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Collagen (NeuraGen® ) nerve conduits and stem cells for peripheral nerve gap repair
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Pietro G. di Summa a,∗ , Paul J. Kingham b , Corrado C. Campisi c , Wassim Raffoul a , Daniel F. Kalbermatten d a
Department of Plastic, Reconstructive Surgery, University Hospital of Lausanne (CHUV), Rue du Bugnon 46, 1011 Lausanne, Switzerland Section for Anatomy, Department of Integrative Medical Biology, Umeå universitet, hus H, Biologihuset, SE-901 87 Umeå, Sweden c Department of Plastic, Reconstructive Surgery, University Hospital of Genova, Ospedale S. Martino, Largo Rossana Benzi 10, 16132 Genova, Italy d Department of Plastic, Reconstructive Surgery, University Hospital of Basel, Spitalstrasse 21, CH-4031 Basel, Switzerland b
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h i g h l i g h t s • • • • •
FDA-approved collagen nerve guides are efficient scaffold for cell delivery. Schwann cells show affinity for collagen, influencing distal cell infiltration. Proximal regeneration at 2-weeks was not influenced by regenerative cells. Endogenous Schwann cells influence sprouting dynamics of growth fronts. Cell neurotrophic potential may be improved by material functionalization.
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Article history: Received 25 February 2014 Received in revised form 7 April 2014 Accepted 17 April 2014
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Keywords: Peripheral nerve regeneration FDA-approved collagen conduits Adipose-derived stem cells Schwann cell-like differentiation
Collagen nerve guides are used clinically for peripheral nerve defects, but their use is generally limited to lesions up to 3 cm. In this study we combined collagen conduits with cells as an alternative strategy to support nerve regeneration over longer gaps. In vitro cell adherence to collagen conduits (NeuraGen® nerve guides) was assessed by scanning electron microscopy. For in vivo experiments, conduits were seeded with either Schwann cells (SC), SC-like differentiated bone marrow-derived mesenchymal stem cells (dMSC), SC-like differentiated adipose-derived stem cells (dASC) or left empty (control group), conduits were used to bridge a 1 cm gap in the rat sciatic nerve and after 2-weeks immunohistochemical analysis was performed to assess axonal regeneration and SC infiltration. The regenerative cells showed good adherence to the collagen walls. Primary SC showed significant improvement in distal stump sprouting. No significant differences in proximal regeneration distances were noticed among experimental groups. dMSC and dASC-loaded conduits showed a diffuse sprouting pattern, while SC-loaded showed an enhanced cone pattern and a typical sprouting along the conduits walls, suggesting an increased affinity for the collagen type I fibrillar structure. NeuraGen® guides showed high affinity of regenerative cells and could be used as efficient vehicle for cell delivery. However, surface modifications (e.g. with extracellular matrix molecule peptides) of NeuraGen® guides could be used in future tissue-engineering applications to better exploit the cell potential. © 2014 Published by Elsevier Ireland Ltd.
1. Introduction
∗ Corresponding author. Tel.: +41 0 21 314 22 11; fax: +41 0 21 314 25 30. E-mail addresses: [email protected], [email protected] (P.G. di Summa), [email protected] (P.J. Kingham), campisi [email protected] (C.C. Campisi), [email protected] (D.F. Kalbermatten).
Autografts are currently the gold standard in nerve repair, but have the drawback requiring sacrifice of a functional nerve. A few nerve conduits (NC), tubular structures designed to bridge the nerve gap created after nerve injury, have received approval for mass production from the US Food and Drug Administration (FDA) and Conformit Europe (CE) reviewed in [2,17]. These include synthetic non-resorbable polyvinyl alcohol hydrogels (SaluBridgeTM )
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Please cite this article in press as: P.G. di Summa, et al., Collagen (NeuraGen® ) nerve conduits and stem cells for peripheral nerve gap repair, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.029
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and polymer tubes with various degradation times (Neurotube® , made of polyglycolide acid and Neurolac® , made of poly d,l-lactide-caprolactone). Two of the NC are made from collagen, NeuraGen® nerve guide and NeuroMatrix, Neuroflex® . While commercial nerve guides represent a promising alternative to autografts when direct tensionless repair is not possible, their use is limited to lesions up to 3 cm [8]. For this reason, researchers have focused on different biomaterials and the inclusion of coatings, growth factors, intraluminal structures as well as the incorporation of supportive cells. The rationale of cell transplantation into neural conduits is that the presence of a cellular supportive platform within the NC can secrete a constant flow of neurotrophic factors and matrix proteins to assist nerve repair. The beneficial effects of in vivo transplantation of Schwann cells (SC) are widely reported [2]. Problems do however exist in clinical applications of autologous SC, with limited tissue availability, time consuming derivation and expansion ex vivo and donor site morbidity [20]. Adult stem cells such as bone marrow-derived mesenchymal stem cells (MSC) and adipose-derived stem cells (ASC) have shown profound plasticity, showing in vitro differentiation into non-mesenchymal fates, including SC-like phenotypes [14,24]. Our group firstly described the in vivo transplantation and effects of dASC using biodegradable fibrin nerve conduits [11], showing significant improvements in regeneration rates, similar to the autografts [9]. To our knowledge, besides their growing applicability in tissue engineering and nerve regeneration, dASC have never been tested with a commercially available conduit. In this study we evaluated the interactions of both dASC and SC-like differentiated bone marrow derived MSC (dMSC) with the Neuragen® conduit in vitro. The in vivo early nerve regeneration across empty Neuragen® conduits and Neuragen® conduits coupled with dMSC, dASC and SC was assessed using a 1-cm rat sciatic nerve gap in a two-weeks experiment.
