Cell Transplantation, Vol. 22, pp. 2029–2039, 2013 Printed in the USA. All rights reserved. Copyright 2013 Cognizant Comm. Corp.
0963-6897/13 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368912X658953 E-ISSN 1555-3892 www.cognizantcommunication.com
Sciatic Nerve Regeneration by Cocultured Schwann Cells and Stem Cells on Microporous Nerve Conduits Lien-Guo Dai,*† Guo-Shiang Huang,‡ and Shan-hui Hsu‡§¶ *Department of Chemical Engineering, National Chung Hsing University, Taichung, Taiwan †Department of Orthopedics, Kuang Tien General Hospital, Taichung, Taiwan ‡Institute of Polymer Science and Engineering, National Taiwan University, Taipei, Taiwan §Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan ¶Institute of Biomedical Engineering, National Chung Hsing University, Taichung, Taiwan
Cell transplantation is a useful therapy for treating peripheral nerve injuries. The clinical use of Schwann cells (SCs), however, is limited because of their limited availability. An emerging solution to promote nerve regeneration is to apply injured nerves with stem cells derived from various tissues. In this study, different types of allogeneic cells including SCs, adipose-derived adult stem cells (ASCs), dental pulp stem cells (DPSCs), and the combination of SCs with ASCs or DPSCs were seeded on nerve conduits to test their efficacy in repairing a 15-mm-long critical gap defect of rat sciatic nerve. The regeneration capacity and functional recovery were evaluated by the histological staining, electrophysiology, walking track, and functional gait analysis after 8 weeks of implantation. An in vitro study was also performed to verify if the combination of cells led to synergistic neurotrophic effects (NGF, BDNF, and GDNF). Experimental rats receiving conduits seeded with a combination of SCs and ASCs had the greatest functional recovery, as evaluated by the walking track, functional gait, nerve conduction velocity (NCV), and histological analysis. Conduits seeded with cells were always superior to the blank conduits without cells. Regarding NCV and the number of blood vessels, conduits seeded with SCs and DPSCs exhibited better values than those seeded with DPSCs only. Results from the in vitro study confirmed the synergistic NGF production from the coculture of SCs and ASCs. It was concluded that coculture of SCs with ASCs or DPSCs in a conduit promoted peripheral nerve regeneration over a critical gap defect. Key words: Schwann cells (SCs); Adipose-derived adult stem cells (ASCs); Dental pulp stem cells (DPSCs); Peripheral nerve injury; Nerve regeneration; Conduits
INTRODUCTION Peripheral nerve injuries, often caused by trauma, are treated extensively under surgical intervention. The surgical technique named as “gold standard” refers to the use of autologous nerve in bridging a critical gap defect but still has some drawbacks such as the secondary surgery, painful neuroma formation, and difficulty in obtaining nerve of suitable size from patients (2,4,36). Besides, the nerve functionality may not be completely recovered due to several factors, including the loss of targeted tissue or the longer nerve gap. Schwann cells (SCs) can participate in remyelination of the bridged nerves (5). These cells have been demonstrated to promote regrowth of injured axons and to cause the functional recovery based on animal models. The addition of exogenous SCs can also guide the axonal regrowth across the nerve gap (11,31). Although transplantation of SCs has been considered as a biologically effective
strategy (16,17), it also has disadvantages such as the limited expansion potential and lengthy culture times of SCs as well as the possible morbidity associated with nerve harvested to obtain a sufficient number of SCs for transplantation. Therefore, a number of stem cells have been explored as alternatives for use in nerve regeneration (27). Adipose-derived stem cells (ASCs) have been shown to successfully differentiate into neuronal lineages and SCs in vitro and may be of benefit for treatment of peripheral nerve injuries (13,24,51). This may be also related to the fact that a number of nerve growth factors, including insulinlike growth factor (IGF) and fibroblast growth factor (FGF), are secreted by ASCs (37,55). Dental pulp consists of ecto-mesenchymal components, containing neural crestderived cells, which display plasticity and multipotential capabilities (43). Dental pulp stem cells (DPSCs) also have the ability to differentiate into neuronal-like cells, which makes them a potential alternative for the treatment
Received April 3, 2012; final acceptance September 7, 2012. Online prepub date: November 27, 2012. Address correspondence to Shan-hui Hsu, Institute of Polymer Science and Engineering, National Taiwan University, No. 1, Sec. 4 Roosevelt Road, Taipei 10617, Taiwan, R.O.C. Tel: +886-2-33665313; Fax: +886-2-33665237; E-mail:
[email protected]
