Functional recoveries of sciatic nerve regeneration by combining chitosan-coated conduit and neurosphere cells induced from adipose-derived stem cells

Biomaterials xxx (2013) 1e11

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Functional recoveries of sciatic nerve regeneration by combining chitosan-coated conduit and neurosphere cells induced from adiposederived stem cells Yuan-Yu Hsueh a, b, Ya-Ju Chang c, d, Tzu-Chieh Huang e, Shih-Chen Fan f, Duo-Hsiang Wang a, Jia-Jin Jason Chen g, h, Chia-Ching Wu c, d, g, h, *, Sheng-Che Lin a, ** a Division of Plastic Surgery, Department of Surgery, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, No. 1, University Rd., Tainan 701, Taiwan b Institute of Clinical Medicine, National Cheng Kung University, No. 1, University Rd., Tainan 701, Taiwan c Department of Cell Biology and Anatomy, National Cheng Kung University, No. 1, University Rd., Tainan 701, Taiwan d Institute of Basic Medical Sciences, National Cheng Kung University, No. 1, University Rd., Tainan 701, Taiwan e Department of Occupational Therapy, National Cheng Kung University, No. 1, University Rd., Tainan 701, Taiwan f Department of Occupational Therapy, I-Shou University, No.1, Sec.1, Syuecheng Rd., Dashu District, Kaouhsiung City 84001, Taiwan g Department of Biomedical Engineering, National Cheng Kung University, No. 1, University Rd., Tainan 701, Taiwan h Medical Device Innovation Center, National Cheng Kung University, No. 1, University Rd., Tainan 701, Taiwan

a r t i c l e i n f o

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Article history: Received 18 September 2013 Accepted 27 November 2013 Available online xxx

Suboptimal repair occurs in a peripheral nerve gap, which can be partially restored by bridging the gap with various biosynthetic conduits or cell-based therapy. In this study, we developed a combination of chitosan coating approach to induce neurosphere cells from human adipose-derived stem cells (ASCs) on chitosan-coated plate and then applied these cells to the interior of a chitosan-coated silicone tube to bridge a 10-mm gap in a rat sciatic nerve. Myelin sheath degeneration and glial scar formation were discovered in the nerve bridged by the silicone conduit. By using a single treatment of chitosan-coated conduit or neurosphere cell therapy, the nerve gap was partially recovered after 6 weeks of surgery. Substantial improvements in nerve regeneration were achieved by combining neurosphere cells and chitosan-coated conduit based on the increase of myelinated axons density and myelin thickness, gastrocnemius muscle weight and muscle fiber diameter, and step and stride lengths from gait analysis. High expressions of interleukin-1b and leukotriene B4 receptor 1 in the intra-neural scarring caused by using silicone conduits revealed that the inflammatory mechanism can be inhibited when the conduit is coated with chitosan. This study demonstrated that the chitosan-coated surface performs multiple functions that can be used to induce neurosphere cells from ASCs and to facilitate nerve regeneration in combination with a cells-assisted coated conduit. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Chitosan Sciatic nerve injury Nerve conduit Adipose-derived stem cell Spheroid Neurosphere

1. Introduction Despite advances in neuroscience and microsurgery, the process of repairing peripheral nerve injuries remains suboptimal, particularly when a nerve gap is present [1]. After the injury occurs, the distal stump undergoes axon demyelination and degradation, causing the atrophy of effector muscles and the impairment of normal functions, such as gait [2]. The most widely used technique

* Corresponding author. Tel.: þ886 6 2353535x5327; fax: þ886 6 2093007. ** Corresponding author. Tel.: þ886 6 2673861; fax: þ886 6 3366361. E-mail addresses: [email protected] (C.-C. Wu), [email protected]. tw (S.-C. Lin).

for bridging the gap in an injured nerve is using an autologous nerve graft to guide the regenerating nerve [3]. However, the functional results of using a nerve graft are variable and the lack of a donor nerve also impedes the application of the nerve graft in a clinical setting [4]. Three types of conventional bridging material have been used in the past few decades. The first type is biological conduits such as blood vessels, skeletal muscles, and tendons, but the length of the conduit is limited because of problems related to tubular collapse, poor regeneration, scar tissue proliferation, and adhesion. The second type is nonbiologic synthetic conduits such as silicone tubes and nylon tubes. The results remain poor because that the cytotoxicity might cause a subsequent foreign body reaction and prolonged inflammation [5]. The third type is biodegradable ones including poly(lactide-co-glycolide), poly(phosphoester),

0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.11.081

Please cite this article in press as: Hsueh Y-Y, et al., Functional recoveries of sciatic nerve regeneration by combining chitosan-coated conduit and neurosphere cells induced from adipose-derived stem cells, Biomaterials (2013), http://dx.doi.org/10.1016/j.biomaterials.2013.11.081