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2.1. Experimental animals
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All animal protocols were approved by the local veterinary commission, in accordance with the European Community Council directive 86/609/ECC for the care and use of laboratory animals. Male Sprague–Dawley rats (Janvier, France) weighing 250 g were used for this study.
2.2. Cell harvest and adult stem cells mesodermal differentiation Adult Schwann cells (SC) were isolated from rat sciatic nerves as previously described [11] and maintained in Dulbecco’s Modified Eagle’s Medium plus Glutamax (DMEM, Invitrogen, UK) containing 10% fetal bovine serum (FBS), 1% penicillin–streptomycin (P/S) and supplemented with 14 M forskolin (Sigma, UK) and 100 ng/ml neuregulin-1 1 (R&D Systems, UK). Adipose-derived stem cells (ASC) were harvested as described previously [14] and maintained in Modified Eagle Medium (␣-MEM; Invitrogen, UK) containing 10% (v/v) FBS, and 1% (v/v) P/S solution. Bone marrowderived mesenchymal stem cells (MSC) were harvested from adult Sprague-Dawley rat femoral bones as previously described [3] and maintained in the same growth medium as for ASC. In order to confirm stem cell multi-potency, bone marrow MSC and ASC were incubated with specific media to induce differentiation into the three mesodermal-derived lineages as in previous literature, using adipogenic, osteogenic and chondrogenic induction media for three weeks [21].
2.3. Adult stem cells differentiation to a Schwann cell phenotype At early passages (P2–P3), when cells were sub-confluent, bone marrow-derived MSC and ASC were treated with differentiation medium (DM) containing 5 ng/mL platelet-derived growth factor, 10 ng/mL basic fibroblast growth factor (both from PeproTech Ltd.), 14 mM forskolin (Sigma) and 200 ng/ml of neuregulin-1 1 (R&D Systems, UK) as previously described [11,14]. Cultures were incubated under these conditions for at least 2 weeks with DM changes every 48 h. Stem cell differentiation into SC-like cells was assessed by immunocytochemistry for typical SC markers S100 and GFAP in parallel to SC as positive controls [3,14]. Negative controls with lack of primary antibodies were also included. 2.4. Scanning electron microscope in vitro imaging To study cell-conduit interactions in vitro 1 × 106 cells (either Schwann cells or dASC) were suspended in 50 l of respective medium and seeded into longitudinally cut NeuraGen® conduits. Top up of media was performed after 2 h. After 48 h incubation, conduits were washed with PBS and fixed in 2.5% glutaraldehyde in PBS for 1 h at 4 ◦ C. After fixation, further washing in PBS and dehydration was performed using increasing concentrations of ethanol, followed by washes in hexamethylsidilazane (HMDS). After dehydration, samples were mounted on stubs and gold sputtered for scanning electron microscopy qualitative analysis (VPSEM, Zeiss EVO60, Zeiss, Germany, up to 1000×; Phenom, G2 pro desktop, Lambda Photometrics, UK, up to 5000×). 2.5. Cell seeding for in vivo experiments Prior to implantations in rats, the different regenerative cells were detached from the flask by trypsinization. After centrifugation and aspiration of the supernatant, 2 × 106 cells were resuspended in 50 l of DM (or SCGM for the SC group) and carefully pipetted into the NeuraGen® conduits. The conduits without cells contained just 50 l of DM. To avoid cell or medium dispersion during this procedure, one side of the collagen tube was temporarily sealed using a vascular clip. Conduits with cells were kept at 37 ◦ C with 5% CO2 until surgical implantation (for a maximum of 3 h). Before implantation the vascular clips were cut away, leaving a conduit of 14 mm length. 2.6. Microsurgical technique Five conduits were implanted for each different group involved in the study (empty nerve guide, guide seeded with SC, dASC, dMSC) for a total n = 20. The operation was performed on the left sciatic nerve under aseptic conditions as previously described [9]. The sciatic nerve was transected 1 cm proximal to its distal branches. Proximal and distal nerve stumps were inserted 2 mm into the NeuraGen® conduit, thus leaving a 10 mm gap, and fixed to it by a single epineural suture at each end (9/0 Prolene, Ethicon). Muscles and fascia layers were closed with single resorbable stitches (4/0 Softcat, Braun, Germany) and the skin by a continuous running suture (4/0 Prolene, Ethicon, Germany). 2.7. Conduits harvesting and immunohistochemistry Animals were sacrificed by CO2 euthanasia followed by cervical dislocation. Harvested conduits where fixed according to a previously published protocol [11] and embedded in OCT freezing media (Tissue-tek, Sakura, Japan), frozen with dry ice and stored at −80 ◦ C. Longitudinal cryo-sections (14 m) were prepared onto slides (Superfrost plus, Menzel-Gläser, Germany). A total of 60 sections were taken for each conduit explanted and stored at −20 ◦ C.
