Collagen scaffolds combined with collagen-binding ciliary neurotrophic factor facilitate facial nerve repair in mini-pigs Chao Lu,1* Danqing Meng,2,3* Jiani Cao,2* Zhifeng Xiao,2 Yi Cui,2,4 Jingya Fan,1 Xiaolong Cui,3 Bing Chen,2 Yao Yao,1 Zhen Zhang,1 Jinling Ma,1 Juli Pan,1 Jianwu Dai2 1
Department of VIP Service, School of Stomatology, Capital Medical University, Beijing 10050, China State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 3 Nanyitiao, Zhongguancun, Beijing 100190, China 3 Graduate School, Chinese Academy of Sciences, Beijing 100190, China 4 Reproductive and Genetic Center of National Research Institute for Family Planning, Beijing 100081, China 2
Received 20 June 2014; revised 28 July 2014; accepted 31 July 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35305 Abstract: The preclinical studies using animal models play a very important role in the evaluation of facial nerve regeneration. Good models need to recapitulate the distance and time for axons to regenerate in humans. Compared with the most used rodent animals, the structure of facial nerve in mini-pigs shares more similarities with humans in microanatomy. To evaluate the feasibility of repairing facial nerve defects by collagen scaffolds combined with ciliary neurotrophic factor (CNTF), 10-mm-long gaps were made in the buccal branch of mini-pigs’ facial nerve. Three months after surgery, electrophysiological assessment and histological examination were performed to evaluate facial nerve regeneration. Immunohistochemistry
and transmission electron microscope observation showed that collagen scaffolds with collagen binding (CBD)-CNTF could promote better axon regeneration, Schwann cell migration, and remyelination at the site of implant device than using scaffolds alone. Electrophysiological assessment also showed higher recovery rate in the CNTF group. In summary, combination of collagen scaffolds and CBD-CNTF showed promising effects on facial nerve regeneration in mini-pig C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: models. V 00A:000–000, 2014.
Key Words: facial nerve regeneration, collagen scaffold, ciliaryneurotrophic factor, mini-pig
How to cite this article: Lu C, Meng D, Cao J, Xiao Z, Cui Y, Fan J, Cui X, Chen B, Yao Y, Zhang Z, Ma J, Pan J, Dai J. 2014. Collagen scaffolds combined with collagen-binding ciliary neurotrophic factor facilitate facial nerve repair in mini-pigs. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
Facial nerve injuries caused by infection, tumor excision, traffic accident, and war trauma could lead to facial paralysis which are both physiologic and mental burden for the patients.1 After the injury, the distal segment of the facial nerve went through Wallerian degeneration completely, whereas the proximal stump underwent only a limited extent degeneration as far as to the first node of Ranvier.2,3 Afterwards, the proximal stump developed excess axonal sprouts than original to maximize the chances of target reinnervation.4 During regeneration, Schwann cells and fibroblasts from both ends proliferate and migrate out of the nerve stumps to guide sprouting axons across the wound.5 In the late stage of the regeneration, the axon/
Schwann interaction provides signals for Schwann cells to redifferentiate and remyelinate the newly formed axons.6–10 Among facial nerve trauma, transection is a more severe injury, which breaks down the structural continuity of facial nerve. Transection, which leaves relatively long gaps, needs to be bridged by grafts or nerve conduits.11–13 The structural and functional defects of facial nerve need to be recovered by good axonal regeneration and remyelination, and the targeted muscles need to be correctly reinnervated. To achieve these goals, at least four basic elements need to be concerned: (1) Protecting neurons from apoptosis. (2) Building up an optimal environment for axonal regeneration. (3) Enhancing the regeneration ability of axons. (4) Guiding axons to regenerate toward their original targets.14,15
*These authors contributed equally to this work. Correspondence to: Juli Pan, Department of VIP Service, Beijing Stomatological Hospital, Capital Medical University, Beijing 100050, China; e-mail: [email protected] and Jianwu Dai, State key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 3 Nanyitiao, Zhongguancun, Beijing 100190, China Contract grant sponsor: The “Strategic Priority Research Program of the Chinese Academy of Sciences”; contract grant number: XDA01030000 Contract grant sponsor: National High Technology Research and Development Program (“863” Program) of China; contract grant number: 2012AA020501 Contract grant sponsor: National Nature Science Foundation; contract grant number: 81372086
C 2014 WILEY PERIODICALS, INC. V
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FIGURE 1. The collagen scaffolds used for facial nerve repair and surgery procedure. A: The longitudinal view of a collagen tube. B: Image of the liner-order collagen fibers used for nerve guidance. C: Gross view obtained immediately after the scaffolds implanted at the injury site. D: Regenerated nerve obtained 3 months after the surgery. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
