PPY Fibrous Conduits: Promoting Peripheral

Jul 28, 2016 - Peripheral nerve injuries represent a great challenge for surgeons. The conductive neural scaffold has experienced increasing interest ...
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Electrospinning of PELA/PPY Fibrous Conduits: Promoting Peripheral Nerve Regeneration in Rats by Self-Originated Electrical Stimulation Zi-Fei Zhou,†,‡,∥ Fan Zhang,†,∥ Jian-Guang Wang,† Quan-Chi Chen,† Wei-Zhi Yang,† Ning He,† Ying-Ying Jiang,§ Feng Chen,*,§ and Jun-Jian Liu*,† †

Department of Orthopedic Surgery, Shanghai Tenth People’s Hospital, Tongji University, Shanghai 200072, China Department of Orthopedic Surgery, Shanghai East Hospital, Tongji University, Shanghai 200072, China § State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China ‡

S Supporting Information *

ABSTRACT: Peripheral nerve injuries represent a great challenge for surgeons. The conductive neural scaffold has experienced increasing interest because of its good biocompatibility and similar electrical properties as compared to those of a normal nerve. Herein, nerve conduits made from poly(D,L-lactide)-co-poly(ethylene glycol) and polypyrrole (20%, 30%, and 50%) (PELA−PPY) were prepared by electrospinning, and used in regeneration of peripheral nerve defects. The results of an in vitro experiment indicated a high biocompatibility for the as-prepared materials, supporting the attachment and proliferation of a rat pheochromocytoma PC-12 cell. Furthermore, the PELA−PPY nerve conduit implanted in the sciatic nerve defects (10 mm) of the Spraguee−Dawley rats for 12 weeks showed similar results with the autograft, while it demonstrated a better outcome than the PELA nerve conduit in electrophysiological examination, sciatic function index, total amount of regenerated myelinated nerve fibers, axon diameter, myelin thickness, and several immunohistochemistry indices (S-100, laminin, neurofilament, bromodeoxyuridine, and glial fibrillary acidic portein). We supposed that the bioactivity is mainly generated by the PPY in composite nanofibers which could transmit self-originated electrical stimulation between cells. Due to the facile preparation and excellent in vivo performance, the PPY−PELA nerve conduit is promising for use as a bioengineered biomaterial for peripheral nerve regeneration. KEYWORDS: polypyrrole, nerve regeneration, tissue engineering, electrospinning, fibrous conduit

1. INTRODUCTION

Currently, conductive nerve tissue engineering scaffolds may provide a promising substitute for autograft in the repair of the damaged nerve. Generally, an outstanding nerve conduit requires good mechanical bearing performance and electrochemical activity, which could promote the growth of neurons and axons, and prevent fibrous scar tissue ingrowth.20,21 Electrical stimulation can significantly promote the PC-12 cellular ability of adhesion, migration, proliferation, and neurite outgrowth on the PPY-containing membrane in vitro, supporting that PPY can efficiently transfer electrical stimulation signals between nerve cells, so as to regulate and maintain the bioactivities of nerve cells.15,22 At present, most studies focused on the electrical conduction of PPY-containing biomaterials as well as their influence after electrical stimulation on PC-12 or Schwann cells.23−25 The in vivo experiment is still urgently needed to further explain the important role of PPY in nerve regeneration. It will be better to demonstrate the results of nerve regeneration in vivo, if there are more indices regarding

Peripheral nerve defect is a common clinical disease with poor prognosis, because of the complicated treatment and high morbidity. If a traumatic injury causes a serious nerve defect, excessive hyperplasia of nerve elements and connective tissue may result in neurofibroma or nonregeneration, thus leading to social and psychological impacts on the patient’s quality of life.1,2 Up to now, an autograft remains the gold standard to restore the structure and function of injured nerve, but there are many limitations including donor nerve inavailability, local tissue adhesion, and repeated operations.3−6 Great efforts have been made to synthesize safe and effective nerve conduits, rather than to retain the normal nerve in the donor sites.7,8 Biodegradable materials have been gradually developed and employed to both in vivo and in vitro studies, such as polylactic acid,9 polycaprolactone,10 chitosan,11 gelatin,12 and so on. Recently, polypyrrole (PPY) which can be synthesized simply and exhibits excellent biocompatibility and electrical stimuli in Schwann cells has attracted much attention.13−17 It has aroused wide concern among medical workers in terms of repairing peripheral nerve detects by substituting autologous nerve grafts.18,19 © XXXX American Chemical Society

