Fabrication of Chitosan/Polypyrrole‐coated poly(L‐lactic acid)/Polycaprolactone aligned fibre films for enhancement of neural cell compatibility and neurite growth

Abstract Objective Chitosan (CS) and polycaprolactone (PCL) were added into a nerve scaffold of poly(L‐lactide acid) (PLLA)/polypyrrole (PPy)‐based fibre films to solve the unmatch with the nerve strength and the aseptic inflammation from PLLA. Methods Poly (L‐lactide acid)‐polycaprolactone (PLLA/PCL) fibre films coated with chitosan (CS) and polypyrrole (PPy) were prepared by electrospinning of aligned PLLA/PCL fibres, electrochemical deposition of PPy nanoparticles and in situ doping of CS in PPy. PC12 cells were electrically stimulated with 100 mV for 2 hours every day via CS/PPy‐PLLA/PCL fibre film to promote the neurite growth. Results The surface conductivity and tensile strength of CS/PPy‐PLLA/PCL fibre films were 1.03 s/m and 13 MPa, respectively. CS content in fibre films was about 7.5 mg/cm2, improving the pH value (reached to 5.1) of immersion solution of the fibre film at 16 days. Compared with PPy‐PLLA/PCL fibre film, more and longer axons were grown out from PC12 cells cultured on CS/PPy‐PLLA/PCL fibre film, indicating the positive effect of CS in fibre film on axon growth. The cell differentiation rate and neurite length on CS/PPy‐PLLA/PCL fibre film reached to 38% and 75 μm, respectively. These results suggest the promotion of electrical stimulation on neurite growth and alignment. Conclusions A synergistic mechanism about the promotion of CS, electrical stimulation and aligned fibres on PC12 cells differentiation, axon outgrowth was proposed. These results indicated the potential application of CS/PPy‐PLLA/PCL fibre film in the field of the nerve repair and regeneration.


| INTRODUC TI ON
For nerve cell adhesion, migration and subsequent tissue regeneration, supporting materials have been widely applied in tissue engineering (TE). These materials include natural macromolecules, synthetic polymers and their composite materials. [1][2][3] Due to the particularity of nerve plerosis, the repair scaffold material should possess several properties as follows: good biocompatibility, aligned structure for the guidance of axon growth, 4 electrical conductivity for the stimulation of axon growth, 5 appropriate mechanical property, 6 biodegradability for avoiding two times the surgery and aseptic inflammation. 7 Recently, some reported materials have met above two-three targets. For instance, poly(L-lactic acid) (PLLA) conduits presented the better results of nerve regeneration in the rat sciatic nerve model. 8 Yang et al reported that neuronal stem cells could grow along the aligned direction of PLLA fibre. 9 However, these aligned PLLA fibres did not possess electrical conductivity and could not electrically stimulate the axon growth. The application of conductive polymers in the peripheral nerve repair was widely investigated, due to their good bio-compatibility and the ability of transmitting electrical signal to the contacted tissue. [10][11][12][13] Polypyrrole (PPy), as a commonly used conductive polymer with good biocompatibility, has been investigated for the application in nerve repair and regeneration. 14 Rivers et al reported that electrical stimulation (ES) through PPy film could accelerate neurite outgrowth. 15 George et al prepared a PPy tube, which could be placed in transected rat sciatic nerves and supported nerve regeneration for over 8 weeks. 16 However,this conduit could not guide axon growth during the nerve repair. These reports suggested that the single material could not meet all requirements of the nerve repair. Therefore, composite materials were prepared for nerve plerosis. Zhou et al cultured PC12 cells (a cell line derived from a pheochromocytoma of the rat adrenal medulla with an embryonic origin from the neural crest) 17 on conductive PPy/PLLA fibre films, showing good results of neurite adhesion, alignment and elongation. 18 Some reports also indicated that PPy-coated fibre films could be applied in neural tissue engineering, due to their biocompatibility, degradation, aligned structure and electrical conductivity. 19,20 As a degradable scaffold, PLLA was easy to be electrospun into fibre films with unique regular topography. 21,22 However, its ductility was lower than normal nerve tissue. 23 Moreover, the lactic acids from PLLA degradation in the implanted site maybe induce aseptic inflammation. 7 These results indicated that PLLA fibre films could not completely meet the requirements of nerve repair. Therefore, PLLA fibre film need to be integrated with other materials to improve their combination property, such as polycaprolactone (PCL) and Chitosan (CS).
Some studies demonstrated that PLLA/PCL scaffold prepared via electrospinning could allow cells adhesion and proliferation. 24,25 Moreover, immune-histochemical examination showed that PLLA/ PCL fibres provoked only a modest inflammatory response as nerve repair material. 26 Due to its toughness, 27 PCL was mixed with PLLA to toughen PLLA scaffold and did not decrease the biodegradability of the scaffold. 28 In addition, the report of Cooper et al indicated that aligned PCL/CS fibre films could be also used in tissue engineering. 29 Chitosan has gradually become well-accepted biomaterial for recent years, due to its good biocompatibility and biodegradability. 30,31 Previous studies demonstrated that CS was neural cyto-compatible. 32 Moreover, CS coating could promote the growth of the dorsal root ganglion cell on scaffold material. 33 Zhao et al reported a study on the degradation products of CS, chitooligosaccharides, which could promote nerve repair. 34 Furthermore, CS could improve neural cell adhesion under weak acidic condition, 29,35 because the products of CS degradation were weakly alkal. 36 In this work, a conductive CS/PPy-coated PLLA/PCL fibre scaffold was prepared, in order to improve the mechanical matching of PLLA fibres with nerve tissue and the micro-environment of neurite growth in PLLA-based scaffolds via PCL addition and the in situ doping of CS. Furthermore, the conductivity and degradation of CS/ PPy-coated PLLA/PCL films were investigated, and their effect on neurite adhesion and elongation was evaluated via neural cell test.