So far, most researchers used either rats or rabbits as injury models to test their strategies.16–18 But there are still many differences in facial nerve between rodent animals and human beings.14 For the extratemporal part of human facial nerve, the perifascicular, interfascicular, and intrafascicular connective tissue can occupy almost half of the whole cross-section with an average of 14 nerve fascicles disperse in it.19–21 But in rodent animals, there are few fascicles (usually one major fascicles accompanied with some small ones) with scarce connective tissue surrounding them as described previously.22 Besides, in rodent models, regeneration occurs over a relatively short distance compared with that in humans.14 The previous studies implied that mini-pigs are a suitable experimental model for the preclinical research in facial nerve repair, for pig facial nerve has very similar anatomic structures with that of humans, which allows long gaps model as well.23 Although autologous grafts (such as nerve, artery, vein, epineurial sheaths, and tendon) or allografts processed by immunosuppression or decellularization have exhibited encouraging results in peripheral nerve repair,15,24–26 their drawbacks of sacrificing donor tissue and ill-fitness in diameter and length limited their clinical uses. Thus, nerve conduits made of or modified with extracellular matrix such as collagen, laminin, and fibronectin become candidates to substitute tissue grafts.10,27–29
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It has been demonstrated in our previous study that collagen conduits filled with linear-ordered collagen (LOC) fibers (which had adsorbed collagen-binding ciliary neurotrophic factor [CBD-CNTF] and brain-derived neurotrophic factor [CBD-BDNF]) could promote facial nerve regeneration in a 4-mm rat facial nerve trunk gap model.22 To test whether this strategy could also achieve promising results in longer gap models in larger animals, 10-mm gap in the buccal branch (BB) of mini-pigs’ facial nerve was created and the structural and functional recovery indexes were examined. METHODS AND MATERIALS
Preparation of collagen nerve conduit The collagen nerve conduits [Fig. 1(A)] were prepared as described previously and cut into 10-mm long before use.22 In brief, 10-mm-long uniform conduits, with a 4.5-mm lumen and 1-mm wall thickness, were made by scrolling collagen membrane around glass molds and let them airdried. Then, they were treated overnight with crosslinking solution (30 mM 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide and 10 mM N-hydroxysuccin-imide in 50 mM 2-(Nmorpholino)ethanesulfonic acid solution, pH 5.5) at room temperature. The crosslinking agent was washed with NaH2PO4 (0.1M) and phosphate-buffered saline (PBS) for at least 10 times each. Finally, the collagen conduits were
FACIAL NERVE REPAIR IN MINI-PIGS BY COLLAGEN SCAFFOLDS WITH CNTF
ORIGINAL ARTICLE
FIGURE 2. HE staining of the facial nerve. A: Transverse section of a normal nerve. A bundle of axons (black arrowhead) was wrapped by perineurium (black arrow). Several axon fascicles together with connective tissue and blood vessels (white arrowhead) are surrounded by epineurium (white arrow). Scale bar: 500 mm. Longitudinal section showed a normal axon fascicle in (B) and the regenerated nerve in the PBS (C) and CBD-CNTF (D) group. Scale bar: 50 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
removed from the molds and freeze dried. The conduits were sterilized by radiation using 8-K gray before used in the animal experiments. Production of LOC fibers and CBD-CNTF As described previously,22 LOC fibers were prepared from bovine aponeurosis [Fig. 1(B)].30 The recombined protein CBD-CNTF-his6 was expressed in Escherichia coli, purified by Nickel chelating Sepharose (Amersham Biosciences, Piscataway, NJ). The purity of CBD-CNTF was confirmed by SDS-PAGE followed by Coomassie Brilliant Blue staining, whereas the identity was confirmed by Western blotting with anti-his6 primary antibody. The bioactivity of CBDCNTF was tested by 3-(4,5-dimethylthiazol-2-yl)22,5-diphenyltetrazolium bromide of dorsal nerve ganglia cell culture.10 The protein was lyophilized and stored at 280 C. Before the start of the experiment, CBD-CNTF was dissolved in ddH2O, and a bundle of LOC fibers (occupied about half lumen space of the collagen conduit) was incubated with 100 mg of CBD-CNTF (in 100 mL) for 30 min at 37 C and then inserted into the conduit. Surgical procedures of facial nerve transection Experiments were performed in accordance with the US National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals published by the US National Academy of Sciences (http://oacu.od.nih.gov/regs/index. htm) and approved by Animal Care and Use Committee, Beijing, China. Twelve female mini-pigs of 13 months old (weight, 35–40 kg) were anesthetized by intramuscularly injection of ketamine (10 mg/kg) and xylazine (2 mg/kg). After sterilization, an incision of 3–5 cm was made subcutaneously to expose the trunk of the right BB of facial nerve.