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polyurethanes, chitosan, and decellularized biomatrices. The controllable degradation can be tailored by altering their molecular weight and composition. In addition, biofunctional modification including incorporation or surface-tethering of neurotrophic factors potentiates its clinical application [6]. In addition to conduits, using neural stem cell (NSC) therapy is another approach to facilitate peripheral nerve regeneration [7]. NSCs are primarily produced by isolating them from the fetal central nervous system or by deriving the NSCs from embryonic stem cells (ESCs), which raises critical ethical concerns and increases the risk of tumor formation [8]. The adipose-derived stem cells (ASCs) are sources of multipotent stem cells that can be differentiated into several distinct lineages [9]. The ASCs and bone marrow-derived stem cells (BMSCs) possess similar characteristics regarding multipotency and molecular signatures, and also share common genetic signals [10,11]. The advantages of using ASCs are the abundance of these cells, the ease of harvesting the cells by performing minimally-invasive procedures, and an autologous origin that requires no immunosuppression [12]. The ASCs can trans-differentiate into the neural lineage and form free-floating spheroid bodies in various neurotrophic media [13,14]. Similar genetic expression profiles are present in spheroid bodies derived from ASCs, BMSCs, and NSCs [15]. These spheroid bodies, called neurospheres, are capable of self-renewal and clonal isolation, and possess great potential for generating neuronal [16], glial [17], and oligodendrocyte [18] cells. Numerous biomaterials have been proposed to guide peripheral nerve regeneration [6,19]. Among these biomaterials, chitosan tubes may facilitate and guide axon regeneration. Chitosan is a naturally occurring polysaccharide and is non-cytotoxic and highly biodegradable. Chitosan has been widely used in gene delivery [20,21], cell culture [22e24], and tissue engineering [25,26]. In addition, chitosan produces anti-inflammatory effects against neutrophil infiltration into organs, tumor necrosis factor alpha (TNF-a) and interleukin-1b (IL-1b) levels in serum, as well as antioxidative properties [27]. Several studies have demonstrated that a chitosan conduit can act as a favorable scaffold biomaterial characterized by minimal cytotoxicity and high biodegradability to promote nerve regeneration when combined with Schwann cells [28], NSCs [29,30] or BMSCs [31e33]. Only a few studies has reported that chitosan was combined with ASCs to facilitate sciatic nerve regeneration [34,35]. In our previous studies, the neurogenic potential of human ASCs was induced and formed neurosphere on a chitosan-coated surface [36]. The current study designed several functional evaluations for illustrating the therapeutic outcomes of sciatic nerve regeneration to bridge a 10-mm gap by using conduits coated with or without chitosan, and by combining neurosphere cells derived from human ASCs. We also investigated the underlying mechanisms of therapeutic effects produced by a chitosan-coated conduit with or without performing the cell-based treatments. 2. Materials and methods 2.1. Preparation of the chitosan-coated plate and conduit The preparation of cell culture plates and nerve conduits coated with chitosan (the degree of deacetylation was 85%; 417963, SigmaeAldrich, St. Louis, MO, USA) was conducted in accordance with the process used in our previous study [36]. In summary, 1% w/v of chitosan powder was dissolved in 1 M of acetic acid, and the impurities were then removed using vacuum filtration (Nunc, Roskilde, Denmark). The chitosan solution was added to the tissue culture plates (Corning Inc., NY, USA), which were then dried at 60  C for 24 h. After a thin film formed on a culture plate, the surface was neutralized by applying 1 N of aqueous NaOH for 30 min and then washed thoroughly using distilled water. The chitosan-coated plates were exposed to ultraviolet light overnight before the cells were seeded. Similar coating methods were applied to the nerve conduits. A medical-grade silicone tube (1.5 mm inner diameter, 15 mm in length) (VersilicÒ 760110, SaintGobain, Courbevoie, France) was filled with the chitosan solution (1%). The silicone tube was placed upright, and the chitosan was evenly dried on the inner wall

