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Sciatic nerve repair by microgrooved nerve conduits made of chitosan-gold nanocomposites
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Surgical Neurology 70 (2008) S1:9 – S1:18 www.surgicalneurology-online.com
Peripheral Nerves
Sciatic nerve repair by microgrooved nerve conduits made of chitosan-gold nanocomposites Yi-Lo Lin, DVM, PhD a , Jui-Chi Jen, MSc a,b , Shan-hui Hsu, PhD a,c,d,⁎, Ing-Ming Chiu, PhD e a
Center of Tissue Engineering and Stem Cells Research, National Chung Hsing University, Taichung 402, Taiwan, ROC b Department of Veterinary Microbiology, National Chung Hsing University, Taichung 402, Taiwan, ROC c Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan, ROC d Institute of Biomedical Engineering, National Chung Hsing University, Taichung 402, Taiwan, ROC e Center of Stem Cell Research, National Health Research Institutes, Miaoli County 350, Taiwan, ROC Received 11 July 2007; accepted 28 January 2008
Abstract
Background: To better direct the repair of peripheral nerve after injury, an implant consisting of a multicomponent micropatterned conduit seeded with NSC was designed. Methods: The mechanical properties of the chi-Au nanocomposites were tested. In vitro, the effect of chi-Au on cell behavior (NSC and glial cell line C6) and the influence of micropattern on cell alignment were evaluated. In vivo, the micropatterned conduits with/without the preseeded NSC were implanted to bridge a 10-mm–long defect of the sciatic nerve in 9 male Sprague-Dawley rats. The repair outcome was investigated 6 weeks after the surgery. Results: Based on the dynamic modulus, chitosan with 50 ppm or more gold was a stronger material than others. In vitro, gold at 25 or 50 ppm led to better cell performance for NSC; and gold at 50 ppm gave better cell performance for C6. On the microgrooved substrate, the NSC had elongated processes oriented parallel to the grooves, whereas the NSC on the nonpatterned surfaces did not exhibit a particular bias in alignment. In vivo, the number of regenerated axons, the regenerated area, and the number of blood vessels were significantly higher in the NSC-preseeded conduit. Conclusion: Modification of the chitosan matrix by gold nanoparticles not only provides the mechanical strength but also affects the cellular response. The preliminary in vivo data demonstrated that the biodegradable micropatterned conduits preseeded with NSC provided a combination of physical and biological guidance cues for regenerating axons at the cellular level and offered a better alternative for repairing sciatic nerve transactions. © 2008 Elsevier Inc. All rights reserved.
Keywords:
Peripheral nerve regeneration; Micropatterned conduit; Chitosan; Gold
Abbreviations: ANOVA, 1-way analysis of variance; BDNF, brainderived neurotrophic factor; cDNA, complementary DNA; chi-Au, chitosangold; DAPI, 4,6-diamidino-2-phenylindole dihydrochloride; DMA, dynamic mechanical analysis; DMEM, Dulbecco modified Eagle medium; E, experimental; ECM, extracellular matrix; FBS, fetal bovine serum; GDNF, glial cell line–derived neurotrophic factor; GFP, green fluorescent protein; IT, intermediate toe spreading; N, normal; NGF, nerve growth factor; NSC, neural stem cells; PCR, polymerase chain reaction; PDMS, poly(dimethylsiloxane); PL, footprint length; SEM, scanning electron microscopy; SFI, sciatic functional index; Tg, transition temperature; TS, toe spreading. ⁎ Corresponding author. Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan, ROC. Tel.: +886 422840510 711; fax: +886 422854734. E-mail address: [email protected] (S. Hsu). 0090-3019/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.surneu.2008.01.057
1. Introduction Peripheral nerve injuries are common in clinical practice because of trauma. The regeneration of the injured nerve is slow and can result in complicated rehabilitation. In mature neurons, there is almost no cellular replication activity, although nerve has an intrinsic capacity of inducing regeneration. When a nerve defect or gap is longer, implantation of a graft is often necessary to bridge the proximal and distal nerve stumps for promoting nerve regeneration. The most widely used material to bridge a peripheral nerve defect is the autologous nerve. An attractive alternative to the autograft
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consists of bridging the 2 nerve stumps by a biodegradable conduit, which has the advantage of directing axonal growth properly [1,13,29,41]. To further improve nerve regeneration, the conduit could be loaded with biologically active compounds such as neuronal supporting cells and NGFs [3,16,40]. Schwann cells are closely associated with, and play key roles in, the development, maintenance, and regeneration of peripheral neurons [15]. After the injury, the orientation of Schwann cells may guide the regenerating axons. The conduits containing Schwann cells were shown to promote axonal growth and improve the nerve regeneration in vivo [19,38]. However, the immunogenicity and the source of Schwann cells were regarded as the limitations for clinical applications. With the growing applicability of NSC, most of the work has been focused in the central nervous system [37]. Recently, the neural progenitor cells have been transplanted; and their differentiation abilities have promoted the axonal regeneration through a nerve defect in the rat model [28]. The advantage of using NSC is that pluripotent stem cells may allow multiple differentiation paths, creating an environment that supports axon regeneration. The microenvironment of the conduit mimicking the biological situation is a vital issue in tissue regeneration. Chitosan, a natural polysaccharide, has structural characteristics similar to glycosaminoglycans and seems to mimic the functional behavior of glycosaminoglycans. The previous research has also revealed that chitosan has bacteriostatic [10,42] and hemostatic [26] properties. Furthermore, chitosan is enzymatically degraded to absorbable oligosaccharides. Chitosan, however, has low mechanical strength under physiological conditions [25], which has limited their use as nerve guidance channels for clinical applications [43]. Because gold has high mechanical strength with good biocompatibility, the mixture of chitosan with gold nanoparticles, or the chi-Au nanocomposites, might bring about advantages to the composite biomaterial [23] for nerve conduits. Apart from the biomaterial of the conduit, a promising strategy providing instructive environments involves some special microarchitectures that allow structural support for axonal regrowth and affect cellular orientation and the presentation of ECM proteins to the cells [30]. The micropatterned inner surface is expected to direct the cell growth during the nerve reconstruction and subsequently to induce neuron alignment and effective repair. This study was designed as a preliminary study to evaluate the effect of NSC-seeded tissue-engineered nerve conduit on nerve regeneration. In vitro, the effects of chiAu composite materials on cell proliferation, gene expression, and directing cell alignment were evaluated. In vivo, the feasibility for peripheral nerve implantation was explored. The conduit was implanted to bridge the rat sciatic nerve across a 10-mm–long defect. The repair outcome was investigated 6 weeks after the surgery. It was expected that the multicomponent, biodegradable conduit
of micropatterned architecture seeded with NSC might direct cell replacement, facilitate regeneration, and guide repair to create a more physiologically relevant structure. 2. Materials and methods 2.1. Preparation of chi-Au nanocomposite materials One percent chitosan (Sigma, St Louis, Mo) solution was dissolved in 0.5 mol/L acetic acid for 12 hours at room temperature. The gold nanoparticles in solution were supplied by Global NanoTech, Taipei, Taiwan. The solution contained only pure-gold fine particles (5 nm) in water (50 ppm/mL). After filtering the chitosan solution, a certain amount of gold nanoparticle solution was added; so the final concentration in the polymer was 25, 50, or 100 ppm. To make the substrates for biological tests, 100 μL of the chi-Au solution was coated onto coverslips (15-mm diameter; Matsunami, Osaka, Japan) and air-dried for 48 hours. 2.2. DMA of chi-Au materials Changes in dynamic properties of chi-Au materials were examined by DMA using a TA Instruments DMA 2980 (New Castle, DE). To prepare the samples for mechanical testing, chi-Au solution was cast on a glass mold and peeled after being dried. Sample strips with a geometry of about 30 mm long × 5.0 mm wide × 0.07 mm thick were run in tension at 1 Hz and an amplitude of 10 μm. Samples were mounted in the DMA at room temperature and then run from 30°C to 220°C at a ramp rate of 5°C/minute. The dynamic modulus was expressed as storage modulus (in megapascals), and the tendency to dissipate energy was indicated by the parameter tan δ. 2.3. Cell maintenance Rat glioma cell line C6 (BCRC-60046; Bioresources Collection and Research Center, Hsinchu, Taiwan) and murine NSC were applied for in vitro tests. The NSC were isolated from 2-month–old mouse brain and were transfected with F1B-GFP (US Patents 7045678 and 6984518, and pending US Patent 11/375889) [6]. The stable cell lines were obtained by selection with 200 μg/mL Geneticin (11811; Gibco, Los Angeles, CA). The Geneticinresistant NSC were pooled and expanded. The GFP-positive mouse brain cells were enriched using fluorescenceactivated cell sorter (FACS Aria; BD Biosciences, Palo Alto, Calif) repeatedly until greater than 95% purity was reached. The C6 cells were cultured in DMEM supplemented with 44 mmol/L NaHCO3 (Sigma), 10% FBS, streptomycin-penicillin (50 U/mL), and 1% sodium pyruvate (59203-100M; JRH, Lenexa, KS). DMEM/F12 (11330, Gibco, Carlsbad, CA) supplemented with 10% FBS, 400 μg/mL Geneticin, 20 ng/mL human FGF1, and streptomycin-penicillin (100 U/mL) was used for NSC culture medium. Cultures were incubated in a humidified incubator with 5% CO2 at 37°C. The medium was refreshed twice weekly.