Please cite this article in press as: P.G. di Summa, et al., Collagen (NeuraGen® ) nerve conduits and stem cells for peripheral nerve gap repair, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.029
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Q4 Fig. 1. Adult stem cell multi-potency. ASC obtained from fat (A) and bone marrow-derived MSC (E) of adult rats were successfully differentiated into three cell lineages: adipogenic (Oil Red O staining of fat droplets, B and F), chondrogenic (toluidine blue staining of proteoglycans, C and G) and osteogenic (Alizarin Red S staining for calcium deposition, D and H). Scale bar = 100 m. Lower row: differentiated Schwann cell like ASC (dASC) and primary Schwann cells (SC) immunostained for S100 (green), GFAP (red) and DAPI (blue). Scale bar = 20 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Immunostaining on frozen sections using primary antibodies rabbit anti-S100 and rabbit anti-PGP 9.5 (both 1:500, Dako, UK) was performed as previously described [11]. The sections were examined under fluorescence microscope. Using a micro-grid, the regenerating front directed toward the distal end was measured in each section [11,24]. Axonal regeneration distance (PGP 9.5) and S100 positive cell distribution inside the conduit were evaluated. The four highest regeneration values for each conduit were selected and the average of these was recognized as representative of the maximal length of the regeneration cone.
3.2. Cell seeding and conduit properties Prior to in vivo experiments, we tested the NeuraGen® conduit and its capacity to retain cells within. Two conduits were seeded with either dASC or SC, and then fixed after 48 h. Cell retention was qualitatively judged with scanning electron microscopy. A good cell distribution was found throughout the conduits and there was a satisfactory adhesion of the cells to collagen walls (Fig. 2). During surgery the collagen conduits were easy to handle and micro-surgical suture was uneventful. 3.3. Postoperative complications and autotomy
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2.8. Data analysis One-way analysis of variance (ANOVA) with Bonferroni multiple comparison test was used to statistically analyze data (GraphPad Prism). Significance was determined as *p < 0.05.
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All animals survived; none of the animals developed infections or surgical complications. As expected, at the time of wound reopening after 2 weeks in vivo, collagen conduits showed no signs of resorbtion. Hematoma, infection, scarring or excess inflammatory reactions were not detected. Lower limb autotomy was present in 1 out of 20 animals (5%). A supplementary animal was operated to re-establish the numbers per group. 3.4. Axon regeneration and Schwann cells infiltration
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ASC obtained from fat and bone marrow-derived MSC of adult rats showed a flattened fibroblast-like morphology and both showed multi-lineage differentiation potential (Fig. 1). MSC and ASC also differentiated into a SC-like phenotype as previously described [11,14]. The stem cells showed obvious morphological changes and exhibited the spindle-like shape typical of SC; a significant number of the cells co-expressed the SC markers (S100, GFAP, and p75) (Fig. 1, lower row).
Axonal regeneration distance was assessed proximally by PGP 9.5 immunohistochemistry (Fig. 3). Immunohistochemistry on longitudinal sections showed the growing patterns of the regenerating nerves. The growth regeneration front profile was generally better defined in the NeuraGen® + SC group when compared to the other experimental groups (empty, dASC, dMSC), in which there was occasional mis-directional growth. Moreover, SC displayed a particular affinity to type I collagen, with two lateral sprouts along the collagen walls accompanying the central growth
Please cite this article in press as: P.G. di Summa, et al., Collagen (NeuraGen® ) nerve conduits and stem cells for peripheral nerve gap repair, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.029
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Fig. 2. Scanning electron micrographs of the collagen tube at increasing magnifications. Longitudinal sections of collagen tube, seeded with SC (A–C) and dASC (D–F) exhibiting a network aspect and semi-permeability. Scale bars: A, D =200 m; B, C, E, F = 20 m.