after the tube was incubated in an oven at 60  C for 24 h. The coated tube was then neutralized and cut to 10 mm in length for nerve conduit. The conduits were exposed to ultraviolet light overnight before being implanted into an animal. 2.2. ASC culture and spheroid formation Human ASCs were obtained from healthy donors with informed consent and with the approval of the Review Board of National Cheng Kung University (NCKU) Hospital. The ASC isolation protocols followed those of Dr. Zuk and Dr. Hedrick at the University of California, Los Angeles [9]. In summary, the raw lipoaspirates were washed to remove debris, and were treated with 0.075% collagenase (type I; Sigmae Aldrich) for 30 min at 37  C. The collagenase was inactivated using an equal volume of Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Inc., Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), and the infranatant was centrifuged for 10 min at low speed. The pelleted stromal vascular fraction (SVF) was separated from the floating population of mature adipocytes. The SVF consisted of a heterogeneous cell population, and this was enriched in preadipocytes by using plastic adherence. All SVF cells were cultured and passaged in DMEM containing 10% FBS at least 3 times before use. The multipotency ability of isolated ASCs was assessed by performing osteogenesis, adipogenic, and chondrogenic induction, as in a previous study [36]. On Passage 3, ASC cells were seeded onto the chitosan-coated plate with 2  104 cells per cm2 for 48 h to develop free-floating spheroids [36]. 2.3. Sciatic nerve transection and treatments In the process of peripheral nerve repair using conventional nerve tubes, a length limit or critical gap exists between the proximal and distal stumps of a cut nerve, above which regeneration does not occur. This distance is approximately 10 mm in the sciatic nerve of rats [37]. Therefore, we designed a rat sciatic nerve transection model containing a 10-mm gap. The experimental procedures used for this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at NCKU. Male Sprague-Dawley rats weighing 200e250 g (6e7 weeks old) were anesthetized using Zoletil (50 mg/kg intraperitoneally) (Virbac, Carros, France). An incision was made from the right sciatic notch to the distal thigh. The subcutaneous tissue was bluntly dissected under the skin to expose the biceps femoris muscle. The sciatic nerve was freed from the investing fascia and prepared for further manipulation (Fig. 1A, left panel). The animals were randomly assigned to the following 5 groups: the sham operation group (Sham) explored the sciatic nerve without any damage to the nerve tissue; the silicone group (S) used a silicone conduit to bridge the 10-mm gap (Fig. 1A, middle panel); and in the chitosan group (C), the gap was bridged using a chitosan-coated conduit. The neurospheres formed by seeding the ASCs on a chitosan-coated plate were dissociated into single cells and 1  105 cells were then applied to the silicone conduit (S þ N) or chitosan-coated conduit (C þ N). The wounds were closed by suturing the skin. After surgery, the animals were housed at 21  0.5  C in cages with free access to food and water. Six weeks after surgery, the rats were euthanized and the surgical sites were subsequently opened to harvest the nerve tissue (Fig. 1A, right panel). 2.4. Examination of myelin sheath The nerve tissues were isolated and fixed overnight in 4% paraformaldehyde. In each animal, three 2-mm segments were cut in the proximal section of the anastomosis (proximal), the middle portion of the nerve gap (middle), and the distal section of the anastomosis (distal) (Fig. 2A). The nerve segments were fixed again overnight in 1% OsO4 (Merck KGaA, Darmstadt, Germany). These three segments were separately dehydrated in a gradient of ethanol (EtOH) (50%, 75%, 85%, 95%, and 100%). During the dehydration process, gradient mixtures of EPON (Ladd Research Industries, Inc., Burlington, VT, USA) in 2-propanol were prepared (33%, 66%, and 100%). Tissues were treated with gradient EPON solutions and maintained in pure EPON overnight. Tissues were then embedded in EPON in an oven at 60  C overnight. Semi-thin sections (0.5 mm) of the nerve explants were cut on a microtome (Reichert Jung 1130, Leica, Wetzlar, Germany). The myelin sheath was stained with toluidine blue (0.1%, SigmaeAldrich). The images were captured using a digital camera (Sony, Japan) attached to a microscope (Eclipse TE300, Nikon, Japan) running ImageProÒ software (Media Cybernetics, L.P., Silver Spring, MD, USA). The number of myelinated axons was counted using a high-power field objective lens (40, Nikon, Japan) with 5 separate fields in each slice, 3 separate slices in each segment in random by three single-blinded students who were taught to identify the re-myelinated axons under the same criteria. The nerve regeneration was evaluated by examining the axon density and axon ratio from proximal to distal segments. To determine axon density, the number of myelinated axons per visual field was counted in the middle segments. The myelinated axons contained in the distal and proximal segments were counted, and the percentage of the distal myelinated axons to proximal myelinated axons ratio was then calculated. To observe the ultrastructure of the myelin sheath, the nerve explants embedded in EPON were sectioned at 80 nm on a microtome (Leica). The sections were then placed on a copper mesh (Pleco, Clovis, CA, USA). The nuclear acid inside the sections was stained with 1% uranyl acetate (TAAB, Berkshire, UK), and the proteins were stained with 1% lead citrate (Ladd). After brief air-drying, the samples

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Fig. 1. Regeneration in the rat sciatic nerve transection model. (A) The sciatic nerve was explored from the point of emergence from the spinal cord to the sciatic notch (left panel). At the midpoint, the nerve was transected to create a 10-mm gap and bridged with a silicone conduit (middle panel). Nerve regeneration took place within the conduit, and the whole segment of the sciatic nerve was harvested 6 weeks after surgery (right panel). (B) Examples of regenerated sciatic nerves from control (Sham), silicon conduit (S), chitosancoated conduit (C), neurosphere cells in a silicone conduit (S þ N), and neurosphere cells combined with a chitosan-coated conduit (C þ N) groups. Scale bar, 2 mm.