Y.-L. Lin et al. / Surgical Neurology 70 (2008) S1:9–S1:18 Table 1 Dynamic mechanical properties of chi-Au
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2.7. Cell alignment
ppm
Storage modulus (MPa) (37°C)
Tg (fsC)
0 25 50 100
2830 2787 3456 3593
161.60 160.86 160.43 162.88
2.4. Assessment of cell proliferation Before seeding the cells, the composite substrates on coverslips were soaked in 70% ethanol, rinsed with PBS, and then placed into 24-well culture plates. To assess the effect of gold nanoparticles at different concentrations, cells at a density of 104 per well for C6 and 5 × 104 per well for NSC were seeded. The cell number was counted at 24, 48, and 72 hours by the hemocytometer. 2.5. Gene expression of neurotrophic factors Total RNA was extracted from C6 or NSC grown on chiAu nanocomposites after culture for 72 hours. Trizol reagent (15596-018; Invitrogen, Carlsbad, CA) was added after the cells were trypsinized. Five micrograms of total RNA was reverse-transcribed with the first-strand cDNA synthesis kit (Fermentas, St Leon-Rot, Germany) following the manufacturer's instructions. Polymerase chain reaction was performed in a 25-μL reaction volume containing 1 μL of the cDNA, 0.5 μL of 10 μmol/L each primer (Table 2), and 5 μL of 5× PCR Master Mix buffer (Gene Mark, Tainan, Taiwan). Polymerase chain reaction was carried out in a GeneAmp PCR system 2700 thermal cycler (ABI, Foster CA, USA). The cycling parameters of cDNA were 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension at 72°C for 7 minutes. β-Actin was used to confirm fidelity of the PCR reaction and as an internal control for semiquantitative analysis. The amplified products were analyzed by electrophoresis on 1.5% agarose-TAE (10 mmol/L Tris [pH 7.5], 5.7% glacial acetic acid, and 1 mmol/L EDTA) gels and visualized by ethidium bromide staining. 2.6. Fabrication of microgrooves on chi-Au Conventional photolithographic techniques were used to fabricate silicon wafers with the desired micropatterns that were then transferred to the PDMS submaster by casting PDMS on the silicon wafer [21]. To transfer microgrooves on the chi-Au substrates, the solution containing 50 ppm gold nanoparticles was cast on the PDMS submasters. After being air-dried, the substrates were then immersed in alcohol and detached from the PDMS submasters. To study the physical guidance of NSC on micropatterned chi-Au substrates, the pattern dimensions were 20/20/3 μm (groove width/spacing/depth), based on the previous studies involving the optimal alignment of Schwann cells [21].
To evaluate cell alignment, the cultured NSC (3 × 104 cells per square centimeter) on the substrates were examined at 24 and 72 hours after culture. After staining with methylene blue, the orientation of the NSC on the substrates was measured quantitatively using an image analysis system (Image-Pro Lite; Media Cybernetics, Silver Spring, MD) [21]. Those cells with an orientation angle between −10° and 10° were identified as the aligned cells, and the proportion of the aligned cells in the population was calculated. Control data were taken from measurements made on NSC on nonpatterned chi-Au substrates. 2.8. Cytoskeleton observation To evaluate the cytoskeleton of NSC on the substrates, cells cultured after 24 and 72 hours were fixed in 4% paraformaldehyde. After several rinses with PBS, 500 μL of 0.5% Triton X-100 (t-octylphenoxypolyethoxyethanol; Sigma, T9284) was added for 10 minutes. The samples were rinsed, and 300 μL of 0.03% phalloidin (P2141, Sigma) was added in the dark for 30 minutes. To stain the cell nuclei, cells were rinsed; and 300 μL of 0.01% DAPI (D9542, Sigma) was added in the dark for 30 minutes. Samples were rinsed and then mounted onto microscope slides. The cytoskeleton was observed by the fluorescence microscope (Eclipse 80i; Nikon, Tokyo, Japan). 2.9. Conduits for implantation The micropatterned substrates were rolled into conduits by a 1.5-mm–diameter glass mandrel and adhered by a small amount of chitosan solution. Conduits with 1.95-mm internal diameter and 2.22-mm external diameter were sectioned into 12-mm–long segments. The suspension of GFP-positive mouse NSC (5 × 104/mL) was injected into the conduit, and then the conduit was sealed immediately at both ends and rotated at 1 rpm for 72 hours before implantation. The structure of microgrooved conduit (without cell seeding) was imaged using SEM. After mounting, conduits were gold-coated using a Hitachi coating unit IB-2 coater. Coated samples were examined by a scanning electron microscope (S-3000N, Hitachi, Tokyo, Japan).
Table 2 Primers used in this study Gene
Sequence
Size (base pairs)
BDNF
F–GAC TCT GGA GAG CGT GAA T R–CCA CTC GCT AAT ACT GTC AC F–ACC TCT TCG GAC ACT CTG GA R–GTC CGT GGC TGT GGT CTT AT F–CCC GAA GAT TAT CCT GAC CA R–TAG CCC AAA CCC AAG TCA GT F–TCC TGT GGC ATC CAC GAA ACT R–GGA GCA ATG ATC CTG ATC TTC
325
NGF GDNF β-Actin
168 242 185
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2.10. Animal implantation of microgrooved nerve conduits Nine male Sprague-Dawley rats weighing 250 to 300 g were used for the preliminary animal test (n = 3 for each group). The 2 experimental groups received the grooved surface conduits seeded with and without NSC, respectively. The other group received reversed autografts and served as controls. The animals were housed in standard laboratory cages under temperature-controlled conditions at 24°C ± 1°C and 45% humidity with 12-hour light cycles, with free access to standard rat chow and water. All procedures followed the guidelines for the animal care, and the Ethical Committee of National Chung Hsing University had approved the experiment. Animals were anesthetized with Citosol (6 mg/100g intraperitoneally (Shinlin Sinseng Pharmaceutical Co, Taipei, Taiwan)), and the left hind legs were used to perform the surgery. A skin incision from the left knee to the hip was made to expose the underlying muscles, which were then retracted to reveal the sciatic nerve. The microgrooved conduit, with or without GFP-positive mouse NSC, or the reversed autograft was interposed into the 10-mm sciatic nerve defect. The nerve stumps were anchored inside to a length of approximately 1 mm of the conduit by 7-0 nylon microsutures. The operation site was sutured in layers using 3-0 Dexon (Unik Surgical Sutures MFG Co, Taipei, Taiwan). Six weeks after implantation, the rats implanted with conduits or controls were euthanized.
ANOVA. Bonferroni post hoc analysis was used for multiple group comparisons to determine the statistical significance of the results. P b .05 was considered as statistically significant. 3. Results A technique for fabricating microgrooves on chi-Au substrates was developed, and the effects on the in vitro behaviors of NSC and C6 and the in vivo implantation were investigated. 3.1. DMA of chi-Au materials The dynamic mechanical properties of chi-Au were investigated using DMA. From the overlay of DMA scans on individual chi-Au concentration, there was a concentration-dependent increase in the dynamic modulus except for the values of 25 ppm before 82°C, which were slightly lower than those of 0 ppm. At 37°C, the dynamic modulus was 2787 MPa at 25 ppm, whereas it was 2830 MPa at 0 ppm. The overall patterns were similar at 0 and 25 ppm. At 50 ppm, there was a higher dynamic modulus compared with 0 and 25 ppm. Chitosan with 100 ppm gold, as expected, was a stronger material than others, as could be seen by the
2.11. Functional assessment of nerve regeneration Walking track analysis was performed after 3 and 6 weeks. For the calculation of SFI, the following footprint parameters were measured: the distance to the PL, TS, and IT. Data were collected for both the N and the E hind legs. The SFI was calculated based on the formula used by Bain et al [2] expressed below. An index of zero reflected normal function, and −100 theoretically represented complete loss of function. SFI ¼ 38:3½ð EPL NPLÞ=NPL þ 109:5½ð ETS NTS Þ=NTS þ 13:3½ð EIT NIT Þ=NIT 8:8 2.12. Histologic analysis The tissues were harvested, fixed overnight in 4% paraformaldehyde, stained with 1% osmium tetroxide (Polysciences, Warrington, Pa), embedded in paraffin, thin-sectioned (4 μm), and stained with toluidine blue. The number of myelinated axons and the regenerated area were determined using an image analysis system (ImagePro Lite, Media Cybernetics). The number of blood vessels was also counted. 2.13. Statistical analysis Data from the experiments were expressed as mean ± standard deviation. Statistical differences were analyzed by
Fig. 1. Dynamic mechanical analysis of chi-Au. Chitosan composites containing 0 (control), 25, 50, and 100 ppm of gold were measured. A: Storage modulus; B: tan δ. Values were measured from 30°C to 220°C.
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highest dynamic modulus (Fig. 1A, Table 1). Chitosan-gold 0 and 25 ppm had higher tan δ than chi-Au 50 and 100 ppm. Glass Tg indicated by the peak of tan δ was slightly reduced at 50 ppm (Fig. 1B, Table 1). 3.2. Effect of chi-Au substrates on cell proliferation and gene expression The cytocompatibility of different chi-Au composites was tested in vitro by analyzing cell proliferation and gene expression of both C6 cells and NSC. Gold nanoparticles had a stimulatory effect on cell proliferation that was concentration dependent (Fig. 2A, B). For NSC, cell proliferation was significantly increased, compared with the control (0 ppm), at the concentration of 25 or 50 ppm. However, when the gold concentration was increased to 100 ppm, cell proliferation was similar to the control. Results showed that the nanocomposites containing 25 or 50 ppm were superior to those at other concentrations in the proliferation of both C6 and NSC. Results from the semiquantitative analysis of neurotrophic gene expression for C6 and NSC cultured on various chi-Au substrates are summarized in Fig. 2. The effects of
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gold on gene expression of C6 and NSC were different. In general, higher levels of both BDNF and GDNF genes were expressed than the NGF gene expression. Comparing chi-Au of different gold concentrations, higher NGF expression in C6 was detected at 50 ppm of gold, whereas the amount of BDNF and GDNF increased with the concentration of gold (Fig. 2C). On the contrary, there was no significant concentration dependence in the gene expression of NGF, BDNF, and GDNF in the NSC (Fig. 2D). For NGF and GDNF, the gene expression on chitosan alone was slightly higher than that at other concentrations; but the difference did not reach significance. For BDNF, the gene expression on chi-Au of all concentrations was similar. The only significance was that the GDNF gene expression at 25 ppm of gold was lower than that on chitosan alone. From the results of cell proliferation and gene expression, the concentration of gold at 25 and 50 ppm showed better cell compatibilities than at other concentrations. Although chi-Au 25 ppm showed better cell proliferation for the NSC, the modulus was weak and even lower than that of chitosan alone at 37°C. Considering both cell compatibilities and mechanical strength for the conduit, chi-Au 50 ppm would be a better choice for in vivo environment.
