Bio-Medical Materials and Engineering 23 (2013) 545–554 DOI 10.3233/BME-130775 IOS Press
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Biocompatibility evaluation of electrospun silk fibroin nanofibrous mats with primarily cultured rat hippocampal neurons Yahong Zhao a , Weijia Zhao a , Shu Yu a , Yibing Guo b , Xiaosong Gu a and Yumin Yang a,∗ a b
Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, China Surgical Comprehensive Laboratory, Affiliated Hospital of Nantong University, Nantong, China
Received 18 August 2011 Accepted 3 June 2013 Abstract. In this study, electrospinning was performed to fabricate silk fibroin (SF) nanofibrous mats, which were used as substrates for in vitro culture of rat hippocampal neurons. The light and electron micrographs demonstrated that the electrospun SF nanofibrous mat supported the survival and growth of the attached hippocampal neurons. MTT assay and immunocytochemistry in couple with Western blot analysis respectively indicated there was no significant difference in both the cell viability and expression levels of some proteins, including GAP-43, MAP-2, NF, and β-tubulin, between hippocampal neurons cultured in the electrospun SF nanofibrous mat extract and in plain neuronal medium. Our results indicated that electrospun SF nanofibrous mats were biocompatible to primary culture of hippocampal neurons without cytotoxic effects on the cell phenotype and functions, raising a potential possibility of using these mats for CNS therapeutic applications. Keywords: Biomaterials, nanofiber, biocompatibility, silk fibroin, hippocampal neurons
1. Introduction Since the adult mammalian central nervous system (CNS) fails to spontaneously regenerate after injuries and degeneration, developing CNS therapy strategies has attracted research interests [1]. Recently, cell implantation and controlled release of neutrophic factors are accepted as two promising approaches to aiding CNS regeneration. They often require the use of suitable biomaterial-based matrices as the carrier to present biochemical cues [2–4]. To use any new matrix, therefore, an investigation of its biocompatibility with CNS cells or tissues becomes an important prerequisite. With recent development of nanotechnology, fibrous matrices at nano-scale level are now becoming potential candidates for tissue engineering applications because they mimic the topography of natural extracellular matrix (ECM) [5]. Many novel manufacturing methods, including electrospinning, phase separation, and self-assembly, have been used for fabrication of such biofunctional nanofibrous matrices. Of which electrospun fibrous matrices have a high surface-area-to-volume ratio suitable for cell *
Address for correspondence: Yumin Yang, Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, JS 226001, China. Tel./Fax: +86 513 85511585; E-mail: [email protected]. 0959-2989/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved
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attachment and growth as well as a possibility of mimicking the ECM architecture favorable to tissue regeneration [6,7]. Silk fibroin (SF)-based biomaterials possess favorable physicochemical and biological properties [8–11], and have found increasing applications in biomedical fields [12–15]. More instriguingly, SF-based materials have been used for preparing nanofibrous matrices, for example via electrospining, and the resulting matrices have been applied in various areas of tissue engineering. However, they have not been extensively studied for nerve regeneration, especially CNS injury repair [5,16–20], in depth studies are thus needed to further examine the feasibility of SF-based matrices with the nanofibrous configuration for nerve repair applications. On the other hand, although the biocompatibility of SF-based materials with CNS cells have been reported [13], the issue of whether the matrices made up of SF-based biomaterials via electrospining fabrication could keep the neuroaffinity is still worthy to be explored. In this study, we aimed at investigating the possibility of electrospun SF nanofibrous mats used as a candidate matrix for CNS therapeutic applications. The primarily cultured hippocampal neurons of developing rat brains, which usually serve as a well-defined cell model [21], were cultured on the substrate consisting of electrospun SF nanofibrous mats for the in vitro examination of the biocompatibility of the substrate with CNS cells. The survival and growth characteristics of hippocampal neurons on electrospun SF nanofibrous mats were investigated by morphological observation, MTT analysis, immunocytochemistry and Western blot analysis.
2. Materials and methods 2.1. Preparation of electrospun SF nanofibrous mats Raw silk fibers (from Bombyx mori cocoons) were bought from Xinyuan sericulture company, Hai’an, Jiangsu, China. The SF aqueous solution was prepared as previously described [15], and then spread on stainless steel dishes to generate the air-dried regenerated SF membranes which were further dissolved in 98% (wt/wt) formic acid to obtain 13% (wt/wt) SF spinning solution. A home-made electrospinning setup, used in this study, was made up of a high voltage supplier, a capillary needle anode, and a grounded collector cathode. A high electric potential (20 kV) was applied to the tip of the needle anode (ID 0.9 mm), into which the droplet of SF spinning solution was loaded. The electrospun nanofibrous mat was generated on the stainless steel meshes. The distance between the needle tip and the steel collector ranged from 7 to 13 cm. A constant volume flow rate (0.3 ml/h) was maintained by a syringe pump, which kept the SF spinning solution in the needle. After electrospun SF nanofibrous mats were removed from the stainless steel meshes, they were inserted into 100% alcohol for 10 min to induce conformational transition [22], followed by wash with distilled water at 37◦ C for 72 h to remove residual formic acid. The treated mats had to be sterilized with a hyperbaric method, and then equilibrated in DMEM culture medium (Gibco) for 30 min prior to use. 2.2. Characterization of electrospun SF mats For chemical characterization, the mats were ground, and pelleted with dried KBr, followed by analysis with a model Nexus 870 Fourier transform infrared (FT-IR) spectrophotometer (Nicolet Instruments Co., Madison, WI) at a range of 400–4000 cm−1 .