were observed under a transmission electron microscope (JEM-1400, JEOL, Tokyo, Japan) at 120 KV and 64 mA. 2.5. Histological assessments Several histological stainings were performed to observe the tissue morphology and protein expressions [38]. The paraformaldehyde-fixed nerve segments were dehydrated and then embedded in paraffin. The nerve tissues were sectioned transversely at 10 mm. A hematoxylin and eosin (H&E) stain was applied to observe tissue morphology. In summary, the tissue sections were immersed in xylene to remove the paraffin, and were then rehydrated using an ethanol gradient and double-distilled H2O (ddH2O). The tissue sections were stained with hematoxylin (Merck KGaA) for 4 min, and then stained with eosin (Leica Microsystems) for 20 s. The stained samples were rinsed with tap water, dehydrated in an EtOH gradient, and then mounted using xylene gel (SigmaeAldrich). Immunohistochemistry (IHC) staining was performed to detect specific expression patterns exhibited by proteins. The primary antibodies were used with anti-neural filament heavy chain (NFH, 1:200; Millipore, Billerica, MA, USA) to identify neuron and anti-glial fibrillary acidic protein (GFAP, 1:400; Millipore) in glia cells. The paraffin sections were de-waxed in xylene, rehydrated in an EtOH gradient, treated with 3% H2O2 (SigmaeAldrich) for 30 min, and then treated with 0.25% trypsin (Invitrogen) for 20 min. Non-specific primary antibody binding was blocked using 5% fetal bovine serum (Invitrogen) diluted in 0.02% Tris-buffered saline with Tween 20 (TBST) (SigmaeAldrich). The tissue was incubated with primary antibodies and stored overnight at 4  C to detect inflammatory protein. After rinsing with 0.02% TBST, the primary antibodies were bound by secondary antibodies (1:500, Abcam, Cambridge, UK) for 60 min and then labeled using an AB reagent (Vector, Burlingame, CA, USA) for 30 min to couple with DAB. Tissue sections were counterstained with hematoxylin and then mounted using a mounting medium. The sections were examined under a light microscope. To detect the inflammatory responses in various treatments and elucidate the underlying signaling mechanism, the IHC staining of inflammatory proteins against the IL1-b and TNF-a was performed in the nerve sections contained in the middle segments. The expression levels of these proteins were assessed using specific primary antibodies against IL1-b (1:100, Abcam) and TNF-a (1:100, Abcam). Cyclooxygenase (COX) and 5-lipoxygenase (5-LO) are 2 major pathways that are used to convert arachidonic acid (AA) into various inflammatory products [38]. COX-2 is a key enzyme in the COX family that produces prostaglandin E2 (PGE2) to regulate inflammatory responses and pain signals [39]. The expression levels of COX-2 were detected using specific primary antibodies against COX-2 (1:100, Cayman, Ann Arbor, MI, USA). The prostaglandin receptors 1 and 4 (EP1 and EP4) were also studied to assess the activation of PGE2-induced tissue inflammation caused by specific

antibodies of EP1 (1:100, Abcam) and EP4 (1:100, Abcam). The 5-LO converted AA into a different inflammatory factor named leukotriene. The leukotriene B4 receptor 1 (BLT1) antibody (1:100, Cayman), which is related to the 5-LO receptor, was also used in the high-affinity detection of leukotriene B4 expression levels. 2.6. Innervated muscle weight and remodeling of muscle fibers The gastrocnemius is the largest muscle innervated by the sciatic nerve in rats and starts to atrophy after nerve injury [40]. To assess the nerve re-innervation, the relative gastrocnemius muscle weight (RGMW) and fiber diameter were measured immediately after the rats were sacrificed. The gastrocnemius (excluding the soleus) of both limbs were harvested from the bone attachments. The RGMW was calculated from the ratio of the muscle weight on the experimental side to that of the contralateral side. Immediately after measuring the muscle weight, the muscle tissues were fixed and histologically assessed by applying the same methods used to assess the nerve tissues. H&E staining was used to examine the morphology of muscle fibers. For each section, 5 dissimilar fields were chosen randomly using a 20 light microscope. All diameters of muscle fiber in the field were measured and displayed as a mean value, which was presented as the ratio compared with the uninjured side. The muscle fibers were categorized into fast and slow types of muscle fibers by presenting various types of myosin (fast or slow). Thus, the IHC staining was applied to identify various types of muscle fibers by using specific primary antibodies against fast myosin heavy chain (MHC-fast) (1:100, Abcam) and slow myosin heavy chain (MHC-slow) (1:100, SigmaeAldrich). 2.7. Gait analysis A walking track equipped with a video-based system was modified from that used in a previous study [41]. The apparatus consisted of a Plexiglas chamber that was 80 cm long, 6 cm wide, and 12 cm high with a mirror tilted at 45 underneath the walking track. The tilted mirror reflected the image of the rat’s paws, which was conveniently recorded using an EX-F1 digital camera (Casio, Tokyo, Japan). The camera was set to simultaneously record a direct lateral view and a reflected view from below the walking track. Before the walking test, the rats were shaved and the skin of both hindlimbs was marked with red color for lateral kinematical data acquisition. The use of colored landmarks on the lateral malleolus and the fifth metatarsal head provided a simple method for determining the stance and swing phases of the gait cycle from heel contact to toe-off. The task was repeated until 5 or 6 satisfactory walks of at least 4 steps without pause were obtained. In this study, only the hindlimb stepping patterns were analyzed. The digital images obtained from each trial were processed with a threshold setting to detect the boundary of the soles, and critical points for

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Fig. 2. Morphology of the myelin sheath located in various cross sections of regenerated nerves. (A) Toluidine blue staining demonstrated the disappearance of the myelin sheath in a silicone conduit (S) and the axon re-myelination that occurred in other nerve segments. The representative segments in the proximal, middle, and distal sections of the nerve gap were collected for analysis (indicated by squares). Scale bar, 50 mm. (B) The density of re-myelinated axons in the middle nerve segments (middle portion of conduit) increased in the C and C þ N group compared with that of the S group. (C) A similar pattern was also observed in the ratio of re-myelinated axons in the distal segment to those in the proximal segments. (D) Electron microscopy images revealed the degradation of the myelin sheath in the S and S þ N groups (arrows). *p < 0.05 compared with the S group; #p < 0.05 compared with the C group. determining the derivation of paw indices were determined using Matlab software (MathWorks, Natick, MA, USA). After identifying sequential footprints, 2 basic spatial parameters, step and stride lengths, were determined (Fig. 5A). The step length was the distance between each step to the contralateral hindlimb. The stride length was the distance of advance in the ipsilateral hindlimb. Each parameter was averaged from at least 20 footsteps. 2.8. Statistics The data are expressed as the mean  SD. Statistical analysis was performed using a one-way analysis of variance (ANOVA) and the Scheffe post hoc test. Values of p < 0.05 were considered statistically significant.