Fig. 2. Effects of chi-Au on proliferation and gene expression of C6 and NSC. Cells were grown on biomaterials in 24-well plates, and the effects of gold at 0 (control), 25, 50, and 100 ppm were compared. Values are expressed as mean; error bars represent SD; a indicates significantly (P b .05) different from the value at 0 ppm. A and B: Cell proliferation. Data were measured at 24, 48, and 72 hours. C and D: Gene expression. Data were measured at 72 hours.
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Fig. 3. The orientation of NSC on the microgrooved substrates. A: Actin and DAPI staining for NSC on the nonpatterned substrate (a) and the grooved chi-Au substrate (b). B: NSC alignment (percentage of cells aligned) at 24 and 72 hours. Values are expressed as mean ± SD. Scale bar: 20 μm.
3.3. Effect of guidance cues on the alignment of NSC The morphology and the alignment of the NSC on the micropatterned substrates are shown in Fig. 3. The growth and orientation of NSC on the micropatterned surface were influenced by the 3-dimensional topography of the substrate.
Most of the NSC appeared multipolar on the nonpatterned substrates (panel A, a) and bipolar on the microgrooved substrates (panel A, b). The NSC on the nonpatterned surfaces did not exhibit a particular bias in alignment, but extended processes in a radial fashion. However, the morphology of NSC on the micropatterned surfaces had elongated processes oriented parallel to the grooves of the patterned substrate. The degree of cell orientation on the micropatterned substrates after 24 and 72 hours of culturing was displayed in panel B. The results showed that, after 24 hours, 78.3% of the NSC were aligned on the chitosan substrate and 83.7% of the NSC were aligned on the chi-Au substrate (P b .05). After 72 hours in culture, over 90% of cells were aligned in both substrates; and the alignment on chi-Au
Table 3 The sciatic function index of the 2 experimental groups
At 3 wk At 6 wk Fig. 4. The structure of the inner surface for the microgrooved conduits. The image was obtained by SEM.
Chi-Au
Chi-Au + NSC
−72.62 ± 2.79 −63.39 ± 10.10 ⁎
−69.32 ± 4.16 −48.74 ± 12.80⁎,⁎⁎
The values represent means ± standard deviations. ⁎ Significantly (P b .05) greater than at 3 weeks. ⁎⁎ Significantly (P b .05) greater than chi-Au.
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Fig. 5. Light micrographs of the regenerated nerve. The cross sections were through the midpoint of the nerve conduit. A: Chitosan-Au microgrooved conduit. B: Chitosan-Au microgrooved conduit preseeded with NSC. C: Autograft. D: Normal sciatic nerve.
substrate was slightly better than on chitosan alone (not significant) (Fig. 3B). 3.4. Morphology of the conduit The SEM observations clearly showed that the microgrooves on the lumen surface of the conduits remained visible after the rolling procedure (Fig. 4). The featured dimensions were also well kept. 3.5. Functional assessment of nerve regeneration The SFI values are listed in Table 3. The values at 3 weeks after operation were similar in both chi-Au groups with or without NSC. At the sixth week, the values were significantly greater than those at the third week for both groups. Especially, the SFI value in the NSC-seeded conduits was significantly greater than the value in the nonseeded chiAu conduits. 3.6. Retrieving in vivo implantation and nerve regeneration No animals died after 6 weeks of implantation process. Macroscopically, the implant appeared as a discreetly enlarged area integrated in the sciatic nerve. The conduit was not yet degraded. The nerve was successfully connected; a white tubular substance through the conduit was observed. The histologic sections showed that the number of myelinated axons and the regenerated area were significantly greater in the NSC-preseeded conduit than in the conduit alone (Fig. 5 and Table 4). Angiogenesis was remarkable around the nerve fibers in the NSC-preseeded conduit.
4. Discussion When the nerve injury is severe and the nerve function is lost, the portion of the distal stump dies and degenerates; the proximal stump may be able to regenerate and reestablish the nerve function. However, it is difficult for the growth cone of the regenerating axon to reconstruct larger nerve gaps. To better direct the repair after peripheral nerve injury, a graft implant between the proximal and the distal nerve stumps as a guide for the regeneration axon is required. To develop an optimal artificial nerve graft for clinical applications, mechanical support, functional cells, growth factors, and ECM of the conduit should be considered. In this study, it was focused on the effects of nerve regeneration by the incorporation of these factors that attempted to modify the microenvironment of neuronal physiology in vivo. The interaction of the implanted cells and the biomaterial is complicated, and it depends on the cell type and the characteristics of the biomaterial. Recently, various biodegradable materials have been used as scaffolds for nerve regeneration, including polyglycolide, polylactide, poly(ɛTable 4 Data of histologic analyses at the midconduit from the 2 experimental groups
2
Regenerated area (mm ) No. of myelinated axons No. of blood vessels
Chi-Au
Chi-Au + NSC
0.298 ± 0.148 453 ± 29 18 ± 5
1.030 ± 0.424 ⁎ 1938 ± 287 ⁎ 55 ± 11 ⁎
The values represent means ± standard deviations. ⁎ Significantly (P b .05) greater than chi-Au.
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caprolactone), and chitosan [8,39]. Our previous study showed that gene expression for neurotrophic factors in Schwann cells was up-regulated on chitosan substrate compared with that on polylactide [22]. Chitosan also showed a good affinity for nerve cells and promoted the survival and neurite outgrowth of nerve cells in vitro [14,17], which suggested that chitosan might be applicable as a scaffold for axonal regeneration in peripheral nerves. Because the mechanical strength of the conduit under physiological conditions is very crucial for the guidance and supporting of nerve regeneration, the mechanical property of chi-Au nanocomposites was tested. From the results of DMA, gold played an important role in the mechanical property of the chitosan material. The mechanical strength increased with the amount of gold in the chitosan film except at 25 ppm, where the mechanical strength was even lower than that in chitosan alone. With increasing temperature, the dynamic modulus of chi-Au 25 ppm became higher than that of the original chitosan. However, considering the in vivo environment (37°C), chi-Au 25 ppm would not be a good option. The tan δ patterns also showed concentration dependence of gold, and it was found that chi-Au at lower gold concentrations was softer (larger tan δ). The sharp transitions might represent changes in crystalline domains within the chitosan matrix. Although the tan δ Tg was close among chi-Au, as would be expected because the main component of all nanocomposites contained chitosan in their makeup, each nanocomposite had its own transition curve and inherent characteristics unique to its material morphology. The results might suggest that the chitosan matrix and hence its DMA signature have been significantly modified by gold, which might facilitate nucleophilic attack, via solvation, of the polymer backbone. To determine the suitable gold concentration of the chitosan matrix, this study was first conducted by culturing C6 and NSC in vitro, comparing the biomaterial properties in different chi-Au nanocomposites and the behavior of both types of cells on the biomaterials. Because gold nanoparticles could change the microstructure of the biomaterial, it was possible that gold nanoparticles could modulate the cellular response through the physical features. Alterations in cell proliferation and gene expression could be detected as expected, depending on the amount of gold nanoparticles. There was concentration dependence of the gold nanoparticles on both types of cells. This indicated that different gold concentrations affected the behavior of both C6 and NSC in culture. This was in line with our previous findings for in vitro endothelial cell response to the polyurethane-gold nanocomposites [23]. There was a clear effect of amount of gold nanoparticles on the results of the present study. The overall patterns in cell proliferation and gene expression were similar for both C6 and NSC. Adding a certain amount of gold nanoparticles seemed to have a stimulating effect on cell proliferation and gene expression. However, if a higher amount of gold nanoparticles was used, the result was not much different from that of the simple
chitosan. In particular, our findings suggested that the stimulatory effects on cell proliferation and gene expression decreased with the amount of gold nanoparticles more than 50 ppm. The decreasing effect at higher gold concentrations was also shown in polyurethane-gold nanocomposites [23]. The results indicated that 25 and 50 ppm gave better cell performance for NSC, whereas 50 ppm led to better results in C6. Because 50 ppm of gold was better for both types of cells, this concentration was chosen to prepare the conduits for the in vivo implantation. The mechanism of gold nanoparticles on cells is not clear. There may be an effect through the interaction between cells and the nanofeatured surfaces. It has been reported that fibroblast response to surface nanoislands was enhanced [11]. Mediation of cell marker expression on leukocytes by nanostructured surfaces was observed [5]. Results from these studies might point out a possibility of modified cellular to tissue responses by the nanocomposite surfaces. This might also imply that cells are capable of detecting external surface changes by nanometric size. Higher density of nanoparticles in the surface on the other hand may interfere with cell growth. Nanotechnology holds great promise for many applications, including biomedical uses. Some in vitro studies showed that gold nanoparticles could be taken up by human cells, but there was no acute cytotoxicity [7,9]. Our in vitro data showed that there was no uptake of gold nanoparticles by NSC (data not shown). However, until now, very little is known regarding the in vivo toxicity especially for a longer term. In this study, we have not tracked the destiny of gold nanoparticles in vivo; but this has been planned. The preliminary in vivo results showed that the conduits preseeded with NSC were able to support regeneration of the sciatic nerve better than the empty conduits. The recovery degree of sciatic nerve function was accessed by measuring the SFI that provided a noninvasive and easily quantifiable method in the rat model [2]. As SFI compared the experimental and the normal prints, each animal acted as its own control. Our data showed that, at 3 weeks after operation, the function was still poor in both groups. At the sixth week, an increasing ability of walking became evident. A better and faster functional recovery was shown in the NSC-grafted conduits. In contrast, the functional recovery was in a slower progress in the chi-Au group. Quantitative histology demonstrated that the total number of myelinated axons was 4-fold higher in the conduits containing NSC. The total number of myelinated axons in the chi-Au conduits with and without NSC corresponded to approximately 25% and 6%, respectively, of the total number of myelinated axons present in the normal sciatic nerve [33]. More angiogenesis formation was also found in the NSCpreseeded group, which provided nutrient transport to the regenerating axons [32]. The degree of myelination may be related to the maturity of the axon, but may not measure the nerve function and the integration of central nervous system. The SFI gives general information of nerve regeneration and