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2.3. Cell culture and treatment Sprague–Dawley embryonic rats were obtained from the experimental animal center of Nantong University. All experimental procedures involving animals were conducted as per Institutional Animal Care guidelines and approved ethically by the administration committee of experimental animals, Jiangsu Province, China. The rat hippocampal neurons were isolated as described previously [23] with minor modifications. After E 18 embryonic rats were sacrificed by cervical dislocation under anesthesia, their brains were quickly removed and the hippocampi were harvested on a cold stage. The procured hippocampal tissues were mechanically and enzymatically dissociated into a single-cell suspension, which was then plated onto poly-lysine-coated plates at a density of 1×105 per cm2 in DMEM supplemented with 10% F12 and 10% FBS (Gibco, Grand Island, NY) for 4 h. The medium was replaced by neuronal culture medium, and cells were incubated at 37◦ C in a humidified atmosphere of 95% air and 5% CO2 for 7–8 days, the time required for maturation of hippocampal neurons. Half of the culture medium was replaced every 2 days. The glial content of cultured cells was measured to be only 0.5% of the total cell population. Primarily cultured hippocampal neurons were seeded onto the poly-L-lysine (Sigma) coated coverslips, or the substrate made up of electrospun SF nanofibrous mats, which had been placed onto a 24-well culture plate, followed by soaking in plain neuronal culture medium. Another portion of primarily cultured hippocampal neurons was randomized into two groups for treatment with different mediums that were plain neuronal culture medium (positive control), and electrospun SF nanofibrous mats extract, respectively. 2.4. Light and electron microscopy After rat hippocampal neurons were cultured for designated times, the cell growth was observed under an inverted microscope (Olympus, Tokyo, Japan), and photographs were taken. In order to determine the average and total length of neurites for the hippocampal neurons cultured on the different substrates for 5 days, five fields were randomly selected for measurements of each photograph with Q550 IW image analysis system (Leica Imaging Systems Ltd., Cambridge, England). All measurements were performed in triplicate. After 3- or 7-day culture on the poly-lysine-coated coverslips, or the substrate made up of electrospun SF nanofibrous mats, the hippocampal neurons were washed twice with phosphate buffer saline (PBS, pH 7.2) and fixed in 4% glutaraldehyde. They were then post-fixed with 1% OsO4 , dehydrated stepwise in increasing concentrations of ethanol, and dried in a critical point drier (Hitachi, Tokyo, Japan), followed by coating with gold in a JFC-1100 unit (Jeol Inc., Japan) and observation under a scanning electron microscope (JEM-T300, Jeol Inc., Japan). 2.5. MTT assay After incubation in two different extract fluid, the viability of hippocampal neurons was assessed by MTT assay as previously described [24,25]. 2.6. Immunocytochemistry After 7-day incubation, the samples were fixed and subjected to immunocytochemistry. Goat anti-growth associated protein-43 (GAP-43, 1:200 dilution, Santa Cruz, CA), mouse monoclonal antimicrotubule-associated protein 2 (MAP2, 1:1,000 dilution, Sigma), rabbit anti-neurofilament (NF)
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200 IgG fraction of antiserum (1:200 dilution, Sigma), and rabbit anti β-tubulin III (1:50 dilution, Sigma), as primary antibody, and Cy3-labeled goat antirabbit IgG (1:200 dilution, Santa Cruz), FITClabeled donkey anti-goat IgG (1:200 dilution, Santa Cruz) and TRITC-labeled donkey anti-mouse IgG (1:200 dilution, Santa Cruz), as secondary antibody, were used. The samples were also stained with 5 µg/ml Hoeechst 33342 (Sigma). Finally, the samples were observed under a confocal laser scanning microscope (TCS SP2, Leica Microsystems, Germany). 2.7. Western blot analysis After 12, 24, 48 or 72 h treatment in two different mediums, the hippocampal neurons were analyzed for protein expression levels of GAP-43 and β-tubulin III by using Western blotting as described previously [13]. In brief, the hippocampal neurons were washed with PBS and lysed with lysis buffer containing protease inhibitors (Promega, Madison, WI) at designated times. Protein concentration was detected by BCA method (calibrated on bovine serum albumin) to maintain the same loads. Protein extracts were heat denatured at 100◦ C for 10 min, electrophoretically separated on a 10% SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat dry milk in TBST buffer (50 mM Tris–HCl, 100 mM NaCl, and 0.1% Tween-20, pH 7.4) and incubated with a 1:500 dilution of goat anti-GAP43 polyclonal antibody and 1:1,500 dilution of rabbit anti β-tubulin III antibody in 5% non-fat dry milk in TBST buffer at 4◦ C overnight. The membranes were washed with TBST buffer (5 min × 3), and further incubated with a 1:5,000 dilution of donkey anti-goat IgG and 1:10,000 dilution of donkey anti-rabbit IgG at room temperature for 2 h. After the membrane was washed, the image was scanned with Odyssey (LI-COR, Lincoln, NE), and the data of optical density were analyzed using PDQuest 7.2.0 software (Bio-Rad). GAPDH (1: 500) was used as an internal control. 2.8. Statistical analysis The data were expressed as means ± SD, and analyzed by one-way ANOVA. Statistical significance was accepted at the 0.05 confidence level. 3. Results and discussion 3.1. Morphological observation SEM showed that the electrospun SF mat was composed of a large number of randomly oriented fibers, and the diameter of most fibers ranged from 200 to 800 nm, although a few fibers were over 1 µm in diameter (Fig. 1(A)). Figure 1(B) provides the comparison of FT-IR spectra among two SF-based matrices. The three FT-IR curves (a, b) are similar to each other in shape, confirming an identical chemical composition for them. However, slight differences in the peak wave number are noted for two FT-IR curves, due to conformation transitions that take place during the fabrication process of electrospun SF mats. 3.2. Hippocampal neuron cell affinity of electrospun SF nanofibrous mats As revealed by light microscopy for visualizing the cell growth of hippocampal neurons cultured on the poly-lysine-coated coverslips, or the substrate made up of electrospun SF nanofibrous mats. Hippocampal neurons were found to adhere to electrospun SF nanofibrous mats immediately after inocu-
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Fig. 1. Nanofiber diameter distribution (A) of electrospun SF mats and the FT-IR spectra (B) of air-dried SF membranes (a), and electrospun SF mats (b).
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(B)
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Fig. 2. Light (A), (B) and scanning electron (C), (D) micrographs of hippocampal neurons cultured on electrospun SF nanofibrous mats for 3 days (A), (C) or 7 days (B), (D), respectively. Scale bar: 20 µm. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-130775.)
lation, and the cells showed a spherical shape with smooth surface and obvious refraction. After 3-day culture, the neurites of hippocampal neurons adhered to and extended along the substrate (Fig. 2(A)), and after 7-day culture the cells with long and dense neurites were found to form neural networks (Fig. 2(B)).