3. Results 3.1. Improvement of nerve regeneration by using various conduits and cell therapies Six weeks after surgery, most of the treatment groups demonstrated gross nerve regeneration within the conduits (chitosan conduits or silicone containing with neurosphere cells). The diameter of the regenerated nerve was the smallest in the silicon conduit (S) (Fig. 1B). In the S group, 2 out of 12 sciatic nerves did not regenerate within the conduits. The proximal ends of nonregenerated nerves bulged as neuroma with excessive fibrosis formation. The application of neurosphere cells to silicone conduit

(S þ N) resulted in a thicker regenerative nerve compared with that of the S group, and all nerves could grow across the gap after surgery for 6 weeks. Regarding the nerve gaps that received chitosancoated conduit (C) or combination of neurosphere cells within chitosan-coated conduit (C þ N), the regenerative nerves showed thick diameters and a dense white color that typically occurs in normal peripheral nerve tissues.

3.1.1. Chitosan conduit enhances axon regeneration and remyelination The toluidine blue stain revealed the myelin sheaths of sciatic nerve in sham rats, and these rats received dissimilar treatments for 6 weeks after surgery (Fig. 2A). In the sham group, the myelin sheaths formed an insulating envelope that surrounded the core of the axon in the proximal, middle, and distal segments. The nerve gap bridged by silicone conduit (S) displayed few myelinated circles in the middle of the conduit. The myelin sheaths were barely present in the distal nerve segments of the S group, which indicated Wallerian degeneration and myelin degradation. Applying cells from neurospheres to the silicone conduit (S þ N) did not improve nerve regeneration. The axon density in the middle nerve segments was calculated, and the results indicated a substantial increase of density in the nerve gaps treated by the chitosan-coated conduit

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without (C) or with (C þ N) neurosphere cells (Fig. 2B). Although the size of the myelin sheaths observed in the C þ N group was smaller than that of the sham group, the axon density observed in the C þ N group was similar to that of the sham group. The relative ratio of myelinated axons in the distal segment to those in the proximal provided an index of axonal sprouting. Almost 80% of the axons crossed the critical gap to the distal segment in the C þ N group (Fig. 2C). In addition, electron microscopy revealed the degradation of the myelin sheath in both the S and S þ N groups, but not in the C and C þ N groups (Fig. 2D). 3.1.2. Chitosan conduit or cell therapy reduces glial scar formation To further investigate the cell types existing within the regenerated nerve, IHC staining was used on the middle nerve segments (Fig. 3). H&E staining revealed the transverse section of the nerve in the middle segments (Fig. 3, first and second panels). No substantial difference was observed among all of the groups when the neurofilaments were stained using an antibody against NFH (Fig. 3, third panel). The number of glial cells detected using GFAP staining increased markedly, as signified by the brown color representing in S group (Fig. 3, fourth panel). This indicates that using a silicone conduit may cause intra-neural scarring in the nerve gap. The expression of GFAP protein decreased in the C, S þ N, and C þ N groups. Thus, the chitosan conduit or assisted cells derived from neurospheres can reduce intra-neural scarring within the regenerated nerve.

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3.2. Prevention of effector muscle atrophy and maintenance of muscle fibers After nerve transection, the gastrocnemius atrophied and then regained innervation after nerve regeneration. Six weeks after surgery, the gastrocnemius muscles were isolated from both hind legs of rats from different groups (Fig. 4A). The muscle obtained from the rats treated with silicone conduit (S) demonstrated a slim gross morphology. The relative gastrocnemius muscle weight (RGMW) increased in the re-innervated muscles of C, S þ N, and C þ N groups compared with that of the S group (Fig. 4B). In addition, the muscle weight observed in the C þ N group significantly increased compared with that of the C group. This result revealed an additional benefit of applying the neurosphere cells. H&E staining was performed on the transverse section of the gastrocnemius muscles. The cross section of the individual muscle fibers was observed in the sham group (Fig. 4C). The atrophy muscle demonstrated degradations of muscle fibers and became fragments in the S group. In the muscles that were subjected to either the chitosan-coated conduit or cell-assisted therapy, the diameter of the muscle fibers was regained after nerve re-innervation. Morphometric analysis of the regenerated muscles revealed that the increase in muscle fiber diameter was similar to the increase in muscle weight (Fig. 4D).