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functional recovery because it represents the outcome of the repair process. To further understand the specific process during functional recovery, nerve conduction velocity will be considered in our next step because there is significant correlation between electrophysiological and morphologic parameters (ie, conduction velocity and axon diameters/ myelination) [12]. The GFP-positive NSC were shown to have the selfrenewal property and could differentiate into astrocytes, neurons, and oligodendrocytes in vitro (Chiu et al, unpublished data). Astrocytes have been shown to produce a variety of cytokines and neurotrophic factors, which stimulate the viability and proliferation of distinct cells [31]. Apparently, the inclusion of neuronal supporting cells is critical for axonal proliferation. On the contrary, the regeneration in empty conduits had poorer results, which might be due to the absence of aligned supporting cells within the conduit, leading to the early degeneration of the elongated axons [4]. It has been shown that Schwann-like supportive cells were derived in the nerve graft filled with the neuronal progenitor cells graft, but the number was too small for sciatic nerve regeneration by itself [28]. On the other hand, NSC might remain undifferentiated to contribute the trophic support rather than differentiate into functional cells [37]. Because the graft NSC played an important part of the regeneration, further investigation, for example, immunocytochemistry or cell prelabeling, will be performed to trace the fates of the grafted NSC and to clarify the associated mechanism. Neurotrophic factors play important roles in the maintenance and survival of peripheral neuronal cells. The supply of retrogradely transported neurotrophic factors was disrupted after peripheral nerve injury, leading to neuronal cell death and poor regeneration. Administration of exogenous neurotrophic factors after nerve injury has been shown to mimic the effect of target organ–derived trophic factors on neuronal cells. Seeding the conduits with NSC before implantation may provide a living, interactive source of neurotrophic factors that increases the chance of neuronal outgrowth and enhances peripheral nerve regeneration. Our results showed that the NGF, BDNF, and GDNF genes were expressed by NSC; and these factors might promote nerve regeneration [16,18,24]. NGF and BDNF act directly to promote survival and indirectly on regenerated axons by activating nonneuronal cells, which might release growthpromoting molecules and enhance the regeneration of all neuronal populations [20,36]. GDNF and BDNF support motoneuron survival in vitro, prevent axotomy-induced motoneuron degeneration in vivo [34,44], have a strong effect on motor fiber regeneration [16], and improve the conduction velocity of motoneurons after regeneration [27]. Therefore, integrating the multiple stimuli to direct the secretion of neurotrophic factors by NSC offered opportunities to mimic the natural in vivo environment. Moreover, the surface of the implant, which was microgrooved, provided more than just mechanical support
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but also the positive influence on cell alignment. Our results demonstrated that the patterned substrate directed the NSC to extend processes along the inside of the groove and influenced the cells to spread in the groove direction (Fig. 3). The results were in line with the finding that Schwann cells could be successfully aligned by the microgrooves [6]. In vivo, the presence of the longitudinally aligned NSC appeared to effectively promote the oriented growth of the damaged peripheral nerve. Special microarchitecture allows the optimal structural support for axonal regrowth and affects cellular orientations. It was possible that the patterned substrate increased NSC proliferation, leading to the enhanced neuronal differentiation and neurogenesis [30,35]. Further evaluation is yet needed to analyze the fates of grafted NSC and clarify the mechanism of nerve regeneration as mentioned. Providing a grooved support enables the temporal guidance of the complex processes of tissue formation and regeneration. In conclusion, the results in this study demonstrated a strategy for enhancing adult NSC proliferation and alignment that had important applications in guided peripheral nerve regeneration. In vitro results showed that chitosan was biocompatible to nerve cells and could be used as the conduit material to facilitate nerve cell proliferation and growth. Modification of the chitosan matrix by gold nanoparticles not only provided the mechanical strength but also affected the cellular response. Seeding the micropatterned conduits with NSC before implantation may provide a living, interactive source of neurotrophic factors. Finally, the combination of physical and biological cues enables optimistic potential for improving the rate of functional recovery. Although the results of the present study are still far from conclusive for clinical applications yet, they certainly warrant further study for bridging longer gap and investigation on retrograde labeling, cell fate tracing, and motor and sensory nerve function recovery to give a more in-depth view. Acknowledgments This work was supported by grants from Ministry of Education, National Science Council, and National Health Research Institutes. The authors would like to thank Global NanoTech for the supply of gold nanoparticles and Dr Han Chang (Chung Shan Medical University) for the help in analyzing the histologic sections. References [1] Archibald SJ, Krarup C, Shefner J, Li ST, Madison RD. A collagenbased nerve guide conduit for peripheral nerve repair: an electrophysiological study of nerve regeneration in rodents and nonhuman primates. J Comp Neurol 1991;306:685-96. [2] Bain JR, Mackinnon SE, Hunter DA. Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat. Plast Reconstr Surg 1989;83:129-38. [3] Bryan DJ, Wang KK, Chakalis-Haley DP. Effect of Schwann cells in the enhancement of peripheral-nerve regeneration. J Reconstr Microsurg 1996;12:439-46.
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[4] Buti M, Verdu E, Labrador RO, Vilches JJ, Fores J, Navarro X. Influence of physical parameters of nerve chambers on peripheral nerve regeneration and reinnervation. Exp Neurol 1996;137:26-33. [5] Buttiglieri S, Pasqui D, Migliori M, Johnstone H, Affrossman S, Sereni L, Wratten ML, Barbucci R, Tetta C, Camussi G. Endothelization and adherence of leucocytes to nanostructured surfaces. Biomaterials 2003;24:2731-8. [6] Chang CJ, Hsu SH. The effects of low-intensity ultrasound on peripheral nerve regeneration in poly(DL-lactic acid-co-glycolic acid) conduits seeded with Schwann cells. Ultrasound Med Biol 2004;30:1079-84. [7] Chithrani BD, Ghazani AA, Chan WC. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett 2006;6:662-8. [8] Ciardelli G, Chiono V. Materials for peripheral nerve regeneration. Macromol Biosci 2006;6:13-26. [9] Connor EE, Mwamuka J, Gole A, Murphy CJ, Wyatt MD. Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity. Small 2005;1:325-7. [10] Cuero RG. Antimicrobial action of exogenous chitosan. Exs 1999;87:315-33. [11] Dalby MJ, Giannaras D, Riehle MO, Gadegaard N, Affrossman S, Curtis AS. Rapid fibroblast adhesion to 27 nm high polymer demixed nano-topography. Biomaterials 2004;25:77-83. [12] Dellon AL, Mackinnon SE. Selection of the appropriate parameter to measure neural regeneration. Ann Plast Surg 1989;23:197-202. [13] Den Dunnen WF, Van der Lei B, Schakenraad JM, Blaauw EH, Stokroos I, Pennings AJ, Robinson PH. Long-term evaluation of nerve regeneration in a biodegradable nerve guide. Microsurgery 1993;14:508-15. [14] Dillon GP, Yu X, Sridharan A, Ranieri JP, Bellamkonda RV. The influence of physical structure and charge on neurite extension in a 3D hydrogel scaffold. J Biomater Sci Polym Ed 1998;9:1049-69. [15] Feneley MR, Fawcett JW, Keynes RJ. The role of Schwann cells in the regeneration of peripheral nerve axons through muscle basal lamina grafts. Exp Neurol 1991;114:275-85. [16] Fine EG, Decosterd I, Papaloizos M, Zurn AD, Aebischer P. GDNF and NGF released by synthetic guidance channels support sciatic nerve regeneration across a long gap. Eur J Neurosci 2002;15:589-601. [17] Freier T, Montenegro R, Shan Koh H, Shoichet MS. Chitin-based tubes for tissue engineering in the nervous system. Biomaterials 2005;26: 4624-32. [18] Gavazzi I, Kumar RD, McMahon SB, Cohen J. Growth responses of different subpopulations of adult sensory neurons to neurotrophic factors in vitro. Eur J Neurosci 1999;11:3405-14. [19] Guenard V, Kleitman N, Morrissey TK, Bunge RP, Aebischer P. Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration. J Neurosci 1992;12:3310-20. [20] Houle JD. Regeneration of dorsal root axons is related to specific nonneuronal cells lining NGF-treated intraspinal nitrocellulose implants. Exp Neurol 1992;118:133-42. [21] Hsu SH, Chen CY, Lu PS, Lai CS, Chen CJ. Oriented Schwann cell growth on microgrooved surfaces. Biotechnol Bioeng 2005;92:579-88. [22] Hsu SH, Lu PS, Ni HC, Su CH. Fabrication and evaluation of microgrooved polymers as peripheral nerve conduits. Biomedical Microdevices 2007;9:665-74. [23] Hsu SH, Tang CM, Tseng HJ. Biocompatibility of poly(ether)urethanegold nanocomposites. J Biomed Mater Res A 2006;79:759-70. [24] Lindsay RM. Nerve growth factors (NGF, BDNF) enhance axonal regeneration but are not required for survival of adult sensory neurons. J Neurosci 1988;8:2394-405.
