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Bone marrow stromal cells and resorbable collagen guidance tubes enhance sciatic nerve regeneration in mice
Experimental Neurology 198 (2006) 457 – 468 www.elsevier.com/locate/yexnr
Bone marrow stromal cells and resorbable collagen guidance tubes enhance sciatic nerve regeneration in mice Fátima Rosalina Pereira Lopes a , Lenira Camargo de Moura Campos a , José Dias Corrêa Jr. e , Alex Balduino a , Silvano Lora c , Francesco Langone b , Radovan Borojevic a,d , Ana Maria Blanco Martinez a,⁎ a
Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Brasil b Departamento de Fisiologia e Biofísica, Universidade Estadual de Campinas, São Paulo, Brasil c Istituto per la Sintesi Organica e la Fotoreattività, CNR, Legnaro-Padova, Italy d APABCAM, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Brasil e Departamento de Morfologia. Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Brasil Received 21 July 2005; revised 23 November 2005; accepted 14 December 2005 Available online 20 February 2006
Abstract We evaluated peripheral nerve regeneration using a tubular nerve guide of resorbable collagen filled with either bone marrow-derived cells (BMDCs) in Dulbecco's cell culture medium (DMEM) or with DMEM alone (control). The control group received just the culture medium (vehicle). The left sciatic nerves of ten isogenic mice were transected and the tubular nerve guides were sutured to the end of the proximal and distal nerve stumps. Motor function was tested at 2, 4 and 6 weeks after surgery using the walking track test. The pawprints were analyzed and the print lengths (PL) were measured to evaluate functional recovery. After 6 weeks, mice were anesthetized, perfused transcardially with fixative containing aldehydes, and the sciatic nerves and tubes were dissected and processed for scanning and transmission electron microscopy. Scanning electron microscopy of the collagen tube revealed that the tube wall became progressively thinner after surgery, proving that the tube can be resorbed in vivo. Quantitative analysis of the regenerating nerves showed that the number of myelinated fibers and the myelin area were significantly increased in the experimental group. Also, motor function recovery was faster in animals that received the cell grafts. These results indicate that the collagen tube filled with BMDCs provided an adequate and favorable environment for the growth and myelination of regenerating axons compared to the collagen tube alone. © 2005 Elsevier Inc. All rights reserved. Keywords: Peripheral nerve regeneration; Bone marrow-derived cells; Collagen tube; Schwann cells; Transplantation; Electron microscopy
Introduction When compared to the central nervous system (CNS), peripheral nervous system (PNS) has a greater capacity for regeneration, although complete repair is rare and, in severe injuries, functional recovery is poor. In cut injuries, the most common procedure is to suture the two nerve stumps together ⁎ Corresponding author. Av. Brig. Trompowsky s/n, Cidade Universitária, Ilha do Fundão, Centro de Ciências da Saúde, Instituto de Ciências Biomédicas, Bloco F, Departamento de Histologia e Embriologia, 21941-540, Rio de Janeiro, RJ, Brasil. Fax: +55 21 25626480. E-mail address: [email protected] (A.M. Blanco Martinez). 0014-4886/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.12.019
(Madison et al., 1992; Ide, 1996). However, when there are large gaps, a scaffold or accessory structures are generally required to guide and protect the nerve during growth, and to provide a tension-free full regeneration of the nerve. Although nerve allografts and autografts are indicated in these cases, this strategy is usually avoided because of the high tissue morbidity (Heath and Rutkowski, 1998; Evans, 2000), and artificial tubes have been used to guide nerve regeneration (Terzis et al., 1997; Nakamura et al., 2004). Recently, several groups have tried to develop synthetic biodegradable polymers to build tubular prostheses to connect the proximal and distal stumps ((Keeley et al., 1993; Archibald et al., 1995; Langone et al., 1995; Wang et al., 2001; Ngo et al., 2003; Nakamura et al., 2004). Their use
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has the advantage of avoiding a second surgery to relieve chronic nerve compression and the unavoidable tissue reaction from the implanted tissue. Neurotrophic factors such as neural growth factor (NGF) and brain-derived-neurotrophic factor (BDNF), and/or transforming growth factor-beta (TGFβ), or extracellular matrix (ECM) molecules, such as laminin and fibronectin, have also been combined with the prosthesis, enhancing and improving axonal sprouting and guidance towards the target (Lindsay, 1988; Aebischer et al., 1989; Rich et al., 1989; Menei et al., 1998; Tom et al., 2004; Yu and Shoichet, 2005). Transplantation of Schwann cells has also been tested, since these cells are essential for nerve regeneration and development (Aguayo et al., 1977; Frerichs et al., 2002; Mosahebi et al., 2002; Fansa et al., 2003). However, cell number and donor site availability are two limiting factors to the use of Schwann cells for cell therapy. Therefore PNS regeneration with successful functional recovery remains a challenge to physicians and neuroscientists. Support of bone marrow-derived cells has been used recently in several models of tissue regeneration. Bone marrow harbors two distinct types of stem cells that are closely associated and interacting: blood and mesenchymal cell progenitors (Bianco and Robey, 2000). The mesenchymal stem cells (MSC) have the ability to differentiate into other cells of either mesenchymal or epithelial origin, such as endothelial cells, striated skeletal muscle cells, cardiomyocytes, and hepatocytes (Kawada et al., 2004; Sato et al., 2005; Bossolasco et al., 2005; Lee et al., 2005). This capacity makes MSCs particularly promising for cell therapy and tissue engineering. Multipotent MSCs can be isolated from the subendosteal region, in which they sustain proliferation of hematopoietic cells and interact with blood vessels and the subendosteal nerve plexus (Balduino et al., 2005). In vitro induction of molecular markers of glia and neurons in MSCs has been reported (Sánchez-Ramos et al., 2000). In vivo studies have also shown that they can improve nerve regeneration, by differentiating into Schwann-like cells and supporting nerve fiber growth and myelination (Dezawa et al., 2001; Cuevas et al., 2002; Choi et al., 2005). Therefore, we have chosen MSCs in order to promote simultaneously growth and differentiation of nerve fibers, blood vessels and the supportive connective tissue. Differently from the hematopoietic stem cells (HSCs), the exact niche of the MSC is not clear, although a few observations suggest that they reside around arterioles and capillaries with a phenotype close to pericytes (Shi and Gronthos, 2003). Recent studies showed that multipotent MSCs can be isolated from endosteal surface rich in osteoblasts, which are similar to those isolated from the bone marrow (Tuli et al., 2003). Endosteal bone-lining cells participate in HSC niche formation (Taichman and Emerson, 1998; Calvi et al., 2003; Zhang et al., 2003) together with subendosteal reticular cells (Balduino et al., 2005), controlling HSC quiescence, expansion and/or differentiation. MSCs have been described recently as promoters of axonal regeneration and myelination (Dezawa et al., 2001; Cuevas et al., 2002; Choi et al., 2005). Working with a population of in vitro differentiated MSCs, Dezawa et al. (2001) have
reported that these cells have an important regenerative potential after being transplanted into the stumps of transected sciatic nerves. Cuevas et al. (2002) assessed the therapeutic potential of unmanipulated MSCs on peripheral nerve regeneration, and also reported a functional and morphological improvement after sciatic nerve transection. More recently Choi et al. (2005) have reported that, when MSC were added to a vein conduit they promoted an increase in the number and diameter of myelinated axons that regenerated across a 15-mm nerve gap in rabbits. In the present study, we tested two strategies for the improvement of sciatic nerve regeneration after surgical transection: (1) a biodegradable collagen tube was used to bridge the nerve gap; (2) the tube was filled with a selected population of MSCs from the endosteal surface, hereafter referred to as bone marrow-derived cells (BMDCs). The combination of these two strategies has proven to be very encouraging since it results in regenerating nerves with an overall better organization and vascularization. In addition, the nerves supplemented with BMDCs showed a significant increase in the number of myelinated nerve fibers, which displayed a thicker myelin sheath than the nerves without BMDCs. Materials and methods Culture of bone marrow-derived cells (BMDCs) Inbred Balb/c mice were sacrificed, and bone marrow stromal cells were harvested following the previously described protocol (Balduino et al., 2005). Briefly, 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 cell culture medium in order to wash out 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 min each, at 37°C. Cells were harvested from the second digestion, washed twice in cold BSS, suspended and plated in Dulbecco's medium (DMEM-Sigma) containing antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin) and 10% fetal bovine serum (FBS-Cultilab, Campinas, SP, Brazil). To identify these cells later, they were labeled with 5 μg/mL of bisbenzamide (Sigma-Hoechst 33258), prior to surgical implantation. Reverse transcriptase polymerase chain reaction (RT-PCR) RNA was extracted using the TRIzol (Invitrogen LifeTechnologies, São Paulo, SP, Brazil) from stromal cells isolated from the bone marrow as described above and maintained in culture for 3–7 days. One μg was used to prepare cDNA. PCR (25 μL), was carried out with Taq-DNA Polymerase (Invitrogen) and cycles were established as