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regeneration cone (Fig. 3). No statistically significant differences were noticed in proximal axonal sprouting (Fig. 4). By using alternate sections stained with PGP 9.5 (axons) and S100 (Schwann cells) a clear interposed pattern between regenerating axons and Schwann cells sprouting at the proximal stump was observed (Figs. 3 and 4). Statistical analysis showed that the collagen conduits seeded with SC significantly improved SC infiltration at the distal nerve stump compared with the empty conduit (NeuraGen® +SC: 5.18 ± 0.29 mm, empty NeuraGen® 3.85 ± 0.26 mm, *p < 0.05, all values mean ± SEM). No significant difference was noticed between the SC group and stem cells groups (dASC, dMSC) with either type of antibody staining.
4. Discussion The potential advantages of a nerve conduit over autologous nerve sensory graft in nerve repair include reduction of collateral sprouting, no donor site morbidity, and operational time saving, especially in multiple nerve injuries [17]. However, nerve conduits have limited benefit in treating long gaps. Cell transplantation into the conduits has been studied as a possible solution to improve the conductive microenvironment of the hollow nerve guides. Electron microscope images after 48 h in vitro incubation showed effective cells retaining into the conduits; this was particularly evident when seeding with Schwann cells, suggesting an effective adhesion to
Fig. 3. PGP 9.5 staining shows axonal regeneration into the collagen conduits either empty or seeded with different regenerative cells. (A) Empty collagen conduit. (B) Collagen conduit + dMSC. (C) Collagen conduit + dASC. (D) Collagen conduit + SC. Right column: detail of SC infiltration into the collagen conduit, suggesting a particular affinity of transplanted SC to the collagen walls. Nerve stumps regrowth from left to right in all images. Scale bar = 1 mm.
Please cite this article in press as: P.G. di Summa, et al., Collagen (NeuraGen® ) nerve conduits and stem cells for peripheral nerve gap repair, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.029
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Fig. 4. S100 staining showing SC cell migration across the collagen conduits either empty or seeded with different regenerative cells. (A) Empty collagen conduit. (B) Collagen conduit + dMSC. (C) Collagen conduit + dASC. (D) Collagen conduit + SC. Nerve stumps regrowth from left to right in all images. Scale bar = 1 mm. Lower row: quantification of regeneration distances measured with PGP 9.5 and S100 cell staining (proximal and distal infiltration, respectively). Values are mean distances (mm) ± SEM (*p < 0.05).
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collagen, consistent with previous literature showing good Schwann cells affinity to collagen substrates [23]. All conduits (empty or cell-seeded) supported early axonal regeneration. No signs of conduit resorbtion were noticed at 2 weeks consistent with the expected complete degradation period of 4 years [7]. Empty NeuraGen® conduits displayed a particularly good performance with over 5 mm of average axonal regeneration after 2 weeks, which compares favorably with other experimental conduits [11,18]. In all groups axonal regeneration distances overlapped SC migration patterns, suggesting that regenerating axons in the proximal stump were associated to Schwann cells, confirming the “SC-axon partnership” during nerve regrowth, which has been previously described as a key sign of effective early regeneration [4]. However, transplanted cells did not significantly improve the regeneration distances. At first glance, the generally similar proximal regeneration patterns (Fig. 3) suggest that the important local cells infiltration, allowed by the semi-permeable conduit, could have concealed the effect of delivered cells. Semi-permeability of
collagen material is one of the main characteristics of NeuraGen® guides [16] and has been identified as an important feature to promote nerve regeneration by allowing the beneficial passage into the lumen of bioactive factors and local cells such as proliferating macrophages, Schwann cells and fibroblasts, which are involved in the first phases of nerve regeneration [5]. Although previous experiments revealed initial beneficial effects of transplanted regenerative cells [11,13], it may be that longer time points are required to detect significant improvements, as shown previously when using Schwann cells [18]. What was interesting was the significantly better SC infiltration at the distal stump in the NeuraGen® conduits seeded with SC. We cannot rule out the possibility that the transplanted SC migrated from the walls of the conduit to the distal stump but the pattern of staining is consistent with an infiltration of endogenous SC. SC are the main actors of this regeneration trigger serving as scaffolds for regenerating axons by expressing surface adhesion molecules, and attracting them while proliferating and releasing neurotrophic factors [4].