[25] Madihally SV, Matthew HW. Porous chitosan scaffolds for tissue engineering. Biomaterials 1999;20:1133-42. [26] Malette WG, Quigley HJ, Gaines RD, Johnson ND, Rainer WG. Chitosan: a new hemostatic. Ann Thorac Surg 1983;36:55-8. [27] Munson JB, McMahon SB. Effects of GDNF on axotomized sensory and motor neurons in adult rats. Eur J Neurosci 1997;9: 1126-9. [28] Murakami T, Fujimoto Y, Yasunaga Y, Ishida O, Tanaka N, Ikuta Y, Ochi M. Transplanted neuronal progenitor cells in a peripheral nerve gap promote nerve repair. Brain Res 2003;974:17-24. [29] Nicoli Aldini N, Perego G, Cella GD, Maltarello MC, Fini M, Rocca M, Giardino R. Effectiveness of a bioabsorbable conduit in the repair of peripheral nerves. Biomaterials 1996;17:959-62. [30] Recknor JB, Sakaguchi DS, Mallapragada SK. Directed growth and selective differentiation of neural progenitor cells on micropatterned polymer substrates. Biomaterials 2006;27:4098-108. [31] Ridet JL, Malhotra SK, Privat A, Gage FH. Reactive astrocytes: cellular and molecular cues to biological function. Trends Neurosci 1997;20:570-7. [32] Rosen JM, Padilla JA, Nguyen KD, Padilla MA, Sabelman EE, Pham HN. Artificial nerve graft using collagen as an extracellular matrix for nerve repair compared with sutured autograft in a rat model. Ann Plast Surg 1990;25:375-87. [33] Schmalbruch H. Loss of sensory neurons after sciatic nerve section in the rat. Anat Rec 1987;219:323-9. [34] Sendtner M, Holtmann B, Kolbeck R, Thoenen H, Barde YA. Brainderived neurotrophic factor prevents the death of motoneurons in newborn rats after nerve section. Nature 1992;360:757-9. [35] Song HJ, Stevens CF, Gage FH. Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons. Nat Neurosci 2002;5:438-45. [36] Taniuchi M, Clark HB, Johnson Jr EM. Induction of nerve growth factor receptor in Schwann cells after axotomy. Proc Natl Acad Sci U S A 1986;83:4094-8. [37] Teng YD, Lavik EB, Qu X, Park KI, Ourednik J, Zurakowski D, Langer R, Snyder EY. Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. Proc Natl Acad Sci U S A 2002;99:3024-9. [38] Thompson DM, Buettner HM. Schwann cell response to micropatterned laminin surfaces. Tissue Eng 2001;7:247-65. [39] Toba T, Nakamura T, Shimizu Y, Matsumoto K, Ohnishi K, Fukuda S, Yoshitani M, Ueda H, Hori Y, Endo K. Regeneration of canine peroneal nerve with the use of a polyglycolic acid-collagen tube filled with laminin-soaked collagen sponge: a comparative study of collagen sponge and collagen fibers as filling materials for nerve conduits. J Biomed Mater Res 2001;58:622-30. [40] Tohill M, Terenghi G. Stem-cell plasticity and therapy for injuries of the peripheral nervous system. Biotechnol Appl Biochem 2004;40: 17-24. [41] Tountas CP, Bergman RA, Lewis TW, Stone HE, Pyrek JD, Mendenhall HV. A comparison of peripheral nerve repair using an absorbable tubulization device and conventional suture in primates. J Appl Biomater 1993;4:261-8. [42] Tsai GJ, Su WH. Antibacterial activity of shrimp chitosan against Escherichia coli. J Food Prot 1999;62:239-43. [43] Yamaguchi I, Itoh S, Suzuki M, Sakane M, Osaka A, Tanaka J. The chitosan prepared from crab tendon I: the characterization and the mechanical properties. Biomaterials 2003;24:2031-6. [44] Yan Q, Elliott J, Snider WD. Brain-derived neurotrophic factor rescues spinal motor neurons from axotomy-induced cell death. Nature 1992;360:753-5.
Surgical Neurology 70 (2008) S1:9 – S1:18 www.surgicalneurology-online.com
Peripheral Nerves
Sciatic nerve repair by microgrooved nerve conduits made of chitosan-gold nanocomposites Yi-Lo Lin, DVM, PhD a , Jui-Chi Jen, MSc a,b , Shan-hui Hsu, PhD a,c,d,⁎, Ing-Ming Chiu, PhD e a
Center of Tissue Engineering and Stem Cells Research, National Chung Hsing University, Taichung 402, Taiwan, ROC b Department of Veterinary Microbiology, National Chung Hsing University, Taichung 402, Taiwan, ROC c Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan, ROC d Institute of Biomedical Engineering, National Chung Hsing University, Taichung 402, Taiwan, ROC e Center of Stem Cell Research, National Health Research Institutes, Miaoli County 350, Taiwan, ROC Received 11 July 2007; accepted 28 January 2008
Abstract
Background: To better direct the repair of peripheral nerve after injury, an implant consisting of a multicomponent micropatterned conduit seeded with NSC was designed. Methods: The mechanical properties of the chi-Au nanocomposites were tested. In vitro, the effect of chi-Au on cell behavior (NSC and glial cell line C6) and the influence of micropattern on cell alignment were evaluated. In vivo, the micropatterned conduits with/without the preseeded NSC were implanted to bridge a 10-mm–long defect of the sciatic nerve in 9 male Sprague-Dawley rats. The repair outcome was investigated 6 weeks after the surgery. Results: Based on the dynamic modulus, chitosan with 50 ppm or more gold was a stronger material than others. In vitro, gold at 25 or 50 ppm led to better cell performance for NSC; and gold at 50 ppm gave better cell performance for C6. On the microgrooved substrate, the NSC had elongated processes oriented parallel to the grooves, whereas the NSC on the nonpatterned surfaces did not exhibit a particular bias in alignment. In vivo, the number of regenerated axons, the regenerated area, and the number of blood vessels were significantly higher in the NSC-preseeded conduit. Conclusion: Modification of the chitosan matrix by gold nanoparticles not only provides the mechanical strength but also affects the cellular response. The preliminary in vivo data demonstrated that the biodegradable micropatterned conduits preseeded with NSC provided a combination of physical and biological guidance cues for regenerating axons at the cellular level and offered a better alternative for repairing sciatic nerve transactions. © 2008 Elsevier Inc. All rights reserved.
Keywords:
Peripheral nerve regeneration; Micropatterned conduit; Chitosan; Gold
Abbreviations: ANOVA, 1-way analysis of variance; BDNF, brainderived neurotrophic factor; cDNA, complementary DNA; chi-Au, chitosangold; DAPI, 4,6-diamidino-2-phenylindole dihydrochloride; DMA, dynamic mechanical analysis; DMEM, Dulbecco modified Eagle medium; E, experimental; ECM, extracellular matrix; FBS, fetal bovine serum; GDNF, glial cell line–derived neurotrophic factor; GFP, green fluorescent protein; IT, intermediate toe spreading; N, normal; NGF, nerve growth factor; NSC, neural stem cells; PCR, polymerase chain reaction; PDMS, poly(dimethylsiloxane); PL, footprint length; SEM, scanning electron microscopy; SFI, sciatic functional index; Tg, transition temperature; TS, toe spreading. ⁎ Corresponding author. Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan, ROC. Tel.: +886 422840510 711; fax: +886 422854734. E-mail address: [email protected] (S. Hsu). 0090-3019/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.surneu.2008.01.057
1. Introduction Peripheral nerve injuries are common in clinical practice because of trauma. The regeneration of the injured nerve is slow and can result in complicated rehabilitation. In mature neurons, there is almost no cellular replication activity, although nerve has an intrinsic capacity of inducing regeneration. When a nerve defect or gap is longer, implantation of a graft is often necessary to bridge the proximal and distal nerve stumps for promoting nerve regeneration. The most widely used material to bridge a peripheral nerve defect is the autologous nerve. An attractive alternative to the autograft
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consists of bridging the 2 nerve stumps by a biodegradable conduit, which has the advantage of directing axonal growth properly [1,13,29,41]. To further improve nerve regeneration, the conduit could be loaded with biologically active compounds such as neuronal supporting cells and NGFs [3,16,40]. Schwann cells are closely associated with, and play key roles in, the development, maintenance, and regeneration of peripheral neurons [15]. After the injury, the orientation of Schwann cells may guide the regenerating axons. The conduits containing Schwann cells were shown to promote axonal growth and improve the nerve regeneration in vivo [19,38]. However, the immunogenicity and the source of Schwann cells were regarded as the limitations for clinical applications. With the growing applicability of NSC, most of the work has been focused in the central nervous system [37]. Recently, the neural progenitor cells have been transplanted; and their differentiation abilities have promoted the axonal regeneration through a nerve defect in the rat model [28]. The advantage of using NSC is that pluripotent stem cells may allow multiple differentiation paths, creating an environment that supports axon regeneration. The microenvironment of the conduit mimicking the biological situation is a vital issue in tissue regeneration. Chitosan, a natural polysaccharide, has structural characteristics similar to glycosaminoglycans and seems to mimic the functional behavior of glycosaminoglycans. The previous research has also revealed that chitosan has bacteriostatic [10,42] and hemostatic [26] properties. Furthermore, chitosan is enzymatically degraded to absorbable oligosaccharides. Chitosan, however, has low mechanical strength under physiological conditions [25], which has limited their use as nerve guidance channels for clinical applications [43]. Because gold has high mechanical strength with good biocompatibility, the mixture of chitosan with gold nanoparticles, or the chi-Au nanocomposites, might bring about advantages to the composite biomaterial [23] for nerve conduits. Apart from the biomaterial of the conduit, a promising strategy providing instructive environments involves some special microarchitectures that allow structural support for axonal regrowth and affect cellular orientation and the presentation of ECM proteins to the cells [30]. The micropatterned inner surface is expected to direct the cell growth during the nerve reconstruction and subsequently to induce neuron alignment and effective repair. This study was designed as a preliminary study to evaluate the effect of NSC-seeded tissue-engineered nerve conduit on nerve regeneration. In vitro, the effects of chiAu composite materials on cell proliferation, gene expression, and directing cell alignment were evaluated. In vivo, the feasibility for peripheral nerve implantation was explored. The conduit was implanted to bridge the rat sciatic nerve across a 10-mm–long defect. The repair outcome was investigated 6 weeks after the surgery. It was expected that the multicomponent, biodegradable conduit