Received: June 17, 2016 Accepted: July 28, 2016

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DOI: 10.1021/acsbiomaterials.6b00335 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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2.4. Cell Culture in Vitro. The poorly differentiated rat adrenal pheochromocytoma PC-12 cells purchased from the cell bank of the Chinese Academy of Sciences were cultured in RPMI-1640 (Gibco) supplemented with NaHCO3 1.5 g/L, glucose 2.5 g/L, sodium pyruvate 0.11g/L, and 10% fetal bovine serum (Sigma) and maintained in a humid incubator (37 °C, 5% CO2, Thermo). The CCK8 analysis was applied to detect the proliferation of a PC-12 cell in normal medium and lixivium of the PELA and PELA−PPY nanofibers. The PC-12 cells were seeded at a regular density (104 cells per hole) on the glass slides, with PELA and PELA−PPY nanofibers which were placed in a 6 well cell culture cluster (Corning). After 7 days’ culture, the cells were fixed in 4% paraformaldehyde for 15 min. Then, the glass slide and membranes were washed gently by the PBS solution. Cytoskeleton was labeled by iFluor 594-phalloidine (AAT Bioquest), and DAPI was applied for nuclear staining. 2.5. Surgical Procedure. Male Spraguee−Dawley (SD) rats (200−250 g) were employed to assess the repairing effect of autograft and different nerve conduits. The rates were maintained under standard conditions, and all the surgery procedures were performed in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals (http://oacu.od.nih.gov/regs/ index.htm). All the animals were divided into 5 groups, each with 12 rats: group A, autograft group working as a positive control; group B, PELA nerve conduits; group C, PELA−20% PPY nerve conduits; group D, PELA−30%PPY nerve conduits; group E: PELA−50%PPY nerve conduits. Anesthetized by pentobarbital sodium (50 mg/kg), the rats were fixed on the operating table, with the operating region of the right hind limb shaved and disinfected with iodine. After the skin incision, muscles on the right femur were bluntly dissected, and the right sciatic nerve was exposed.20,26 Nerve tissue (10 mm) at the center of the right sciatic nerve was separated and removed so as to generate an artificial nerve defect. An artificial nerve defect was generated by separating and removing 10 mm nerve tissue at the center of the right sciatic nerve. Then, the nerve conduit (11 mm) was secured with 8-0 nylon to both the proximal and distal segments of the severed nerve, with a depth around 0.5 mm into the conduits. Thus, the nerve gap between two stumps was 10 mm long. As to the autograft group, the severed sciatic nerve (11 mm) was reversed and sutured back to the original region by epineurial coaptation serving leaving the same 10 mm gap. The implanted site was carefully sterilized and irrigated, with the muscle layer sutured by 4−0 chronic gut sutures and skin by 2-0 silk sutures. 2.6. Walking Track Analysis. According to the formula by Bain et al., the sciatic function index (SFI) was introduced and calculated to evaluate the recovery function after surgery at 4, 8, and 12 weeks.27 The process was repeated until there were at least three times of distinct footmarks. Both hind plantars were dipped in red ink, and the footprints were obtained when the rats moved on the surface of the white paper. The figures of toe spread (TS), paw length (PL), and intermediary toe spread (IT) were collected, both in the normal (N) and experimental (E) legs. SFI equals zero in normal rats for the normal sciatic nerve, while the number equals −100 in the case of rats with completely severed sciatic nerve. The SFI formula was as follows:

the nerve fiber, such as immunohistochemistry S-100 and/or neurofilament protein. In order to overcome the existing difficulties and deficiency, we have studied the in vivo performance of PPY based nerve conduit using a series of PELA−PPY composite fibrous conduits. The PELA−PPY nerve conduits with tubular structure were prepared by a simple electrospinning process, which displayed high biocompatibility with PC-12 cell in vitro and excellent performance to promote peripheral nerve regeneration in Spraguee−Dawley rat models. We have verified that the performances of PELA−PPY conduits in nerve regeneration are almost as same as in the autograft method which is the gold standard to restore the structure and function of an injured nerve. Therefore, we further suppose that the PPYs play key roles in the promotion of a nerve regeneration process.

