite is not a proper hydrophilic structure; while, incorporating chitosan nanoparticles into the PPy-Alg structure make it completely hydrophilic. Since the wettability of the nanochitosan/ PPy-Alg scaffold was very higher than the produced PPy-Alg composite, we showed the wettability (water absorption) in a video (Supplementary Video, Media 1). It seems that the use of the nanochitosan in the scaffold increases the specific surface area and surface energy and makes the scaffold more hydrophilic. Consistent to this result, the other studies have reported that due to hydrophilic groups which present on nanochitosan surface, its incorporation increases the absorption of water 27,28 . Swelling evaluation. Water uptake behavior of the scaffold was measured in PBS solution at 37 °C. Swelling of the scaffolds involves the uptake of body fluids which facilitates the transfer of nutrients and causes cellular penetration into the scaffold 31 . As shown in Fig. 5a, the results of the swelling test indicated that nanochitosan/ PPy-Alg scaffold increased the swelling during the time period of 21 days, which is appropriate for further cell adhesion and cell penetration into the scaffold.   www.nature.com/scientificreports/ might be due to proliferation of the cells. Overall we concluded that the prepared scaffold is biocompatible and non-cytotoxic.

Study of adhesion of OLN-93 neural cells. SEM images shows that the OLN-93 neural cells were
attached well on the nanochitosan/PPy-Alg scaffold surface after 24 h (Fig. 7). These results showed that nanochitosan/PPy-Alg scaffold promoted OLN-93 neural cells adhesion and the cells expressed their characteristic morphology. Previous studies have shown that the surface characteristics of scaffolds significantly affect cell adhesion and hydrophilic surface of scaffolds were favorable for attachment of the cells to scaffolds 35 . Moreover, chitosan nanoparticles in scaffolds probably have a role in nanoparticles-dependent attachment via involving in the cell membrane.
Histology studies. Figure 8 shows the optical image of OLN-93 with uniform distribution of the cells. The nanochitosan/PPy-Alg scaffolds were stained with H&E in which cell nuclei appeared in purple and scaffold in red. The histological results showed that the neural cells attached well to the scaffold and no cell cluster was obvious in the figure.

Conclusion
Polypyrrole-alginate composites were synthesized with different wt% of pyrrole and alginate. The results show PPy-Alg composite with ratio 2:10 possesses high conductivity which served as a suitable material, which was incorporated with nanochitosan to fabricate nanochitosan/PPy-Alg scaffold for tissue engineering applications.  www.nature.com/scientificreports/