The trunk of buccal facial nerve is 4 cm long in average before branching. The 12 mini-pigs are randomly divided into two groups (n 5 6/each): (1) CNTF group, a segment of 8-mm nerve was removed by sharp microsurgery scissors, leaving a 10-mm gap after retraction of the nerve ends. The proximal and distal nerve stumps were bridged by 10-mm collagen conduit, the conduit was packed with LOC fibers which had been incubated with CNTF as described above. Four 9/0 monofilament nylon interrupted epineurium sutures were applied at each end of the anastomosis [Fig. 1(C)]. Another 100 mg of CBD-CNTF was injected into the proximal and distal nerve stumps with 50 mg at each end. (2) PBS group (control): equal volume of PBS was applied instead of CBD-CNTF. The mini-pigs were applied with penicillin after 3 days of the operation. One month later, the wound was reopened and 200 mg of CBD-CNTF was injected to the gap site in the CNTF group, whereas equal volume of PBS was injected in the PBS group. Gross observation The nerve conduit degradation, as well as surrounding tissue and regenerating nerve, was examined by gross 3 months after the operation in all animals. Histological analysis and transmission electron microscope observation The histological assessments were performed 3 months after operation. In each group, half of the nerve samples (randomly chosen from three mini-pigs) were used for transversal study and the rest for longitudinal study and transmission electron microscope (TEM) observation. The regenerated nerves were isolated and divided into proximal and distal segments (5 mm each), and fixed in 4% v/v of
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FIGURE 3. Transverse section of the regenerated nerve stained with NF body. A: (a1, a2) Represent the transections of a normal facial nerve. The regenerated nerve of the PBS group (b1, b2, d1, d2) and CNTF group (c1, c2, e1, e2) were divided into proximal segments (b1, b2, c1, c2) and distal segments (d1, d2, e1, e2) to illustrate the regeneration process. The magnifications of selected areas in the left panel (a1–e1) are shown in the right-hand side (a2–e2). Scale bar: 100 mm. B,C: The statistical analysis of NF-positive area. n 5 3, *p < 0.05. Nor: normal. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
formaldehyde for 48 h. Then, the segments were embedded in paraffin. Sections of 5 mm in thickness were cut from each segment and examined by immunohistochemistry using antibodies to neurofilament (anti-NF, 1:1000 dilution; Abcam) and S100 (1:2,000 dilution; Sigma). Using ImagePro Plus software (Media Cybernetics), the percentage of NF-positive and S100-positive area (positive staining area/ total area) was quantified at a magnification of 2003. The sections were also stained by Luxol fast blue (Solvent blue 38; S3382, Sigma) according to a previously published protocol10 to give an overall view of remyelinated axons. The ultrastructure of newly formed myelin was observed by TEM at a magnification of 40003. The number, diameter, and the thickness of the newborn myelin sheaths were evaluated by Image-Pro Plus software (Media Cybernetics). Electrophysiological assessment As for electrophysiological tests, the regenerated facial nerve was re-exposed under general anesthesia at 3 months postsurgery. The electrophysiological test was evaluated by an electromyography system (RM6240, Chengdu, China). The stimulating electrode was placed in the proximal end of
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buccal facial nerve, two recording electrodes were placed upstream (R1) and downstream (R2) of the regenerated segment, and the reference electrode was inserted into the ipsilateral fore limb subcutaneously. The ratio of action potential recorded by R2 divided by that recorded by R1 was used to evaluate the facial nerve functional recovery. Statistical analysis The Graphpad Prism 5 was used for the statistical analysis. All values are presented as mean 6 standard deviation. Comparisons between PBS and CNTF groups were performed using Student’s t-test. The p-value of 1 cm. There are still some improvements needed to be made in addition to electrophysiological evaluation to the min-pig facial nerve model, such as functional evaluation in mini-pig. CONCLUSIONS
In conclusion, a 10-mm-long gap model in mini-pigs’ facial nerve was used in this study to test the effectiveness of CBD-CNTF-modified collagen scaffolds in nerve regeneration. The collagen scaffolds facilitated oriented Schwann cell migration and axon regeneration, whereas the application of CBD-CNTF promotes axon sprout and myelin formation, indicating that our CBD-CNTF-modified collagen scaffolds may repair 10-mm injury in the facial nerve. REFERENCES 1. Sun F, Zhou K, Mi W, Qiu J. Repair of facial nerve defects with decellularized artery allografts containing autologous adiposederived stem cells in a rat model. Neurosci Lett 2011;499:104–108. 2. Boivin A, Pineau I, Barrette B, Filali M, Vallie`res N, Rivest S, Lacroix S. Toll-like receptor signaling is critical for Wallerian degeneration and functional recovery after peripheral nerve injury. J Neurosci 2007;27:12565–12576.