| Preparation of electrospun PLLA/PCL
PLLA/PCL (75/25 w/w, Mv~230 000, see Data S1) block copolymer was dissolved in hexafluoroisopropanol (HFIP) as spinning solution with a concentration of 0.1 g/mL. The receiver was a cylinder with indium-tin oxide (ITO) glass sheets (2 × 3 cm 2 ), which could rotate with the roller at 500 rpm to collect the aligned fibres, as shown in Figure S1. The spinning time was 1.5 hours under 10 kV to obtain aligned PLLA/PCL fibre film on ITO sheet.

| Characterization of CS/PPy-PLLA/PCL fibre films
The morphology of the fibre films was observed with scanning electron microscopy (SEM, HITACHI S4800, Japan). The chemical composition of the samples was analysed with Fourier Transform Infrared Spectra (FTIR). In addition, there is a characteristic peak at 465 nm in absorption spectra to identify the interaction of Chitosan and Zincon (ZCN). Thus, the interaction was used to quantitatively analysed CS content by ultraviolet-visible spectrophotometer. 38,39 Conductivity of the fibre films was measured with linear four-point

| Cell differentiation and neurite growth
Undifferentiated PC12 cells purchased from Shanghai institute of biochemistry and biology were cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum (Gibco) and 5% horse serum (Hyclone) at 37°C and 5% CO 2 . Prior to seeding, the cells were pre-cultured with NGF of 50 ng/mL for 3 days to obtain their differentiation. 41 At the same time, CS/PPy-PLLA/PCL and PPy-PLLA/ PCL fibre films were placed in the 24-well plate and disinfected with 75% alcohol for 0.5 hour and UV lights for 12 hours.
To observe the neurites from differentiated PC12 cells on two fibre films, pre-cultured cells were cultured (1 × 10 4 per well) with 50 ng/mL NGF and 50 ng/mL NT-3 (Biovision) on these fibre films for 1, 3 and 5 days. In this process, NGF could maintain nerve survival, promote axon differentiation and improve nerve regeneration. 42,43 NT-3 could maintain neuron growth and promote synapse formation. 44 During the culture period, CS/PPy-PLLA/PCL groups were exerted electrical stimulation (ES) with two electrodes (see Figure   S2) at 100 mV for 2 hours every day. 45 Then, phalloidin and 4′, 6-diamidino-2-phenylindole (DAPI)-stained cells on fibre films were observed under the inverted microscope. There were three parameters to be analysed as follows: cell differentiation rate, the neurite length and neurite alignment. (a) The cell with at least one neurite longer than the cell body was regarded as a differentiated cell. 46,47 The related protein, microtubule-associated protein-2 (MAP-2), was measured by enzyme linked immunosorbent assay (ELISA) kit of MAP-2.
(b) The neurite length was measured from the tip of the neurite to the cell body by Image Pro Plus software. (c) The cell alignment was based on the angle between fibre axis and cell neurite. In this study, when electrical stimulation was exerted on cells with aligned fibre film, the electric field direction is parallel to the fibre axis. When the angle between fibre axis and cell neurite is less than 10°, the cell was regarded to be aligned along the fibre axis. These cells which aligned along the fibres were called "normalized cells". and then, Trk receptors were able to regulate synaptic growth and cells differentiation through the activation pathway. 52 Thus, the two proteins could be used to analyse the influence of electrical stimulation.