Please cite this article in press as: P.G. di Summa, et al., Collagen (NeuraGen® ) nerve conduits and stem cells for peripheral nerve gap repair, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.029
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Thus, the improved distal SC infiltration may secondarily enhance proximal regeneration at later time points. The amplified SC infiltration at the distal stump is consistent with the sharper growth cone pattern in the NeuraGen® + SC group, when compared with the other experimental groups. Transplanted SC seemed to have a preferential affinity to the collagen walls (Figs. 3 and 4), building connections to the distal stump along the collagen walls. This goes in line with previous literature describing expression of a number of integrin and non-integrin receptors on the surface of SC which interact with the extracellular matrix (ECM) molecules, including collagen fibrils, during axon regrowth [6]. The preferential affinity of SC for collagen may also explain the increased distal cell infiltration, which could contribute to enhanced neurotrophic factor release by the cells [1]. It would be interesting to know if the distal cell infiltration would provide benefits in cases of longer, critical distance gap injuries. In these situations, it is hypothesized that with prolonged denervation the distal stump is progressively less supportive to receiving the axons which eventually cross the gap. By transplanting Schwann cells in the conduits they might enhance endogenous distal Schwann cell survival, proliferation, migration and alignment to provide a more effective pathway for the regenerating axons. The signals transmitted between the transplanted cells and endogenous cells need to be elucidated to further optimize the use of the more clinically relevant stem cells. The stem cells did not play a significant role in term of axonal regeneration in this study, despite showing effective in vitro differentiation toward a Schwann cell phenotype. These initial findings are in contrast with previous reports of our research group and others [7,9,12] using various experimental conduits. A few studies have described the application of SC cells and MSC to collagen guides [19,23], but to our knowledge no literature exists on the association of ASC with collagen nerve guides. Recently, Ladak and coworkers seeded NeuraGen® with SC and MSC to cross a 12 mm gap after total sciatic nerve transection in rats [15]. The authors reported a significant increase in retrogradely labeled motoneurons in the NC groups seeded with dMSC and SC. However, compared with empty conduits no functional improvements were shown in SC or dMSC groups [15]. This may suggest that, even if collagen remains one of the most versatile and effective conduits for peripheral nerve repair, it requires further modifications to allow it to be coupled with therapeutic cells [15,23]. Future studies will have to consider the use of multiple approaches to guarantee the best conduit design to combine ideal mechanical properties with the optimal cell performance. We previously reported the positive effect of extracellular matrix molecules such as laminin and fibronectin on neurotrophic activity and cell viability of dASC [10]. Surface functionalization by laminin could improve the intraluminal guidance and directionality [4,22] and at the same time help preserve the neurotrophic effect of the SC-like differentiated stem cells, thereby supporting regeneration across nerve gaps.
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This study confirms the good performance of commercially available collagen tubes for peripheral nerve repair, with substantial axonal regeneration at 2 weeks post implantation. The supportive environment of the conduits appears to be enhanced by addition of Schwann cells. Whether significant differences can be observed among the various cell types at longer time points will be assessed in future studies. This preliminary report suggests that tissue engineering refinements such as extracellular matrix coatings of the collagen scaffolds might be needed to enhance the effect
of the delivered stem cell, improving cell-material interactions and, as a result, regeneration across nerve gaps. References [1] S.J. Armstrong, M. Wiberg, G. Terenghi, P.J. Kingham, ECM molecules mediate both Schwann cell proliferation and activation to enhance neurite outgrowth, Tissue Eng. 13 (2007) 2863–2870. [2] J.H. Bell, J.W. Haycock, Next generation nerve guides: materials, fabrication, Q3 growth factors, and cell delivery, Tissue Eng. Part B: Rev. (2011). [3] J. Caddick, P.J. Kingham, N.J. Gardiner, M. Wiberg, G. Terenghi, Phenotypic and functional characteristics of mesenchymal stem cells differentiated along a Schwann cell lineage, Glia 54 (2006) 840–849. [4] Y.Y. Chen, D. McDonald, C. Cheng, B. Magnowski, J. Durand, D.W. Zochodne, Axon and Schwann cell partnership during nerve regrowth, J. Neuropathol. Exp. Neurol. 