2. EXPERIMENTAL SECTION 2.1. Materials. The chloroform, absolute ethyl alcohol, absolute methyl alcohol, FeCl3·6H2O, and sodium dodecyl benzenesulfonatec (SDBS) were analytical grade and were purchased from Sinopharm Group Company (Shanghai), China. Pyrrole and trifluoroethanol were purchased from Aladdin Reagents (Shanghai) Co., LTD, China. Polyethylene glycol−polylactic acid (PELA) (Mw of PEG, 5000; Mw of PLA, 145000) was purchased from Jinan Daigang Biomaterial Co., Ltd. 2.2. Preparation of PELA and PELA−PPY Nerve Conduits. A typical experiment for preparation of PELA−PPY (50%) is as follows: 2 g of PELA was dissolved in 40 mL of chloroform under continuous stirring. Meanwhile, 2 g of pyrrole and 1 g of SDBS were dissolved in a mixed solvent of 1.8 mL of absolute methyl alcohol and 0.8 mL of deionized water. Then, the two solutions were mixed together under continuous stirring to obtain a homogeneous solution. Thereafter, 1 g of FeCl3·6H2O dissolved in 1 mL of deionized water was added dropwise into the above solution, and the resulting solution was continuously stirred for 12 h. Finally, the resulting solution was precipitated using absolute ethyl alcohol, and washed with deionized water and absolute ethyl alcohol repeatedly. The product of PELA− PPY composite bulk was collected by centrifugation and dried at 60 °C. The content of PPY in the composite bulk was described by the additional amount of pyrrole, and could be adjusted from 50% to 30% and 20% by reducing the content of pyrrole in the reaction solution in the process of polymerization. For the preparation of pure PELA and PELA−PPY composite nanofibers, 0.8 g of PELA or PELA−PPY (20%, 30%, and 50%) was dissolved in 10 mL of trifluoroethanol under magnetic stirring for 6 h. Then, the resulting solutions were separately filled into a 10 mL syringe with a stainless steel needle (inner diameter 0.2 mm) for electrospinning. The electrospinning process was carried out at room temperature with a constant voltage of 20 kV (BGG6-358, BMEI, China) and feeding rate of 1 mL h−1 using an electrospinning device (Changsha Nayi Instrument Technology Co., Ltd., China). A rotating stainless steel rod (300 rpm) with diameter of 2 mm was used as the collector for the fibrous tubular conduits. The distance between the needle tip and collector was 15 cm. 2.3. Characterization of Nerve Conduits. The scanning electron microscopy (SEM) micrographs were obtained with a JEOL JSM-6700 field-emission scanning electron microscope. Fourier transform infrared (FTIR) spectra were taken on an FTIR spectrometer (FTIR-7600, Lambda Scientific, Australia). The mechanical properties including tensile and compressive properties were taken on electronic tensile test machine (Drick DRK101A, China), at a rate of 10 mm per min. The thermogravimetry (TG) and differential scanning calorimetric (DSC) curves were taken in flowing air with a heating rate of 10 °C min−1 by a STA 409/PC thermal analyzer (Netzsch, Germany). Conductivity of the electrospun PELA−PPY fibrous conduits was tested with the ST2258C multifunction digital four probe tester (Suzhou Jingge Electronic Co., Ltd.).