| Composition, structure and property of CS/ PPy-PLLA/PCL fibre films
As shown in the image of Figure 1A, the electrospun PLLA/PCL fibres were aligned and almost paralleled to each other. Their average diameter was 0.57 μm. After CS/PPy was electro-deposited on naked PLLA/PCL, the fibres in the deposition film remained aligned structure, as shown in Figure 1B  These results suggest that CS/PPy-PLLA/PCL fibre films were promising to reduce local aseptic inflammation risk caused by PLLA degradation, because the dedoped CS balanced pH value of solution.
Hydrogen ions could induce the cleavage of ester bonds, accelerating the hydrolysis of PLLA and its copolymer, thus the dedoped CS could decrease the degradation rate of fibre films. 54 The weight changes of fibre films immersed in 0.7% NaCl solution for 16 days are shown in Figure S4, indicating that the residue weights of PPy-PLLA/PCL fibre films in 2nd-16th day were less than those of CS/ PPy-PLLA/PCL fibre films. As shown in Figure 2B, there was no significant difference between the maximum stresses of two groups at the 16th day, meaning that the culture medium did not significantly affect tensile property for CS/PPy-PLLA/PCL and CS/PPy-PLLA fibrefilms. The maximum stress for CS/PPy-PLLA/PCL fibre films levelled off at around 13.4 MPa, which was close to biomechanics of elbow joint ulnar nerve of rabbits, 55 larger than around 9.15 MPa of CS/PPy-PLLA fibre films. This result indicated that the addition of PCL increased the strength of copolymer fibres (see Figure S3).
The results of Figure 2C show suggesting that CS could dissolve into the solution, increasing conductive shells defects. CV curves in Figure 2D show that the electrochemical property of CS/PPy-PLLA/PCL fibre film could maintain well stability for 16 days, because the charge-discharge process of PPy coating corresponded with the adsorption and disadsorption of doped Cl − ions, respectively. 56 The weak peaks at −0.4 V and −0.5 V represented the intercalation and de-intercalation behaviour of Cl − , respectively. In addition, the electric capacity of the prepared film was slowly decreased with the immersion time.

| Cell compatibility
MTT results of L929 cells cultured on culture plates (control groups), CS/PPy-PLLA/PCL and PPy-PLLA/PCL fibre films are shown in Figure S5a. It is observed that the cyto-viability in CS/PPy-PLLA/PCL groups was significantly higher than that in control groups (P < 0.05), while PPy-PLLA/PCL fibre films limited the cell viability (P < 0.01 or P < 0.05). The MTT results of PC12 cells ( Figure S5b) show that the cyto-viability in PPy-PLLA/PCL groups was significantly lower than that of control groups at 3 days (P < 0.01), while the cyto-viability in CS/PPy-PLLA/PCL groups was significantly higher than that of control groups (P < 0.05). These results suggest that CS doping in the composite fibres could promote the viability of cells. Furthermore, there was significant difference between the two treated groups of films with/without CS (P < 0.01), meaning that CS could enhance the cyto-compatibility of the prepared fibre films.