Fig. 3. Histological and immunohistochemical staining of neural markers in regenerated nerves. The middle segments of the nerve were harvested to understand the neural pattern of regeneration. The tissue morphologies were observed using hematoxylin and eosin (H&E) staining. The expression of neurofilament heavy chain (NFH) staining did not differ among the groups. Positive staining of the glial fibrillary acid protein (GFAP) was expressed in the S group, which indicated glial scar formation, and was suppressed in the other 3 treatment groups.

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Fig. 4. Effector muscle atrophy and re-innervation. (A) The gastrocnemius muscles of both hindlimbs were harvested 6 weeks after surgery. (B) Compared with the S group, the relative gastrocnemius muscle weight (RGMW) of the operated side to the non-operated side significantly increased in the C, S þ N, and C þ N groups. In addition, the value produced by the C þ N group was greater than that of the C group. (C) H&E staining revealed that the muscle fiber atrophied in the S group. (D) A similar re-innervation pattern was confirmed by examining the diameter of muscle fibers. (E) Immunohistochemistry staining of the fast and slow types of myosin heavy chain (MHC) revealed an increase in the number of slow fibers in the re-innervated muscles, but indicated no changes in the number of fast fibers. Scale bar, 100 mm. *p < 0.05 compared with the S group; #p < 0.05 compared with the C group.

The types of muscle fiber were further investigated using IHC staining and specifically targeting the fast and slow types of MHC. In the sham group, the innervated gastrocnemius muscle exhibited a mixed population of fast and slow muscle fibers (Fig. 4E). In the healthy gastrocnemius muscle, the major population is fast muscle fiber [42], which was also observed in the muscle of sham operation. The degenerated muscle fibers in the S group could not detect the protein expression for both types of MHC. IHC staining revealed the loss of fast muscle fiber after 6 weeks of using chitosan-coated conduits and/or cell-assisted therapies. Significant increases of slow-type MHC protein expressions were observed in these treatment groups, particularly in the C þ N, as compared with the results of using a silicone conduit (Fig. 4E). Therefore, combining a chitosan-coated conduit and neurosphere cells facilitated effector muscle regeneration in terms of weight and fiber size by maintaining the slow-acting muscle fibers.

which suggests the post-surgery nerve stock on motor functions (Fig. 5B and C). The step length measured in the C and C þ N groups was greater than that measured in the S group after 3 weeks, and was approach to that of the sham group at the end of 6 weeks (Fig. 5B). An abnormal gait pattern was observed in the rats subjected to the silicone conduit for 6 weeks (Supplementary movie 1). However, the gait pattern observed in the C þ N group indicated that the surgical hindlimb performed normal movements compared with limb on the contralateral side (Supplementary movie 2). The stride length underwent a similar pattern of recovery, with a substantial increase in the stride lengths observed in the C and C þ N groups (Fig. 5C). Comparing the gait performances within the silicone or chitosan groups revealed no evident beneficial effect of the therapeutic cells on gait recovery. Taken together, the gait functions primarily improved when chitosan-coated conduits were applied. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2013.11.081.

3.3. Chitosan conduit and neurosphere cells improve motor function To evaluate the overall function of neuromuscular unit and sensory-motor coordination, the walking gait of rats was monitored using a video-assisted analysis system after the rats received various treatments for 1, 3, and 6 weeks (Fig. 5A). Both the step and stride length were similar among the various groups on the first week,

3.4. Anti-inflammation signal mechanisms for improving nerve regeneration Based on the abundance of glia cells discovered in the silicone conduit (Fig. 3), the inflammatory signals were investigated to distinguish the underlying mechanism of inflammation-induced

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A

LStepL

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Week 6

Fig. 5. Functional assessments of dynamic gait analysis. (A) The step and stride lengths were acquired using a high-speed video recording system to observe the rats when receiving various treatments for 1, 3, and 6 weeks. (B, C) At the end of 6 weeks after surgery, both the step and stride lengths increased in the C and C þ N groups compared with those of the S group. *p < 0.05 compared with the S group.

glial scar formation. We tested several inflammatory signals by using IHC staining specifically targeting the TNF-a, IL-1b, COX-2, and 5-LO pathways. The expression levels of TNF-a protein did not change in the middle nerve segments among various treatments (Supplementary Fig. 1). Significant increases in IL-1b protein expressions were discovered in the S and S þ N groups (Fig. 6, first panel). Using the chitosan-coated conduit reduced the IL-1b proteins in the C and C þ N groups. To illustrate the major pathway that converted the AA into various inflammatory products, the COX and 5-LO pathways were investigated by examining the protein expression levels of COX-2 and BLT1. The expression levels of the COX-2 proteins did not differ among the groups after 6 weeks of surgery (Fig. 6, second panel). On the other hand, the BLT1 protein expression was observed in the S and S þ N groups, but it was reduced in the C and C þ N groups (Fig. 6, third panel). To exclude the possibility of a COX-2 signaling pathway, the prostaglandin receptors (EP1 and EP4) that could be activated by the COX-2 product (PGE2) were tested, and the results indicated that neither EP1 and EP4 were involved in nerve inflammation (Supplementary Fig. 1). Therefore, the chitosan-coated conduit might reduce inflammatory signals by inhibiting the IL-1b and BLT1 pathways to promote axon regeneration.