SFI = 109.5(ETS‐NTS)/NTS‐38.3(EPL‐NPL) /NPL‐13.3(EIT‐NIT)/NIT − 8.8 2.7. Triceps Weight Analysis. The moist weights of the triceps surae muscle (TSM) were measured at 12 weeks. The TSMs of both ends in each rat were dissected, and blood attached on the surface of the muscles was gently blotted with bibulous paper. Promptly, the moist weight was collected in each rat, both in the experimental side and the healthy side. The TSM weight percentage was calculated as follows:

TSM weight (%) = TSM(operated leg)/TSM(nonoperated leg) 2.8. Electrophysiological Evaluation. Before the rats were sacrificed, electrophysiological tests (MYTO, Esaote, Italy) were performed on all rats at 12 weeks postoperatively as described previously.28,29 Under anesthetization, the right sciatic nerve was B

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Figure 1. Electrospun PELA/PPY fibrous conduits and the procedures of the in vivo implantation for nerve regeneration. (A) SEM micrographs of PELA fibers and PELA−PPY composite fibers. (B) General photographs of the PELA−PPY nerve conduits and the autograft. The gross observation of the sciatic nerve is given immediately after surgical procedures in transected sciatic nerve of the SD rat model. A 11 mm segment of the nerve was transected, and this defect was bridged by the autogenous nerve or PELA, PELA-20%PPY, PELA-30%PPY, and PELA-50%PPY nerve conduit. (C) Illustrated sketch of the process for promoting peripheral nerve regeneration in rats by self-originated electrical stimulation using the electrospun PELA/PPY fibrous conduits. software (student version for windows). Only p < 0.05 was regarded as statistically significant.

exposed, and two electric poles were placed; the stimulating one was placed at the proximal end of the regenerated nerve or autograft, and the recording one was placed in gastrocnemius belly. Nerve conduction velocity (NCV) and peak amplitude (PA) were recorded. 2.9. Morphological Analysis. The implanted conduits were harvested fixed with 2.5% glutaraldehyde in 4 °C for 2 h, and washed in PBS. Then, the samples were fixed in 1% osmium tetraoxide in 4 °C for 2 h. After being washed by PBS, the samples were dehydrated using ethanol step by step, following poxypropane replacement. Subsequently, the samples were embedded in epoxy resin and cut into semithin cross sections (1 mm thick) in the middle portion of the specimens. Slices were stained with toluidine blue and observed under a light microscope (Leica, Germany). The samples were also cut into ultrathin sections (50 nm thick), and then were stained with lead citrate and uranylacetate. Also, the CM-120 transmission electron microscope (PHILIP, Netherlands) was employed to view and photograph the specimen. 2.10. Immunohistochemistry. All the mice were sacrificed after 12 weeks’ implantation. The regenerated nerves and normal nerves were dissected and fixed in 4% paraformaldehyde for 24 h. After being embedded in paraffin and cut into 4 mm sections, the regenerated and normal nerves were stained with hematoxylin-eosin, and several immunohistochemistry antibodies. All the procedures were performed according to the standard immunohitochemical protocol. Briefly, after permeation and blocking of nonspecific binding, sections were incubated in two primary antibodies: rabbit antilaminin (1:100, Abcam), mouse antibromodeoxyuridine (Brdu, 1:200, CST), rabbit anti-200kD neurofilament heavy antibody (1:100, Abcam), mouse antiglial fibrillary acidic portein (GFAP, 1:100, CST), and rabbit anti S-100 antibody (1:200, Sigma). Finally, the immunostained samples were viewed under an optical microscope (Leica). For quantitative analysis of the immunohistochemical staining, positive cells were counted using ImageJ software (National Institute of Health, Bethesda, MD). 2.11. Statistical Analysis. The results were given as mean ± standard deviation (SD). When the significant differences among different groups were compared, a one-way analysis of variance (ANOVA) was used, followed by Tukey’s post-test using SPSS 11.5