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Fig. 3. The total length of neurites for hippocampalneurons cultured on Pll-coated coverslips and the substrate made up of electrospun SF nanofibrous mats for 5 days. No significant differences in length was found between two different substrates (P > 0.05).
This result was further evidenced by the comparisons in the length of neuritis (Fig. 3) for hippocampal neurons cultured on two substrates without significant difference detected. Visual inspection under scanning electron microscopy further revealed that after 3-day culture on the substrate made up of electrospun SF nanofibrous mats, a majority of hippocampal neurons adherent to electrospun SF nanofibrous mats were oval- or spindle-shaped, and they connected to each other via extending neuritis (Fig. 2(C)); and after 7-day culture a dense array of neurons connected to each other via their neurites, the cells with long and dense axons and dendrites took compact arrangements, and they interweaved mutually to form neural networks (Fig. 2(D)). Also interestingly, the above-mentioned morphological features were close to those for hippocampal neurons cultured under normal conditions (data not shown). Taken together, histological observations collectively suggest that primarily cultured hippocampal neurons were able to adhere to and grow on the electrospun SF nanofibrous mats substrate, and the above observations are comparable with those for hippocampal neurons that were cultured on the poly-lysine coated coverslips, few visible differences were detected between them, despite geometric divergence of the different substrates. 3.3. Immunocytochemical observation of hippocampal neurons onto electrospun SF nanofibrous mats After 7-day culture on the electrospun SF nanofibrous mats-based substrates, the hippocampal neurons underwent immunocytochemistry with antibodies against GAP-43, MAP2, NF200 and β-tubulin III, respectively, because the expression of these four important neuronal marker proteins presents an effective standard for biocompatibility evaluation of electrospun SF nanofibrous mats-based substrates. GAP-43 and NF function as specific neuronal markers,and GAP-43 is a neuron-specic phospho protein which promotes survival and maintenance of function of neurons [26–28]. MAP2, as the major microtubule associated protein of brain tissue, co-localized and potentially interacts with Gap-43, and it promotes microtubule assembly and forms side-arms on microtubules [29]. As was shown by GAP43 (green) and MAP2 (red) double-labeled staining, the cells (double-stained) were noted to expand on electrospun SF nanofibrous mats fibers (Auto-fluorescence, blue), while axons (stained by GAP-43) and dendrites (stained by MAP2) were found to attach to electrospun SF nanofibrous mats fibers to form neural networks (Fig. 4(A)). Anti-NF immunocytochemistry following 7-day culture demonstrated that NF immunopositive hippocampal neurons attached to and expanded on the electrospun SF nanofibrous mats fibers to form cell nets as characterized by the relatively strong red fluorescence emission (Fig. 4(C)).
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Fig. 4. Light micrographs either following GAP-43 and MAP2 double-labeled (A) staining of hippocampal neurons after 7-day culture on electrospun SF nanofibrous mats, in which MAP2 immunopositive cell bodies and dendrites were seen in (a), GAP-43 immunopositive cell bodies and axons were seen in (b), and (c) is the merge of (a) and (b); or following anti-β-tubulin III (B) or anti-NF-200 (C) immunocytochemistry and Hoechst 33342 staining of hippocampal neurons after 7-day culture on electrospun SF nanofibrous mats, in which β-tubulin III or NF immunopositive neurons were seen in (a), Hoechst-labeled cell nuclei of neurons and electrospun SF nanofibrous mat auto-fluorescence were seen in (b), and (c) is the merge of (a) and (b). Scale bars, 20 µm. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-130775.)
Beta-tubulin III, as a member of tubulin family, is found in the brain and root ganglia and localized to neurons in the central and peripheral nervous system, where its expression seems to increase during axonal outgrowth [30]. After 7-day culture on electrospun SF nanofibrous mats, β-tubulin III immunopositive hippocampal neurons were observed to attach to and expand on the electrospun SF nanofibrous mats and connect to each other, thus interconnecting to form cell nets, as characterized by the relatively strong red fluorescence emission (Fig. 4(B)). Collectively, immunocytochemistry indicated that the expression and distribution of GAP-43, MAP2, NF200 and β-tubulin III in hippocampal neurons culture on the electrospun SF nanofibrous mats-based substrates were similar to those in hippocampal neurons cultured on poly-lysine coated coverslips. These data provide further evidence that the electrospun SF nanofibrous mats, as well as the poly-lysine coated coverslips, are biocompatible to hippocampal neurons in vitro. 3.4. Cell viability test for hippocampal neurons cultured in electrospun SF nanofibrous mats extract MTT, as a quantitative measure of cell viability, was used to test the possible cytotoxic effects of the electrospun SF nanofibrous mat. It was found that the cell viability of hippocampal neurons cultured in electrospun SF nanofibrous mats was not significantly different from that in plain neuronal medium at all time points of culture (Fig. 5).
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Fig. 5. The changes in cell viability of hippocampal neurons, expressed by the OD value, after hippocampal neurons were cultured in plain neuronal culture medium or electrospun SF nanofibrous mat extract for 12, 24, 48 or 72 h, respectively. ANOVA was used for statistical analysis. All p values are larger than 0.05.
Fig. 6. The protein levels (relative to GAPDH) of GAP-43 (a, b) or Tubulin (c, d) in the hippocampal neurons that were treated with plain neuronal culture medium (A), or electrospun SF nanofibrous mats extract (B) for 12, 24, 48 and 72 h, respectively. There were no significant differences between the two mediums at the same time point (P > 0.05). The representative Western blot is also shown.