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3. Lee SK, Wolfe SW. Peripheral nerve injury and repair. J Am Acad Orthop Surg 2000;8:243–252. 4. Ngeow WC. Scar less: A review of methods of scar reduction at sites of peripheral nerve repair. Oral Surg Oral Med Oral Pathol Oral Radiol Endodontol 2010;109:357–366. 5. Parrinello S, Napoli I, Ribeiro S, Wingfield Digby P, Fedorova M, Parkinson DB, Doddrell RD, Nakayama M, Adams RH, Lloyd AC. EphB signaling directs peripheral nerve regeneration through Sox2-dependent Schwann cell sorting. Cell 2010;143:145–155. 6. Fancy SP, Chan JR, Baranzini SE, Franklin RJ, Rowitch DH. Myelin regeneration: A recapitulation of development? Annu Rev Neurosci 2011;34:21–43. 7. Fricker FR, Antunes-Martins A, Galino J, Paramsothy R, La Russa F, Perkins J, Goldberg R, Brelstaff J, Zhu N, McMahon SB, Orengo C, Garratt AN, Birchmeier C, Bennett DL. Axonal neuregulin 1 is a rate limiting but not essential factor for nerve remyelination. Brain 2013;136:2279–2297. 8. Fricker FR, Lago N, Balarajah S, Tsantoulas C, Tanna S, Zhu N, Fageiry SK, Jenkins M, Garratt AN, Birchmeier C, Bennett DL. Axonally derived neuregulin-1 is required for remyelination and regeneration after nerve injury in adulthood. J Neurosci 2011;31: 3225–3233. 9. Stassart RM, Fledrich R, Velanac V, Brinkmann BG, Schwab MH, Meijer D, Sereda MW, Nave K. A role for Schwann cell-derived neuregulin-1 in remyelination. Nat Neurosci 2013;16:48–54. 10. Cao J, Sun C, Zhao H, Xiao Z, Chen B, Gao J, Zheng T, Wu W, Wu S, Wang J, Dai J. The use of laminin modified linear ordered collagen scaffolds loaded with laminin-binding ciliary neurotrophic factor for sciatic nerve regeneration in rats. Biomaterials 2011;32:3939–3948. 11. Lin HY, Liu F, Zhang C, Zhang Z, Kong Z, Zhang X, Hoffman RM. Characterization of nerve conduits seeded with neurons and Schwann cells derived from hair follicle neural crest stem cells. Tissue Eng A 2011;17:1691–1698. 12. Xue CB, Hu N, Gu Y, Yang Y, Liu Y, Liu J, Ding F, Gu X. Joint use of a chitosan/PLGA scaffold and MSCs to bridge an extra large gap in dog sciatic nerve. Neurorehabil Neural Repair 2012;26:96–106. 13. Sun F, Zhou K, Mi W, Qiu J. Combined use of decellularized allogeneic artery conduits with autologous transdifferentiated adipose-derived stem cells for facial nerve regeneration in rats. Biomaterials 2011;32:8118–8128. € ke A. Mechanisms of disease: What factors limit the success of 14. Ho peripheral nerve regeneration in humans? Nat Clin Pract Neurol 2006;2:448–454. 15. Schmidt CE, Leach JB. Neural tissue engineering: Strategies for repair and regeneration. Ann Rev Biomed Eng 2003;5:293–347. 16. Wang X, Luo E, Li Y, Hu J. Schwann-like mesenchymal stem cells within vein graft facilitate facial nerve regeneration and remyelination. Brain Res 2011;1383:71–80. 17. Knox CJ, Hohman MH, Kleiss IJ, Weinberg JS, Heaton JT, Hadlock TA. Facial nerve repair. Laryngoscope 2013;123:1618– 1621. 18. Moran LB, Graeber MB. The facial nerve axotomy model. Brain Res Rev 2004;44:154–178.