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follows: initial denaturation at 95°C for 10 min and 35 cycles of PCR (95° for 30 s, 60°C for 30 s, and 72°C for 30 s), followed by an extra extension period of 10 min, at 72°C. D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primer pairs used were: GAPDH sense—TGC ACC ACC AAC TGC TTA G; GAPDH antisense—GAT GCA GGG ATG ATG TTC; BDNF sense— CTG AGC AAA GCC GAA CTT CT; BDNF anti-sense— GCC TTC ATG CAA CCG AAG TA; NGFb sense—GTC TGG GCC CAA TAA AGG TT; NGFb anti-sense—CCT GTA CGC CGA TCA AAA AC. Collagen tube The artificial nerve conduit was a biodegradable tube obtained from 18-month-old calf peritoneal sheath removed just after death. The parietal layer of the peritoneal sheath was removed and washed in ethyl alcohol, rinsed with chloroform and dried at room temperature. The parietal layer was examined to select and eliminate regions with irregular macroscopic morphological features. The remaining material was compressed until an adequate and regular thickness was obtained. After that, rectangular fragments with a surface of 100 mm2 were treated with proteolytic enzymes, washed in distilled water and wrapped around a 1.0-mm-diameter stainless-steel rod. The preparation was immersed in an aqueous solution of a cross-linking agent for 2 h. The tubes were dried at 37°C, stripped from the rods, washed in water to remove the unreacted cross-linking agent, dried again, packed in vials and then sterilized by gamma radiation at 25 kGy. No changes in mechanical properties and morphologic structure of the tubes were observed. Biocompatibility tests were performed in a previous study by in vitro cell culture and in vivo subcutaneous implants in mice (Lora et al., 1997). Effectiveness of the tubes as a nerve guide was also tested by using them to repair transected sciatic nerves in mice (Langone et al., 1997). Surgical procedure Ten female inbred adult Balb/c mice weighing 20–25 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. Subsequently, BMDCs were injected into the tube in the gap area. The experimental group (n = 5) 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/μL for each animal. The control group (n = 5) received the collagen tube filled with DMEM (1.0 μL). After 6 weeks of regeneration, mice were anesthetized and perfused transcardially with fixative solution containing aldehydes (4% paraformaldehyde and 2.5% glutaraldehyde
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in 0.1 M phosphate buffer, pH 7.4). The left sciatic nerves were exposed and the mid-portions of the growing nerves (corresponding to the previous gap area) were harvested and processed for electron microscopy. Scanning electron microscopy For scanning electron microscopy (SEM), collagen tubes were fixed in 3% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), postfixed in 1% OsO4 for 2 h, washed and dehydrated in ethanol series. The samples were dried at critical point (CPD 030, Balzers Instruments, Liechtenstein) and gold sputtered (FL-9496 Balzers Union Coater). Scanning electron micrographs were obtained with a JEOL JSM-5310 scanning microscope. Transmission electron microscopy After fixation by perfusion, the segments of the nerves were immersed for 2 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), washed in the same buffer followed by 0.1 M cacodylate buffer (pH 7.4), postfixed for 90 min in 1% osmium tetroxide containing 0.8% potassium ferrocianide and 5 nM calcium chloride in 0.1 M cacodylate buffer (pH 7.4). The segments were washed in 0.1 M cacodylate buffer (pH 7.4) and distilled water and stained in 1% uranyl acetate overnight, dehydrated in graded acetone, infiltrated with Poly/Bed 812 resin (Polysciences, Inc., Washington, PA) and polymerized at 60°C for 48 h. Five hundred nanometer semi-thin cross-sections were obtained for light microscopy studies, and stained with toluidine blue. Ultra-thin cross-sections (60–70 nm) for transmission electron microscopy (TEM) analysis were collected on copper grids and contrasted in uranyl acetate and lead citrate. TEM studies were carried out using a Zeiss 900 transmission electron microscope operated at 80 kV. Functional analysis—walking tracks All animals were subjected to a postoperative walking track analysis, based on the protocol described by Inserra et al. (1998), 2, 4 and 6 weeks after surgery. Briefly, pawprints were recorded by painting the hind paws of each animal with China ink. The animals were allowed to walk along a 45 × 6.5 cm track on a white paper (Canson A4 140 g/m2). The pawprints were analyzed and the distance between the third toe and the hind pad – print length (PL) – was measured in the left paws, for normal and operated animals. Differences between the groups were assessed using the ANOVA and Kruskal–Wallis tests. A P value b0.05 was considered statistically significant. Quantitative analysis For the sampling procedure, we photographed in the transmission electron microscope 8 random fields in each nerve cross-section at a magnification of 4400×. All crosssectional areas of the nerve were scanned and sampled, avoiding a sample bias. A total of 40 pictures were obtained
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for each experimental group. Since we had two experimental conditions (sectioned treated with DMEM and sectioned treated with DMEM + BMDC), the total number of pictures was 80. All negatives were scanned and subjected to quantitative analysis using the Image Pro Plus program (Media Cybernetics). Based on the qualitative observations, we calculated and compared the following parameters between the two groups: total number of nerve fibers, number of non-myelinated fibers, number of myelinated fibers, nerve fiber area, axon area and myelin area. The results of these quantifications were analyzed statistically using Prism software (Graph Pad Inc.) and the Mann– Whitney or Kruskal–Wallis tests. Results were considered significant for P b 0.05. Results General observations None of the animals developed any serious post-surgical complication. Wounded tissues healed spontaneously and there were no trophic ulcerations on the operated legs.
Collagen tubes are progressively degraded in vivo Fig. 1 shows the scanning electron microscopy of a collagen tube before surgery (Figs. 1A–D), and 6–10 weeks after being implanted (Figs. 1E–F). Fig. 1A presents the tube at a low magnification. At a higher magnification, we can observe that the tube wall is composed of highly ordered and parallel layers of collagen fibers (Figs. 1B–D). The outer surface of the tube exhibits a network organization (Fig. 1B), while the inner surface displays alignment of the collagen fibers (Fig. 1D). The thickness of the tube wall can be seen in Fig. 1C. Modifications of the tube wall after 6 and 10 weeks of engraftment were also evaluated by scanning electron microscopy. Fig. 1E shows a transverse section of the tube wall interacting with the growing tissue inside it, 6 weeks after surgery. The tube wall is thinner at some areas and there is a small amount of cell reaction around the tube. At 10 weeks after surgery the tube wall is much thinner (Fig. 1F) than the intact tube and is in intimate contact with the tissue growing inside. Blood vessels filled with blood cells are seen running inside the tube, indicating that they are functional.
Fig. 1. Scanning electron micrographs of the collagen tube. (A) View of the empty collagen tube. (B) External surface of the tube wall exhibiting a network aspect; (C) View of the tube wall; (D) internal surface displaying a longitudinal alignment of the collagen fibers. (E, F) Transverse section of the tube wall at 6 (E) and 10 weeks (F) after implantation. Observe that the thickness of the tube wall is much thinner 10 weeks after implantation; blood vessels are also observed in F. Areas between arrows represent the thickness of the tube wall.
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BMDC treatment provides a better overall organization of regenerating nerves
Fig. 2. RT-PCR analysis of BMDC population. Expression of GAPDH, BDNF, and NGFβ was evaluated. Figure shows the agarose electrophoresis analysis of the PCR-product.
BMDCs express NGFβ Expression of two important neural growth factors, BDNF and NGFβ, in bone marrow subendosteal cells was monitored by RT-PCR. Cells obtained from cultures, expressed NGFβ but not BDNF, as shown in Fig. 2.
After 14 days' culture BMDCs were examined by phase microscopy (Fig. 3A). By this time, the cells had reached confluence and formed a compact layer in which individual cells displayed a polygonal shape. Since we labeled the BMDCs with Hoeschst, the cells' nuclei can be seen in blue (Fig. 3B). A longitudinal section of the sciatic nerve proximal stump, 1 week after engraftment, shows rows of Hoeschstlabeled cells (Fig. 3C and insert). These cells invade the surviving proximal stump in a very organized fashion, suggesting that the implanted BMDCs are able to migrate and interact with the peripheral nerve. Semi-thin sections of regenerating nerves after 6 weeks revealed extensive structural differences between the control and the experimental groups (Fig. 4). In the latter, the growing nerve had a well-defined perineurium, with parallel layers of fibroblastoid cells (Figs. 4A and B). Very few inflammatory cells were observed in the surrounding space.