3. RESULTS AND DISCUSSION 3.1. Characterization of PELA−PPY Composite Nerve Conduits. The PELA and PELA−PPY composite fibers and nerve conduits were easily fabricated by electrospinning, which had porous structure and could mimic natural extracellular matrix (ECM). The PPY polymer used for electrospinning was prepared by an in situ polymerization reaction in PELA solution by a catalyst of ferric chloride. With the increment of PPY content from 20% to 50%, the composite fibers still maintained good fibrous morphology (Figure 1A and Figure S1). The addition of PPY has an obvious influence on the diameter of the composite fibers. The average diameter of pure PELA was 250 nm, while the PELA−PPY (20%, 30%, and 50%) composite fibers’ average diameters were 503, 389, and 270 nm after adding PPY (Figure S2). The average diameters of the PELA− PPY composite fibers obviously increased after adding 20% PPY into the composite fibers, and then decreased after a further increase in the content of PPY (30% and 50%). It has been well-studied that the diameter of the electrospun fibers usually depends on the surface tension, flow rate, and electrical conductivity of the polymer solution.30,31 Due to the insoluble properties and the conductivity, the addition of PPY into the PELA solution could lead to changed physicochemical properties of the electrospinning solution. As shown in Figure S3, the FTIR spectrum of the PELA− PPY composite fibers exhibits absorption peaks at around 1629, 1274, 2929, 1386, and 1457 cm−1, which originate from the stretching vibration of CO, COC, CH, CC, and CN, respectively. The increasing content of PPY could give rise to the stretching vibrations, indicating that the PPY was successfully composited into the fibers. TG and DSC curves for the as-prepared pure PELA fiber and the PELA−PPY fibers are C

DOI: 10.1021/acsbiomaterials.6b00335 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering displayed in Figure S4. The sharp weight losses can be observed in the curves at 300−400 °C, due to the thermal oxidative decomposition of the pure PELA fiber and the PELA−PPY fibers in air atmosphere. Meanwhile, the peak position of oxidation decomposition varies with the increased levels of PPY in DSC curves. The highest oxidation-exothermic peak of pure PELA fibers locates at 376 °C; however, the second oxidationexothermic peak (approximately 422 °C) appears with the addition of PPY. Both results show that the polymer PPY is successfully compounded into the composite fibers in the electrospinning process. The mechanical properties of the PELA and PELA−PPY fibers have also been studied (Figure S5). The stress−strain curves of pure PELA and PELA−PPY composite fibers with similar elastic and plastic deformations are obtained. With the increase of PPY in the composite fibers, the plastic deformation reduces gradually, and the elastic deformation takes the main part. With the increasing content of PPY, the tensile failure stresses change slightly, and the average fracture stress of the PELA and PELA−PPY (20−50%) fibers are 8.1, 7.8, 7.4, and 4.9 MPa, demonstrating that PPY has a small effect on the maximum fracture stress of the composite. Meanwhile, the content of PPY displays an obvious influence on the tensile strain of composite fibers. The maximum tensile strain is as high as 49.8%, and declines rapidly to 17.0%, 16.3%, and 9.7%, when PPY content was 20%, 30%, and 50%, respectively. 3.2. Cytocompatibility and Cytoskeletal Assessment. Then, the biocompatibility of PELA−PPY (20−50%) fibrous conduits is investigated using PC-12 cells. The CCK8 analysis demonstrates similar proliferative activity in both control and experimental groups when they were cultured in leach liquor of the PELA and PELA−PPY nerve conduit at day 1, 3, and 5 (Figure 2A). After 7 days of culture, the PC-12 cells attach and proliferate well on the glass slide and PELA and PELA−PPY fiber membranes (Figure 2B). There is no significant difference in cellular morphology between the control group and experiment groups. Both the CCK8 results and the phalloidine-staining images determine good ability of PC-12 cells in their attachment and proliferation on the fibrous membrance. These results obviously suggest the nontoxic property of the PELA−PPY fibers, which pose no harm to the normal cells both in the proliferation and adhesion. Thus, the good cytocompatibility facilitates the PELA−PPY fiber to be a good nerve conduit. 3.3. Histological Assessment. Furthermore, we investigate the in vivo performance of different fibrous conduits in peripheral nerve regeneration. All of the surgical procedures go well, and the SD rats survive without any operative complications (Figure 1B). We observe the gross morphology of regenerated nerves by methylene blue staining. Numerous bunches of regenerated nerves can be found in all nerve conduit groups and the autograft group (Figure 3). The structures of myelinated fibers in the PELA−PPY conduits are obviously more compact and unified than that of the PELA group, and are very similar to that of the autograft group. Additionally, a transmission electron microscope is applied to the midportion sections of the regenerated nerve after 3 months’ implantation to evaluate the total number, diameters, and myelin thickness of regenerative axons (Figure 4). Quantitative analysis shows that the PELA nerve conduit is no match for the PELA−PPY conduit for the nerve regeneration regarding the total number of regenerated myelinated nerve fibers, average myelin thickness, and axon diameter. There is no significant difference