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of neurological disease (3,22,29,30). Like ASCs, DPSCs also produce neurotrophic factors, including nerve growth factor (NGF), glial cell line–derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF) (32). Recently, the coculture system of SCs and stem cells was also reported. The coculture of SCs and ASCs induced the ASCs to differentiate into Schwann-like cells in vitro, which may help create a microenvironment that promotes nerve regeneration (49). However, there is little study regarding the synergistic effect of SCs and stem cells on peripheral nerve regeneration in vivo. Artificial nerve conduits have been used to replace autografts in the peripheral nerve surgery. Poly(d,l-lactide) (PLA) is easily handled with proper biodegradation rate and a low inflammatory response (46). Novel microporous PLA conduits were previously developed to promote peripheral nerve regeneration (20,21). In this study, the candidate cells including SCs, ASCs, DPSCs, and the combination of SCs with ASCs or DPSCs were analyzed in vitro for neurotrophic gene expression by RT-PCR at 0, 3, and 7 days and protein expression by ELISA at 7 days. The ability of cell-seeded conduits to regenerate rat sciatic nerve over a 15-mm-long defect was evaluated. Histological staining, functional gait analysis, and electrophysiology were used to contrast the effects on nerve regeneration from different types of cells. MATERIALS AND METHODS Isolation and Culture of ASCs All protocols involving the use of experimental animals were approved by the institutional review board. Isolation of rat ASCs followed that described in literature (6). ASCs were extracted from the subcutaneous fat positioned at the hind leg and side abdominal region of two female Sprague– Dawley rats (weight from 350 to 500 g) purchased from the National Laboratory Animal Center (Taipei, Taiwan). The adipose tissues were minced and added with 200 U/ml type I collagenase (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS; Invitrogen, Carlsbad, CA, USA) for degradation at 37°C with gentle agitation for 30 min. Following mechanical and enzymatic treatment, the tissues were homogenized in the medium containing low-glucose Dulbecco’s modified Eagle’s medium/ Ham’s F12 (1:1) (Gibco, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco). After homogenization, cells were cultured in an incubator (37°C/5% CO2, 95% relative humidity). The culture medium was refreshed two times every week. Cells of the 2nd to 5th passages were used in this study. Isolation and Culture of DPSCs Rat DPSCs were isolated according to a method modified from a previous study (54). Rat dental pulp was obtained
from the mandibular incisors using two female 2-weekold neonatal Sprague–Dawley rats purchased from the National Laboratory Animal Center and washed two times with calcium- and magnesium-free PBS (Gibco). The teeth were cut into several pieces and incubated with gentle shaking at 37°C for 60 min in 3 ml of a sterile enzyme solution containing 0.3% collagenase type IA (Sigma) and dipase II (Roche, Basel, Switzerland) in PBS. The digested medium containing DPSCs was filtered by a 40-mM mesh (BD Falcon, Becton-Dickinson, Heidelberg, Germany) and washed with PBS three times. The cell pellets were resuspended with Eagle’s a-minimal essential medium (a-MEM) (Invitrogen) supplemented with 10% FBS and 100 U/ml penicillin– streptomycin, and cultured in T75 flasks (BD Falcon) in the incubator. The culture medium was refreshed every 2 days. Cells of the 2nd to 5th passages were used in this study. Culture of SCs Rat SCs derived from culture of rat primary cells (RSC96, ATCC: CRL-2765, Manassas, VA, USA) were purchased and cultured in T75 flasks with Dulbecco’s modified Eagle’s medium (containing 4 mM l-glutamine, 1.5 g/L sodium bicarbonate, and 4.5 g/L glucose; Gibco), supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FBS in an incubator. The culture medium was refreshed every 2 days. Cells of the 2nd to 5th passages were used in this study. Analysis of Surface Markers and Cell Morphology The surface markers for ASCs and DPSCs were identified by flow cytometric analysis of cluster of differentiation 29 (CD29), CD31, CD34, CD44, CD45, CD105 (all from BioLegend, San Diego, CA, USA), CD73 (BD Pharmingen, San Diego, CA, USA), and CD90 (Serotec, Raleigh, NC, USA) antibodies. A total of 5 ´ 105 cells were used and washed twice with PBS supplemented with 1% FBS. Cells were then resuspended in 100 ml of PBS/1% FBS and incubated with monoclonal antibodies for 30 min at 4°C, before further washing with PBS. Fluorescein iso thiocyanate (FITC)-conjugated CD29 (CD29-FITC), phycoerythrin (PE)-conjugated CD31 (CD31-PE), CD44-FITC, and CD90-PE were operated for direct staining; mouse monoclonal anti-CD34 (Santa Cruz Biotechnology), mouse monoclonal anti-CD45 (Santa Cruz Biotechnology), mouse monoclonal anti-CD73 (BD Biosciences), and mouse monoclonal anti-CD105 (BD Biosciences) were purchased for indirect staining. The corresponding isotypic antibodies including mIgG1-FITC and mIgG1-PE (Serotec) were used for direct staining. Secondary antibodies applied in this study were FITC-conjugated goat-anti-mouse antibody (Chemicon). The final concentration was the same between the isotype controls and test antibodies. Fluorescence intensities were determined by a flow cytometer (FACSCalibur, BD, Franklin Lakes, NJ, USA).
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Additionally, the cellular morphology of SCs, ASCs, and DPSCs was observed using an inverted optical microscope (Leica DMIRB, Wetzlar, Germany). Coculture of SCs With ASCs or DPSCs SCs and ASCs (or DPSCs) at 1:1 ratio (1 ´ 104 cells/cm2) were mixed and seeded on six-well culture plates (Nunc, Penfield, NY, USA). SCs, ASCs, and DPSCs (2 ´ 104 cells/ cm2) were also seeded as controls. Cocultured cells were analyzed for neurotrophic gene expression by RT-PCR at 0, 3, and 7 days and protein expression by ELISA at 7 days. For RT-PCR, total RNA was extracted from cells using Trizol reagent (15596-018, Invitrogen, Bangalore, India) after the cells were trypsinized by 0.05% trypsin/EDTA (Gibco). Five micrograms of total RNA was used for reverse transcription with the first-strand cDNA synthesis kit (Fermentas, Waltham, MA, USA). One microliter of the cDNA reaction mixture was applied in each PCR reaction. The PCR reaction was performed using selective forward and reverse primers for b-actin (as an internal standard), GDNF, BDNF, and NGF. The sequences of the primers used were as follows: GDNF forward, 5¢-CCAGAGAAT TCCAGAGGGAAAGGTC-3¢; GDNF reverse, 5¢-CAG ATACATCCACACCGTTTAGCGG-3¢; BDNF forward, 5¢-AGTGGGCAAAGGAGCGG-3¢; BDNF reverse, 5¢-CG CTCATTCATTAGAATCACGT-3¢; NGF forward, 5¢-TCA CTGTGGACCCCAAACT-3¢; NGF reverse, 5¢-TCCTGT GAGTCCTGTTGAAGG-3¢; b-actin forward, 5¢-GTATG CCTCTGGTCGTACCA-3¢; b-actin reverse, 5¢-CTTCTG CATCCTGTCAGC AA-3¢. PCR was performed in a GeneAmp PCR system 2700 thermal cycler (Applied Biosystems, Foster City, CA, USA) for 35 cycles. Cycling parameters were 94°C for 30 s, 60°C (for BDNF and GDNF) or 55°C (for NGF and b-actin) for 30 s, and then 72°C for 30 s, followed by a final extension at 72°C for 7 min. b-Actin was used as an internal control for the semiquantitative analysis and to confirm the fidelity of the PCR reaction. The amplified products were determined by electrophoresis (Gelcompany, San Francisco, CA, USA) on 1.5% agarose-TAE [10 mM Tris (pH 7.5), 5.7% glacial acetic acid, and 1 mM EDTA] gels (Roche) and quantified by ethidium bromide (Sigma) staining. For ELISA, the medium from each well was collected and analyzed using the ChemiKine sandwich ELISA kits (Chemicon, Chandlers Ford, Hampshire, UK) for NGF and Emax immunoassay systems (Promega, Madison, WI, USA) for GDNF and BDNF, according to the manufacturer’s protocols. All samples were analyzed in triplicate, and the absorbance was measured at 450 nm (Hitachi F2500, Tokyo, Japan). Fabrication of Nerve Conduits Nerve conduits were fabricated as previously described (20,21). PLA (8300D, Cargill, Minneapolis, MN, USA)
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was dissolved in 1,4-dioxane solvent (10% concentration; Qiagen, Germantown, MD, USA) and cast on a dish. The dish (Phytotechnology Laboratories, Shawnee Mission, KS, USA) was then put in 40% alcohol for 24 h, replaced in water for 8 h, and dried in a 40°C oven for 24 h to remove any organic solvent residue. The cast membranes were rolled into conduits by a 1.5-mm-diameter mandrel, and the lateral ends were adhered tightly by a small amount of 1,4-dioxane solvent. Animal Surgery Prior to surgery, cells were seeded onto the inner wall of the conduits dynamically. A total of 1 ´ 106 cells were filled into conduits and sealed at both ends with agarose plugs (Roche). The constructs were placed on a roller that rotated at a fixed speed (0.16 rpm) at 37°C for 3 days (12), which enabled uniform attachment of cells on the lumen surface of the conduits. In the cocultured groups, each type of cells at half the number was seeded. Thirty adult male Sprague– Dawley rats (250–300 g), purchased from the National Laboratory Animal Center, were divided into six groups. Group 1 received blank conduits (without cells). Group 2 received conduits with SCs. Group 3 received conduits with ASCs. Group 4 received conduits with DPSCs. Group 5 received conduits with ASCs and SCs. Group 6 received conduits with DPSCs and SCs. Rats were anesthetized with isoflurane (Baxter, Deerfield, IL, USA) during the surgery. The sciatic nerve was exposed by skin and muscle splitting incision. The left sciatic nerve was cut and removed near the obturator tendon in midthigh with the aid of an operation microscope. A 17-mm nerve conduit (1.53-mm inner diameter, 0.21-mm thickness) was used to bridge the 15-mm-long nerve gap. The conduit ends were left 1 mm for anchoring the proximal and distal nerve stumps by 7-0 nylon microsutures (UNIK Surgical Sutures Mfg. Co., Taipei, Taiwan). The wound was subsequently closed in layers using 2-0 Dexon sutures (UNIK) after surgical treatment. The animals were given food and water ad libitum and sacrificed at 8 weeks after surgery. The time schedule allowed sufficient time for nerve regeneration with respect to the length of the gap. Walking Track Analysis The walking track was recorded on all animals weekly before the animals were sacrificed at 8 weeks. Before the experiment, animals were trained to walk down in a 150 ´ 8-cm track set on a darkened enclosure. The sciatic functional index (SFI) used in evaluating the functional muscle reinnervation was obtained based on the walking track analysis (18). The equation was expressed as SFI = -38.3 (PLF) + 109.5 (TSF) + 13.3 (ITF) - 8.8, where PLF (print length function) = (experimental PL - normal PL)/normal PL; TSF (toe spread function) = (experimental
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TS - normal TS)/normal TS (first to fifth toe); and ITF (intermedian toe spread function) = (experimental IT - normal IT)/normal IT (second to fourth toe). Functional Gait Analysis The procedure for functional gait analysis followed the protocol described previously (34). First, digital videos of experimental rats were captured weekly from 1 to 8 weeks after sciatic nerve surgery. The photographs of gait cycle of walking movement were recorded from a confining walkway until the steady and continuous walking patterns were obtained for tested rats. Each individual frame was further analyzed by the Ulead Video Software (Torrance, CA, USA). The terminal stance phase angle of the rat gait cycle was calculated as the angle (in degrees) of the intersection of the lines extending from the knee to the ankle joint and from the ankle joint to the metatarsal head detected from the images. The angle was used as an index for the functional gait analysis. Electrophysiological Examination Rats were anesthetized by sodium pentobarbital (4%, 40 mg/kg; Sigma-Aldrich), and the regenerated sciatic nerves were carefully exposed at 8 weeks postimplantation. The nerve conduction velocity (NCV) was measured using a recording system (ADInstruments Pty. Ltd., Castle Hill, NSW, Australia), where the associated signals were recorded by the software for data capture and analysis. The NCV across the regenerated nerve was calculated by evoking the compound action potential and dividing the distance between electrodes by conduction latency (8). Histological Analysis The tissues of regenerative sciatic nerve were harvested after surgery and immediately fixed in cold 3% glutaraldehyde solution (Amresco, Solon, OH, USA). After 2 days, the nerve conduits were transected at medium segment. For successful connection of sciatic nerve in a conduit, a thin white tubular substance that connected the two anastomoses ends was observed. The success rate was determined for each group. Tissue samples with conduits were then washed in PBS. They were then postfixed in 1% osmium tetroxide (Polysciences, Warrington, PA, USA), dehydrated in the gradient concentrations of ethanol solutions, and finally embedded. The embedded samples were sliced into 3-mm-thick sections and then stained with 1% toluidine blue (Amresco), which did not stain the conduits. All sliced sections were observed under an optical microscope, and photographs were taken using a digital camera (Nikon H666L, Tokyo, Japan). The cross-sectional area of the regenerated nerve, as well as the number of myelinated axons and blood vessels, was calculated using an image analysis system (Image-Pro Lite, Media Cybernetics, Silver Spring, MD, USA).
Figure 1. Neurotrophic gene expression of the five different cell culture groups. The gene expressions of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF) from five cultures including Schwann cell (SCs), adipose-derived stem cells (ASCs), dental pulp stem cells (DPSCs), SC + ASC, and SC + DPSC determined by PCR amplification after 0, 3, and 7 days of incubation. The experiments were conducted in triplicate. *p conduits without cells. Nerve Conduction The average maximum values of NCV, derived from each group, are demonstrated in Figure 5. The normal sciatic nerve had significantly greater value of maximum NCV compared with the injured nerve (p SC + DPSC ~ ASC > SC ~ DPSC > conduits without cells (Fig. 7C). DISCUSSION The migration and proliferation of SCs play a crucial role in the cellular phase of successful nerve repair (11,53). SCs can secrete neurotrophic factors, which is important for nerve regeneration (1,42). SCs, however, are not easy to obtain for adequate amount of cells, and their efficacy in bridging a longer gap may not be as satisfactory. Therefore, stem cell-based therapies are considered as an alternative for repairing the long nerve defect (44). We compared in this study two stem cells and two combinations of SCs with stem cells in bridging sciatic nerve injury over a critical gap by the use of nerve conduits. From the success rate of nerve connection, electrophysiology,
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Figure 6. Histology of the nerve sections at the mid-conduit after 8 weeks of implantation. (A) Conduit, (B) SC, (C) ASC, (D) DPSC, (E) SC + ASC, and (F) SC +DPSC. For abbreviations, refer to Figure 3. Scale bar: 100 mm.
functional recovery, and histological analysis, conduits seeded with cells had better performance than the blank conduits. The addition of either SCs or combination of SCs with stem cells (half the number each) was beneficial for peripheral nerve repair, although the effect from SCs alone was not as remarkable. SCs and stem cells may help create a microenviroment that promoted axon extension gap. Previous studies showed that ASCs could differentiate into neuron-like cells and SC-like cells in vitro (38,51). ASCs can secrete a few nerve growth factors such as IGF and FGF (55). ASCs can be differentiated into cells with a Schwannlike phenotype that benefited neurite extension (24). ASCs in Matrigel were reported to enhance the regeneration of nerves and induce axon growth in sciatic nerve defects (15,26). On the other hand, the differentiation of DPSCs
into neuron-like, SC-like, or endothelial cell-like cells was also reported in literature (14,23,41). DPSCs from both rats and humans expressed NGF, BDNF, and GDNF genes in vitro (32). The paracrine effect as well as the differentiation possibility of the implanted stem cells may contribute to nerve regeneration. DPSCs also promoted facial nerve regeneration in vivo (40). The positive effect of SCs has often been reported in literature. In our study, conduits cocultured with SCs and ASCs were superior to other groups including SC-seeded conduits. On the other hand, functional recovery based on walking track and functional gait analyses showed that rats receiving conduits seeded with SCs, ASCs, DPSCs alone, or SCs plus DPSCs had similar performance with no statistical difference among the groups. Silicone tubes
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Figure 7. Quantitative data from histological analyses of the transplant area. (A) Area of regenerated nerve; (B) number of myelinated axons; (C) number of blood vessels. For abbreviations, refer to Figure 3. *p