Fig. 3. (A) Phase microscopy of BMDCs (arrows) in culture, showing their typical morphological characteristics; (B) The nuclei of the Hoeschst-labeled cells are seen in blue (arrows); (C) Longitudinal section of the sciatic nerve proximal stump, 1 week after engraftment, exhibiting rows of Hoeschst-labeled cells (arrows). Scale bar: A–C: 40 μm. Insert: 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Semi-thin cross-sections of regenerating sciatic nerve (Toluidine blue stain) 6 weeks after injury. (A–B) Experimental (BMDC) group; (A) this section displays an organized regenerating nerve; (B) many clusters of regenerating nerve fibers (arrows) and newly formed blood vessels are seen (arrowheads). (C–D) Control (DMEM) group; (C) the clusters of regenerating nerve fibers are not so obvious and the tissue is less organized. (D) Observe large and empty newly formed blood vessels (arrows). Scale bar: 40 μm.
The regenerating nerve was divided into discrete axon bundles, surrounded by well-formed endoneurium with thin cells and abundant extracellular matrix. In the group that did not receive BMDCs, the perineural space was occupied by a thick layer of poorly organized cells, and numerous nonadherent inflammatory cells (Figs. 4C and D). There was no endoneurium, and the space between growing axons contained only loose unorganized material. Simultaneously, the experimental group contained numerous blood vessels. Capillaries were present in the endoneurium and perineurium, and larger vessels in the perineurium only. They were parallel to the axis of the growing nerve, indicating a neoformation of an ordered vascular tree. Besides the endothelium, all the vessels contained pericytes and the larger ones had mural cells that sustained the vascular walls and allowed the vessels to maintain a regular oval section, compatible with the controlled internal pressure. Most of the vessels contained erythrocytes, indicating a functional blood supply. Conversely, the control group contained no typical capillaries inside the growing nerve. The blood vessels both inside and around the nerve had a distended lacunar form, without mural cells, and with only very rare erythrocytes, indicating that the blood vascular tree was not sufficiently organized to sustain a functional blood supply.
BMDC treatment enhances axon regeneration and myelination Ultrastructural analysis of the regenerating nerves also revealed important differences between control and experimental groups (Figs. 5 and 6). Regenerating nerves from the experimental group exhibited many regenerating clusters of nerve fibers. These clusters were constituted of myelinated and non-myelinated nerve fibers ensheathed by endoneural cells and their processes. Among the nerve fibers were collagen fibrils forming the endoneurium and nuclei of Schwann cells associated with myelinated fibers or groups of non-myelinated fibers. The axoplasm and myelin sheaths were well preserved and had a normal appearance (Fig. 5A). Fig. 5B shows the periphery of the nerve, where there is a compact perineurium composed of alternating layers of collagen and perineural cells and processes. Healthy-looking blood vessels among or inside the regenerating clusters can also be seen (Fig. 5C). In contrast with these results the control group showed growing nerves with less obvious and smaller regenerating clusters. Also, there was an apparent reduction in the number of myelinated nerve fibers, which looked thinner and more dispersed (Fig. 6A). At the periphery of the nerve, the cell layer corresponding to the perineurium was also observed (Fig. 6B) but it was less compact when compared to the experimental group.
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(including the fibers which were still in the process of myelination) showed that they were significantly more numerous (P = 0.007) in the experimental group than in the control group (Fig. 7A). Concerning the number of non-myelinated fibers, there was no significant difference between the two groups (Fig. 7B). The total number of nerve fibers was greater in the experimental group, even though this difference did not achieve significance (Fig. 7C). With regard to nerve fiber area and myelin sheath area there was a significant increase in the experimental group compared to the control group (Figs. 8A and B). Meanwhile, there was no significant difference in the axon area between the experimental and the control group (Fig. 8C).
Fig. 5. Transmission electron micrographs of regenerating nerve, 6 weeks after the BMDC engraftment. (A) Cross-section of the nerve showing many regenerating clusters consisting of myelinated (thin arrows), myelinating (thick arrow) and non-myelinated (arrowheads) nerve fibers. The axoplasm and myelin sheaths are well preserved. (B) At the periphery of the nerve, a compact and well-organized perineurium is observed (stars). (C) Healthylooking blood vessels (arrow) are seen among or inside the regenerating clusters. Scale bar: 1.7 μm.
BMDC treatment increases both the number of myelinated fibers and the myelin area Morphometric studies were carried out based on the qualitative observations described above. All results are expressed as mean ± S.D. of the replicates. Quantitative analysis of the number of myelinated nerve fibers
Fig. 6. Transmission electron micrographs of the regenerating nerve, 6 weeks after surgery (DMEM-control). (A) Cross-section showing small and poorly developed regenerating clusters, composed of dispersed and thin myelinated nerve fibers (arrows). (B) At the periphery of the nerve, a non-compact perineurium is observed. Scale bar: 1.7 μm.
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Discussion The peripheral nervous system has a great potential for regeneration, particularly when an appropriate microenvironment is supplied. However, current treatments for peripheral nerve injury accompanied by loss of a critical length of the nerve do not provide good functional recovery. In the present work, we combined the use of a biodegradable tube with the bone marrow stromal cells engraftment in order to enhance peripheral nerve regeneration after sciatic nerve injury. The combination of these two strategies resulted in a better regeneration and myelination of sciatic nerve fibers. In addition, the regenerating nerves showed an overall better organization and vascularization when compared to control groups. Nowadays, the most common strategy to solve the problem of peripheral nerve gap after an injury is to use an autologous nerve graft as a bridge (Evans et al., 1995;
Fig. 7. Quantitative analysis of the number of myelinated nerve fibers (A), number of non-myelinated fibers (B) and total number of nerve fibers (C) in control (DMEM) and experimental (BMDC) groups. Values represent mean ± SD. *P = 0.007.
BMDC treatment improves motor function Footprints were monitored at 2, 4 and 6 weeks after surgery. A total of 335 prints were measured for three animal groups: experimental group (BMDCs), control group (DMEM) and a third group (normal, unoperated). This third group was added in order to better characterize and compare the data. Analyzing the footprints we concluded that the most obvious visual difference was seen for the print length (PL), which represents the distance between the third toe and the hind pad (Fig. 9A). All results are expressed as mean ± SD of the replicates. Quantitative analysis of the PL showed a better recovery for the experimental group than for the control group, even though statistical analysis demonstrated no significant difference among the groups (Figs. 9B and C). The return of motor function could be assessed qualitatively by the animals' ability to bear their own weight during the walking track test.
Fig. 8. Analysis of nerve fiber area (A), myelin sheath area (B) and axon area (C) in control (DMEM) and experimental (BMDCs) groups. Values represent mean ± SD. *P = 0.007.
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Fig. 9. Quantitative analysis of print length (PL). (A) Pattern of normal (unoperated) and experimental (after sciatic nerve transection) pawprints. Note the elongation of the PL in operated animals. (B–C) Variation of PL with time shows differences between the groups, but this was not statistically significant. The PL index values are better in animals receiving BMDC grafts than in the control group (DMEM). Compare the results with those for normal (unoperated) animals.