of micropatterned architecture seeded with NSC might direct cell replacement, facilitate regeneration, and guide repair to create a more physiologically relevant structure. 2. Materials and methods 2.1. Preparation of chi-Au nanocomposite materials One percent chitosan (Sigma, St Louis, Mo) solution was dissolved in 0.5 mol/L acetic acid for 12 hours at room temperature. The gold nanoparticles in solution were supplied by Global NanoTech, Taipei, Taiwan. The solution contained only pure-gold fine particles (5 nm) in water (50 ppm/mL). After filtering the chitosan solution, a certain amount of gold nanoparticle solution was added; so the final concentration in the polymer was 25, 50, or 100 ppm. To make the substrates for biological tests, 100 μL of the chi-Au solution was coated onto coverslips (15-mm diameter; Matsunami, Osaka, Japan) and air-dried for 48 hours. 2.2. DMA of chi-Au materials Changes in dynamic properties of chi-Au materials were examined by DMA using a TA Instruments DMA 2980 (New Castle, DE). To prepare the samples for mechanical testing, chi-Au solution was cast on a glass mold and peeled after being dried. Sample strips with a geometry of about 30 mm long × 5.0 mm wide × 0.07 mm thick were run in tension at 1 Hz and an amplitude of 10 μm. Samples were mounted in the DMA at room temperature and then run from 30°C to 220°C at a ramp rate of 5°C/minute. The dynamic modulus was expressed as storage modulus (in megapascals), and the tendency to dissipate energy was indicated by the parameter tan δ. 2.3. Cell maintenance Rat glioma cell line C6 (BCRC-60046; Bioresources Collection and Research Center, Hsinchu, Taiwan) and murine NSC were applied for in vitro tests. The NSC were isolated from 2-month–old mouse brain and were transfected with F1B-GFP (US Patents 7045678 and 6984518, and pending US Patent 11/375889) [6]. The stable cell lines were obtained by selection with 200 μg/mL Geneticin (11811; Gibco, Los Angeles, CA). The Geneticinresistant NSC were pooled and expanded. The GFP-positive mouse brain cells were enriched using fluorescenceactivated cell sorter (FACS Aria; BD Biosciences, Palo Alto, Calif) repeatedly until greater than 95% purity was reached. The C6 cells were cultured in DMEM supplemented with 44 mmol/L NaHCO3 (Sigma), 10% FBS, streptomycin-penicillin (50 U/mL), and 1% sodium pyruvate (59203-100M; JRH, Lenexa, KS). DMEM/F12 (11330, Gibco, Carlsbad, CA) supplemented with 10% FBS, 400 μg/mL Geneticin, 20 ng/mL human FGF1, and streptomycin-penicillin (100 U/mL) was used for NSC culture medium. Cultures were incubated in a humidified incubator with 5% CO2 at 37°C. The medium was refreshed twice weekly.
Y.-L. Lin et al. / Surgical Neurology 70 (2008) S1:9–S1:18 Table 1 Dynamic mechanical properties of chi-Au
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2.7. Cell alignment
ppm
Storage modulus (MPa) (37°C)
Tg (fsC)
0 25 50 100
2830 2787 3456 3593
161.60 160.86 160.43 162.88
2.4. Assessment of cell proliferation Before seeding the cells, the composite substrates on coverslips were soaked in 70% ethanol, rinsed with PBS, and then placed into 24-well culture plates. To assess the effect of gold nanoparticles at different concentrations, cells at a density of 104 per well for C6 and 5 × 104 per well for NSC were seeded. The cell number was counted at 24, 48, and 72 hours by the hemocytometer. 2.5. Gene expression of neurotrophic factors Total RNA was extracted from C6 or NSC grown on chiAu nanocomposites after culture for 72 hours. Trizol reagent (15596-018; Invitrogen, Carlsbad, CA) was added after the cells were trypsinized. Five micrograms of total RNA was reverse-transcribed with the first-strand cDNA synthesis kit (Fermentas, St Leon-Rot, Germany) following the manufacturer's instructions. Polymerase chain reaction was performed in a 25-μL reaction volume containing 1 μL of the cDNA, 0.5 μL of 10 μmol/L each primer (Table 2), and 5 μL of 5× PCR Master Mix buffer (Gene Mark, Tainan, Taiwan). Polymerase chain reaction was carried out in a GeneAmp PCR system 2700 thermal cycler (ABI, Foster CA, USA). The cycling parameters of cDNA were 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension at 72°C for 7 minutes. β-Actin was used to confirm fidelity of the PCR reaction and as an internal control for semiquantitative analysis. The amplified products were analyzed by electrophoresis on 1.5% agarose-TAE (10 mmol/L Tris [pH 7.5], 5.7% glacial acetic acid, and 1 mmol/L EDTA) gels and visualized by ethidium bromide staining. 2.6. Fabrication of microgrooves on chi-Au Conventional photolithographic techniques were used to fabricate silicon wafers with the desired micropatterns that were then transferred to the PDMS submaster by casting PDMS on the silicon wafer [21]. To transfer microgrooves on the chi-Au substrates, the solution containing 50 ppm gold nanoparticles was cast on the PDMS submasters. After being air-dried, the substrates were then immersed in alcohol and detached from the PDMS submasters. To study the physical guidance of NSC on micropatterned chi-Au substrates, the pattern dimensions were 20/20/3 μm (groove width/spacing/depth), based on the previous studies involving the optimal alignment of Schwann cells [21].
To evaluate cell alignment, the cultured NSC (3 × 104 cells per square centimeter) on the substrates were examined at 24 and 72 hours after culture. After staining with methylene blue, the orientation of the NSC on the substrates was measured quantitatively using an image analysis system (Image-Pro Lite; Media Cybernetics, Silver Spring, MD) [21]. Those cells with an orientation angle between −10° and 10° were identified as the aligned cells, and the proportion of the aligned cells in the population was calculated. Control data were taken from measurements made on NSC on nonpatterned chi-Au substrates. 2.8. Cytoskeleton observation To evaluate the cytoskeleton of NSC on the substrates, cells cultured after 24 and 72 hours were fixed in 4% paraformaldehyde. After several rinses with PBS, 500 μL of 0.5% Triton X-100 (t-octylphenoxypolyethoxyethanol; Sigma, T9284) was added for 10 minutes. The samples were rinsed, and 300 μL of 0.03% phalloidin (P2141, Sigma) was added in the dark for 30 minutes. To stain the cell nuclei, cells were rinsed; and 300 μL of 0.01% DAPI (D9542, Sigma) was added in the dark for 30 minutes. Samples were rinsed and then mounted onto microscope slides. The cytoskeleton was observed by the fluorescence microscope (Eclipse 80i; Nikon, Tokyo, Japan). 2.9. Conduits for implantation The micropatterned substrates were rolled into conduits by a 1.5-mm–diameter glass mandrel and adhered by a small amount of chitosan solution. Conduits with 1.95-mm internal diameter and 2.22-mm external diameter were sectioned into 12-mm–long segments. The suspension of GFP-positive mouse NSC (5 × 104/mL) was injected into the conduit, and then the conduit was sealed immediately at both ends and rotated at 1 rpm for 72 hours before implantation. The structure of microgrooved conduit (without cell seeding) was imaged using SEM. After mounting, conduits were gold-coated using a Hitachi coating unit IB-2 coater. Coated samples were examined by a scanning electron microscope (S-3000N, Hitachi, Tokyo, Japan).
Table 2 Primers used in this study Gene
Sequence
Size (base pairs)
BDNF
F–GAC TCT GGA GAG CGT GAA T R–CCA CTC GCT AAT ACT GTC AC F–ACC TCT TCG GAC ACT CTG GA R–GTC CGT GGC TGT GGT CTT AT F–CCC GAA GAT TAT CCT GAC CA R–TAG CCC AAA CCC AAG TCA GT F–TCC TGT GGC ATC CAC GAA ACT R–GGA GCA ATG ATC CTG ATC TTC
325
NGF GDNF β-Actin
168 242 185
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2.10. Animal implantation of microgrooved nerve conduits Nine male Sprague-Dawley rats weighing 250 to 300 g were used for the preliminary animal test (n = 3 for each group). The 2 experimental groups received the grooved surface conduits seeded with and without NSC, respectively. The other group received reversed autografts and served as controls. The animals were housed in standard laboratory cages under temperature-controlled conditions at 24°C ± 1°C and 45% humidity with 12-hour light cycles, with free access to standard rat chow and water. All procedures followed the guidelines for the animal care, and the Ethical Committee of National Chung Hsing University had approved the experiment. Animals were anesthetized with Citosol (6 mg/100g intraperitoneally (Shinlin Sinseng Pharmaceutical Co, Taipei, Taiwan)), and the left hind legs were used to perform the surgery. A skin incision from the left knee to the hip was made to expose the underlying muscles, which were then retracted to reveal the sciatic nerve. The microgrooved conduit, with or without GFP-positive mouse NSC, or the reversed autograft was interposed into the 10-mm sciatic nerve defect. The nerve stumps were anchored inside to a length of approximately 1 mm of the conduit by 7-0 nylon microsutures. The operation site was sutured in layers using 3-0 Dexon (Unik Surgical Sutures MFG Co, Taipei, Taiwan). Six weeks after implantation, the rats implanted with conduits or controls were euthanized.