64 (2005) 613–622. [5] C. Cheng, D.W. Zochodne, In vivo proliferation, migration and phenotypic changes of Schwann cells in the presence of myelinated fibers, Neuroscience 115 (2002) 321–329. [6] M.A. Chernousov, D.J. Carey, Schwann cell extracellular matrix molecules and their receptors, Histol. Histopathol. 15 (2000) 593–601. [7] W. Daly, L. Yao, D. Zeugolis, A. Windebank, A. Pandit, A biomaterials approach to peripheral nerve regeneration: bridging the peripheral nerve gap and enhancing functional recovery, J. R. Soc. Interface 9 (2012) 202–221. [8] D.N. Deal, J.W. Griffin, M.V. Hogan, Nerve conduits for nerve repair or reconstruction, J. Am. Acad. Orthop. Surg. 20 (2012) 63–68. [9] P.G. di Summa, D.F. Kalbermatten, E. Pralong, W. Raffoul, P.J. Kingham, G. Terenghi, Long-term in vivo regeneration of peripheral nerves through bioengineered nerve grafts, Neuroscience 181 (2011) 278–291. [10] P.G. di Summa, D.F. Kalbermatten, W. Raffoul, G. Terenghi, P.J. Kingham, Extracellular matrix molecules enhance the neurotrophic effect of Schwann cell-like differentiated adipose-derived stem cells and increase cell survival under stress conditions, Tissue Eng. Part A 19 (2013) 368–379. [11] P.G. di Summa, P.J. Kingham, W. Raffoul, M. Wiberg, G. Terenghi, D.F. Kalbermatten, Adipose-derived stem cells enhance peripheral nerve regeneration, J. Plast. Reconstr. Aesthet. Surg.: JPRAS 63 (2010) 1544–1552. [12] J.H. Gu, Y.H. Ji, E.S. Dhong, D.H. Kim, E.S. Yoon, Transplantation of adipose derived stem cells for peripheral nerve regeneration in sciatic nerve defects of the rat, Curr. Stem Cell Res. Ther. 7 (2012) 347–355. [13] D.F. Kalbermatten, P.J. Kingham, D. Mahay, C. Mantovani, J. Pettersson, W. Raffoul, H. Balcin, G. Pierer, G. Terenghi, Fibrin matrix for suspension of regenerative cells in an artificial nerve conduit, J. Plast. Reconstr. Aesthet. Surg. 61 (2008) 669–675. [14] P.J. Kingham, D.F. Kalbermatten, D. Mahay, S.J. Armstrong, M. Wiberg, G. Terenghi, Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro, Exp. Neurol. 207 (2007) 267–274. [15] A. Ladak, J. Olson, E.E. Tredget, T. Gordon, Differentiation of mesenchymal stem cells to support peripheral nerve regeneration in a rat model, Exp. Neurol. 228 (2011) 242–252. [16] S.T. Li, S.J. Archibald, C. Krarup, R.D. Madison, Peripheral nerve repair with collagen conduits, Clin. Mater. 9 (1992) 195–200. [17] M.F. Meek, J.H. Coert, US Food and Drug Administration/Conformit Europeapproved absorbable nerve conduits for clinical repair of peripheral and cranial nerves, Ann. Plast. Surg. 60 (2008) 110–116. [18] A. Mosahebi, M. Wiberg, G. Terenghi, Addition of fibronectin to alginate matrix improves peripheral nerve regeneration in tissue-engineered conduits, Tissue Eng. 9 (2003) 209–218. [19] F.R. Pereira Lopes, L. Camargo de Moura Campos, J. Dias Correa Jr., A. Balduino, S. Lora, F. Langone, R. Borojevic, A.M. Blanco Martinez, Bone marrow stromal cells and resorbable collagen guidance tubes enhance sciatic nerve regeneration in mice, Exp. Neurol. 198 (2006) 457–468. [20] L.A. Pfister, M. Papaloizos, H.P. Merkle, B. Gander, Nerve conduits and growth factor delivery in peripheral nerve repair, J. Peripher. Nerv. Syst. 12 (2007) 65–82. [21] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143– 147. [22] N. Rangappa, A. Romero, K.D. Nelson, R.C. Eberhart, G.M. Smith, Laminincoated poly(l-lactide) filaments induce robust neurite growth while providing directional orientation, J. Biomed. Mater. Res. 51 (2000) 625– 634. [23] F. Stang, H. Fansa, G. Wolf, M. Reppin, G. Keilhoff, Structural parameters of collagen nerve grafts influence peripheral nerve regeneration, Biomaterials 26 (2005) 3083–3091. [24] M. Tohill, C. Mantovani, M. Wiberg, G. Terenghi, Rat bone marrow mesenchymal stem cells express glial markers and stimulate nerve regeneration, Neurosci. Lett. 362 (2004) 200–203.
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Neuroscience Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Collagen (NeuraGen® ) nerve conduits and stem cells for peripheral nerve gap repair
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Pietro G. di Summa a,∗ , Paul J. Kingham b , Corrado C. Campisi c , Wassim Raffoul a , Daniel F. Kalbermatten d a
Department of Plastic, Reconstructive Surgery, University Hospital of Lausanne (CHUV), Rue du Bugnon 46, 1011 Lausanne, Switzerland Section for Anatomy, Department of Integrative Medical Biology, Umeå universitet, hus H, Biologihuset, SE-901 87 Umeå, Sweden c Department of Plastic, Reconstructive Surgery, University Hospital of Genova, Ospedale S. Martino, Largo Rossana Benzi 10, 16132 Genova, Italy d Department of Plastic, Reconstructive Surgery, University Hospital of Basel, Spitalstrasse 21, CH-4031 Basel, Switzerland b
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FDA-approved collagen nerve guides are efficient scaffold for cell delivery. Schwann cells show affinity for collagen, influencing distal cell infiltration. Proximal regeneration at 2-weeks was not influenced by regenerative cells. Endogenous Schwann cells influence sprouting dynamics of growth fronts. Cell neurotrophic potential may be improved by material functionalization.