Foidart-Dessalle et al., 1997; Gordon et al., 2003; Murakami et al., 2003). Most of the experimental and clinical data support the view that an autologous nerve with blood vessels is the best material to bridge the gaps (Millesi et al., 1972), but the length of available nerve grafts is limited and a transplanting nerve donor is required. Another possibility to bridge nerve gaps is the use of tubes made with nonhomotopic biological material such as decalcified bone, or artificial materials such as nylon fiber, silicone or polyurethane (Lundborg et al., 1982; Danielsen et al., 1993). Despite the fact that they can provide tubular support for nerve regeneration, they cannot be degraded or absorbed in vivo, requiring a second surgical procedure to withdraw them. With the development of tissue engineering, tubular prostheses using biological materials have become the focus of interest in the last few years. Therefore, biodegradable
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nerve guides promise to be a successful alternative, since they not only direct the outgrowing nerve fibers towards the distal stump but also prevent neuroma formation and ingrowth of fibrous tissue inside the nerve gap (Nakamura et al., 2004). Collagen is the major component of the extracellular matrix and is known to promote cellular proliferation and tissue regeneration. It has been reported that the biological features of collagen make it a very good choice for use as a nerve guide tube and to promote peripheral nerve regeneration (Archibald et al., 1995; Kitahara et al., 1999; Yoshii and Oka, 2001; Keilhoff et al., 2003), especially when in association with engraftment of specialized cells. In accordance with these studies, we have used in the present work collagen tubes for axonal elongation and regeneration. Our results are very promising since the tube-wall became thinner with time, proving that the collagen was gradually resorbed; there was also evidence of high immunocompatibility, as almost no inflammatory reaction was observed around the tubes or in cell contact with their internal surface. The number of myelinated fibers, the nerve fiber diameter and the myelin sheath area are probably the most reliable parameters of nerve regeneration. In those animals that received collagen tubes with BMDCs, the number of myelinated fibers as well as the myelin sheath area increased, probably due to the presence of the implanted cells. Several proposals can be raised to explain the role of BMDCs in peripheral nerve regeneration. It is well known that some molecules play an important role in nerve fiber regeneration after an injury. The main neurotrophins that participate in nerve regeneration include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF) and ciliary neurotrophic factor (CNTF). They are released from the distal nerve stump during Wallerian degeneration, and are primarily produced by Schwann cells (Ide, 1996; Fu and Gordon, 1997). Although the pattern of expression of these factors is not the same, they seem to work in a complementary way to each other during peripheral nerve regeneration, in order to promote the survival and outgrowth of sensory, sympathetic and motor neurons (Lindsay, 1988; Rich et al., 1989; Brown et al., 1991; Sendtner et al., 1992). Recent data describe that when MSCs are differentiated in vitro by treatment with neurotrophic factors and implanted at the proximal end of sciatic nerves, they are capable of supporting nerve fiber regeneration (Dezawa et al., 2001; Mimura et al., 2004). The subendosteal region is rich in nerves, and BMDCs used here expressed NGFβ, but not BDNF in vitro. Despite the fact that we did not provide evidence that the BMDCs are secreting NGFβ in vivo, our data suggest that this molecule may play an important role in nerve regeneration. In addition, the same cells produce several cytokines and chemokines important for hematopoiesis (Balduino et al., 2005). Interleukin-6 and stem cell factor secreted by subendosteal cells are potent inducers of Schwann cells proliferation and differentiation, and also play a role in Schwann cell migration (Hirota et al., 1996; Haggiag et al., 2001). These data suggest that BMDCs injected into the collagen tube may induce increased numbers of Schwann cells, indirectly improving nerve fiber myelination and regeneration.
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Another possibility is the transdifferentiation of stroma cells with myofibroblast phenotype into Schwann cells. Bone marrow MSCs can differentiate into cardiomyocytes (Kawada et al., 2004), striated muscle cells (Lee et al., 2005), neurons (Bossolasco et al., 2005), and Schwann-like cells (Dezawa et al., 2001; Cuevas et al., 2002). When injected into the collagen tube, neurotrophic factors produced and secreted by the proximal stump may induce BMDCs to undergo differentiation into Schwann-like cells, increasing the number of myelinated fibers, and improving nerve regeneration. The third factor that could have improved the myelination is the activation of the Jagged-Notch signaling pathway. The subendosteal cells used in the present study express Jagged-1 (Balduino et al., 2005). In the bone marrow subendosteal niche, the endosteal cells can control the development and fate of hematopoietic stem cells through the activation of the JaggedNotch signaling pathway (Calvi et al., 2003; Zhu and Emerson, 2004). The proteins of the Notch family are cell-surface receptors that are activated by the Delta and Jagged ligands. The Notch signaling pathway has been known to influence cell fate in the developing nervous system. Recent data suggest that rather than simply inhibiting neuronal differentiation and maintaining a neural progenitor state, Notch may promote the acquisition of a glial fate in the PNS (Gaiano et al., 2000; Morrison et al., 2000; Scheer et al., 2001; Joseph et al., 2004). In the mammalian PNS the neural crest is a multipotent precursor population that gives rise to a great variety of cell types including neurons and Schwann cells. Morrison et al. (2000) have reported that Notch irreversibly commits neural crest stem cells to Schwann cell fate. It is well known that Schwann cells play a major role in peripheral nerve regeneration. Since we have used cells that express Jagged-1, it is reasonable to suggest that the increased axonal myelination, as seen in our experimental group that received the BMDC grafts, was due to enhancement of Schwann cell differentiation through activation of the Notch-Jagged signaling pathway. Besides the axon growth and improved myelination, a striking feature of our model was regeneration of ordered internal structure of the growing nerve with a functional vascular tree. Neoangiogenesis depends upon proliferation of endothelia and mobilization of perivascular myofibroblasts that differentiate into pericytes and mural cells (Carmeliet, 2000). The appropriate connective tissue cell support is also required for the ingrowth of new vessels into the regenerating tissues. Both were present in the regenerating nerve with BMDCs, but not in nerve regeneration without these cells. Since a proper and functional blood supply is required for the longstanding regeneration and function of tissues, the reestablishment of the blood vessel tree may explain the relative improvement of regeneration under condition in which the full neoangiogenesis was granted. Motor function recovery is the ultimate result to be obtained in nerve regeneration. We found that BMDC treatment supported the recovery of motor function as demonstrated by the better PL values after a 6-week postoperative period, when compared to the control group. This result is probably explained by the increased number of myelinated nerve fibers and the
myelin sheath area in the BMDC-treated group. The presence of many clusters of regenerating axons, among which we observed axons in different stages of the process of myelination, indicates that the process of regeneration could be continued for a longer period of time. Therefore, better recovery would be expected if the animals were allowed to survive for a longer postoperative period. In conclusion, the association of a biodegradable collagen tube filled with bone marrow-derived cells induced a better regeneration of peripheral nerve fibers across a 3-mm nerve gap. Our results open the possibility of a novel form of treatment for patients suffering from peripheral nerve lesions, since recent results showed that this population of bone marrow cells can be easily obtained from humans (Sakaguchi et al., 2004). Collagen tubes can be stored and made available for long periods of time. In the future, the use of cells from a particular niche and/or the regulation of known signaling pathways may provide additional tools for therapy in several neurodegenerative diseases. Acknowledgments We are grateful to Jorge Luís da Silva and Roberto da Silva Ferreira for excellent technical assistance and to Carlos Eduardo Vicentini and Márcio de Oliveira Peixoto for their help at the initial stages of this work. 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. Aguayo, A.J., Kassarhan, J., Skamene, E., Kongshavn, P., Bray, G.M., 1977. Myelination of mouse axons by Schwann cells transplanted from normal and abnormal human nerve. Nature 268, 753–755. Archibald, S.J., Shefner, J., Krarup, C., Madison, R.D., 1995. Monkey median nerve repaired by nerve graft or collagen nerve guide tube. J. Neurosci. 15, 4109–4123. Balduino, A., Hurtado, S.P., Frazao, P., Takiya, C.M., Alves, L.M., Nasciutti, L.E., El-Cheikh, M.C., Borojevic, R., 2005. Bone marrow subendosteal microenvironment harbours functionally distinct haemosupportive stromal cell populations. Cell Tissue Res. 319 (2), 255–266. Bianco, P., Robey, G.P., 2000. Marrow stromal stem cells. J. Clin. Invest. 12, 1663–1668. Bossolasco, P., Cova, L., Calzarossa, C., Rimoldi, S.G., Borsotti, C., Deliliers, G.L., Silani, V., Soligo, D., Polli, E., 2005. Neuro-glial differentiation of human bone marrow stem cells in vitro. Exp. Neurol. 193 (2), 312–325. Brown, M.C., Perry, V.H., Lunn, E.R., Gordon, S., Heumann, R., 1991. Macrophage dependence of peripheral nerve sensory regeneration: possible involvement of nerve growth factor. Neuron 6, 359–370. Calvi, L.M., Adams, G.B., Weibrecht, K.W., Weber, J.M., Olson, D.P., Knight, M.C., Martin, R.P., Schipani, E., Divieti, P., Bringhurst, F.R., Milner, L.A., Kronenberg, H.M., Scadden, D.T., 2003. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425, 841–846. Carmeliet, P., 2000. Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 6 (4), 389–395. Choi, B.H., Zhu, S.J., Kim, B.Y., Huh, J.Y., Lee, S.H., Jung, J.H., 2005. Transplantation of cultured bone marrow stromal cells to improve peripheral nerve regeneration. Int. J. Oral Maxillofac. Surg. 34, 537–542. Cuevas, P., Carceller, F., Dujovny, M., Garcia-Gómez, I., Cuevas, B., González-Corrochano, R., Diaz-González, D., Reimers, D., 2002. Peripheral nerve regeneration by bone marrow stromal cells. Neurol. Res. 24, 634–638.