| Cell differentiation and neurite growth
PC12 cells were cultured on CS/PPy-PLLA/PCL and PPy-PLLA/PCL fibre films with or without ES for 1, 3, 5 days, and the images are shown in Figure S6. Figure 3 is the images of PC12 cells cultured for 5 days on two kinds of fibre films with or without ES. The differentiated PC12 cells and the extended neurites could be observed clearly. The orange arrows represent direction of fibres axis. The neurites in each group were almost aligned to grow, and neurite length from the differentiated cells was increased with the cultivation time.
Compared with ES groups (shown in Figure 3B,D), no ES groups ( Figure 3A,C) showed less and shorter neurites. In addition, the cells cultured on CS/PPy-PLLA/PCL fibre films outgrew more and longer neurites ( Figure 3C,D), compared with those on PPy-PLLA/PCL fibre films ( Figure 3A,B). Especially, the image of a single cell cultured on CS/PPy-PLLA/PCL fibre films in Figure S7 shows that the axon length was more than 100 μm and parallel with the direction of fibres axis.
It is found in Figure 4A Figure 4A) were too short to extend along the fibres, leading to the unnoticeable orientation. In Figure 4D was used as control groups. As shown in Figure 4A, B, the results of neurite length and cell differentiation rate in the electrical stimulation group were slightly higher than those without ES, except for the neurite lengths results in the 1st day. These results suggested that, the electrical stimulation of 100 mV could slightly promote PC12 cells to protrude longer neurites. However, ES did not prominently influence the neurite alignment ( Figure 4C). The results in Figure 4D show that the FAK concentration in ES group increased faster in the first 3 days (from ~387 pg/mL at 1 day to ~502 pg/mL at 3 days), then reaches to ~598 pg/mL at 5 days, higher than those of groups without ES, indicating that ES could improve adhesion of PC12.
Moreover, it is seen in SEM images of Figure 5A,B that, the fibrefilms maintain their aligned structures and smooth surfaces and could guide neurite growth along the fibre axis (see the magnified images of Figure 5A,B). Neurites attached to fibre surface or even grew into strip fibrous septa. As shown in Figure 5A Figure 4C shows the angle between fibre axis and cell neurite those at the 1st day (P < 0.01), and then, there was a little increase at the 5rd day. These results further indicate that ES from fibre films could significantly improve differentiation of PC12 cells and axon outgrowth in the first 3 days.  Figure 6A. The filopodia could perceive extracellular guidance cues 57,58 and extend along fibres ( Figure 6B). Because the exposed CS chains have positive charge, they can be combined with cell membrane with negative charge to promote cells adherence ( Figure 6C) [61][62][63] and repeated cycles of self-assembly and disassembly 57 (marked by green arrows in Figure 6c). In this process, growth cone collapsed at the neck to form new cytoplasmic domain of the axon (marked by black arrows). 57 Neurofilament protein (NF) was closely related to the acceleration of axoplasmic transport. 56 The amounts of three kinds of proteins under ES were slightly increased, compared with those without ES ( Figure 6D and In addition, ES provides energy to the dynein assembly and disassembly, 57,68 resulting in axon outgrowth. Thus, when PC12 cells are exerted with ES, they would produce galvanotaxis. 69 With the increase of FAK, Trk A and NF-L, PC12 cells present better differentiation and axon outgrowth.

| CON CLUS IONS
CS/PPy-PLLA/PCL fibre films with good cyto-compatibility were prepared through the electro-deposition of CS-doped PPy on aligned PLLA/PCL fibre films. The content of CS on the conductive shell was 7.5 mg/cm 2 . The tensile strength of this fibre film was around 13 MPa, which was close to that of elbow joint ulnar nerve of rabbits. 48 After the immersion in saline for 16 days, the immersion solution of CS/PPy-PLLA/PCL fibre films could still maintain stable pH of about 5.1 (higher than 4.7 of the sample without CS), indicating that CS doping could reduce local acid property from the degradation product. Due to its surface conductivity of about 1.03 s/m, ES was available to promote the growth and differentiation of PC12 cells, and to support the directional growth of neurites. A mechanism on the synergistic effect of CS, aligned fibre films and ES to promote cell adhesion and axon growth was proposed. Therefore, the CS/PPy-coated PLLA/PCL fibre films would be applicable to the nerve tissue engineering in future.

ACK N OWLED G EM ENTS
This work has been supported by the National Natural Science