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19. Captier G, Canovas F, Bonnel F, Seignarbieux F. Organization and microscopic anatomy of the adult human facial nerve: Anatomical and histological basis for surgery. Plast Reconstr Surg 2005;115: 1457–1465. 20. Phillips CD, Bubash LA. The facial nerve: anatomy and common pathology. Semin Ultrasound CT MR 2002;23:202–217. 21. Amorim AA Jr, Amorim MJAAL, Santos CRO, Nery-Brito LT, Melo IFR, Gabriel RBL. Extratemporal fascicular study of facial nerve in human. Braz J Morphol Sci 2012;29:79–81. 22. Cao J, Xiao Z, Jin W, Chen B, Meng D, Ding W, Han S, Hou X, Zhu T, Yuan B, Wang J, Liang W, Dai J. Induction of rat facial nerve regeneration by functional collagen scaffolds. Biomaterials 2013;34:1302–1310. 23. Sasaki R, Watanabe Y, Yamato M, Aoki S, Okano T, Ando T. Surgical anatomy of the swine face. Lab Anim 2010;44:359–363. 24. Barcelos AS, Rodrigues AC, Silva MDP, Padovani CR. Inside-out vein graft and inside-out artery graft in rat sciatic nerve repair. Microsurgery 2003;23:66–71. 25. Lavasani M. Gehrmann S, Gharaibeh B, Clark KA, Kaufmann RA, ault B, Goitz RJ, Huard J. Venous graft-derived cells participate Pe in peripheral nerve regeneration. PLoS One 2011;6:e24801. 26. Nishiura Y, Brandt J, Nilsson A, Kanje M, Dahlin L. Addition of cultured Schwann cells to tendon autografts and freeze-thawed muscle grafts improves peripheral nerve regeneration. Tissue Eng 2004;10:157–164. 27. Okamoto H, Hata K, Kagami H, Okada K, Ito Y, Narita Y, Hirata H, Sekiya I, Otsuka T, Ueda M. Recovery process of sciatic nerve defect with novel bioabsorbable collagen tubes packed with collagen filaments in dogs. J Biomed Mater Res A 2010;92:859–868. 28. Chen YS, Hsieh CL, Tsai CC, Chen TH, Cheng WC, Hu CL, Yao CH. Peripheral nerve regeneration using silicone rubber chambers filled with collagen, laminin and fibronectin. Biomaterials 2000;21: 1541–1547. n-Sa nchez M, Rudra S, Mukhopadaya S, 29. Mukhatyar VJ, Salmero Barker TH, Garcıa AJ, Bellamkonda RV. Role of fibronectin in topographical guidance of neurite extension on electrospun fibers. Biomaterials 2011;32:3958–3968. 30. Lin H, Chen B, Wang B, Zhao Y, Sun W, Dai J. Novel nerve guidance material prepared from bovine aponeurosis. J Biomed Mater Res A 2006;79:591–598. 31. Cerri F, Salvatore L, Memon D, Martinelli Boneschi F, Madaghiele M, Brambilla P, Del Carro U, Taveggia C, Riva N, Trimarco A, Lopez ID, Comi G, Pluchino S, Martino G, Sannino A, Quattrini A. Peripheral nerve morphogenesis induced by scaffold micropatterning. Biomaterials 2014;35:4035–4045. 32. Matsumoto K, Ohnishi K, Kiyotani T, Sekine T, Ueda H, Nakamura T, Endo K, Shimizu Y. Peripheral nerve regeneration across an 80mm gap bridged by a polyglycolic acid (PGA)–collagen tube filled with laminin-coated collagen fibers: A histological and electrophysiological evaluation of regenerated nerves. Brain Res 2000; 868:315–328. 33. Yao L, de Ruiter GC, Wang H, Knight AM, Spinner RJ, Yaszemski MJ, Windebank AJ, Pandit A. Controlling dispersion of axonal regeneration using a multichannel collagen nerve conduit. Biomaterials 2010;31:5789–5797.