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Bone marrow stromal cells and resorbable collagen guidance tubes enhance sciatic nerve regeneration in mice Fátima Rosalina Pereira Lopes a , Lenira Camargo de Moura Campos a , José Dias Corrêa Jr. e , Alex Balduino a , Silvano Lora c , Francesco Langone b , Radovan Borojevic a,d , Ana Maria Blanco Martinez a,⁎ a
Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Brasil b Departamento de Fisiologia e Biofísica, Universidade Estadual de Campinas, São Paulo, Brasil c Istituto per la Sintesi Organica e la Fotoreattività, CNR, Legnaro-Padova, Italy d APABCAM, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Brasil e Departamento de Morfologia. Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Brasil Received 21 July 2005; revised 23 November 2005; accepted 14 December 2005 Available online 20 February 2006
Abstract We evaluated peripheral nerve regeneration using a tubular nerve guide of resorbable collagen filled with either bone marrow-derived cells (BMDCs) in Dulbecco's cell culture medium (DMEM) or with DMEM alone (control). The control group received just the culture medium (vehicle). The left sciatic nerves of ten isogenic mice were transected and the tubular nerve guides were sutured to the end of the proximal and distal nerve stumps. Motor function was tested at 2, 4 and 6 weeks after surgery using the walking track test. The pawprints were analyzed and the print lengths (PL) were measured to evaluate functional recovery. After 6 weeks, mice were anesthetized, perfused transcardially with fixative containing aldehydes, and the sciatic nerves and tubes were dissected and processed for scanning and transmission electron microscopy. Scanning electron microscopy of the collagen tube revealed that the tube wall became progressively thinner after surgery, proving that the tube can be resorbed in vivo. Quantitative analysis of the regenerating nerves showed that the number of myelinated fibers and the myelin area were significantly increased in the experimental group. Also, motor function recovery was faster in animals that received the cell grafts. These results indicate that the collagen tube filled with BMDCs provided an adequate and favorable environment for the growth and myelination of regenerating axons compared to the collagen tube alone. © 2005 Elsevier Inc. All rights reserved. Keywords: Peripheral nerve regeneration; Bone marrow-derived cells; Collagen tube; Schwann cells; Transplantation; Electron microscopy
Introduction When compared to the central nervous system (CNS), peripheral nervous system (PNS) has a greater capacity for regeneration, although complete repair is rare and, in severe injuries, functional recovery is poor. In cut injuries, the most common procedure is to suture the two nerve stumps together ⁎ Corresponding author. Av. Brig. Trompowsky s/n, Cidade Universitária, Ilha do Fundão, Centro de Ciências da Saúde, Instituto de Ciências Biomédicas, Bloco F, Departamento de Histologia e Embriologia, 21941-540, Rio de Janeiro, RJ, Brasil. Fax: +55 21 25626480. E-mail address: [email protected] (A.M. Blanco Martinez). 0014-4886/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.12.019
(Madison et al., 1992; Ide, 1996). However, when there are large gaps, a scaffold or accessory structures are generally required to guide and protect the nerve during growth, and to provide a tension-free full regeneration of the nerve. Although nerve allografts and autografts are indicated in these cases, this strategy is usually avoided because of the high tissue morbidity (Heath and Rutkowski, 1998; Evans, 2000), and artificial tubes have been used to guide nerve regeneration (Terzis et al., 1997; Nakamura et al., 2004). Recently, several groups have tried to develop synthetic biodegradable polymers to build tubular prostheses to connect the proximal and distal stumps ((Keeley et al., 1993; Archibald et al., 1995; Langone et al., 1995; Wang et al., 2001; Ngo et al., 2003; Nakamura et al., 2004). Their use
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has the advantage of avoiding a second surgery to relieve chronic nerve compression and the unavoidable tissue reaction from the implanted tissue. Neurotrophic factors such as neural growth factor (NGF) and brain-derived-neurotrophic factor (BDNF), and/or transforming growth factor-beta (TGFβ), or extracellular matrix (ECM) molecules, such as laminin and fibronectin, have also been combined with the prosthesis, enhancing and improving axonal sprouting and guidance towards the target (Lindsay, 1988; Aebischer et al., 1989; Rich et al., 1989; Menei et al., 1998; Tom et al., 2004; Yu and Shoichet, 2005). Transplantation of Schwann cells has also been tested, since these cells are essential for nerve regeneration and development (Aguayo et al., 1977; Frerichs et al., 2002; Mosahebi et al., 2002; Fansa et al., 2003). However, cell number and donor site availability are two limiting factors to the use of Schwann cells for cell therapy. Therefore PNS regeneration with successful functional recovery remains a challenge to physicians and neuroscientists. Support of bone marrow-derived cells has been used recently in several models of tissue regeneration. Bone marrow harbors two distinct types of stem cells that are closely associated and interacting: blood and mesenchymal cell progenitors (Bianco and Robey, 2000). The mesenchymal stem cells (MSC) have the ability to differentiate into other cells of either mesenchymal or epithelial origin, such as endothelial cells, striated skeletal muscle cells, cardiomyocytes, and hepatocytes (Kawada et al., 2004; Sato et al., 2005; Bossolasco et al., 2005; Lee et al., 2005). This capacity makes MSCs particularly promising for cell therapy and tissue engineering. Multipotent MSCs can be isolated from the subendosteal region, in which they sustain proliferation of hematopoietic cells and interact with blood vessels and the subendosteal nerve plexus (Balduino et al., 2005). In vitro induction of molecular markers of glia and neurons in MSCs has been reported (Sánchez-Ramos et al., 2000). In vivo studies have also shown that they can improve nerve regeneration, by differentiating into Schwann-like cells and supporting nerve fiber growth and myelination (Dezawa et al., 2001; Cuevas et al., 2002; Choi et al., 2005). Therefore, we have chosen MSCs in order to promote simultaneously growth and differentiation of nerve fibers, blood vessels and the supportive connective tissue. Differently from the hematopoietic stem cells (HSCs), the exact niche of the MSC is not clear, although a few observations suggest that they reside around arterioles and capillaries with a phenotype close to pericytes (Shi and Gronthos, 2003). Recent studies showed that multipotent MSCs can be isolated from endosteal surface rich in osteoblasts, which are similar to those isolated from the bone marrow (Tuli et al., 2003). Endosteal bone-lining cells participate in HSC niche formation (Taichman and Emerson, 1998; Calvi et al., 2003; Zhang et al., 2003) together with subendosteal reticular cells (Balduino et al., 2005), controlling HSC quiescence, expansion and/or differentiation. MSCs have been described recently as promoters of axonal regeneration and myelination (Dezawa et al., 2001; Cuevas et al., 2002; Choi et al., 2005). Working with a population of in vitro differentiated MSCs, Dezawa et al. (2001) have
reported that these cells have an important regenerative potential after being transplanted into the stumps of transected sciatic nerves. Cuevas et al. (2002) assessed the therapeutic potential of unmanipulated MSCs on peripheral nerve regeneration, and also reported a functional and morphological improvement after sciatic nerve transection. More recently Choi et al. (2005) have reported that, when MSC were added to a vein conduit they promoted an increase in the number and diameter of myelinated axons that regenerated across a 15-mm nerve gap in rabbits. In the present study, we tested two strategies for the improvement of sciatic nerve regeneration after surgical transection: (1) a biodegradable collagen tube was used to bridge the nerve gap; (2) the tube was filled with a selected population of MSCs from the endosteal surface, hereafter referred to as bone marrow-derived cells (BMDCs). The combination of these two strategies has proven to be very encouraging since it results in regenerating nerves with an overall better organization and vascularization. In addition, the nerves supplemented with BMDCs showed a significant increase in the number of myelinated nerve fibers, which displayed a thicker myelin sheath than the nerves without BMDCs. Materials and methods Culture of bone marrow-derived cells (BMDCs) Inbred Balb/c mice were sacrificed, and bone marrow stromal cells were harvested following the previously described protocol (Balduino et al., 2005). Briefly, 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 cell culture medium in order to wash out 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 min each, at 37°C. Cells were harvested from the second digestion, washed twice in cold BSS, suspended and plated in Dulbecco's medium (DMEM-Sigma) containing antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin) and 10% fetal bovine serum (FBS-Cultilab, Campinas, SP, Brazil). To identify these cells later, they were labeled with 5 μg/mL of bisbenzamide (Sigma-Hoechst 33258), prior to surgical implantation. Reverse transcriptase polymerase chain reaction (RT-PCR) RNA was extracted using the TRIzol (Invitrogen LifeTechnologies, São Paulo, SP, Brazil) from stromal cells isolated from the bone marrow as described above and maintained in culture for 3–7 days. One μg was used to prepare cDNA. PCR (25 μL), was carried out with Taq-DNA Polymerase (Invitrogen) and cycles were established as
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follows: initial denaturation at 95°C for 10 min and 35 cycles of PCR (95° for 30 s, 60°C for 30 s, and 72°C for 30 s), followed by an extra extension period of 10 min, at 72°C. D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primer pairs used were: GAPDH sense—TGC ACC ACC AAC TGC TTA G; GAPDH antisense—GAT GCA GGG ATG ATG TTC; BDNF sense— CTG AGC AAA GCC GAA CTT CT; BDNF anti-sense— GCC TTC ATG CAA CCG AAG TA; NGFb sense—GTC TGG GCC CAA TAA AGG TT; NGFb anti-sense—CCT GTA CGC CGA TCA AAA AC. Collagen tube The artificial nerve conduit was a biodegradable tube obtained from 18-month-old calf peritoneal sheath removed just after death. The parietal layer of the peritoneal sheath was removed and washed in ethyl alcohol, rinsed with chloroform and dried at room temperature. The parietal layer was examined to select and eliminate regions with irregular macroscopic morphological features. The remaining material was compressed until an adequate and regular thickness was obtained. After that, rectangular fragments with a surface of 100 mm2 were treated with proteolytic enzymes, washed in distilled water and wrapped around a 1.0-mm-diameter stainless-steel rod. The preparation was immersed in an aqueous solution of a cross-linking agent for 2 h. The tubes were dried at 37°C, stripped from the rods, washed in water to remove the unreacted cross-linking agent, dried again, packed in vials and then sterilized by gamma radiation at 25 kGy. No changes in mechanical properties and morphologic structure of the tubes were observed. Biocompatibility tests were performed in a previous study by in vitro cell culture and in vivo subcutaneous implants in mice (Lora et al., 1997). Effectiveness of the tubes as a nerve guide was also tested by using them to repair transected sciatic nerves in mice (Langone et al., 1997). Surgical procedure Ten female inbred adult Balb/c mice weighing 20–25 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. Subsequently, BMDCs were injected into the tube in the gap area. The experimental group (n = 5) 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/μL for each animal. The control group (n = 5) received the collagen tube filled with DMEM (1.0 μL). After 6 weeks of regeneration, mice were anesthetized and perfused transcardially with fixative solution containing aldehydes (4% paraformaldehyde and 2.5% glutaraldehyde
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in 0.1 M phosphate buffer, pH 7.4). The left sciatic nerves were exposed and the mid-portions of the growing nerves (corresponding to the previous gap area) were harvested and processed for electron microscopy. Scanning electron microscopy For scanning electron microscopy (SEM), collagen tubes were fixed in 3% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), postfixed in 1% OsO4 for 2 h, washed and dehydrated in ethanol series. The samples were dried at critical point (CPD 030, Balzers Instruments, Liechtenstein) and gold sputtered (FL-9496 Balzers Union Coater). Scanning electron micrographs were obtained with a JEOL JSM-5310 scanning microscope. Transmission electron microscopy After fixation by perfusion, the segments of the nerves were immersed for 2 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), washed in the same buffer followed by 0.1 M cacodylate buffer (pH 7.4), postfixed for 90 min in 1% osmium tetroxide containing 0.8% potassium ferrocianide and 5 nM calcium chloride in 0.1 M cacodylate buffer (pH 7.4). The segments were washed in 0.1 M cacodylate buffer (pH 7.4) and distilled water and stained in 1% uranyl acetate overnight, dehydrated in graded acetone, infiltrated with Poly/Bed 812 resin (Polysciences, Inc., Washington, PA) and polymerized at 60°C for 48 h. Five hundred nanometer semi-thin cross-sections were obtained for light microscopy studies, and stained with toluidine blue. Ultra-thin cross-sections (60–70 nm) for transmission electron microscopy (TEM) analysis were collected on copper grids and contrasted in uranyl acetate and lead citrate. TEM studies were carried out using a Zeiss 900 transmission electron microscope operated at 80 kV. Functional analysis—walking tracks All animals were subjected to a postoperative walking track analysis, based on the protocol described by Inserra et al. (1998), 2, 4 and 6 weeks after surgery. Briefly, pawprints were recorded by painting the hind paws of each animal with China ink. The animals were allowed to walk along a 45 × 6.5 cm track on a white paper (Canson A4 140 g/m2). The pawprints were analyzed and the distance between the third toe and the hind pad – print length (PL) – was measured in the left paws, for normal and operated animals. Differences between the groups were assessed using the ANOVA and Kruskal–Wallis tests. A P value b0.05 was considered statistically significant. Quantitative analysis For the sampling procedure, we photographed in the transmission electron microscope 8 random fields in each nerve cross-section at a magnification of 4400×. All crosssectional areas of the nerve were scanned and sampled, avoiding a sample bias. A total of 40 pictures were obtained
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for each experimental group. Since we had two experimental conditions (sectioned treated with DMEM and sectioned treated with DMEM + BMDC), the total number of pictures was 80. All negatives were scanned and subjected to quantitative analysis using the Image Pro Plus program (Media Cybernetics). Based on the qualitative observations, we calculated and compared the following parameters between the two groups: total number of nerve fibers, number of non-myelinated fibers, number of myelinated fibers, nerve fiber area, axon area and myelin area. The results of these quantifications were analyzed statistically using Prism software (Graph Pad Inc.) and the Mann– Whitney or Kruskal–Wallis tests. Results were considered significant for P b 0.05. Results General observations None of the animals developed any serious post-surgical complication. Wounded tissues healed spontaneously and there were no trophic ulcerations on the operated legs.
Collagen tubes are progressively degraded in vivo Fig. 1 shows the scanning electron microscopy of a collagen tube before surgery (Figs. 1A–D), and 6–10 weeks after being implanted (Figs. 1E–F). Fig. 1A presents the tube at a low magnification. At a higher magnification, we can observe that the tube wall is composed of highly ordered and parallel layers of collagen fibers (Figs. 1B–D). The outer surface of the tube exhibits a network organization (Fig. 1B), while the inner surface displays alignment of the collagen fibers (Fig. 1D). The thickness of the tube wall can be seen in Fig. 1C. Modifications of the tube wall after 6 and 10 weeks of engraftment were also evaluated by scanning electron microscopy. Fig. 1E shows a transverse section of the tube wall interacting with the growing tissue inside it, 6 weeks after surgery. The tube wall is thinner at some areas and there is a small amount of cell reaction around the tube. At 10 weeks after surgery the tube wall is much thinner (Fig. 1F) than the intact tube and is in intimate contact with the tissue growing inside. Blood vessels filled with blood cells are seen running inside the tube, indicating that they are functional.
Fig. 1. Scanning electron micrographs of the collagen tube. (A) View of the empty collagen tube. (B) External surface of the tube wall exhibiting a network aspect; (C) View of the tube wall; (D) internal surface displaying a longitudinal alignment of the collagen fibers. (E, F) Transverse section of the tube wall at 6 (E) and 10 weeks (F) after implantation. Observe that the thickness of the tube wall is much thinner 10 weeks after implantation; blood vessels are also observed in F. Areas between arrows represent the thickness of the tube wall.
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BMDC treatment provides a better overall organization of regenerating nerves
Fig. 2. RT-PCR analysis of BMDC population. Expression of GAPDH, BDNF, and NGFβ was evaluated. Figure shows the agarose electrophoresis analysis of the PCR-product.
BMDCs express NGFβ Expression of two important neural growth factors, BDNF and NGFβ, in bone marrow subendosteal cells was monitored by RT-PCR. Cells obtained from cultures, expressed NGFβ but not BDNF, as shown in Fig. 2.
After 14 days' culture BMDCs were examined by phase microscopy (Fig. 3A). By this time, the cells had reached confluence and formed a compact layer in which individual cells displayed a polygonal shape. Since we labeled the BMDCs with Hoeschst, the cells' nuclei can be seen in blue (Fig. 3B). A longitudinal section of the sciatic nerve proximal stump, 1 week after engraftment, shows rows of Hoeschstlabeled cells (Fig. 3C and insert). These cells invade the surviving proximal stump in a very organized fashion, suggesting that the implanted BMDCs are able to migrate and interact with the peripheral nerve. Semi-thin sections of regenerating nerves after 6 weeks revealed extensive structural differences between the control and the experimental groups (Fig. 4). In the latter, the growing nerve had a well-defined perineurium, with parallel layers of fibroblastoid cells (Figs. 4A and B). Very few inflammatory cells were observed in the surrounding space.
Fig. 3. (A) Phase microscopy of BMDCs (arrows) in culture, showing their typical morphological characteristics; (B) The nuclei of the Hoeschst-labeled cells are seen in blue (arrows); (C) Longitudinal section of the sciatic nerve proximal stump, 1 week after engraftment, exhibiting rows of Hoeschst-labeled cells (arrows). Scale bar: A–C: 40 μm. Insert: 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Semi-thin cross-sections of regenerating sciatic nerve (Toluidine blue stain) 6 weeks after injury. (A–B) Experimental (BMDC) group; (A) this section displays an organized regenerating nerve; (B) many clusters of regenerating nerve fibers (arrows) and newly formed blood vessels are seen (arrowheads). (C–D) Control (DMEM) group; (C) the clusters of regenerating nerve fibers are not so obvious and the tissue is less organized. (D) Observe large and empty newly formed blood vessels (arrows). Scale bar: 40 μm.
The regenerating nerve was divided into discrete axon bundles, surrounded by well-formed endoneurium with thin cells and abundant extracellular matrix. In the group that did not receive BMDCs, the perineural space was occupied by a thick layer of poorly organized cells, and numerous nonadherent inflammatory cells (Figs. 4C and D). There was no endoneurium, and the space between growing axons contained only loose unorganized material. Simultaneously, the experimental group contained numerous blood vessels. Capillaries were present in the endoneurium and perineurium, and larger vessels in the perineurium only. They were parallel to the axis of the growing nerve, indicating a neoformation of an ordered vascular tree. Besides the endothelium, all the vessels contained pericytes and the larger ones had mural cells that sustained the vascular walls and allowed the vessels to maintain a regular oval section, compatible with the controlled internal pressure. Most of the vessels contained erythrocytes, indicating a functional blood supply. Conversely, the control group contained no typical capillaries inside the growing nerve. The blood vessels both inside and around the nerve had a distended lacunar form, without mural cells, and with only very rare erythrocytes, indicating that the blood vascular tree was not sufficiently organized to sustain a functional blood supply.
BMDC treatment enhances axon regeneration and myelination Ultrastructural analysis of the regenerating nerves also revealed important differences between control and experimental groups (Figs. 5 and 6). Regenerating nerves from the experimental group exhibited many regenerating clusters of nerve fibers. These clusters were constituted of myelinated and non-myelinated nerve fibers ensheathed by endoneural cells and their processes. Among the nerve fibers were collagen fibrils forming the endoneurium and nuclei of Schwann cells associated with myelinated fibers or groups of non-myelinated fibers. The axoplasm and myelin sheaths were well preserved and had a normal appearance (Fig. 5A). Fig. 5B shows the periphery of the nerve, where there is a compact perineurium composed of alternating layers of collagen and perineural cells and processes. Healthy-looking blood vessels among or inside the regenerating clusters can also be seen (Fig. 5C). In contrast with these results the control group showed growing nerves with less obvious and smaller regenerating clusters. Also, there was an apparent reduction in the number of myelinated nerve fibers, which looked thinner and more dispersed (Fig. 6A). At the periphery of the nerve, the cell layer corresponding to the perineurium was also observed (Fig. 6B) but it was less compact when compared to the experimental group.
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(including the fibers which were still in the process of myelination) showed that they were significantly more numerous (P = 0.007) in the experimental group than in the control group (Fig. 7A). Concerning the number of non-myelinated fibers, there was no significant difference between the two groups (Fig. 7B). The total number of nerve fibers was greater in the experimental group, even though this difference did not achieve significance (Fig. 7C). With regard to nerve fiber area and myelin sheath area there was a significant increase in the experimental group compared to the control group (Figs. 8A and B). Meanwhile, there was no significant difference in the axon area between the experimental and the control group (Fig. 8C).
Fig. 5. Transmission electron micrographs of regenerating nerve, 6 weeks after the BMDC engraftment. (A) Cross-section of the nerve showing many regenerating clusters consisting of myelinated (thin arrows), myelinating (thick arrow) and non-myelinated (arrowheads) nerve fibers. The axoplasm and myelin sheaths are well preserved. (B) At the periphery of the nerve, a compact and well-organized perineurium is observed (stars). (C) Healthylooking blood vessels (arrow) are seen among or inside the regenerating clusters. Scale bar: 1.7 μm.
BMDC treatment increases both the number of myelinated fibers and the myelin area Morphometric studies were carried out based on the qualitative observations described above. All results are expressed as mean ± S.D. of the replicates. Quantitative analysis of the number of myelinated nerve fibers
Fig. 6. Transmission electron micrographs of the regenerating nerve, 6 weeks after surgery (DMEM-control). (A) Cross-section showing small and poorly developed regenerating clusters, composed of dispersed and thin myelinated nerve fibers (arrows). (B) At the periphery of the nerve, a non-compact perineurium is observed. Scale bar: 1.7 μm.
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Discussion The peripheral nervous system has a great potential for regeneration, particularly when an appropriate microenvironment is supplied. However, current treatments for peripheral nerve injury accompanied by loss of a critical length of the nerve do not provide good functional recovery. In the present work, we combined the use of a biodegradable tube with the bone marrow stromal cells engraftment in order to enhance peripheral nerve regeneration after sciatic nerve injury. The combination of these two strategies resulted in a better regeneration and myelination of sciatic nerve fibers. In addition, the regenerating nerves showed an overall better organization and vascularization when compared to control groups. Nowadays, the most common strategy to solve the problem of peripheral nerve gap after an injury is to use an autologous nerve graft as a bridge (Evans et al., 1995;
Fig. 7. Quantitative analysis of the number of myelinated nerve fibers (A), number of non-myelinated fibers (B) and total number of nerve fibers (C) in control (DMEM) and experimental (BMDC) groups. Values represent mean ± SD. *P = 0.007.
BMDC treatment improves motor function Footprints were monitored at 2, 4 and 6 weeks after surgery. A total of 335 prints were measured for three animal groups: experimental group (BMDCs), control group (DMEM) and a third group (normal, unoperated). This third group was added in order to better characterize and compare the data. Analyzing the footprints we concluded that the most obvious visual difference was seen for the print length (PL), which represents the distance between the third toe and the hind pad (Fig. 9A). All results are expressed as mean ± SD of the replicates. Quantitative analysis of the PL showed a better recovery for the experimental group than for the control group, even though statistical analysis demonstrated no significant difference among the groups (Figs. 9B and C). The return of motor function could be assessed qualitatively by the animals' ability to bear their own weight during the walking track test.
Fig. 8. Analysis of nerve fiber area (A), myelin sheath area (B) and axon area (C) in control (DMEM) and experimental (BMDCs) groups. Values represent mean ± SD. *P = 0.007.
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Fig. 9. Quantitative analysis of print length (PL). (A) Pattern of normal (unoperated) and experimental (after sciatic nerve transection) pawprints. Note the elongation of the PL in operated animals. (B–C) Variation of PL with time shows differences between the groups, but this was not statistically significant. The PL index values are better in animals receiving BMDC grafts than in the control group (DMEM). Compare the results with those for normal (unoperated) animals.