Figure 2. CCK 8 analysis and cocultured PC-12 cells on the glass glide and different fibrous conduits. (A) OD values of CCK8 in the 405 nm channel after 1, 3, and 5 days’ PC-12 culture in the normal medium (NC, negative control) and lixivium of the PELA and PELA−PPY fibrous conduits. (B) Immunofluorescence images of PC12 cells stained by cytoskeleton (phalloidin, red) and nuclei (DAPI, blue) after 7 days’ incubation on the glass glide and different fibrous conduits. Scale bar: 50 μm.

Figure 3. Representative images of toluidine blue staining in sections (1 mm thick) of the regenerative nerve at 3 month postsurgery in the autograft and nerve conduit groups.

observed between the autograft group and the PELA−PPY nerve conduit groups in terms of all the measured parameters. 3.4. Hematoxylin-Eosin Staining and Immunohistochemistry. To evaluate the proliferative activity in autograft and all types of nerve conduit, two immunohistochemical D

DOI: 10.1021/acsbiomaterials.6b00335 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 4. Transmission electron microscope was applied to observe the ultrathin sections (50 nm thick) of the normal nerve and regenerative tissues at 3 month postsurgery (n = 6, **p < 0.05). Parts A and B illustrated the same position with different magnification times. Black arrows tag the same axon. Parts C−E demonstrated the statistical analysis of total number, myelin thickness, and axon diameters of the regenerative myelinated nerve fibers.

GFAP is another protein which is an important component for the nerve cells’ cytoskeletal reorganization, myelination maintenance, and cell adhesion.35 Neurofilament is an important intermediate filament in neurons, and its functions include structural support and regulation of the regenerative axons. And, heavy neurofilament is regarded as an essential marker for the mature axons. In all, these three factors, namely, S-100, GFAP, and heavy neurofilament, are the superior markers for detection of regenerative tissues. Results of these three immunohistochemical experiments manifest the nerve return in all the conduit groups and the autograft group at the histological level. The regenerative tissues from the longitudinal sample section demonstrate that the inner layers of the nerve conduits are suitable for the tissue regeneration (Figure 6A). Additionally, gross observations from the immunohistochemistry in Figure 6B−D indicate that the positive expression can be found in all tissues, though there are some differences among various groups. Thus, quantitative analysis of these three immunohistochemistry indices is performed to obtain an accurate assessment (Figure 7). For these indices, the amount of positive labeled cells in the autograft group and PELA−PPY groups are much more than that in the PELA group. Compared with the autograft group, the PELA−PPY groups show an approximate total number for the neurofilament and S-100 indices (Figure 6A,C), and a relatively smaller number for the

indices, laminin and Brdu, are applied to all of the samples (Figure 5A,B). Laminin plays an essential role in the migration and adhesion of most cells, for it acts as an active part of the basal lamina.32 BrdU is a widely used marker for the detection of proliferative cells in tissues.33 Both markers are labeled, and the histological examination demonstrates that the PELA with 20%PPY or 30%PPY groups exhibits significantly more positive cells than the PELA group, which can in turn benefit the proliferation, adhesion, and migration of the Schwann cells or other crucial cells, while positive numbers in these two groups are approximately that in the autologous nerve graft group. Statistical analysis is performed and shows that the regenerative tissue in PELA conduit exhibits a lower rate compared with the autograft and the PELA−PPY groups (Figure 5 C,D). Meanwhile, no obvious difference of Brdu parameters can be observed among the three PELA−PPY groups (Figure 5D), and PELA−20%PPY and PELA−30% PPY are slightly better than PELA−50%PPY in terms of laminin parameters. Figure 6 shows the regular hematoxylin-eosin (HE) staining of different tissue slices and three immunohitochemical indices, namely, GFAP, neurofilament, and S-100. S-100 protein normally presents neurotrophic effects at the central and peripheral nervous system.34 Thus, during the process of nerve repair, reparative Schwann cells can be detected by labeling the S-100 protein, which stimulates cell proliferation and migration. E

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Figure 5. Immunohistochemical staining of Laminin and Brdu in the regenerative tissue of different groups at 3 months postsurgery. (A) Lamininpositive and (B) Brdu-positive cells were presented under the optical microscope. Accordingly, the relative percentage of positive cells was calculated in parts C and D, respectively. All experiments were repeated three times. **p < 0.05.