FACIAL NERVE REPAIR IN MINI-PIGS BY COLLAGEN SCAFFOLDS WITH CNTF
Department of VIP Service, School of Stomatology, Capital Medical University, Beijing 10050, China State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 3 Nanyitiao, Zhongguancun, Beijing 100190, China 3 Graduate School, Chinese Academy of Sciences, Beijing 100190, China 4 Reproductive and Genetic Center of National Research Institute for Family Planning, Beijing 100081, China 2
Received 20 June 2014; revised 28 July 2014; accepted 31 July 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35305 Abstract: The preclinical studies using animal models play a very important role in the evaluation of facial nerve regeneration. Good models need to recapitulate the distance and time for axons to regenerate in humans. Compared with the most used rodent animals, the structure of facial nerve in mini-pigs shares more similarities with humans in microanatomy. To evaluate the feasibility of repairing facial nerve defects by collagen scaffolds combined with ciliary neurotrophic factor (CNTF), 10-mm-long gaps were made in the buccal branch of mini-pigs’ facial nerve. Three months after surgery, electrophysiological assessment and histological examination were performed to evaluate facial nerve regeneration. Immunohistochemistry
and transmission electron microscope observation showed that collagen scaffolds with collagen binding (CBD)-CNTF could promote better axon regeneration, Schwann cell migration, and remyelination at the site of implant device than using scaffolds alone. Electrophysiological assessment also showed higher recovery rate in the CNTF group. In summary, combination of collagen scaffolds and CBD-CNTF showed promising effects on facial nerve regeneration in mini-pig C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: models. V 00A:000–000, 2014.
Key Words: facial nerve regeneration, collagen scaffold, ciliaryneurotrophic factor, mini-pig
How to cite this article: Lu C, Meng D, Cao J, Xiao Z, Cui Y, Fan J, Cui X, Chen B, Yao Y, Zhang Z, Ma J, Pan J, Dai J. 2014. Collagen scaffolds combined with collagen-binding ciliary neurotrophic factor facilitate facial nerve repair in mini-pigs. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
Facial nerve injuries caused by infection, tumor excision, traffic accident, and war trauma could lead to facial paralysis which are both physiologic and mental burden for the patients.1 After the injury, the distal segment of the facial nerve went through Wallerian degeneration completely, whereas the proximal stump underwent only a limited extent degeneration as far as to the first node of Ranvier.2,3 Afterwards, the proximal stump developed excess axonal sprouts than original to maximize the chances of target reinnervation.4 During regeneration, Schwann cells and fibroblasts from both ends proliferate and migrate out of the nerve stumps to guide sprouting axons across the wound.5 In the late stage of the regeneration, the axon/
Schwann interaction provides signals for Schwann cells to redifferentiate and remyelinate the newly formed axons.6–10 Among facial nerve trauma, transection is a more severe injury, which breaks down the structural continuity of facial nerve. Transection, which leaves relatively long gaps, needs to be bridged by grafts or nerve conduits.11–13 The structural and functional defects of facial nerve need to be recovered by good axonal regeneration and remyelination, and the targeted muscles need to be correctly reinnervated. To achieve these goals, at least four basic elements need to be concerned: (1) Protecting neurons from apoptosis. (2) Building up an optimal environment for axonal regeneration. (3) Enhancing the regeneration ability of axons. (4) Guiding axons to regenerate toward their original targets.14,15
*These authors contributed equally to this work. Correspondence to: Juli Pan, Department of VIP Service, Beijing Stomatological Hospital, Capital Medical University, Beijing 100050, China; e-mail: [email protected] and Jianwu Dai, State key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 3 Nanyitiao, Zhongguancun, Beijing 100190, China Contract grant sponsor: The “Strategic Priority Research Program of the Chinese Academy of Sciences”; contract grant number: XDA01030000 Contract grant sponsor: National High Technology Research and Development Program (“863” Program) of China; contract grant number: 2012AA020501 Contract grant sponsor: National Nature Science Foundation; contract grant number: 81372086
C 2014 WILEY PERIODICALS, INC. V
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FIGURE 1. The collagen scaffolds used for facial nerve repair and surgery procedure. A: The longitudinal view of a collagen tube. B: Image of the liner-order collagen fibers used for nerve guidance. C: Gross view obtained immediately after the scaffolds implanted at the injury site. D: Regenerated nerve obtained 3 months after the surgery. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