ANOVA. Bonferroni post hoc analysis was used for multiple group comparisons to determine the statistical significance of the results. P b .05 was considered as statistically significant. 3. Results A technique for fabricating microgrooves on chi-Au substrates was developed, and the effects on the in vitro behaviors of NSC and C6 and the in vivo implantation were investigated. 3.1. DMA of chi-Au materials The dynamic mechanical properties of chi-Au were investigated using DMA. From the overlay of DMA scans on individual chi-Au concentration, there was a concentration-dependent increase in the dynamic modulus except for the values of 25 ppm before 82°C, which were slightly lower than those of 0 ppm. At 37°C, the dynamic modulus was 2787 MPa at 25 ppm, whereas it was 2830 MPa at 0 ppm. The overall patterns were similar at 0 and 25 ppm. At 50 ppm, there was a higher dynamic modulus compared with 0 and 25 ppm. Chitosan with 100 ppm gold, as expected, was a stronger material than others, as could be seen by the
2.11. Functional assessment of nerve regeneration Walking track analysis was performed after 3 and 6 weeks. For the calculation of SFI, the following footprint parameters were measured: the distance to the PL, TS, and IT. Data were collected for both the N and the E hind legs. The SFI was calculated based on the formula used by Bain et al [2] expressed below. An index of zero reflected normal function, and −100 theoretically represented complete loss of function. SFI ¼ 38:3½ð EPL NPLÞ=NPL þ 109:5½ð ETS NTS Þ=NTS þ 13:3½ð EIT NIT Þ=NIT 8:8 2.12. Histologic analysis The tissues were harvested, fixed overnight in 4% paraformaldehyde, stained with 1% osmium tetroxide (Polysciences, Warrington, Pa), embedded in paraffin, thin-sectioned (4 μm), and stained with toluidine blue. The number of myelinated axons and the regenerated area were determined using an image analysis system (ImagePro Lite, Media Cybernetics). The number of blood vessels was also counted. 2.13. Statistical analysis Data from the experiments were expressed as mean ± standard deviation. Statistical differences were analyzed by
Fig. 1. Dynamic mechanical analysis of chi-Au. Chitosan composites containing 0 (control), 25, 50, and 100 ppm of gold were measured. A: Storage modulus; B: tan δ. Values were measured from 30°C to 220°C.
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highest dynamic modulus (Fig. 1A, Table 1). Chitosan-gold 0 and 25 ppm had higher tan δ than chi-Au 50 and 100 ppm. Glass Tg indicated by the peak of tan δ was slightly reduced at 50 ppm (Fig. 1B, Table 1). 3.2. Effect of chi-Au substrates on cell proliferation and gene expression The cytocompatibility of different chi-Au composites was tested in vitro by analyzing cell proliferation and gene expression of both C6 cells and NSC. Gold nanoparticles had a stimulatory effect on cell proliferation that was concentration dependent (Fig. 2A, B). For NSC, cell proliferation was significantly increased, compared with the control (0 ppm), at the concentration of 25 or 50 ppm. However, when the gold concentration was increased to 100 ppm, cell proliferation was similar to the control. Results showed that the nanocomposites containing 25 or 50 ppm were superior to those at other concentrations in the proliferation of both C6 and NSC. Results from the semiquantitative analysis of neurotrophic gene expression for C6 and NSC cultured on various chi-Au substrates are summarized in Fig. 2. The effects of
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gold on gene expression of C6 and NSC were different. In general, higher levels of both BDNF and GDNF genes were expressed than the NGF gene expression. Comparing chi-Au of different gold concentrations, higher NGF expression in C6 was detected at 50 ppm of gold, whereas the amount of BDNF and GDNF increased with the concentration of gold (Fig. 2C). On the contrary, there was no significant concentration dependence in the gene expression of NGF, BDNF, and GDNF in the NSC (Fig. 2D). For NGF and GDNF, the gene expression on chitosan alone was slightly higher than that at other concentrations; but the difference did not reach significance. For BDNF, the gene expression on chi-Au of all concentrations was similar. The only significance was that the GDNF gene expression at 25 ppm of gold was lower than that on chitosan alone. From the results of cell proliferation and gene expression, the concentration of gold at 25 and 50 ppm showed better cell compatibilities than at other concentrations. Although chi-Au 25 ppm showed better cell proliferation for the NSC, the modulus was weak and even lower than that of chitosan alone at 37°C. Considering both cell compatibilities and mechanical strength for the conduit, chi-Au 50 ppm would be a better choice for in vivo environment.
Fig. 2. Effects of chi-Au on proliferation and gene expression of C6 and NSC. Cells were grown on biomaterials in 24-well plates, and the effects of gold at 0 (control), 25, 50, and 100 ppm were compared. Values are expressed as mean; error bars represent SD; a indicates significantly (P b .05) different from the value at 0 ppm. A and B: Cell proliferation. Data were measured at 24, 48, and 72 hours. C and D: Gene expression. Data were measured at 72 hours.
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Fig. 3. The orientation of NSC on the microgrooved substrates. A: Actin and DAPI staining for NSC on the nonpatterned substrate (a) and the grooved chi-Au substrate (b). B: NSC alignment (percentage of cells aligned) at 24 and 72 hours. Values are expressed as mean ± SD. Scale bar: 20 μm.
3.3. Effect of guidance cues on the alignment of NSC The morphology and the alignment of the NSC on the micropatterned substrates are shown in Fig. 3. The growth and orientation of NSC on the micropatterned surface were influenced by the 3-dimensional topography of the substrate.
Most of the NSC appeared multipolar on the nonpatterned substrates (panel A, a) and bipolar on the microgrooved substrates (panel A, b). The NSC on the nonpatterned surfaces did not exhibit a particular bias in alignment, but extended processes in a radial fashion. However, the morphology of NSC on the micropatterned surfaces had elongated processes oriented parallel to the grooves of the patterned substrate. The degree of cell orientation on the micropatterned substrates after 24 and 72 hours of culturing was displayed in panel B. The results showed that, after 24 hours, 78.3% of the NSC were aligned on the chitosan substrate and 83.7% of the NSC were aligned on the chi-Au substrate (P b .05). After 72 hours in culture, over 90% of cells were aligned in both substrates; and the alignment on chi-Au
Table 3 The sciatic function index of the 2 experimental groups
At 3 wk At 6 wk Fig. 4. The structure of the inner surface for the microgrooved conduits. The image was obtained by SEM.
Chi-Au
Chi-Au + NSC
−72.62 ± 2.79 −63.39 ± 10.10 ⁎
−69.32 ± 4.16 −48.74 ± 12.80⁎,⁎⁎
The values represent means ± standard deviations. ⁎ Significantly (P b .05) greater than at 3 weeks. ⁎⁎ Significantly (P b .05) greater than chi-Au.
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Fig. 5. Light micrographs of the regenerated nerve. The cross sections were through the midpoint of the nerve conduit. A: Chitosan-Au microgrooved conduit. B: Chitosan-Au microgrooved conduit preseeded with NSC. C: Autograft. D: Normal sciatic nerve.
substrate was slightly better than on chitosan alone (not significant) (Fig. 3B). 3.4. Morphology of the conduit The SEM observations clearly showed that the microgrooves on the lumen surface of the conduits remained visible after the rolling procedure (Fig. 4). The featured dimensions were also well kept. 3.5. Functional assessment of nerve regeneration The SFI values are listed in Table 3. The values at 3 weeks after operation were similar in both chi-Au groups with or without NSC. At the sixth week, the values were significantly greater than those at the third week for both groups. Especially, the SFI value in the NSC-seeded conduits was significantly greater than the value in the nonseeded chiAu conduits. 3.6. Retrieving in vivo implantation and nerve regeneration No animals died after 6 weeks of implantation process. Macroscopically, the implant appeared as a discreetly enlarged area integrated in the sciatic nerve. The conduit was not yet degraded. The nerve was successfully connected; a white tubular substance through the conduit was observed. The histologic sections showed that the number of myelinated axons and the regenerated area were significantly greater in the NSC-preseeded conduit than in the conduit alone (Fig. 5 and Table 4). Angiogenesis was remarkable around the nerve fibers in the NSC-preseeded conduit.
4. Discussion When the nerve injury is severe and the nerve function is lost, the portion of the distal stump dies and degenerates; the proximal stump may be able to regenerate and reestablish the nerve function. However, it is difficult for the growth cone of the regenerating axon to reconstruct larger nerve gaps. To better direct the repair after peripheral nerve injury, a graft implant between the proximal and the distal nerve stumps as a guide for the regeneration axon is required. To develop an optimal artificial nerve graft for clinical applications, mechanical support, functional cells, growth factors, and ECM of the conduit should be considered. In this study, it was focused on the effects of nerve regeneration by the incorporation of these factors that attempted to modify the microenvironment of neuronal physiology in vivo. The interaction of the implanted cells and the biomaterial is complicated, and it depends on the cell type and the characteristics of the biomaterial. Recently, various biodegradable materials have been used as scaffolds for nerve regeneration, including polyglycolide, polylactide, poly(ɛTable 4 Data of histologic analyses at the midconduit from the 2 experimental groups
2
Regenerated area (mm ) No. of myelinated axons No. of blood vessels
Chi-Au
Chi-Au + NSC
0.298 ± 0.148 453 ± 29 18 ± 5
1.030 ± 0.424 ⁎ 1938 ± 287 ⁎ 55 ± 11 ⁎
The values represent means ± standard deviations. ⁎ Significantly (P b .05) greater than chi-Au.