GFAP index (Figure 7). The histological recoveries of the regenerated nerve in the PELA−PPY groups are better than that in PELA group, and were approximate to the gold standard of the autograft group. 3.5. Assessment of Nervous Function. Furthermore, the assessment of regenerated nervous function is measured by walking track analysis, triceps weight analysis, and electrophysiological evaluation at 12 weeks postoperatively (Figure 8). Significantly, three PELA−PPY groups show good sciatic function index (SFI) which is close to the autograft group and obviously better than the PELA control (Figure 8A). Figure 8B illustrates recovery rate of triceps surae muscle (TSM) weight of all the control and experiment groups after 3 months’ postimplantation. There is a significant difference among the PELA group and each PELA−PPY group, while no significant difference can be observed among each PELA−PPY group and the autograft group. Both TSM weights and SFI are frequently performed in the assessment of the motor function of sciatic nerve regeneration.36,37 In this study, partial return of motor function is observed, and the motor function return in the PELA−PPY groups is similar to that in the native nerve repair group and significantly better than that in the pure PELA group. After 12 weeks’ implantation of the autograft and nerve conduits, peak amplitude (PA) and nerve conductive velocity (NCV) are measured to evaluate the nerve conduction from electromyography (Figure 8C,D). In comparison with the

PELA group, PELA−PPY groups exhibit obviously better PA and conductive velocity. In these three experimental groups, the PELA−20%PPY and the PELA−30%PPY groups illustrate better peak amplitude and conductive velocity than the PELA− 50%PPY group. Nerve conduction function is often assessed by NCV and PA by an electrophysiological test which is performed for all groups 12 weeks after surgery.28,29 There are similar higher trends of the NCV and PA when compared with the PELA group, indicating a good recovery of nerve conduction function for the PELA−PPY nerve group. As for these two factors, the PELA−20%PPY group exhibits a better outcome than the other two groups. Several factors have an impact on the NCV and PA, such as the axon diameter, myelin sheath thickness, the total number of the regenerated nerves, and internode length.38 The in vivo test results reveal a good recovery in the motor and nerve conduction function in all the PELA−PPY groups, which is comparable to the autograft group. On the basis of the consistent results in vitro and in vivo, we suggest that the PELA−PPY conduits exhibited high biocompatibility and bioactivity which are excellent and suitable for peripheral nerve repairing. The electrospinning is a facile manner to produce polymer fibers and mimic the natural ECM.39 In this study, the electrospun PELA−PLA fibers also provide a biomimic ECM environment for cell growth during nerve regeneration. Meanwhile, the PELA−PPY conduits are bioactive, promoting the proliferation and migration of the F

DOI: 10.1021/acsbiomaterials.6b00335 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 6. (A) HE staining and (B) immunohistochemical staining of GFAP, (C) neurofilament, and (D) S100 in the regenerative tissue of different groups at 3 months postsurgery. The black arrows in HE images indicate the residual PPY polymers. Images were acquired the optical microscope.