4. Discussion Many types of biomaterial and cell-based therapy have been proposed to guide peripheral nerve regeneration, but several results were controversial. In current study, we used various assessments of global and detail nerve tissue morphologies, neural marker expressions, innervate muscle mass and structures, and functional gait movements to compare the outcomes of various treatments (Table 1). Based on these results, we believe that the discrepancies in the previous findings may be due to the different assessments performed in various studies. The most common method for assessing nerve regeneration is to measure axon regeneration [19]. Although we did not observe the beneficial effects on axon regeneration (Fig. 2) and gait analysis (Fig. 5) in the S þ N group, the reduction of glial scar formation (Fig. 3) and the maintenance of muscle structure (Fig. 4) were achieved by applying cell-assisted therapy in the silicone conduit. Similar evidence of the benefits on axon repair was revealed when investigating cell-based therapies that use ASCs to treat the sciatic nerve crush injuries [43]. Several studies have demonstrated the advantage of using a chitosan conduit to improve axon regeneration [31,32,34], muscle mass [31,32], and footprint [18,31,32]. In this study, we further

Please cite this article in press as: Hsueh Y-Y, et al., Functional recoveries of sciatic nerve regeneration by combining chitosan-coated conduit and neurosphere cells induced from adipose-derived stem cells, Biomaterials (2013), http://dx.doi.org/10.1016/j.biomaterials.2013.11.081

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Fig. 6. Inflammation profile and a possible inflammatory signaling pathway in regenerated nerves. Immunohistochemical staining revealed an increase in inflammation induced by Interleukin-1b (IL1-b) protein expressions, and a possible signal pathway mediated by 5-lipoxygenase with exhibiting a high intensity of leukotriene B4 receptor 1 (BLT1) staining in the S group. The involvement of another common inflammatory pathway, cyclooxygenase (COX), was ruled out because no significant changes in the COX-2 signals were observed. The decrease in IL1-b and BLT1 staining revealed the anti-inflammation effects and benefits of using a chitosan-coated conduit in the C and C þ N groups. Scale bar, 100 mm.

conducted the application of combined neurosphere cells derived from ASCs with the chitosan-coated tube, which led to the most favorable outcomes to facilitate rat sciatic nerve regeneration (Table 1). The chitosan-coated surface on a cell culture dish or a 3dimensional culture membrane affects cell differentiation,

Table 1 Summary of the beneficial effects among different treatments.

Axon myelination Muscle weight Myosin expression Gait recovery Anti-inflammation

Sham

S

C

SþN

CþN

þþþ þþþ þþþ þþþ N/A

    

þ þ þ þþ þþ

 þ þ  

þþ þþ þþ þþ þþ

particularly in mesenchymal stem cells [44]. Free-floating spheroid cells are formed spontaneously on the chitosan surface, which is possibly affected by the Rho and Rho-associated kinase signaling pathway [45,46]. The RhoA kinase inhibition in other mesenchymal-like stem cells, such as human placenta-derived multipotent cells, also promotes the neural phenotype that is primarily mediated by the ROCK2 isoform and the respective downstream target myosin II [47]. In addition to the enhanced differentiation capabilities, the ESC-like property of spheroid cells is also induced by the chitosan surface [48]. These findings imply that the formation of spheroids in mesenchymal stem cells has great potential for cell-based therapies. The goal of neural stem cell therapy is to restore neuronal functions or to protect neurons from damage [49]. After peripheral nerve injury occurs, a process of axonal degeneration, myelin degradation, and glial cell proliferation is initiated in a complex

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Y.-Y. Hsueh et al. / Biomaterials xxx (2013) 1e11