So far, most researchers used either rats or rabbits as injury models to test their strategies.16–18 But there are still many differences in facial nerve between rodent animals and human beings.14 For the extratemporal part of human facial nerve, the perifascicular, interfascicular, and intrafascicular connective tissue can occupy almost half of the whole cross-section with an average of 14 nerve fascicles disperse in it.19–21 But in rodent animals, there are few fascicles (usually one major fascicles accompanied with some small ones) with scarce connective tissue surrounding them as described previously.22 Besides, in rodent models, regeneration occurs over a relatively short distance compared with that in humans.14 The previous studies implied that mini-pigs are a suitable experimental model for the preclinical research in facial nerve repair, for pig facial nerve has very similar anatomic structures with that of humans, which allows long gaps model as well.23 Although autologous grafts (such as nerve, artery, vein, epineurial sheaths, and tendon) or allografts processed by immunosuppression or decellularization have exhibited encouraging results in peripheral nerve repair,15,24–26 their drawbacks of sacrificing donor tissue and ill-fitness in diameter and length limited their clinical uses. Thus, nerve conduits made of or modified with extracellular matrix such as collagen, laminin, and fibronectin become candidates to substitute tissue grafts.10,27–29
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It has been demonstrated in our previous study that collagen conduits filled with linear-ordered collagen (LOC) fibers (which had adsorbed collagen-binding ciliary neurotrophic factor [CBD-CNTF] and brain-derived neurotrophic factor [CBD-BDNF]) could promote facial nerve regeneration in a 4-mm rat facial nerve trunk gap model.22 To test whether this strategy could also achieve promising results in longer gap models in larger animals, 10-mm gap in the buccal branch (BB) of mini-pigs’ facial nerve was created and the structural and functional recovery indexes were examined. METHODS AND MATERIALS
Preparation of collagen nerve conduit The collagen nerve conduits [Fig. 1(A)] were prepared as described previously and cut into 10-mm long before use.22 In brief, 10-mm-long uniform conduits, with a 4.5-mm lumen and 1-mm wall thickness, were made by scrolling collagen membrane around glass molds and let them airdried. Then, they were treated overnight with crosslinking solution (30 mM 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide and 10 mM N-hydroxysuccin-imide in 50 mM 2-(Nmorpholino)ethanesulfonic acid solution, pH 5.5) at room temperature. The crosslinking agent was washed with NaH2PO4 (0.1M) and phosphate-buffered saline (PBS) for at least 10 times each. Finally, the collagen conduits were
FACIAL NERVE REPAIR IN MINI-PIGS BY COLLAGEN SCAFFOLDS WITH CNTF
ORIGINAL ARTICLE
FIGURE 2. HE staining of the facial nerve. A: Transverse section of a normal nerve. A bundle of axons (black arrowhead) was wrapped by perineurium (black arrow). Several axon fascicles together with connective tissue and blood vessels (white arrowhead) are surrounded by epineurium (white arrow). Scale bar: 500 mm. Longitudinal section showed a normal axon fascicle in (B) and the regenerated nerve in the PBS (C) and CBD-CNTF (D) group. Scale bar: 50 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
removed from the molds and freeze dried. The conduits were sterilized by radiation using 8-K gray before used in the animal experiments. Production of LOC fibers and CBD-CNTF As described previously,22 LOC fibers were prepared from bovine aponeurosis [Fig. 1(B)].30 The recombined protein CBD-CNTF-his6 was expressed in Escherichia coli, purified by Nickel chelating Sepharose (Amersham Biosciences, Piscataway, NJ). The purity of CBD-CNTF was confirmed by SDS-PAGE followed by Coomassie Brilliant Blue staining, whereas the identity was confirmed by Western blotting with anti-his6 primary antibody. The bioactivity of CBDCNTF was tested by 3-(4,5-dimethylthiazol-2-yl)22,5-diphenyltetrazolium bromide of dorsal nerve ganglia cell culture.10 The protein was lyophilized and stored at 280 C. Before the start of the experiment, CBD-CNTF was dissolved in ddH2O, and a bundle of LOC fibers (occupied about half lumen space of the collagen conduit) was incubated with 100 mg of CBD-CNTF (in 100 mL) for 30 min at 37 C and then inserted into the conduit. Surgical procedures of facial nerve transection Experiments were performed in accordance with the US National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals published by the US National Academy of Sciences (http://oacu.od.nih.gov/regs/index. htm) and approved by Animal Care and Use Committee, Beijing, China. Twelve female mini-pigs of 13 months old (weight, 35–40 kg) were anesthetized by intramuscularly injection of ketamine (10 mg/kg) and xylazine (2 mg/kg). After sterilization, an incision of 3–5 cm was made subcutaneously to expose the trunk of the right BB of facial nerve.