3.5. Expression of GAP-43 and β-tubulin III by hippocampal neurons treated with electrospun SF nanofibrous mats extract Figure 6 compare the protein level of GAP-43 and β-tubulin III in the hippocampal neurons cultured in plain neuronal medium, and electrospun SF nanofibrous mats, respectively. No significant differences were detected between the two mediums, suggesting no inhibitory effects of electrospun SF nanofibrous mats substrate on the biological functions of primary culture of hippocampal neurons. 4. Conclusion In this study, we prepared SF nanofibrous mats by an electrospinning technique. These electrospun mats were found to possess a porous network structure and a three-dimensional nanofibrous feature, which are favorable to cell attachment and growth. We noted that electrospun SF mats supported the survival and growth of hippocampal neurons, and the mat extract showed no significant cytotoxic effects on the proliferation of hippocampal neurons. These results suggest that SF-based materials
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could be processed into electrospun nanofibous mats which were a promising matrix used for CNS repair. Acknowledgements The financial supports of Hi-Tech Research and Development Program of China (863 Program, Grant no. 2006AA02A128), Nature Science Foundation of China (Grant no. 30970713), Basic Research Program of Jiangsu Education Department (Grant no. BK2009518; 07KJA31025) and The Priority Academic Program Development of Jiangsu Higher Education Institutions are gratefully acknowledged. References [1] T.M. Barchet and M.M. Amiji, Challenges and opportunities in CNS delivery of therapeutics for neurodegenerative diseases, Expert. Opin. Drug. Deliv. 6(3) (2009), 211–225. [2] J.B. Recknor, D.S. Sakaguchi and S.K. Mallapragada, Directed growth and selective differentiation of neural progenitor cells on micropatterned polymer substrates, Biomaterials 27(22) (2006), 4098–4108. [3] P. Prang, R. Muller, A. Eljaouhari, K. Heckmann, W. Kunz, T. Weber, C. Faber, M. Vroemen, U. Bogdahn and N. Weidner, The promotion of oriented axonal regrowth in the injured spinal cord by alginate-based anisotropic capillary hydrogels, Biomaterials 27(19) (2006), 3560–3569. [4] N. Popovic and P. Brundin, Therapeutic potential of controlled drug delivery systems in neurodegenerative diseases, Int. J. Pharm. 314(2) (2006), 120–126. [5] H. Cao, T. Liu and S.Y. Chew, The application of nanofibrous scaffolds in neural tissue engineering, Adv. Drug. Deliv. Rev. 61(12) (2009), 1055–1064. [6] S.Y. Chew, R. Mi, A. Hoke and K.W. Leong, Aligned protein-polymer composite fibers enhance nerve regeneration: A potential tissue-engineering platform, Adv. Funct. Mater. 17(8) (2007), 1288–1296. [7] K.K. Jain, Nanobiotechnology-based drug delivery to the central nervous system, Neurodegener. Dis. 4(4) (2007), 287–291. [8] Acharya, C., S.K. Ghosh and S.C. Kundu, Silk fibroin film from non-mulberry tropical tasar silkworms: A novel substrate for in vitro fibroblast culture, Acta Biomater. 5(1) (2009), 429–437. [9] T.N. Salthouse, Biologic response to sutures, Otolaryngol. Head. Neck. Surg. 88(6) 1980, 658–664. [10] Vepari, C. and D.L. Kaplan, Silk as a biomaterial, Prog. Polym. Sci. 32(8,9) (2007), 991–1007. [11] M. Santin, A. Motta, G. Freddi and M. Cannas, In vitro evaluation of the inflammatory potential of the silk fibroin, J. Biomed. Mater. Res. 46(3) (1999), 382–389. [12] G.H. Altman, F. Diaz, C. Jakuba, T. Calabro, R.L. Horan, J. Chen, H. Lu, J. Richmond and D.L. Kaplan, Silk-based biomaterials, Biomaterials 24(3) 2003, 401–416. [13] X. Tang, F. Ding, Y. Yang, N. Hu, H. Wu and X. Gu, Evaluation on in vitro biocompatibility of silk fibroin-based biomaterials with primarily cultured hippocampal neurons, J. Biomed. Mater. Res. A 91A(1) (2008), 166–174. [14] Y. Yang, X. Chen, F. Ding, P. Zhang, J. Liu and X. Gu, Biocompatibility evaluation of silk fibroin with peripheral nerve tissues and cells in vitro, Biomaterials 28(9) (2007), 1643–1652. [15] Y. Yang, F. Ding, J. Wu, W. Hu, W. Liu, J. Liu and X. Gu, Development and evaluation of silk fibroin-based nerve grafts used for peripheral nerve regeneration, Biomaterials 28(36) (2007), 5526–5535. [16] Zhang, X., M.R. Reagan and D.L. Kaplan, Electrospun silk biomaterial scaffolds for regenerative medicine, Adv. Drug. Deliv. Rev. 61(12) (2009), 988–1006. [17] S.Y. Park, C.S. Ki, Y.H. Park, H.M. Jung, K.M. Woo and H.J. Kim, Electrospun silk fibroin scaffolds with macropores for bone regeneration: an in vitro and in vivo study, Tissue Eng. Part A 16(4) (2010), 1271–1279. [18] K. Zhang, X. Mo, C. Huang, C. He and H. Wang, Electrospun scaffolds from silk fibroin and their cellular compatibility, J. Biomed. Mater. Res. A 93(3) (2010), 976–983. [19] A.J. Meinel, K.E. Kubow, E. Klotzsch, M. Garcia-Fuentes, M.L. Smith, V. Vogel, H.P. Merkle and L. Meinel, Optimization strategies for electrospun silk fibroin tissue engineering scaffolds, Biomaterials 30(17) (2009), 3058–3067. [20] B. Marelli, A. Alessandrino, S. Fare, G. Freddi, D. Mantovani and M.C. Tanzi, Compliant electrospun silk fibroin tubes for small vessel bypass grafting, Acta Biomater. 6(10) (2010), 4019–4026. [21] F. Rigato, New Methods for Culturing Cells from Nervous System, P. Poindron, P. Piguet and E. Förster, eds, BioValley Monographs, Vol. 1, P. Poindron, ed., Switzerland, 2005, p. 82.
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[22] H.J. Jin, S.V. Fridrikh, G.C. Rutledge and D.L. Kaplan, Electrospinning Bombyx mori silk with poly(ethylene oxide), Biomacromolecules 3(6) (2002), 1233–1239. [23] Q. He, T. Zhang, Y. Yang and F. Ding, In vitro biocompatibility of chitosan-based materials to primary culture of hippocampal neurons, J. Mater. Sci. Mater. Med. 20(7) (2009), 1457–1466. [24] F. Denizot and R. Lang, Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability, J. Immunol. Methods 89(2) (1986), 271–277. [25] S. Yu, M. Liu, X. Gu and F. Ding, Neuroprotective effects of salidroside in the PC12 cell model exposed to hypoglycemia and serum limitation, Cell. Mol. Neurobiol. 28(8) (2008), 1067–1078. [26] B. Chakravarthy, A. Rashid, L. Brown, L. Tessier, J. Kelly and M. Menard, Association of Gap-43 (neuromodulin) with microtubule-associated protein MAP-2 in neuronal cells, Biochem. Biophys. Res. Commun. 371(4) (2008), 679–683. [27] K. Goslin, D.J. Schreyer, J.H. Skene and G. Banker, Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones, Nature 336(6200) (1988), 672–674. [28] L.I. Benowitz and A. Routtenberg, GAP-43: an intrinsic determinant of neuronal development and plasticity, Trends Neurosci. 20(2) (1997), 84–91. [29] B. Shafit-Zagardo and N. Kalcheva, Making sense of the multiple MAP-2 transcripts and their role in the neuron, Mol. Neurobiol. 16(2) (1998), 149–162. [30] M. Carre, N. Andre, G. Carles, H. Borghi, L. Brichese, C. Briand and D. Braguer, Tubulin is an inherent component of mitochondrial membranes that interacts with the voltage-dependent anion channel, J. Biol. Chem. 277(37) (2002), 33664–33669.
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Biocompatibility evaluation of electrospun silk fibroin nanofibrous mats with primarily cultured rat hippocampal neurons Yahong Zhao a , Weijia Zhao a , Shu Yu a , Yibing Guo b , Xiaosong Gu a and Yumin Yang a,∗ a b
Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, China Surgical Comprehensive Laboratory, Affiliated Hospital of Nantong University, Nantong, China
Received 18 August 2011 Accepted 3 June 2013 Abstract. In this study, electrospinning was performed to fabricate silk fibroin (SF) nanofibrous mats, which were used as substrates for in vitro culture of rat hippocampal neurons. The light and electron micrographs demonstrated that the electrospun SF nanofibrous mat supported the survival and growth of the attached hippocampal neurons. MTT assay and immunocytochemistry in couple with Western blot analysis respectively indicated there was no significant difference in both the cell viability and expression levels of some proteins, including GAP-43, MAP-2, NF, and β-tubulin, between hippocampal neurons cultured in the electrospun SF nanofibrous mat extract and in plain neuronal medium. Our results indicated that electrospun SF nanofibrous mats were biocompatible to primary culture of hippocampal neurons without cytotoxic effects on the cell phenotype and functions, raising a potential possibility of using these mats for CNS therapeutic applications. Keywords: Biomaterials, nanofiber, biocompatibility, silk fibroin, hippocampal neurons
1. Introduction Since the adult mammalian central nervous system (CNS) fails to spontaneously regenerate after injuries and degeneration, developing CNS therapy strategies has attracted research interests [1]. Recently, cell implantation and controlled release of neutrophic factors are accepted as two promising approaches to aiding CNS regeneration. They often require the use of suitable biomaterial-based matrices as the carrier to present biochemical cues [2–4]. To use any new matrix, therefore, an investigation of its biocompatibility with CNS cells or tissues becomes an important prerequisite. With recent development of nanotechnology, fibrous matrices at nano-scale level are now becoming potential candidates for tissue engineering applications because they mimic the topography of natural extracellular matrix (ECM) [5]. Many novel manufacturing methods, including electrospinning, phase separation, and self-assembly, have been used for fabrication of such biofunctional nanofibrous matrices. Of which electrospun fibrous matrices have a high surface-area-to-volume ratio suitable for cell *
Address for correspondence: Yumin Yang, Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, JS 226001, China. Tel./Fax: +86 513 85511585; E-mail: [email protected]. 0959-2989/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved
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attachment and growth as well as a possibility of mimicking the ECM architecture favorable to tissue regeneration [6,7]. Silk fibroin (SF)-based biomaterials possess favorable physicochemical and biological properties [8–11], and have found increasing applications in biomedical fields [12–15]. More instriguingly, SF-based materials have been used for preparing nanofibrous matrices, for example via electrospining, and the resulting matrices have been applied in various areas of tissue engineering. However, they have not been extensively studied for nerve regeneration, especially CNS injury repair [5,16–20], in depth studies are thus needed to further examine the feasibility of SF-based matrices with the nanofibrous configuration for nerve repair applications. On the other hand, although the biocompatibility of SF-based materials with CNS cells have been reported [13], the issue of whether the matrices made up of SF-based biomaterials via electrospining fabrication could keep the neuroaffinity is still worthy to be explored. In this study, we aimed at investigating the possibility of electrospun SF nanofibrous mats used as a candidate matrix for CNS therapeutic applications. The primarily cultured hippocampal neurons of developing rat brains, which usually serve as a well-defined cell model [21], were cultured on the substrate consisting of electrospun SF nanofibrous mats for the in vitro examination of the biocompatibility of the substrate with CNS cells. The survival and growth characteristics of hippocampal neurons on electrospun SF nanofibrous mats were investigated by morphological observation, MTT analysis, immunocytochemistry and Western blot analysis.
2. Materials and methods 2.1. Preparation of electrospun SF nanofibrous mats Raw silk fibers (from Bombyx mori cocoons) were bought from Xinyuan sericulture company, Hai’an, Jiangsu, China. The SF aqueous solution was prepared as previously described [15], and then spread on stainless steel dishes to generate the air-dried regenerated SF membranes which were further dissolved in 98% (wt/wt) formic acid to obtain 13% (wt/wt) SF spinning solution. A home-made electrospinning setup, used in this study, was made up of a high voltage supplier, a capillary needle anode, and a grounded collector cathode. A high electric potential (20 kV) was applied to the tip of the needle anode (ID 0.9 mm), into which the droplet of SF spinning solution was loaded. The electrospun nanofibrous mat was generated on the stainless steel meshes. The distance between the needle tip and the steel collector ranged from 7 to 13 cm. A constant volume flow rate (0.3 ml/h) was maintained by a syringe pump, which kept the SF spinning solution in the needle. After electrospun SF nanofibrous mats were removed from the stainless steel meshes, they were inserted into 100% alcohol for 10 min to induce conformational transition [22], followed by wash with distilled water at 37◦ C for 72 h to remove residual formic acid. The treated mats had to be sterilized with a hyperbaric method, and then equilibrated in DMEM culture medium (Gibco) for 30 min prior to use. 2.2. Characterization of electrospun SF mats For chemical characterization, the mats were ground, and pelleted with dried KBr, followed by analysis with a model Nexus 870 Fourier transform infrared (FT-IR) spectrophotometer (Nicolet Instruments Co., Madison, WI) at a range of 400–4000 cm−1 .
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2.3. Cell culture and treatment Sprague–Dawley embryonic rats were obtained from the experimental animal center of Nantong University. All experimental procedures involving animals were conducted as per Institutional Animal Care guidelines and approved ethically by the administration committee of experimental animals, Jiangsu Province, China. The rat hippocampal neurons were isolated as described previously [23] with minor modifications. After E 18 embryonic rats were sacrificed by cervical dislocation under anesthesia, their brains were quickly removed and the hippocampi were harvested on a cold stage. The procured hippocampal tissues were mechanically and enzymatically dissociated into a single-cell suspension, which was then plated onto poly-lysine-coated plates at a density of 1×105 per cm2 in DMEM supplemented with 10% F12 and 10% FBS (Gibco, Grand Island, NY) for 4 h. The medium was replaced by neuronal culture medium, and cells were incubated at 37◦ C in a humidified atmosphere of 95% air and 5% CO2 for 7–8 days, the time required for maturation of hippocampal neurons. Half of the culture medium was replaced every 2 days. The glial content of cultured cells was measured to be only 0.5% of the total cell population. Primarily cultured hippocampal neurons were seeded onto the poly-L-lysine (Sigma) coated coverslips, or the substrate made up of electrospun SF nanofibrous mats, which had been placed onto a 24-well culture plate, followed by soaking in plain neuronal culture medium. Another portion of primarily cultured hippocampal neurons was randomized into two groups for treatment with different mediums that were plain neuronal culture medium (positive control), and electrospun SF nanofibrous mats extract, respectively. 2.4. Light and electron microscopy After rat hippocampal neurons were cultured for designated times, the cell growth was observed under an inverted microscope (Olympus, Tokyo, Japan), and photographs were taken. In order to determine the average and total length of neurites for the hippocampal neurons cultured on the different substrates for 5 days, five fields were randomly selected for measurements of each photograph with Q550 IW image analysis system (Leica Imaging Systems Ltd., Cambridge, England). All measurements were performed in triplicate. After 3- or 7-day culture on the poly-lysine-coated coverslips, or the substrate made up of electrospun SF nanofibrous mats, the hippocampal neurons were washed twice with phosphate buffer saline (PBS, pH 7.2) and fixed in 4% glutaraldehyde. They were then post-fixed with 1% OsO4 , dehydrated stepwise in increasing concentrations of ethanol, and dried in a critical point drier (Hitachi, Tokyo, Japan), followed by coating with gold in a JFC-1100 unit (Jeol Inc., Japan) and observation under a scanning electron microscope (JEM-T300, Jeol Inc., Japan). 2.5. MTT assay After incubation in two different extract fluid, the viability of hippocampal neurons was assessed by MTT assay as previously described [24,25]. 2.6. Immunocytochemistry After 7-day incubation, the samples were fixed and subjected to immunocytochemistry. Goat anti-growth associated protein-43 (GAP-43, 1:200 dilution, Santa Cruz, CA), mouse monoclonal antimicrotubule-associated protein 2 (MAP2, 1:1,000 dilution, Sigma), rabbit anti-neurofilament (NF)
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200 IgG fraction of antiserum (1:200 dilution, Sigma), and rabbit anti β-tubulin III (1:50 dilution, Sigma), as primary antibody, and Cy3-labeled goat antirabbit IgG (1:200 dilution, Santa Cruz), FITClabeled donkey anti-goat IgG (1:200 dilution, Santa Cruz) and TRITC-labeled donkey anti-mouse IgG (1:200 dilution, Santa Cruz), as secondary antibody, were used. The samples were also stained with 5 µg/ml Hoeechst 33342 (Sigma). Finally, the samples were observed under a confocal laser scanning microscope (TCS SP2, Leica Microsystems, Germany). 2.7. Western blot analysis After 12, 24, 48 or 72 h treatment in two different mediums, the hippocampal neurons were analyzed for protein expression levels of GAP-43 and β-tubulin III by using Western blotting as described previously [13]. In brief, the hippocampal neurons were washed with PBS and lysed with lysis buffer containing protease inhibitors (Promega, Madison, WI) at designated times. Protein concentration was detected by BCA method (calibrated on bovine serum albumin) to maintain the same loads. Protein extracts were heat denatured at 100◦ C for 10 min, electrophoretically separated on a 10% SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat dry milk in TBST buffer (50 mM Tris–HCl, 100 mM NaCl, and 0.1% Tween-20, pH 7.4) and incubated with a 1:500 dilution of goat anti-GAP43 polyclonal antibody and 1:1,500 dilution of rabbit anti β-tubulin III antibody in 5% non-fat dry milk in TBST buffer at 4◦ C overnight. The membranes were washed with TBST buffer (5 min × 3), and further incubated with a 1:5,000 dilution of donkey anti-goat IgG and 1:10,000 dilution of donkey anti-rabbit IgG at room temperature for 2 h. After the membrane was washed, the image was scanned with Odyssey (LI-COR, Lincoln, NE), and the data of optical density were analyzed using PDQuest 7.2.0 software (Bio-Rad). GAPDH (1: 500) was used as an internal control. 2.8. Statistical analysis The data were expressed as means ± SD, and analyzed by one-way ANOVA. Statistical significance was accepted at the 0.05 confidence level. 3. Results and discussion 3.1. Morphological observation SEM showed that the electrospun SF mat was composed of a large number of randomly oriented fibers, and the diameter of most fibers ranged from 200 to 800 nm, although a few fibers were over 1 µm in diameter (Fig. 1(A)). Figure 1(B) provides the comparison of FT-IR spectra among two SF-based matrices. The three FT-IR curves (a, b) are similar to each other in shape, confirming an identical chemical composition for them. However, slight differences in the peak wave number are noted for two FT-IR curves, due to conformation transitions that take place during the fabrication process of electrospun SF mats. 3.2. Hippocampal neuron cell affinity of electrospun SF nanofibrous mats As revealed by light microscopy for visualizing the cell growth of hippocampal neurons cultured on the poly-lysine-coated coverslips, or the substrate made up of electrospun SF nanofibrous mats. Hippocampal neurons were found to adhere to electrospun SF nanofibrous mats immediately after inocu-
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Fig. 1. Nanofiber diameter distribution (A) of electrospun SF mats and the FT-IR spectra (B) of air-dried SF membranes (a), and electrospun SF mats (b).
(A)
(B)
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Fig. 2. Light (A), (B) and scanning electron (C), (D) micrographs of hippocampal neurons cultured on electrospun SF nanofibrous mats for 3 days (A), (C) or 7 days (B), (D), respectively. Scale bar: 20 µm. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-130775.)
lation, and the cells showed a spherical shape with smooth surface and obvious refraction. After 3-day culture, the neurites of hippocampal neurons adhered to and extended along the substrate (Fig. 2(A)), and after 7-day culture the cells with long and dense neurites were found to form neural networks (Fig. 2(B)).
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Fig. 3. The total length of neurites for hippocampalneurons cultured on Pll-coated coverslips and the substrate made up of electrospun SF nanofibrous mats for 5 days. No significant differences in length was found between two different substrates (P > 0.05).
This result was further evidenced by the comparisons in the length of neuritis (Fig. 3) for hippocampal neurons cultured on two substrates without significant difference detected. Visual inspection under scanning electron microscopy further revealed that after 3-day culture on the substrate made up of electrospun SF nanofibrous mats, a majority of hippocampal neurons adherent to electrospun SF nanofibrous mats were oval- or spindle-shaped, and they connected to each other via extending neuritis (Fig. 2(C)); and after 7-day culture a dense array of neurons connected to each other via their neurites, the cells with long and dense axons and dendrites took compact arrangements, and they interweaved mutually to form neural networks (Fig. 2(D)). Also interestingly, the above-mentioned morphological features were close to those for hippocampal neurons cultured under normal conditions (data not shown). Taken together, histological observations collectively suggest that primarily cultured hippocampal neurons were able to adhere to and grow on the electrospun SF nanofibrous mats substrate, and the above observations are comparable with those for hippocampal neurons that were cultured on the poly-lysine coated coverslips, few visible differences were detected between them, despite geometric divergence of the different substrates. 3.3. Immunocytochemical observation of hippocampal neurons onto electrospun SF nanofibrous mats After 7-day culture on the electrospun SF nanofibrous mats-based substrates, the hippocampal neurons underwent immunocytochemistry with antibodies against GAP-43, MAP2, NF200 and β-tubulin III, respectively, because the expression of these four important neuronal marker proteins presents an effective standard for biocompatibility evaluation of electrospun SF nanofibrous mats-based substrates. GAP-43 and NF function as specific neuronal markers,and GAP-43 is a neuron-specic phospho protein which promotes survival and maintenance of function of neurons [26–28]. MAP2, as the major microtubule associated protein of brain tissue, co-localized and potentially interacts with Gap-43, and it promotes microtubule assembly and forms side-arms on microtubules [29]. As was shown by GAP43 (green) and MAP2 (red) double-labeled staining, the cells (double-stained) were noted to expand on electrospun SF nanofibrous mats fibers (Auto-fluorescence, blue), while axons (stained by GAP-43) and dendrites (stained by MAP2) were found to attach to electrospun SF nanofibrous mats fibers to form neural networks (Fig. 4(A)). Anti-NF immunocytochemistry following 7-day culture demonstrated that NF immunopositive hippocampal neurons attached to and expanded on the electrospun SF nanofibrous mats fibers to form cell nets as characterized by the relatively strong red fluorescence emission (Fig. 4(C)).
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Fig. 4. Light micrographs either following GAP-43 and MAP2 double-labeled (A) staining of hippocampal neurons after 7-day culture on electrospun SF nanofibrous mats, in which MAP2 immunopositive cell bodies and dendrites were seen in (a), GAP-43 immunopositive cell bodies and axons were seen in (b), and (c) is the merge of (a) and (b); or following anti-β-tubulin III (B) or anti-NF-200 (C) immunocytochemistry and Hoechst 33342 staining of hippocampal neurons after 7-day culture on electrospun SF nanofibrous mats, in which β-tubulin III or NF immunopositive neurons were seen in (a), Hoechst-labeled cell nuclei of neurons and electrospun SF nanofibrous mat auto-fluorescence were seen in (b), and (c) is the merge of (a) and (b). Scale bars, 20 µm. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-130775.)
Beta-tubulin III, as a member of tubulin family, is found in the brain and root ganglia and localized to neurons in the central and peripheral nervous system, where its expression seems to increase during axonal outgrowth [30]. After 7-day culture on electrospun SF nanofibrous mats, β-tubulin III immunopositive hippocampal neurons were observed to attach to and expand on the electrospun SF nanofibrous mats and connect to each other, thus interconnecting to form cell nets, as characterized by the relatively strong red fluorescence emission (Fig. 4(B)). Collectively, immunocytochemistry indicated that the expression and distribution of GAP-43, MAP2, NF200 and β-tubulin III in hippocampal neurons culture on the electrospun SF nanofibrous mats-based substrates were similar to those in hippocampal neurons cultured on poly-lysine coated coverslips. These data provide further evidence that the electrospun SF nanofibrous mats, as well as the poly-lysine coated coverslips, are biocompatible to hippocampal neurons in vitro. 3.4. Cell viability test for hippocampal neurons cultured in electrospun SF nanofibrous mats extract MTT, as a quantitative measure of cell viability, was used to test the possible cytotoxic effects of the electrospun SF nanofibrous mat. It was found that the cell viability of hippocampal neurons cultured in electrospun SF nanofibrous mats was not significantly different from that in plain neuronal medium at all time points of culture (Fig. 5).
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Fig. 5. The changes in cell viability of hippocampal neurons, expressed by the OD value, after hippocampal neurons were cultured in plain neuronal culture medium or electrospun SF nanofibrous mat extract for 12, 24, 48 or 72 h, respectively. ANOVA was used for statistical analysis. All p values are larger than 0.05.
Fig. 6. The protein levels (relative to GAPDH) of GAP-43 (a, b) or Tubulin (c, d) in the hippocampal neurons that were treated with plain neuronal culture medium (A), or electrospun SF nanofibrous mats extract (B) for 12, 24, 48 and 72 h, respectively. There were no significant differences between the two mediums at the same time point (P > 0.05). The representative Western blot is also shown.
3.5. Expression of GAP-43 and β-tubulin III by hippocampal neurons treated with electrospun SF nanofibrous mats extract Figure 6 compare the protein level of GAP-43 and β-tubulin III in the hippocampal neurons cultured in plain neuronal medium, and electrospun SF nanofibrous mats, respectively. No significant differences were detected between the two mediums, suggesting no inhibitory effects of electrospun SF nanofibrous mats substrate on the biological functions of primary culture of hippocampal neurons. 4. Conclusion In this study, we prepared SF nanofibrous mats by an electrospinning technique. These electrospun mats were found to possess a porous network structure and a three-dimensional nanofibrous feature, which are favorable to cell attachment and growth. We noted that electrospun SF mats supported the survival and growth of hippocampal neurons, and the mat extract showed no significant cytotoxic effects on the proliferation of hippocampal neurons. These results suggest that SF-based materials
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could be processed into electrospun nanofibous mats which were a promising matrix used for CNS repair. Acknowledgements The financial supports of Hi-Tech Research and Development Program of China (863 Program, Grant no. 2006AA02A128), Nature Science Foundation of China (Grant no. 30970713), Basic Research Program of Jiangsu Education Department (Grant no. BK2009518; 07KJA31025) and The Priority Academic Program Development of Jiangsu Higher Education Institutions are gratefully acknowledged. References [1] T.M. Barchet and M.M. Amiji, Challenges and opportunities in CNS delivery of therapeutics for neurodegenerative diseases, Expert. Opin. Drug. Deliv. 6(3) (2009), 211–225. [2] J.B. Recknor, D.S. Sakaguchi and S.K. Mallapragada, Directed growth and selective differentiation of neural progenitor cells on micropatterned polymer substrates, Biomaterials 27(22) (2006), 4098–4108. [3] P. Prang, R. Muller, A. Eljaouhari, K. Heckmann, W. Kunz, T. Weber, C. Faber, M. Vroemen, U. Bogdahn and N. Weidner, The promotion of oriented axonal regrowth in the injured spinal cord by alginate-based anisotropic capillary hydrogels, Biomaterials 27(19) (2006), 3560–3569. [4] N. Popovic and P. Brundin, Therapeutic potential of controlled drug delivery systems in neurodegenerative diseases, Int. J. Pharm. 314(2) (2006), 120–126. [5] H. Cao, T. Liu and S.Y. Chew, The application of nanofibrous scaffolds in neural tissue engineering, Adv. Drug. Deliv. Rev. 61(12) (2009), 1055–1064. [6] S.Y. Chew, R. Mi, A. Hoke and K.W. Leong, Aligned protein-polymer composite fibers enhance nerve regeneration: A potential tissue-engineering platform, Adv. Funct. Mater. 17(8) (2007), 1288–1296. [7] K.K. Jain, Nanobiotechnology-based drug delivery to the central nervous system, Neurodegener. Dis. 4(4) (2007), 287–291. [8] Acharya, C., S.K. Ghosh and S.C. Kundu, Silk fibroin film from non-mulberry tropical tasar silkworms: A novel substrate for in vitro fibroblast culture, Acta Biomater. 5(1) (2009), 429–437. [9] T.N. Salthouse, Biologic response to sutures, Otolaryngol. Head. Neck. Surg. 88(6) 1980, 658–664. [10] Vepari, C. and D.L. Kaplan, Silk as a biomaterial, Prog. Polym. Sci. 32(8,9) (2007), 991–1007. [11] M. Santin, A. Motta, G. Freddi and M. Cannas, In vitro evaluation of the inflammatory potential of the silk fibroin, J. Biomed. Mater. Res. 46(3) (1999), 382–389. [12] G.H. Altman, F. Diaz, C. Jakuba, T. Calabro, R.L. Horan, J. Chen, H. Lu, J. Richmond and D.L. Kaplan, Silk-based biomaterials, Biomaterials 24(3) 2003, 401–416. [13] X. Tang, F. Ding, Y. Yang, N. Hu, H. Wu and X. Gu, Evaluation on in vitro biocompatibility of silk fibroin-based biomaterials with primarily cultured hippocampal neurons, J. Biomed. Mater. Res. A 91A(1) (2008), 166–174. [14] Y. Yang, X. Chen, F. Ding, P. Zhang, J. Liu and X. Gu, Biocompatibility evaluation of silk fibroin with peripheral nerve tissues and cells in vitro, Biomaterials 28(9) (2007), 1643–1652. [15] Y. Yang, F. Ding, J. Wu, W. Hu, W. Liu, J. Liu and X. Gu, Development and evaluation of silk fibroin-based nerve grafts used for peripheral nerve regeneration, Biomaterials 28(36) (2007), 5526–5535. [16] Zhang, X., M.R. Reagan and D.L. Kaplan, Electrospun silk biomaterial scaffolds for regenerative medicine, Adv. Drug. Deliv. Rev. 61(12) (2009), 988–1006. [17] S.Y. Park, C.S. Ki, Y.H. Park, H.M. Jung, K.M. Woo and H.J. Kim, Electrospun silk fibroin scaffolds with macropores for bone regeneration: an in vitro and in vivo study, Tissue Eng. Part A 16(4) (2010), 1271–1279. [18] K. Zhang, X. Mo, C. Huang, C. He and H. Wang, Electrospun scaffolds from silk fibroin and their cellular compatibility, J. Biomed. Mater. Res. A 93(3) (2010), 976–983. [19] A.J. Meinel, K.E. Kubow, E. Klotzsch, M. Garcia-Fuentes, M.L. Smith, V. Vogel, H.P. Merkle and L. Meinel, Optimization strategies for electrospun silk fibroin tissue engineering scaffolds, Biomaterials 30(17) (2009), 3058–3067. [20] B. Marelli, A. Alessandrino, S. Fare, G. Freddi, D. Mantovani and M.C. Tanzi, Compliant electrospun silk fibroin tubes for small vessel bypass grafting, Acta Biomater. 6(10) (2010), 4019–4026. [21] F. Rigato, New Methods for Culturing Cells from Nervous System, P. Poindron, P. Piguet and E. Förster, eds, BioValley Monographs, Vol. 1, P. Poindron, ed., Switzerland, 2005, p. 82.
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