Figure 7. Relative percentage of GFAP-, neurofilament-, and S100-positive cells was calculated in parts A−C, respectively. All experiments were repeated three times. **p < 0.05.

brain (white), and cerebellum are 1.03, 0.47, and 0.89 S/m, respectively.40 There is no obvious advantage of the PELA− PPY composite conduits in long-range electronic conductivity, in comparison with the normal tissue. Therefore, we suppose that the high performance of the as-prepared PELA−PPY composite conduits in peripheral nerve regeneration may be explained by an influence of PPY polymers on the microenvironment in the tissue regeneration process. The PPY may transmit the self-originated electrical stimulation between the cells and promote the cell growth. The self-originated electronic stimulation is the basis for electrical signals in the nerve fibers, and plays a vital role in regeneration of the

Schwann cells or other crucial cells. We suppose that the bioactivity of PELA−PPY conduits is mainly generated by the PPY. We observe that the electrical conductivity of the of PELA−PPY composite increases with increasing PPY content from 0 to 50%, probably as a result of the formation of interlinkage of PPY in the network. The conductivities of the PELA−PPY composite with 30%PPY or 50%PPY are 13.1 ± 3.7 × 10−3 and 6.9 ± 1.6 mS/m, respectively, while the conductivities of the PELA−20%PPY and pure PELA are too low to be measured. As one can see, the conductivity properties of these composites are much lower than that of the normal tissue. For example, the electronic conductivity of brain (gray), G

DOI: 10.1021/acsbiomaterials.6b00335 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 8. Function assessment and electrophysiological examination after 12 weeks’ implantation. (A) Walking track analysis was performed to assess the sciatic function index (SFI) at 12 weeks postsurgery (n = 6, **p < 0.05). (B) Triceps surae muscle (TSM, %) weight analysis at 12 weeks after implantation (n = 6, **p < 0.05). (C) Peak amplitude (PA) detection (mV) at 12 weeks after implantation (n = 6, **p < 0.05). (D) Nerve conductive velocities (NCV, m/s) at 12 weeks after implantation (n = 6, **p < 0.05).

fibers, axon diameter, myelin thickness, and several immunohistochemistry indices (S-100, laminin, neurofilament, bromodeoxyuridine, and glial fibrillary acidic portein), we are surprised to find that the PELA−PPY composite nerve conduit shows similar results with the autograft group which is regarded as the gold standard in repairing peripheral nerve defect currently. Considering the facile preparation and remarkable in vivo effect on the repairing transected sciatic nerve of SD rat model, the PPY−PELA composite nerve conduits represent a potential application in promoting the guidance and regeneration of peripheral nerve defect.

peripheral nerve. As the results of HE staining in Figure 6 show, there are many residual PPY polymers in the regeneration nerves, which are well-embedded in the nerve cells, while the PELA constituent in fibers is not observed, indicating the degradation of the PELA polymer. The PPY may directly interact with nerve cells after being revealed from the fibers and the degradation of PELA polymer. The results in previous reports have indicated a good interaction between the cells and PPY which can significantly promote the growth of neurons and axons.15,22 Therefore, the PPY may modify the living microenvironment of nerve cells through transmitting the selforiginated electrical stimulation between the cells and two nerve stumps. With consideration of the high biocompatibility and remarkable in vivo effect on the repair of peripheral nerve defects in the SD rat model, the PPY−PELA nerve conduits display potential applications in peripheral nerve defect regeneration and tissue engineering.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00335.

4. CONCLUSIONS In summary, the PPY polymer is synthesized in situ with PELA and electrospun into composite fibrous conduits for the regeneration of peripheral nerve defects. The results of the in vitro study indicate that PELA−PPY (0%, 20%, 30%, and 50%) composite conduits support well the attachment and proliferation of rat pheochromocytoma PC-12 cells, proving good biocompatibility of these materials. Furthermore, the in vivo implanted experiments are performed in the sciatic nerve defects (10 mm) of the Spraguee−Dawley rats for 12 weeks. By analyzing results of electrophysiological examination, sciatic function index, total amount of regenerated myelinated nerve

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SEM, FTIR, thermal properties, and mechanical properties (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ∥

Z.-F.Z. and F.Z. contributed equally to this article.

Notes

The authors declare no competing financial interest. H

DOI: 10.1021/acsbiomaterials.6b00335 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

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ACKNOWLEDGMENTS This work was supported by the “National Natural Science Foundation of China” (Grants 31370986, 51472259) and Youth Innovation Promotion Association CAS (2015203). Great gratitude should be expressed to Ke Liu for her sincere suggestion in the process of electrophysiological evaluation.

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DOI: 10.1021/acsbiomaterials.6b00335 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX