Transplantation of bone-marrow-derived cells into a nerve guide resulted in transdifferentiation into Schwann cells and effective regeneration of transected mouse sciatic nerve

Micron 41 (2010) 783–790

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Transplantation of bone-marrow-derived cells into a nerve guide resulted in transdifferentiation into Schwann cells and effective regeneration of transected mouse sciatic nerve Fátima Rosalina Pereira Lopes a,1 , Flávia Frattini a,b,1 , Suelen Adriani Marques a,c , Fernanda Martins de Almeida a , Lenira Camargo de Moura Campos a , Francesco Langone d , Silvano Lora e , Radovan Borojevic a,f , Ana Maria Blanco Martinez a,∗ a

Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Brazil Departamento de Patologia da Faculdade de Medicina, Universidade Federal do Rio de Janeiro, Brazil c Departamento de Neurobiologia, Instituto de Biologia, Universidade Federal Fluminense, Brazil d Departamento de Fisiologia e Biofísica, Universidade Estadual de Campinas, São Paulo, Brazil e Instituto per la Sintesi Orgânica e la Fotoreatività, CNR, Legnaro-Padova, Italy f APABCAM, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Brazil b

a r t i c l e

i n f o

Article history: Received 5 March 2010 Received in revised form 18 May 2010 Accepted 19 May 2010 Keywords: Peripheral nerve Electron Microscopy Bone-marrow-derived cells Motor function Schwann cells

a b s t r a c t Peripheral nerves possess the capacity of self-regeneration after traumatic injury. Nevertheless, the functional outcome after peripheral-nerve regeneration is often poor, especially if the nerve injuries occur far from their targets. Aiming to optimize axon regeneration, we grafted bone-marrow-derived cells (BMDCs) into a collagen-tube nerve guide after transection of the mouse sciatic nerve. The control group received only the culture medium. Motor function was tested at 2, 4, and 6 weeks after surgery, using the sciatic functional index (SFI), and showed that functional recovery was significantly improved in animals that received the cell grafts. After 6 weeks, the mice were anesthetized, perfused transcardially, and the sciatic nerves were dissected and processed for transmission electron microscopy and light microscopy. The proximal and distal segments of the nerves were compared, to address the question of improvement in growth rate; the results revealed a maintenance and increase of nerve regeneration for both myelinated and non-myelinated fibers in distal segments of the experimental group. Also, quantitative analysis of the distal region of the regenerating nerves showed that the numbers of myelinated fibers, Schwann cells (SCs) and g-ratio were significantly increased in the experimental group compared to the control group. The transdifferentiation of BMDCs into Schwann cells was confirmed by double labeling with S100/and Hoechst staining. Our data suggest that BMDCs transplanted into a nerve guide can differentiate into SCs, and improve the growth rate of nerve fibers and motor function in a transected sciatic-nerve model. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Accelerating axonal regeneration to shorten the delay of reinnervation and improve functional recovery after peripheral-nerve injury is a clinical requirement and an experimental challenge. After nerve injury, peripheral axons have the ability to regenerate and, given a proper pathway, reconnect with their targets. Despite this capacity, the functional outcome is often poor, mainly after nerve injuries that sever peripheral nerves far from their targets (Lundborg, 2003). Recovery of function is dependent upon regen-

∗ Corresponding author at: Av. Professor Rodolpho Paulo Rocco, 255, Ilha do Fundão, Centro de Ciências da Saúde, bloco F, sala 12, Instituto de ciências Biomédicas, 21941-902, Brazil. Tel.: +55 21 2562 6431. E-mail address: [email protected] (A.M.B. Martinez). 1 Both authors contributed equally to this work. 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.05.010

eration and correct direction of injured axons, which is a slow process (Udina et al., 2003). In the peripheral nervous system, the major determinants of functional recovery after nerve lesions are the fast and accurate regeneration of axons and the maintenance and survival of the growing axons until they reach the original target organs. A sufficient number of mature axons is an important prerequisite to achieve adequate reinnervation and function (Furey et al., 2007). A reduced rate of nerve regeneration conspires together with atrophy and degeneration of denervated organs, to increase the risk of permanent disability following injury to the peripheral nervous system (PNS). When a nerve is transected and repaired, reinnervation of the distal stump occurs gradually, while regrowth of nerve fibers occurs with high specificity, with former motor fibers preferentially reinnervating muscle (Brushart, 1988). In transection nerve injury, neurorrhaphy is the most useful procedure (Ide, 1996). However, when there is a gap, accessory structures are generally used to guide the nerve towards the distal

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end. Several biodegradable polymers have been used to guide nerve regeneration (Langone et al., 1995; Nakamura et al., 2004; Pereira Lopes et al., 2006; Piquilloud et al., 2007), and their use has the advantage of avoiding a second surgery to remove them, and also preserves a healthy nerve. Many experimental strategies have been developed to promote peripheral-nerve regeneration after trauma: nerve conduits (Nakamura et al., 2004; Pereira Lopes et al., 2006), neurotrophic factors (Aebischer et al., 1989; Tom et al., 2004), electrical fields (Gordon et al., 2003; Baptista et al., 2008), Schwann cells (Frerichs et al., 2002; Fansa et al., 2003), and stem-cell transplantation (Dezawa et al., 2001; Keilhoff et al., 2006; Pereira Lopes et al., 2006). These strategies have usually focused on cell/tissue organization within the gap, rather than maintaining and accelerating the regeneration process. Schwann cells (SCs) are peripheral glial cells that ensheath axons to form myelin. Following nerve injury, SC plays a crucial role in peripheral-nerve regeneration by providing the pathway for regenerating axons and reconstructing myelin, which is indispensable for nerve function (Ide, 1996). Even though cell therapy using SC seems to be effective and promising for the treatment of nerve injuries, there are several drawbacks: a healthy peripheral nerve must be sacrificed, harvesting and expansion to obtain a sufficient amount of cells present technical difficulties, and some time is required for the entire process. Stem cells may be an alternative source for SC. Several populations of stem cells exist in adult tissues, of which the most promising are bone-marrow stromal cells (Bianco et al., 2001). The marrow stroma is a complex tissue that contains cells that are required for lineage commitment of hematopoietic cells. Although initially thought to be primarily hematopoietic supporting cells, the marrow stromal cells also contain nonhematopoietic cells that can differentiate into a variety of mesenchymal and non-mesenchymal cell types, including muscle cells, cardiomyocytes, and hepatocytes (Kawada et al., 2004; Bossolasco et al., 2005; Lee et al., 2005; Sato et al., 2005). Induction of molecular markers of glia and neurons in mesenchymal stem cells has been reported, in vitro (SánchezRamos et al., 2000; Mezey et al., 2003). In vivo studies have also shown that they can improve nerve regeneration (Dezawa et al., 2001; Cuevas et al., 2002; Choi et al., 2005; Keilhoff et al., 2006). Our previous study (Pereira Lopes et al., 2006) indicated that a combination of strategies can be used successfully to enhance sciatic-nerve regeneration after a surgical transection. We used a collagen tube filled with a selected population of bone-marrowderived cells (BMDCs, from the subendosteal surface) to bridge the nerve gap. These cells have previously been characterized by Balduíno et al. (2005), who showed by flow cytometry that they are positive for molecules such as CD44 and CD54. In addition, the RT-PCR analysis showed the mRNA expression of M-CSF, IL-6, IL7, TNF-␣, TGF-␤, SCF, Jagged-1, and Notch-1. In our previous work, we demonstrated beneficial effects in regenerating nerves, with better ultrastructural tissue preservation and an improvement in motor function. Also, our experimental nerves showed a significant increase in the number of myelinated nerve fibers, as well as in nerve-fiber area and myelin-sheath area, compared to control groups (Pereira Lopes et al., 2006). In the present study, we compared the ratio between the number of distal and proximal fibers of growing nerves, addressing the question of the capacity of growing fibers to achieve the distal end and target organs. Our results showed that the BMDCs in a collagen-nerve guide resulted in a significant increase of the axonal growth rates, meaning that the number of axons remained quantitatively uniform throughout the gap; we also found a significant increase in the number of myelinated nerve fibers and Schwann-cell nuclei in the treated group. In this group it was possible to identify BMDCs, pre-labeled with Hoescht, expressing the S100 marker for Schwann cells, indicating that the implanted BMDCs were able to survive and to differenti-

ate into a Schwann-cell-like phenotype. These events resulted in significant return of motor function. 2. Material and methods The mice were housed in cages, and maintained on a 12-h light–dark cycle with free access to water and food. Our experimental study was approved by the Animal Care and Use Committee of the “Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro,” under Protocol number DHEICB003. 2.1. Culture of bone-marrow-derived cells Balb-C mice were killed, and bone-marrow stromal cells were harvested following previously described protocols (Balduíno et al., 2005). Femurs were removed, cleaned of muscle and connective tissue, and treated with 0.125% trypsin and 0.1% collagenase (1:1—5 × 30 min) (Sigma Chemical Company, St. Louis, MO, USA) in order to remove all the adherent cells from the periosteum. Epiphyses were removed and femurs were flushed with the cellculture medium in order to remove the bone-marrow cells. Bones were sectioned into 3–5 mm-long fragments, washed in balanced saline solution (BSS), and treated twice with 0.1% collagenase for 30–40 min each, at 37 ◦ C. Cells were harvested from each digestion, washed twice in cold BSS, suspended, and plated on Dulbecco’s medium (DMEM—Sigma) containing antibiotics (100 U/mL penicillin, 100 ␮g/mL streptomycin) and 10% fetal bovine serum (FBS—Cultilab, Campinas, SP, Brazil). Prior to surgical implantation, these cells were trypsinized and centrifuged, and the pellet was labeled with 5 ␮g/mL bisbenzamide (Sigma-Hoechst 33258) for 30 min. Through this procedure, we could identify these cells later in the tissue. 2.2. Surgical procedure Female adult Balb-C mice (n = 18) weighing about 23 g were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (45 mg/kg). The left sciatic nerve was exposed and transected at the mid-thigh position. The proximal and distal nerve stumps were placed in a tubular collagen-nerve guide (6 mm long), inserted 1.5 mm into it, and sutured to the tube with 10-0 monofilament nylon. A 3-mm gap was left between the nerve stumps. Next, we injected BMDC into the tube, in the gap area (Fig. 1A). The experimental group (n = 9) received the collagen tube filled with Dulbecco’s cell-culture medium (DMEM) supplemented with cells obtained from BMDC cultures, at a final density of 1 × 105 cells/␮L. The control group (n = 9) received the collagen tube filled with DMEM (1.0 ␮L). Another control group (n = 3) received the collagen tube filled with 3T3 cells, a mouse embryonic fibroblast cell line, in order to evaluate the possibility that other cells not related with stem cells, would give results similar to BMDCs. After 2 and 6 weeks of regeneration, the mice were operated again, anesthetized, and transcardially perfused with a fixative solution containing aldehydes (4% paraformaldehyde plus 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). After that, the left sciatic nerves were again exposed, and the regenerating growing nerves were dissected and divided into 2 segments (S1 and S2), as shown in Fig. 1. These segments were taken and processed for electron microscopy and immunohistochemistry, and DAPI stained. For the quantitative analysis, we used animals with 6 weeks of regeneration; and for immunohistochemistry, the survival period was 2 weeks. 2.3. Immunohistochemistry After dissection and post-fixation for 4 h in fresh fixative solution, the segments of the nerves were cryoprotected in increasing

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60 ◦ C for 48 h. Semithin cross-sections for light microscopy studies were cut with an RMC ultramicrotome at a thickness of 500 ␩m, stained in toluidine blue, and observed with a Zeiss Axioskop 2 plus microscope. Ultra-thin cross-sections for transmission electron microscopy (TEM) analysis were cut on a RMC ultramicrotome at a thickness of 60–70 ␩m, collected on copper grids, and contrasted in uranyl acetate and lead citrate. TEM studies were carried out using the Zeiss 900 transmission electron microscope operated at 80 kV. 2.5. Functional analysis—walking tracks

Fig. 1. (A) Schematic view of peripheral-nerve (PN) model of tubulization. Proximal (P) and distal (D) nerve stumps were placed in a collagen-tube (CT—6 mm) nerve guide, supplied with a graft of bone-marrow-derived cells (BMDCs). A 3-mm gap was left between the stumps. (B) After 6 weeks the regenerating nerve (RN) was divided into segment 1 (S1) and segment 2 (S2) and processed for EM.

concentrations of sucrose up to 30%, left in this solution overnight, and then ice-cold embedded in OCT (Tissue Tek). Ten-micrometerthick sections were obtained with a cryostat (Leica CM 1850) and collected on gelatin-coated glass slides. The sections were then washed in 0.1 M saline phosphate buffer (pH 7.4) with Triton X100 (0.3%, washing solution) and bovine serum albumin (3%, Sigma, USA) three times. 10% normal goat serum (Sigma, USA) was used as the blocking solution, for 1 h, so the sections were washed in the previous solution. After that, the slides were incubated overnight with primary antibodies (rabbit policlonal anti S-100 antibody for Schwann cells, 1:100, Sigma, USA; rabbit polyclonal anti Gap 43 antibody for regenerating fibers, 1:50, Santa Cruz Biotechnology Inc., USA). They were then rinsed in the washing solution, followed by incubation with appropriate secondary antibodies (Alexa fluor 488-conjugated goat anti-rabbit IgG and Alexa fluor 546conjugated goat anti-rabbit IgG, 1:600, Molecular Probes, USA) for 2 h. Finally, the sections were rinsed and mounted with n-propyl gallate (250 mg/mL, Sigma, USA). For qualitative analysis, some sections were stained with DAPI nuclear label (0.1 ␮g/mL in 0.9% NaCl, Molecular Probes, USA) for 10 min and then washed and mounted as previously described. Primary antibodies were omitted for negative controls. The sections were observed with a confocal microscope (Olympus IX2-UCB) Plus) and photographed with a Hamamatsu digital camera C10600. 2.4. Transmission electron microscopy After fixation by perfusion, the distal segments of the nerves were immersed for 2 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), then washed in the same buffer followed by 0.1 M cacodylate buffer (pH 7.4), and postfixed for 90 min in 1% osmium tetroxide + 0.8% potassium ferrocyanide and 5 nM calcium chloride in 0.1 M cacodylate buffer (pH 7.4). Next, the segments were washed three times in 0.1 M cacodylate buffer (pH 7.4) and distilled water, and then stained in 1% uranyl acetate overnight. They were then dehydrated in graded acetone, infiltrated in Poly/Bed 812 resin (Polysciences, Inc., Washington, PA, USA), and polymerized at

At 2, 4, and 6 weeks after surgery, all animals were submitted to a postoperative walking-track analysis, based on the protocol described by Inserra et al. (1998). Paw prints were recorded by painting the hindpaws of each animal with India ink. The animals were then allowed to walk along a 45 cm × 6.5 cm track on a white paper (Canson A4 140 g/m2 ). The paw prints were analyzed considering two parameters: toe spread (TS), which is represented by the distance between the first and fifth toes; and print length (PL), the distance between the third toe and the hind pad. All the parameters were measured for the left paw prints, for normal and operated animals. These measurements were calculated according to the Inserra et al. (1998) sciatic functional index (SFI) formula: SFI = 118.9

 ETS − NTS  NTS

− 51.2

 EPL − NPL  NPL

− 7.5

where TS = toe spread in mm, PL = print length in mm, and E and N indicate experimental and normal hind foot, respectively. Differences between the groups were expressed as mean ± S.E. Statistical analysis included analysis of variance (ANOVA) followed by a post hoc Kruskal–Wallis test. A p value <0.05 was considered to be statistically significant. 2.6. Quantitative analysis For the sampling procedure, we photographed in the transmission electron microscope, in a systematic way, 8 microphotographs for each nerve, cross-sectioned at a magnification of 4400×. By this systematic method, the entire cross-sectional area of the nerve was scanned and sampled, thus avoiding sample bias. A total of 80 microphotographs were obtained for each experimental group, 40 for segments 1 and 40 for segment 2. Since we had two experimental conditions (sectioned with DMEM and sectioned treated with DMEM + BMDC), the total number of microphotographs was 160. All negatives were scanned and submitted to quantitative analysis using the Image Pro Plus program (Media Cybernetics). Based on the qualitative observations, we manually counted and compared between the two groups, the following parameters: total number of nerve fibers, number of non-myelinated fibers, number of myelinated fibers, and number of Schwann-cell nuclei. Also, to analyze the growth rate, we found the ratio between the number of myelinated and non-myelinated fibers from the distal and proximal segments of the nerves. Finally, we calculated the g-ratio by dividing the inner axonal diameter by the outer fiber diameter, and stratified the results in ranges of 0.0–0.1, 0.1–0.2, 0.2–0.3, 0.3–0.4, 0.4–0.5, 0.5–0.6, 0.6–0.7, 0.7–0.8, 0.8–0.9 and 0.9–1.0. When ranges were used, the lowest portions were always included and the highest portions excluded (e.g., the 0–2 range includes 0 through 1.99, excluding 2). The results of these quantifications were statistically analyzed using Prism software (Graph Pad Inc.). Data were expressed as means ± standard deviation. A paired 2-tailed t-test for parametric data or a 2-tailed Mann–Whitney U-test for non-parametric data was performed. The results were considered significant for p < 0.05.

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Fig. 2. Immunohistochemical analysis of the grafted cells 2 weeks after transplantation (A–C). Expression of S100, a Schwann-cell marker and Hoechst-labeled cells, a fluorescent nuclear marker. Some Hoechst labeled cells are co-expressing S100 (arrows). Scale bar: (A) 20 ␮m; (B) 10 ␮m; (C) 5 ␮m.

3. Results There were no complications related to the surgery, and all wounds healed well. All animals survived, and no trophic ulcerations on the operated leg were observed over the 6-week regeneration period. All data reported in this study represent the analysis from the distal segment, as illustrated in Fig. 1. Animals treated with 3T3 cells failed to show regenerating nerve fibers inside the collagen tube (Supplementary figure). 3.1.1. BMDCs were able to survive and differentiate into Schwann-like cells, leading to an increase in SC number To address the question whether the BMDCs population survives and differentiates inside the graft, we first analyzed the immunolabeled cells of regenerating fibers, 2 weeks after transplantation. Fig. 2(A–C) shows the results of the immunofluorescence for S100 protein. We found, in the treated group, Hoescht-positive cells throughout the gap, indicating the survival of injected BMDCs. Some of these BMDCs expressed the SC-marker S100, indicating that the implanted BMDCs were able to assume a Schwann-cell-like phenotype. These labeled cells are located around axons, suggesting that they were remyelinating them. In Fig. 3(A and B) we can also observe that BMDC-treated nerves showed DAPI staining higher cellularity in regenerating nerves compared to the DMEM-treated nerves. To evaluate the SC participation in the high celullarity inside the grafts, ultrastructural analyses and quantification were performed. At 6 weeks after surgery (Fig. 3C–E), regeneration was evident in all groups, but the treated group showed better overall tissue organization (Fig. 3C and D). Quantitative analysis of the number of Schwann-cell nuclei showed that they were significantly more numerous (P < 0.01) in the experimental group than in the control group (Fig. 3E). These results suggest that the treatment strategies used in this study were able to positively influence Schwann-cell responses.

to survival effects, the number of myelinated nerve fibers was significantly higher in the treated group (11.62 ± 1.26, p < 0.01) than in the untreated group (2.67 ± 2.04). The number of non-myelinated fibers was also higher in the treated group (17.12 ± 3.87) than in the untreated one (11.84 ± 3.68), but this difference was not significant (p = 0.055). These results are in agreement with quantitative analyses of the nerve proximal segment (previous results). After that, to evaluate the stability and maintenance of the growing fibers at the distal end, we calculated the ratio between the number of distal (segment 2) and proximal (segment 1) myelinated and non-myelinated fibers from the DMEM and BMDCs groups, which resulted in a higher significant rate, in both (Fig. 4E and F) myelinated (0.28 ± 0.21; 1.03 ± 0.13, p < 0.01) and non-myelinated fibers (0.42 ± 0.16; 0.91 ± 0.24, p < 0.05).

3.1.3. Treatment with BMDCs improves functional recovery and g-ratio values Mice were tested to assess the recovery of motor function throughout the study period after injury, at 2, 4, and 6 weeks, using the walking-track analysis (Inserra et al., 1998). A total of 300 prints were measured for both groups (BMDCs and DMEM) to obtain the PL and TS values. The SFI was used to compare the performance of the experimental group. All experimental animals showed functional deficits, with great loss of function in the second week after injury. Animals from both groups showed progressive improvement of motor function as evidenced by SFI values, in the fourth week. However, at the last time studied, we could observe a significant improvement of function in the BMDCs-treated group (p < 0.05), compared to the control group (Fig. 5A). In contrast, the control group stopped recovering motor function at the fourth week, and then began a decline, showing a functional worsening by 6 weeks after lesion. Concerning the G-ratio the BMDCs group also presented better results, since most fibers fell into the optimal range (0.5–0.6, Fig. 5B).

3.1.2. Treatment with BMDCs improves regeneration of nerve fibers and enhances the axonal growth rate

4. Discussion

The immunohistochemistry for GAP 43 (Fig. 4A and B) showed that regenerating axons elongated into the graft, in both groups. A larger number of regenerating nerve fibers successfully reached the distal nerve segment in the BMDCs group. In contrast, very few regenerating nerve fibers were observed at the distal end of the nerves from the control groups. Regenerated myelinated and non-myelinated axons were found, by electron microscopy, at the distal end of the graft (segment 2) in both groups (Fig. 4C and D). The ultrastructural analysis showed that growing nerves from the treated groups exhibited many regenerating clusters of nerve fibers, formed by preserved nerve fibers, indicating better tissue organization. Conversely, the untreated group showed growing nerves with fewer and smaller regenerating clusters. They had smaller-diameter fibers and thinner myelin sheaths. With regard

Axonal regeneration after nerve injury is a prerequisite for functional recovery. The degree of this recovery depends on the number of regenerating axons, the proper reinnervation of target organs, the reestablishment of physiological properties (Fu and Gordon, 1995; Krarup et al., 2002), and the length of time for the process to occur (Weber and Mackinnon, 2005). In this study, we used an adult mouse peripheral-nerve transection and a surgical repair model to demonstrate that BMDCs implanted into a bioresorbable nerve guide were able to survive and to differentiate into a Schwann-celllike phenotype, improve the growth rate of nerve fibers, increase the number of nerve fibers (myelinated and non-myelinated), gratio values and the number of Schwann-cell nuclei distally. We further observed a significant return of motor function in treated animals.

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Fig. 3. (A and B) Micrographs of the nerve, 2 weeks after regeneration. The diameters of regenerating nerves were quite different as shown in DAPI-stained sections. (A) Fewer nuclear-labeled cells are seen in the control group when compared to the experimental group (B). Scale bar: 100 ␮m. (C and D) Ultra-thin cross-sections from the distal segments of the regenerating sciatic nerve, 6 weeks after nerve injury. (C) Control group (DMEM), showing small and poorly developed regenerating clusters exhibiting fibers with a thin myelin sheath (arrows). (D) Experimental group (BMDCs) showing a regenerating cluster consisting of myelinated (arrows), and non-myelinated nerve fibers (arrowheads) surrounded by processes of perineurium-like cells. A Schwann-cell nucleus is observed (asterisk). Many myelinated nerve fibers with well-preserved axoplasm and a proper myelin sheath are seen. Scale bar: 1 ␮m. (E) Quantitative analysis of the number of Schwann-cell nuclei observed in ultra-thin sections from the distal segment. Values represent mean ± SD. ***P = 0.0003.

Different species of animals have been used in peripheral-nerve experimental studies, but the mouse is most frequently used nowadays. Some advantages of using mouse models are: they are small and very prolific, have a short gestation period, and are easy to handle and maintain. According to the CEC (Commission of the European Communities), mouse models are the most useful in scientific research (54%). Also, the mouse model has become the best choice because there are now many kinds of transgenic mice, which allows us to study the role of a particular protein/molecule in the mechanism of degeneration and regeneration (Chorilli et al., 2007).

Regarding transdifferentiation of mesenchymal stem cells into Schwann-like cells, authors have demonstrated that bone-marrow cells are very useful, in vitro (Sánchez-Ramos et al., 2000) and/or in vivo. Keilhoff et al. (2006) were able to transdifferentiate rat mesenchymal stem cells into a Schwann-cell-like phenotype, as characterized by its morphology and expression of the appropriate markers. Shimizu et al. (2007) demonstrated the capability of human bone-marrow stromal cells to differentiate into Schwannlike cells, in a rat sciatic-nerve-injury model. In agreement with these studies, our results demonstrated the plasticity of BMDCs

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Fig. 4. (A and B) Immunohistochemistry for GAP 43 revealed that the regenerated fibers reached the end of the graft in both groups, and evidenced a more intense labeling in the BMDCs group (B). (C and D) Electronmicrographs showing fewer nerve fibers in the DMEM group (C) compared to the BMDCs group (D). (E and F) Quantitative analysis of the distal/proximal ratio of myelinated (E) and non-myelinated (F) regenerating fibers. Values represent mean ± SD. *P < 0.05. **P < 0.01.

and their transdifferentiation into a Schwann-cell-like phenotype. As a result of this transdifferentiation potential, we observed an improvement in the rate of myelination. To ensure the reliability of the results obtained with BMDC, we used 3T3 cells as a second control since they do not have stem-cells characteristics, and are completely different from neural cells. The results showed that 3T3 cells were not capable to induce regeneration and therefore the significant results obtained with BMDC were likely a result of its stem-cell properties. Different conduits have been used to bridge nerve gaps. With the progress in tissue engineering, one of the main strategies for repairing peripheral-nerve defects has focused on creating biological and non-biological tubular nerve guides. The ultimate bioengineered

nerve conduit must be able to enhance regeneration, block invasion of scar tissue, and be biodegradable. During the past few years, studies have concentrated on various conduit materials, particularly on biodegradable polymers (poly-glycolic acid—PGA, PLA, PPE, and others), as well as silicone (Weber and Mackinnon, 2005; Pierucci et al., 2008). As a result of these early studies, some authors believe that the conduit itself does not have a profound effect on the outcome of nerve repair. As a result, nerve repair is now focusing on molecular biological manipulation of the internal conduit features, in order to permit a combination of scaffold-tissue type, trophic factors, and the use of different sources of stem cells (Amado et al., 2008; Johnson and Soucacos, 2008; Luís et al., 2008).

Fig. 5. SFI and g-ratio analyses. (A) Quantitative analysis of SFI values. Variation of SFI with time showed differences between the groups, with a significant improvement of motor function 6 weeks after surgery in the animals receiving BMDCs grafts (B) G-ratio stratified by ranges. The majority of nerve fibers are in the range of 0.5–0.6. Values represent mean ± SD. *P < 0.05.

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The generally poor and variable outcome after traumatic peripheral-nerve lesions (Mackinnon and Dellon, 1990; Gordon et al., 2003; Weber and Mackinnon, 2005; Moldovan et al., 2006; Johnson and Soucacos, 2008) has stimulated the search for improved methods of repair, when loss of nerve tissue results in a gap between the nerve ends. The peripheral nervous system has great regeneration potential, particularly when an appropriate microenvironment is provided (Ide, 1996; Makwana and Raivich, 2005; Chen et al., 2007; Vargas and Barres, 2007). Recent experimental studies have demonstrated the use of different types of stem cells in the process of nerve regeneration. Cui et al. (2008) showed that transplanted neurally induced embryonic stem cells can differentiate into myelin-forming cells and promote nerve regeneration. On the other hand, Luís et al. (2008) demonstrated that the presence of neural cells in the PLGA tube guides did not result in functional recovery. Also, morphometric analysis showed a significantly lower number and size of regenerated nerve fibers, suggesting that this particular type of stem cells has negative effects on nerve regeneration. Regarding the use of bone-marrow cells, the results are more regular in terms of the beneficial effects on regeneration of damaged tissues (Dezawa et al., 2001; Kawada et al., 2004; Bossolasco et al., 2005; Lee et al., 2005; Keilhoff et al., 2006; Pereira Lopes et al., 2006; Chen et al., 2007; Shimizu et al., 2007; Fernandes et al., 2008). It is known that some molecules such as neurotrophins play an important role in nerve-fiber regeneration, and are regarded as axonal growth-promoting factors that stimulate neurite outgrowth and Schwann-cell proliferation and migration (Anton et al., 1994; Chen et al., 2007; Mahay et al., 2008). Among these, one of the most important is the nerve-growth factor (NGF), which is released from the distal nerve end during Wallerian degeneration, and is primarily produced by Schwann cells (Ide, 1996; Fu and Gordon, 1997; Chen et al., 2007; Mahay et al., 2008). The BMDCs used in our study express NGF␤ in vitro (Pereira Lopes et al., 2006). Furthermore, these cells secrete interleukin-6 and stem-cell factors (Balduíno et al., 2005), which are potent inducers of Schwann-cell proliferation, differentiation, and migration (Hirota et al., 1996; Haggiag et al., 2001). Our data suggest that BMDCs implanted into the collagen tube may induce increased numbers of Schwann cells, improving nerve-fiber myelination and regeneration. Nerve regeneration and recovery of target function in humans are often disappointing, even after microsurgical repair of injured nerves (Sunderland, 1978; Terzis et al., 1997). In particular, the regenerative outcome may be discouraging when reinnervation of the denervated target organ is delayed due to either a long distance between the target and lesion site, or delayed nerve repair following major trauma (Sunderland, 1978; Lundborg, 2003). According to Gordon et al. (2003), when the distance between the injury site and the target organ is long, the regenerative potential of the neuron cell bodies and the plasticity of Schwann cells decline with time, and reduce the number of motor axons that regenerate and make functional connections with denervated muscle fibers. Also, Furey et al. (2007) stated that poor functional recovery is not a result of failure to reinnervate targets, but a result of a sufficient number of mature axons failing to reach their respective targets, by being deprived of neurotrophic support in the distal nerve stumps. As a result, poor functional recovery is observed in animal models and human patients when nerve repair is delayed. The reinnervation time is the major important factor that influences the level of outcome after different nerve lesions in monkeys (Krarup et al., 2002). In humans, there is general agreement that functional recovery is improved when the time to reinnervation is short, and that surgical treatment, whenever needed, should be carried out as quickly as possible after a nerve lesion (Lundborg, 2003). Hence, the growth rate and the number of axons may have direct implications for functional recovery. The g-ratio is an index that reflects the opti-

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mal axonal myelination that leads to an appropriate conduction of action potentials. Based on biophysical properties optimal g-ratio values can be defined for the myelinated fibers of the sciatic nerve as 0.55–0.68 (Chomia and Hu, 2009). In our experiment, the animals treated with BMDCs achieved a higher significant growth rate, larger number of nerve fibers (myelinated and non-myelinated) and increased g-ratio values. All these results may characterize an earlier reinnervation of the target organ. GAP-43 is expressed at high levels in the growth cone during axonal regeneration, and is considered a crucial component of an effective regenerative response in the nervous system. Xu et al. (2009) used a silicone nerve guide with neurotrophic factors to enhance sciatic-nerve regeneration, and demonstrated intense GAP-43 immunoreactivity in the regenerating axons. In the present study, even though GAP-43 immunoreactivity was present in the distal stump of both groups, it was more intense in the cell-treated group. These data indicate that these cells promoted axonal regeneration. In this study, we further observed that treatment with BMDCs significantly improved the recovery of motor function in the reinnervated hind limbs, as demonstrated by the better SFI values, from weeks 2 to 6 after nerve repair. Walking-track analysis has been used to reliably determine functional recovery following nerve repair, and the SFI formula is quite successful in describing the functional deficits produced soon after complete nerve lesions (Inserra et al., 1998). The increased number of myelinated nerve fibers, g-ratio values and the maintenance of the growth rate, from the proximal to the distal stump, in the treated group may explain this result. Our experiment confirmed earlier observations concerning the beneficial effects of the use of bone-marrow cells. Furthermore, it demonstrated that the association of a biodegradable collagen tube filled with BMDCs induces transdifferentiation into Schwann-like cells, maintains the growth rate of axons from the lesion site into the distal nerve stump, and consequently promotes a consistent return of motor function. Improving regeneration would counteract the delay in reinnervation of the target organ, which is critical for a successful functional outcome. In addition, this study adds to evidence that these strategies provide a tool for cell-based approaches in peripheral-nerve diseases, in order to overcome the relatively poor outcome of clinical nerve repair. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.micron.2010.05.010. References Aebischer, P., Salessiots, J.M., Winn, S.R., 1989. Basic fibroblast growth factor released from synthetic guidance channels facilitates regeneration across long nerve gaps. J. Neurosci. Res. 23, 282–289. Amado, S., Simões, M.J., Armada da Silva, P.A., Luís, A.L., Shirosaki, Y., Lopes, M.A., Santos, J.D., Fregnan, F., Gambarotta, G., Raimondo, S., Fornaro, M., Veloso, A.P., Varejão, A.S., Maurício, A.C., Geuna, S., 2008. Use of hybrid chitosan membranes and N1E-115 cells for promoting nerve regeneration in an axonotmesis rat model. Biomaterials 29, 4409–4419. Anton, E.S., Weskamp, G., Reichardt, L.F., Matthew, W.D., 1994. Nerve growth factor and its low-affinity receptor promote Schwann cell migration. Proc. Natl. Acad. Sci. U.S.A. 29, 2795–2799. Balduíno, A., Hurtado, S.P., Frazão, P., Takiya, C.M., Alves, L.M., Nasciutti, L.E., ElCheikh, M.C., Borojevic, R., 2005. Bone marrow subendosteal microenvironment harbours functionally distinct haemosupportive stromal cell populations. Cell Tissue Res. 319, 255–266. Baptista, A.F., Gomes, J.R., Oliveira, J.T., Santos, S.M., Vannier-Santos, M.A., Martinez, A.M., 2008. High- and low-frequency transcutaneous electrical nerve stimulation delay sciatic nerve regeneration after crush lesion in the mouse. J. Peripher. Nerv. Syst. 13, 71–80. Bianco, P., Riminucci, M., Gronthos, S., Robey, P.G., 2001. Bone marrow stromal cells: nature, biology and potential applications. Stem Cells 19, 180–192.

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