sequence, which is called Wallerian degeneration. This process is partially mediated by neuroglia and infiltrating inflammatory cells, and is regulated by inflammatory mediators such as cytokines and chemokines [50]. The activation of transcription factors is also associated with the inflammatory response. From the results of using silicone conduit, the high expression of GFAP for glial scarring (Fig. 3) and the decreased number of myelinated axons with ongoing myelin sheath degradation (Fig. 2D) prompted us to further investigate the anti-inflammatory effect in various treatments. Neuroimmune signaling is partially mediated by an innate immune system, including arachidonic acid derivatives such as prostaglandins and leukotrienes. The balance between COX-2 and 5-LO indicates the existence of a complex network that is altered by the activities of various enzymes during bone fracture healing [38,51]. Although the anti-inflammatory effects caused by chitosan material have been reported to prevent neutrophil infiltration into organs and TNF-a and IL-1b levels in serum [27], the downstream signaling mechanism remains unknown. The current study revealed a strong positive staining of IL1-b and BLT1 in the silicone conduit group, and we observed no difference in COX-2, EP1, EP4, and TNF-a among various groups (Fig. 6 and Supplementary Fig. 1). The beneficial anti-inflammatory effects produced by the chitosancoated conduit were discovered on inhibiting the arachidonic acid conversion to the leukotriene by 5-LO pathway, rather than the prostaglandin and COX-2 pathway. The immunomodulation effect of mesenchymal stem cells, including ASCs is due to a combined humoral and cellular effect, mediated both by cytokine expression and regulation of peripheral blood mononuclear cells (PBMCs) [52e55]. The profound immunomodulatory impact of ASCs has already been demonstrated in a variety of experimental models of disease, including spinal cord injury, neurodegenerative diseases, autoimmune diseases, and GvHD [56,57]. In our study, we used human ASCs incorporating chitosan conduit in the immunocompetent Sprague-Dawley rats and did not observe additional inflammatory or immune rejection response. We assumed that the immunomodulatory effect of ASCs might play an important role for different species. An increasing number of studies have emphasized the concept of functional tissue engineering to restore tissue morphology and functions [58e60]. Motion analysis and the analysis of innervated muscle histology are crucial for quantitatively demonstrating the spatial and temporal patterns of motor recovery during nerve regeneration [61]. Regarding muscle histology, we revealed the detailed changes in muscle structure that may be altered by nerve degeneration and re-innervation. The cell-assisted chitosan conduit promoted the re-innervation of effector muscle in the relative muscle weight and fiber regeneration (Fig. 4AeD). The weight of regenerated muscle was approximately 40% in the C þ N group 6 weeks after surgery (Fig. 4B). Long-term therapeutic effects were assessed by harvesting the muscle after 12 weeks, and we observed that the RGMW further increased to 50% (Supplementary Fig. 2). This result is more comprehensive than other studies by using neurotrophic factors combined with chitosan tubes in the same sciatic nerve transection model 12 weeks after surgery [62]. Regarding muscle types, muscle disuse caused by spinal cord injury subsequently caused slow fiber atrophy with a slow-to-fast fiber type shift [63]. The conversion of fiber types in tibialis anterior and soleus muscle was measured after performing spinal cord transection and peripheral nerve transection in rats [64]. Although both spinal cord and peripheral nerve transections resulted in rapid muscle atrophy after 2 weeks, the expressions of slow myosin heavy chain in the soleus muscle decreased after performing spinal cord transection, and increased after completing sciatic nerve transection. After surgery for 6 weeks, we observed decreases of both the fast and slow fibers in atrophied muscle using the silicone

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treatment (Fig. 4E). Increases in the slow-MHC expressions induced by either the chitosan-coated conduit or cell-assisted therapy suggests the beneficial effects for producing slow muscle fibers. However, the underlying mechanisms that protect the muscle switch or promote muscle regeneration still require further investigation by harvesting muscle tissues at various time points. By using walking track analysis, the current study demonstrated substantial increases in the functional performances of rats regarding step and stride length. These results were observed when we implemented treatments that incorporated chitosan-coated conduits (Fig. 5). Cell-based therapies are acknowledged as effective and safe methods for peripheral nerve regeneration [31,32,34,37,65e67]; however, ineffective [68] and even negative [69] effects of these cell-based therapies have also been reported. Although neurosphere cell therapy performed in a chitosan conduit yielded the highest number of regenerating axons, thickest myelin sheath, and largest regenerated muscle, the result of the overall gait analysis did not produced benefits in the cell-assisted groups (S þ N or C þ N). The possible discrepancy between the outcomes concerning muscle structure and gait could be caused by the motion parameters. The sciatic nerve innervates not only the gastrocnemius muscle, but also the soleus, tibialis anterior, and other small muscles to perform complex leg and foot motions. We suggest that future studies should pay closer attention to these muscles, and compare muscle structures that facilitate multiple joint movements. 5. Conclusion We developed a cell-assisted chitosan-coated conduit to bridge a 10-mm gap in the rat sciatic nerve transection model. In the early recovery period, 6 weeks after surgery, beneficial effects that promoted axon re-myelination, reduced intra-neural scarring, enhanced the re-innervation of the effector muscle, and improved overall gait were observed. These functional improvements may have resulted from an anti-inflammatory effect involving the inhibition of IL1-b and leukotriene signaling. We have facilitated peripheral nerve regeneration by combining neurosphere cells induced from human ASCs with a chitosan-coated conduit, giving great potential for clinical application to autologous cell-based therapies for patients with peripheral nerve injury. Acknowledgments This study was supported in part by grants from the National Science Council of Taiwan (NSC 99-2320-B-006-002-MY3 to CCW and NSC 101-2314-B-006-027-MY3 to YYH), the National Health Research Institutes of Taiwan (NHRI EX101-10115BC to CCW), and the National Cheng Kung University Hospital, Tainan, Taiwan R.O.C. (NCKUH-10003008 to YYH). The authors are very grateful to Wallace Academic Editing and Mr. Hans Harn for valuable suggestions. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2013.11.081. References [1] Ruijs AC, Jaquet JB, Kalmijn S, Giele H, Hovius SE. Median and ulnar nerve injuries: a meta-analysis of predictors of motor and sensory recovery after modern microsurgical nerve repair. Plast Reconstr Surg 2005;116:484e94 [discussion 95e96]. [2] Allodi I, Udina E, Navarro X. Specificity of peripheral nerve regeneration: interactions at the axon level. Prog Neurobiol 2012;98:16e37.

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Please cite this article in press as: Hsueh Y-Y, et al., Functional recoveries of sciatic nerve regeneration by combining chitosan-coated conduit and neurosphere cells induced from adipose-derived stem cells, Biomaterials (2013), http://dx.doi.org/10.1016/j.biomaterials.2013.11.081