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Article history: Received 25 February 2014 Received in revised form 7 April 2014 Accepted 17 April 2014
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Keywords: Peripheral nerve regeneration FDA-approved collagen conduits Adipose-derived stem cells Schwann cell-like differentiation
Collagen nerve guides are used clinically for peripheral nerve defects, but their use is generally limited to lesions up to 3 cm. In this study we combined collagen conduits with cells as an alternative strategy to support nerve regeneration over longer gaps. In vitro cell adherence to collagen conduits (NeuraGen® nerve guides) was assessed by scanning electron microscopy. For in vivo experiments, conduits were seeded with either Schwann cells (SC), SC-like differentiated bone marrow-derived mesenchymal stem cells (dMSC), SC-like differentiated adipose-derived stem cells (dASC) or left empty (control group), conduits were used to bridge a 1 cm gap in the rat sciatic nerve and after 2-weeks immunohistochemical analysis was performed to assess axonal regeneration and SC infiltration. The regenerative cells showed good adherence to the collagen walls. Primary SC showed significant improvement in distal stump sprouting. No significant differences in proximal regeneration distances were noticed among experimental groups. dMSC and dASC-loaded conduits showed a diffuse sprouting pattern, while SC-loaded showed an enhanced cone pattern and a typical sprouting along the conduits walls, suggesting an increased affinity for the collagen type I fibrillar structure. NeuraGen® guides showed high affinity of regenerative cells and could be used as efficient vehicle for cell delivery. However, surface modifications (e.g. with extracellular matrix molecule peptides) of NeuraGen® guides could be used in future tissue-engineering applications to better exploit the cell potential. © 2014 Published by Elsevier Ireland Ltd.
1. Introduction
∗ Corresponding author. Tel.: +41 0 21 314 22 11; fax: +41 0 21 314 25 30. E-mail addresses: [email protected], [email protected] (P.G. di Summa), [email protected] (P.J. Kingham), campisi [email protected] (C.C. Campisi), [email protected] (D.F. Kalbermatten).
Autografts are currently the gold standard in nerve repair, but have the drawback requiring sacrifice of a functional nerve. A few nerve conduits (NC), tubular structures designed to bridge the nerve gap created after nerve injury, have received approval for mass production from the US Food and Drug Administration (FDA) and Conformit Europe (CE) reviewed in [2,17]. These include synthetic non-resorbable polyvinyl alcohol hydrogels (SaluBridgeTM )
http://dx.doi.org/10.1016/j.neulet.2014.04.029 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: P.G. di Summa, et al., Collagen (NeuraGen® ) nerve conduits and stem cells for peripheral nerve gap repair, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.029
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and polymer tubes with various degradation times (Neurotube® , made of polyglycolide acid and Neurolac® , made of poly d,l-lactide-caprolactone). Two of the NC are made from collagen, NeuraGen® nerve guide and NeuroMatrix, Neuroflex® . While commercial nerve guides represent a promising alternative to autografts when direct tensionless repair is not possible, their use is limited to lesions up to 3 cm [8]. For this reason, researchers have focused on different biomaterials and the inclusion of coatings, growth factors, intraluminal structures as well as the incorporation of supportive cells. The rationale of cell transplantation into neural conduits is that the presence of a cellular supportive platform within the NC can secrete a constant flow of neurotrophic factors and matrix proteins to assist nerve repair. The beneficial effects of in vivo transplantation of Schwann cells (SC) are widely reported [2]. Problems do however exist in clinical applications of autologous SC, with limited tissue availability, time consuming derivation and expansion ex vivo and donor site morbidity [20]. Adult stem cells such as bone marrow-derived mesenchymal stem cells (MSC) and adipose-derived stem cells (ASC) have shown profound plasticity, showing in vitro differentiation into non-mesenchymal fates, including SC-like phenotypes [14,24]. Our group firstly described the in vivo transplantation and effects of dASC using biodegradable fibrin nerve conduits [11], showing significant improvements in regeneration rates, similar to the autografts [9]. To our knowledge, besides their growing applicability in tissue engineering and nerve regeneration, dASC have never been tested with a commercially available conduit. In this study we evaluated the interactions of both dASC and SC-like differentiated bone marrow derived MSC (dMSC) with the Neuragen® conduit in vitro. The in vivo early nerve regeneration across empty Neuragen® conduits and Neuragen® conduits coupled with dMSC, dASC and SC was assessed using a 1-cm rat sciatic nerve gap in a two-weeks experiment.
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2. Methods
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2.1. Experimental animals
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All animal protocols were approved by the local veterinary commission, in accordance with the European Community Council directive 86/609/ECC for the care and use of laboratory animals. Male Sprague–Dawley rats (Janvier, France) weighing 250 g were used for this study.