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caprolactone), and chitosan [8,39]. Our previous study showed that gene expression for neurotrophic factors in Schwann cells was up-regulated on chitosan substrate compared with that on polylactide [22]. Chitosan also showed a good affinity for nerve cells and promoted the survival and neurite outgrowth of nerve cells in vitro [14,17], which suggested that chitosan might be applicable as a scaffold for axonal regeneration in peripheral nerves. Because the mechanical strength of the conduit under physiological conditions is very crucial for the guidance and supporting of nerve regeneration, the mechanical property of chi-Au nanocomposites was tested. From the results of DMA, gold played an important role in the mechanical property of the chitosan material. The mechanical strength increased with the amount of gold in the chitosan film except at 25 ppm, where the mechanical strength was even lower than that in chitosan alone. With increasing temperature, the dynamic modulus of chi-Au 25 ppm became higher than that of the original chitosan. However, considering the in vivo environment (37°C), chi-Au 25 ppm would not be a good option. The tan δ patterns also showed concentration dependence of gold, and it was found that chi-Au at lower gold concentrations was softer (larger tan δ). The sharp transitions might represent changes in crystalline domains within the chitosan matrix. Although the tan δ Tg was close among chi-Au, as would be expected because the main component of all nanocomposites contained chitosan in their makeup, each nanocomposite had its own transition curve and inherent characteristics unique to its material morphology. The results might suggest that the chitosan matrix and hence its DMA signature have been significantly modified by gold, which might facilitate nucleophilic attack, via solvation, of the polymer backbone. To determine the suitable gold concentration of the chitosan matrix, this study was first conducted by culturing C6 and NSC in vitro, comparing the biomaterial properties in different chi-Au nanocomposites and the behavior of both types of cells on the biomaterials. Because gold nanoparticles could change the microstructure of the biomaterial, it was possible that gold nanoparticles could modulate the cellular response through the physical features. Alterations in cell proliferation and gene expression could be detected as expected, depending on the amount of gold nanoparticles. There was concentration dependence of the gold nanoparticles on both types of cells. This indicated that different gold concentrations affected the behavior of both C6 and NSC in culture. This was in line with our previous findings for in vitro endothelial cell response to the polyurethane-gold nanocomposites [23]. There was a clear effect of amount of gold nanoparticles on the results of the present study. The overall patterns in cell proliferation and gene expression were similar for both C6 and NSC. Adding a certain amount of gold nanoparticles seemed to have a stimulating effect on cell proliferation and gene expression. However, if a higher amount of gold nanoparticles was used, the result was not much different from that of the simple
chitosan. In particular, our findings suggested that the stimulatory effects on cell proliferation and gene expression decreased with the amount of gold nanoparticles more than 50 ppm. The decreasing effect at higher gold concentrations was also shown in polyurethane-gold nanocomposites [23]. The results indicated that 25 and 50 ppm gave better cell performance for NSC, whereas 50 ppm led to better results in C6. Because 50 ppm of gold was better for both types of cells, this concentration was chosen to prepare the conduits for the in vivo implantation. The mechanism of gold nanoparticles on cells is not clear. There may be an effect through the interaction between cells and the nanofeatured surfaces. It has been reported that fibroblast response to surface nanoislands was enhanced [11]. Mediation of cell marker expression on leukocytes by nanostructured surfaces was observed [5]. Results from these studies might point out a possibility of modified cellular to tissue responses by the nanocomposite surfaces. This might also imply that cells are capable of detecting external surface changes by nanometric size. Higher density of nanoparticles in the surface on the other hand may interfere with cell growth. Nanotechnology holds great promise for many applications, including biomedical uses. Some in vitro studies showed that gold nanoparticles could be taken up by human cells, but there was no acute cytotoxicity [7,9]. Our in vitro data showed that there was no uptake of gold nanoparticles by NSC (data not shown). However, until now, very little is known regarding the in vivo toxicity especially for a longer term. In this study, we have not tracked the destiny of gold nanoparticles in vivo; but this has been planned. The preliminary in vivo results showed that the conduits preseeded with NSC were able to support regeneration of the sciatic nerve better than the empty conduits. The recovery degree of sciatic nerve function was accessed by measuring the SFI that provided a noninvasive and easily quantifiable method in the rat model [2]. As SFI compared the experimental and the normal prints, each animal acted as its own control. Our data showed that, at 3 weeks after operation, the function was still poor in both groups. At the sixth week, an increasing ability of walking became evident. A better and faster functional recovery was shown in the NSC-grafted conduits. In contrast, the functional recovery was in a slower progress in the chi-Au group. Quantitative histology demonstrated that the total number of myelinated axons was 4-fold higher in the conduits containing NSC. The total number of myelinated axons in the chi-Au conduits with and without NSC corresponded to approximately 25% and 6%, respectively, of the total number of myelinated axons present in the normal sciatic nerve [33]. More angiogenesis formation was also found in the NSCpreseeded group, which provided nutrient transport to the regenerating axons [32]. The degree of myelination may be related to the maturity of the axon, but may not measure the nerve function and the integration of central nervous system. The SFI gives general information of nerve regeneration and
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functional recovery because it represents the outcome of the repair process. To further understand the specific process during functional recovery, nerve conduction velocity will be considered in our next step because there is significant correlation between electrophysiological and morphologic parameters (ie, conduction velocity and axon diameters/ myelination) [12]. The GFP-positive NSC were shown to have the selfrenewal property and could differentiate into astrocytes, neurons, and oligodendrocytes in vitro (Chiu et al, unpublished data). Astrocytes have been shown to produce a variety of cytokines and neurotrophic factors, which stimulate the viability and proliferation of distinct cells [31]. Apparently, the inclusion of neuronal supporting cells is critical for axonal proliferation. On the contrary, the regeneration in empty conduits had poorer results, which might be due to the absence of aligned supporting cells within the conduit, leading to the early degeneration of the elongated axons [4]. It has been shown that Schwann-like supportive cells were derived in the nerve graft filled with the neuronal progenitor cells graft, but the number was too small for sciatic nerve regeneration by itself [28]. On the other hand, NSC might remain undifferentiated to contribute the trophic support rather than differentiate into functional cells [37]. Because the graft NSC played an important part of the regeneration, further investigation, for example, immunocytochemistry or cell prelabeling, will be performed to trace the fates of the grafted NSC and to clarify the associated mechanism. Neurotrophic factors play important roles in the maintenance and survival of peripheral neuronal cells. The supply of retrogradely transported neurotrophic factors was disrupted after peripheral nerve injury, leading to neuronal cell death and poor regeneration. Administration of exogenous neurotrophic factors after nerve injury has been shown to mimic the effect of target organ–derived trophic factors on neuronal cells. Seeding the conduits with NSC before implantation may provide a living, interactive source of neurotrophic factors that increases the chance of neuronal outgrowth and enhances peripheral nerve regeneration. Our results showed that the NGF, BDNF, and GDNF genes were expressed by NSC; and these factors might promote nerve regeneration [16,18,24]. NGF and BDNF act directly to promote survival and indirectly on regenerated axons by activating nonneuronal cells, which might release growthpromoting molecules and enhance the regeneration of all neuronal populations [20,36]. GDNF and BDNF support motoneuron survival in vitro, prevent axotomy-induced motoneuron degeneration in vivo [34,44], have a strong effect on motor fiber regeneration [16], and improve the conduction velocity of motoneurons after regeneration [27]. Therefore, integrating the multiple stimuli to direct the secretion of neurotrophic factors by NSC offered opportunities to mimic the natural in vivo environment. Moreover, the surface of the implant, which was microgrooved, provided more than just mechanical support
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but also the positive influence on cell alignment. Our results demonstrated that the patterned substrate directed the NSC to extend processes along the inside of the groove and influenced the cells to spread in the groove direction (Fig. 3). The results were in line with the finding that Schwann cells could be successfully aligned by the microgrooves [6]. In vivo, the presence of the longitudinally aligned NSC appeared to effectively promote the oriented growth of the damaged peripheral nerve. Special microarchitecture allows the optimal structural support for axonal regrowth and affects cellular orientations. It was possible that the patterned substrate increased NSC proliferation, leading to the enhanced neuronal differentiation and neurogenesis [30,35]. Further evaluation is yet needed to analyze the fates of grafted NSC and clarify the mechanism of nerve regeneration as mentioned. Providing a grooved support enables the temporal guidance of the complex processes of tissue formation and regeneration. In conclusion, the results in this study demonstrated a strategy for enhancing adult NSC proliferation and alignment that had important applications in guided peripheral nerve regeneration. In vitro results showed that chitosan was biocompatible to nerve cells and could be used as the conduit material to facilitate nerve cell proliferation and growth. Modification of the chitosan matrix by gold nanoparticles not only provided the mechanical strength but also affected the cellular response. Seeding the micropatterned conduits with NSC before implantation may provide a living, interactive source of neurotrophic factors. Finally, the combination of physical and biological cues enables optimistic potential for improving the rate of functional recovery. Although the results of the present study are still far from conclusive for clinical applications yet, they certainly warrant further study for bridging longer gap and investigation on retrograde labeling, cell fate tracing, and motor and sensory nerve function recovery to give a more in-depth view. Acknowledgments This work was supported by grants from Ministry of Education, National Science Council, and National Health Research Institutes. The authors would like to thank Global NanoTech for the supply of gold nanoparticles and Dr Han Chang (Chung Shan Medical University) for the help in analyzing the histologic sections. References [1] Archibald SJ, Krarup C, Shefner J, Li ST, Madison RD. A collagenbased nerve guide conduit for peripheral nerve repair: an electrophysiological study of nerve regeneration in rodents and nonhuman primates. J Comp Neurol 1991;306:685-96. [2] Bain JR, Mackinnon SE, Hunter DA. Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat. Plast Reconstr Surg 1989;83:129-38. [3] Bryan DJ, Wang KK, Chakalis-Haley DP. Effect of Schwann cells in the enhancement of peripheral-nerve regeneration. J Reconstr Microsurg 1996;12:439-46.
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