Foidart-Dessalle et al., 1997; Gordon et al., 2003; Murakami et al., 2003). Most of the experimental and clinical data support the view that an autologous nerve with blood vessels is the best material to bridge the gaps (Millesi et al., 1972), but the length of available nerve grafts is limited and a transplanting nerve donor is required. Another possibility to bridge nerve gaps is the use of tubes made with nonhomotopic biological material such as decalcified bone, or artificial materials such as nylon fiber, silicone or polyurethane (Lundborg et al., 1982; Danielsen et al., 1993). Despite the fact that they can provide tubular support for nerve regeneration, they cannot be degraded or absorbed in vivo, requiring a second surgical procedure to withdraw them. With the development of tissue engineering, tubular prostheses using biological materials have become the focus of interest in the last few years. Therefore, biodegradable
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nerve guides promise to be a successful alternative, since they not only direct the outgrowing nerve fibers towards the distal stump but also prevent neuroma formation and ingrowth of fibrous tissue inside the nerve gap (Nakamura et al., 2004). Collagen is the major component of the extracellular matrix and is known to promote cellular proliferation and tissue regeneration. It has been reported that the biological features of collagen make it a very good choice for use as a nerve guide tube and to promote peripheral nerve regeneration (Archibald et al., 1995; Kitahara et al., 1999; Yoshii and Oka, 2001; Keilhoff et al., 2003), especially when in association with engraftment of specialized cells. In accordance with these studies, we have used in the present work collagen tubes for axonal elongation and regeneration. Our results are very promising since the tube-wall became thinner with time, proving that the collagen was gradually resorbed; there was also evidence of high immunocompatibility, as almost no inflammatory reaction was observed around the tubes or in cell contact with their internal surface. The number of myelinated fibers, the nerve fiber diameter and the myelin sheath area are probably the most reliable parameters of nerve regeneration. In those animals that received collagen tubes with BMDCs, the number of myelinated fibers as well as the myelin sheath area increased, probably due to the presence of the implanted cells. Several proposals can be raised to explain the role of BMDCs in peripheral nerve regeneration. It is well known that some molecules play an important role in nerve fiber regeneration after an injury. The main neurotrophins that participate in nerve regeneration include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF) and ciliary neurotrophic factor (CNTF). They are released from the distal nerve stump during Wallerian degeneration, and are primarily produced by Schwann cells (Ide, 1996; Fu and Gordon, 1997). Although the pattern of expression of these factors is not the same, they seem to work in a complementary way to each other during peripheral nerve regeneration, in order to promote the survival and outgrowth of sensory, sympathetic and motor neurons (Lindsay, 1988; Rich et al., 1989; Brown et al., 1991; Sendtner et al., 1992). Recent data describe that when MSCs are differentiated in vitro by treatment with neurotrophic factors and implanted at the proximal end of sciatic nerves, they are capable of supporting nerve fiber regeneration (Dezawa et al., 2001; Mimura et al., 2004). The subendosteal region is rich in nerves, and BMDCs used here expressed NGFβ, but not BDNF in vitro. Despite the fact that we did not provide evidence that the BMDCs are secreting NGFβ in vivo, our data suggest that this molecule may play an important role in nerve regeneration. In addition, the same cells produce several cytokines and chemokines important for hematopoiesis (Balduino et al., 2005). Interleukin-6 and stem cell factor secreted by subendosteal cells are potent inducers of Schwann cells proliferation and differentiation, and also play a role in Schwann cell migration (Hirota et al., 1996; Haggiag et al., 2001). These data suggest that BMDCs injected into the collagen tube may induce increased numbers of Schwann cells, indirectly improving nerve fiber myelination and regeneration.
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Another possibility is the transdifferentiation of stroma cells with myofibroblast phenotype into Schwann cells. Bone marrow MSCs can differentiate into cardiomyocytes (Kawada et al., 2004), striated muscle cells (Lee et al., 2005), neurons (Bossolasco et al., 2005), and Schwann-like cells (Dezawa et al., 2001; Cuevas et al., 2002). When injected into the collagen tube, neurotrophic factors produced and secreted by the proximal stump may induce BMDCs to undergo differentiation into Schwann-like cells, increasing the number of myelinated fibers, and improving nerve regeneration. The third factor that could have improved the myelination is the activation of the Jagged-Notch signaling pathway. The subendosteal cells used in the present study express Jagged-1 (Balduino et al., 2005). In the bone marrow subendosteal niche, the endosteal cells can control the development and fate of hematopoietic stem cells through the activation of the JaggedNotch signaling pathway (Calvi et al., 2003; Zhu and Emerson, 2004). The proteins of the Notch family are cell-surface receptors that are activated by the Delta and Jagged ligands. The Notch signaling pathway has been known to influence cell fate in the developing nervous system. Recent data suggest that rather than simply inhibiting neuronal differentiation and maintaining a neural progenitor state, Notch may promote the acquisition of a glial fate in the PNS (Gaiano et al., 2000; Morrison et al., 2000; Scheer et al., 2001; Joseph et al., 2004). In the mammalian PNS the neural crest is a multipotent precursor population that gives rise to a great variety of cell types including neurons and Schwann cells. Morrison et al. (2000) have reported that Notch irreversibly commits neural crest stem cells to Schwann cell fate. It is well known that Schwann cells play a major role in peripheral nerve regeneration. Since we have used cells that express Jagged-1, it is reasonable to suggest that the increased axonal myelination, as seen in our experimental group that received the BMDC grafts, was due to enhancement of Schwann cell differentiation through activation of the Notch-Jagged signaling pathway. Besides the axon growth and improved myelination, a striking feature of our model was regeneration of ordered internal structure of the growing nerve with a functional vascular tree. Neoangiogenesis depends upon proliferation of endothelia and mobilization of perivascular myofibroblasts that differentiate into pericytes and mural cells (Carmeliet, 2000). The appropriate connective tissue cell support is also required for the ingrowth of new vessels into the regenerating tissues. Both were present in the regenerating nerve with BMDCs, but not in nerve regeneration without these cells. Since a proper and functional blood supply is required for the longstanding regeneration and function of tissues, the reestablishment of the blood vessel tree may explain the relative improvement of regeneration under condition in which the full neoangiogenesis was granted. Motor function recovery is the ultimate result to be obtained in nerve regeneration. We found that BMDC treatment supported the recovery of motor function as demonstrated by the better PL values after a 6-week postoperative period, when compared to the control group. This result is probably explained by the increased number of myelinated nerve fibers and the
myelin sheath area in the BMDC-treated group. The presence of many clusters of regenerating axons, among which we observed axons in different stages of the process of myelination, indicates that the process of regeneration could be continued for a longer period of time. Therefore, better recovery would be expected if the animals were allowed to survive for a longer postoperative period. In conclusion, the association of a biodegradable collagen tube filled with bone marrow-derived cells induced a better regeneration of peripheral nerve fibers across a 3-mm nerve gap. Our results open the possibility of a novel form of treatment for patients suffering from peripheral nerve lesions, since recent results showed that this population of bone marrow cells can be easily obtained from humans (Sakaguchi et al., 2004). Collagen tubes can be stored and made available for long periods of time. In the future, the use of cells from a particular niche and/or the regulation of known signaling pathways may provide additional tools for therapy in several neurodegenerative diseases. Acknowledgments We are grateful to Jorge Luís da Silva and Roberto da Silva Ferreira for excellent technical assistance and to Carlos Eduardo Vicentini and Márcio de Oliveira Peixoto for their help at the initial stages of this work. 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. Aguayo, A.J., Kassarhan, J., Skamene, E., Kongshavn, P., Bray, G.M., 1977. Myelination of mouse axons by Schwann cells transplanted from normal and abnormal human nerve. Nature 268, 753–755. Archibald, S.J., Shefner, J., Krarup, C., Madison, R.D., 1995. Monkey median nerve repaired by nerve graft or collagen nerve guide tube. J. Neurosci. 15, 4109–4123. Balduino, A., Hurtado, S.P., Frazao, P., Takiya, C.M., Alves, L.M., Nasciutti, L.E., El-Cheikh, M.C., Borojevic, R., 2005. Bone marrow subendosteal microenvironment harbours functionally distinct haemosupportive stromal cell populations. Cell Tissue Res. 319 (2), 255–266. Bianco, P., Robey, G.P., 2000. Marrow stromal stem cells. J. Clin. Invest. 12, 1663–1668. Bossolasco, P., Cova, L., Calzarossa, C., Rimoldi, S.G., Borsotti, C., Deliliers, G.L., Silani, V., Soligo, D., Polli, E., 2005. Neuro-glial differentiation of human bone marrow stem cells in vitro. Exp. Neurol. 193 (2), 312–325. Brown, M.C., Perry, V.H., Lunn, E.R., Gordon, S., Heumann, R., 1991. Macrophage dependence of peripheral nerve sensory regeneration: possible involvement of nerve growth factor. Neuron 6, 359–370. Calvi, L.M., Adams, G.B., Weibrecht, K.W., Weber, J.M., Olson, D.P., Knight, M.C., Martin, R.P., Schipani, E., Divieti, P., Bringhurst, F.R., Milner, L.A., Kronenberg, H.M., Scadden, D.T., 2003. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425, 841–846. Carmeliet, P., 2000. Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 6 (4), 389–395. Choi, B.H., Zhu, S.J., Kim, B.Y., Huh, J.Y., Lee, S.H., Jung, J.H., 2005. Transplantation of cultured bone marrow stromal cells to improve peripheral nerve regeneration. Int. J. Oral Maxillofac. Surg. 34, 537–542. Cuevas, P., Carceller, F., Dujovny, M., Garcia-Gómez, I., Cuevas, B., González-Corrochano, R., Diaz-González, D., Reimers, D., 2002. Peripheral nerve regeneration by bone marrow stromal cells. Neurol. Res. 24, 634–638.
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