The trunk of buccal facial nerve is 4 cm long in average before branching. The 12 mini-pigs are randomly divided into two groups (n 5 6/each): (1) CNTF group, a segment of 8-mm nerve was removed by sharp microsurgery scissors, leaving a 10-mm gap after retraction of the nerve ends. The proximal and distal nerve stumps were bridged by 10-mm collagen conduit, the conduit was packed with LOC fibers which had been incubated with CNTF as described above. Four 9/0 monofilament nylon interrupted epineurium sutures were applied at each end of the anastomosis [Fig. 1(C)]. Another 100 mg of CBD-CNTF was injected into the proximal and distal nerve stumps with 50 mg at each end. (2) PBS group (control): equal volume of PBS was applied instead of CBD-CNTF. The mini-pigs were applied with penicillin after 3 days of the operation. One month later, the wound was reopened and 200 mg of CBD-CNTF was injected to the gap site in the CNTF group, whereas equal volume of PBS was injected in the PBS group. Gross observation The nerve conduit degradation, as well as surrounding tissue and regenerating nerve, was examined by gross 3 months after the operation in all animals. Histological analysis and transmission electron microscope observation The histological assessments were performed 3 months after operation. In each group, half of the nerve samples (randomly chosen from three mini-pigs) were used for transversal study and the rest for longitudinal study and transmission electron microscope (TEM) observation. The regenerated nerves were isolated and divided into proximal and distal segments (5 mm each), and fixed in 4% v/v of
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FIGURE 3. Transverse section of the regenerated nerve stained with NF body. A: (a1, a2) Represent the transections of a normal facial nerve. The regenerated nerve of the PBS group (b1, b2, d1, d2) and CNTF group (c1, c2, e1, e2) were divided into proximal segments (b1, b2, c1, c2) and distal segments (d1, d2, e1, e2) to illustrate the regeneration process. The magnifications of selected areas in the left panel (a1–e1) are shown in the right-hand side (a2–e2). Scale bar: 100 mm. B,C: The statistical analysis of NF-positive area. n 5 3, *p < 0.05. Nor: normal. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
formaldehyde for 48 h. Then, the segments were embedded in paraffin. Sections of 5 mm in thickness were cut from each segment and examined by immunohistochemistry using antibodies to neurofilament (anti-NF, 1:1000 dilution; Abcam) and S100 (1:2,000 dilution; Sigma). Using ImagePro Plus software (Media Cybernetics), the percentage of NF-positive and S100-positive area (positive staining area/ total area) was quantified at a magnification of 2003. The sections were also stained by Luxol fast blue (Solvent blue 38; S3382, Sigma) according to a previously published protocol10 to give an overall view of remyelinated axons. The ultrastructure of newly formed myelin was observed by TEM at a magnification of 40003. The number, diameter, and the thickness of the newborn myelin sheaths were evaluated by Image-Pro Plus software (Media Cybernetics). Electrophysiological assessment As for electrophysiological tests, the regenerated facial nerve was re-exposed under general anesthesia at 3 months postsurgery. The electrophysiological test was evaluated by an electromyography system (RM6240, Chengdu, China). The stimulating electrode was placed in the proximal end of
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buccal facial nerve, two recording electrodes were placed upstream (R1) and downstream (R2) of the regenerated segment, and the reference electrode was inserted into the ipsilateral fore limb subcutaneously. The ratio of action potential recorded by R2 divided by that recorded by R1 was used to evaluate the facial nerve functional recovery. Statistical analysis The Graphpad Prism 5 was used for the statistical analysis. All values are presented as mean 6 standard deviation. Comparisons between PBS and CNTF groups were performed using Student’s t-test. The p-value of 1 cm. There are still some improvements needed to be made in addition to electrophysiological evaluation to the min-pig facial nerve model, such as functional evaluation in mini-pig. CONCLUSIONS
In conclusion, a 10-mm-long gap model in mini-pigs’ facial nerve was used in this study to test the effectiveness of CBD-CNTF-modified collagen scaffolds in nerve regeneration. The collagen scaffolds facilitated oriented Schwann cell migration and axon regeneration, whereas the application of CBD-CNTF promotes axon sprout and myelin formation, indicating that our CBD-CNTF-modified collagen scaffolds may repair 10-mm injury in the facial nerve. REFERENCES 1. Sun F, Zhou K, Mi W, Qiu J. Repair of facial nerve defects with decellularized artery allografts containing autologous adiposederived stem cells in a rat model. Neurosci Lett 2011;499:104–108. 2. Boivin A, Pineau I, Barrette B, Filali M, Vallie`res N, Rivest S, Lacroix S. Toll-like receptor signaling is critical for Wallerian degeneration and functional recovery after peripheral nerve injury. J Neurosci 2007;27:12565–12576.
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FACIAL NERVE REPAIR IN MINI-PIGS BY COLLAGEN SCAFFOLDS WITH CNTF