2.2. Cell harvest and adult stem cells mesodermal differentiation Adult Schwann cells (SC) were isolated from rat sciatic nerves as previously described [11] and maintained in Dulbecco’s Modified Eagle’s Medium plus Glutamax (DMEM, Invitrogen, UK) containing 10% fetal bovine serum (FBS), 1% penicillin–streptomycin (P/S) and supplemented with 14 M forskolin (Sigma, UK) and 100 ng/ml neuregulin-1 1 (R&D Systems, UK). Adipose-derived stem cells (ASC) were harvested as described previously [14] and maintained in Modified Eagle Medium (␣-MEM; Invitrogen, UK) containing 10% (v/v) FBS, and 1% (v/v) P/S solution. Bone marrowderived mesenchymal stem cells (MSC) were harvested from adult Sprague-Dawley rat femoral bones as previously described [3] and maintained in the same growth medium as for ASC. In order to confirm stem cell multi-potency, bone marrow MSC and ASC were incubated with specific media to induce differentiation into the three mesodermal-derived lineages as in previous literature, using adipogenic, osteogenic and chondrogenic induction media for three weeks [21].
2.3. Adult stem cells differentiation to a Schwann cell phenotype At early passages (P2–P3), when cells were sub-confluent, bone marrow-derived MSC and ASC were treated with differentiation medium (DM) containing 5 ng/mL platelet-derived growth factor, 10 ng/mL basic fibroblast growth factor (both from PeproTech Ltd.), 14 mM forskolin (Sigma) and 200 ng/ml of neuregulin-1 1 (R&D Systems, UK) as previously described [11,14]. Cultures were incubated under these conditions for at least 2 weeks with DM changes every 48 h. Stem cell differentiation into SC-like cells was assessed by immunocytochemistry for typical SC markers S100 and GFAP in parallel to SC as positive controls [3,14]. Negative controls with lack of primary antibodies were also included. 2.4. Scanning electron microscope in vitro imaging To study cell-conduit interactions in vitro 1 × 106 cells (either Schwann cells or dASC) were suspended in 50 l of respective medium and seeded into longitudinally cut NeuraGen® conduits. Top up of media was performed after 2 h. After 48 h incubation, conduits were washed with PBS and fixed in 2.5% glutaraldehyde in PBS for 1 h at 4 ◦ C. After fixation, further washing in PBS and dehydration was performed using increasing concentrations of ethanol, followed by washes in hexamethylsidilazane (HMDS). After dehydration, samples were mounted on stubs and gold sputtered for scanning electron microscopy qualitative analysis (VPSEM, Zeiss EVO60, Zeiss, Germany, up to 1000×; Phenom, G2 pro desktop, Lambda Photometrics, UK, up to 5000×). 2.5. Cell seeding for in vivo experiments Prior to implantations in rats, the different regenerative cells were detached from the flask by trypsinization. After centrifugation and aspiration of the supernatant, 2 × 106 cells were resuspended in 50 l of DM (or SCGM for the SC group) and carefully pipetted into the NeuraGen® conduits. The conduits without cells contained just 50 l of DM. To avoid cell or medium dispersion during this procedure, one side of the collagen tube was temporarily sealed using a vascular clip. Conduits with cells were kept at 37 ◦ C with 5% CO2 until surgical implantation (for a maximum of 3 h). Before implantation the vascular clips were cut away, leaving a conduit of 14 mm length. 2.6. Microsurgical technique Five conduits were implanted for each different group involved in the study (empty nerve guide, guide seeded with SC, dASC, dMSC) for a total n = 20. The operation was performed on the left sciatic nerve under aseptic conditions as previously described [9]. The sciatic nerve was transected 1 cm proximal to its distal branches. Proximal and distal nerve stumps were inserted 2 mm into the NeuraGen® conduit, thus leaving a 10 mm gap, and fixed to it by a single epineural suture at each end (9/0 Prolene, Ethicon). Muscles and fascia layers were closed with single resorbable stitches (4/0 Softcat, Braun, Germany) and the skin by a continuous running suture (4/0 Prolene, Ethicon, Germany). 2.7. Conduits harvesting and immunohistochemistry Animals were sacrificed by CO2 euthanasia followed by cervical dislocation. Harvested conduits where fixed according to a previously published protocol [11] and embedded in OCT freezing media (Tissue-tek, Sakura, Japan), frozen with dry ice and stored at −80 ◦ C. Longitudinal cryo-sections (14 m) were prepared onto slides (Superfrost plus, Menzel-Gläser, Germany). A total of 60 sections were taken for each conduit explanted and stored at −20 ◦ C.
Please cite this article in press as: P.G. di Summa, et al., Collagen (NeuraGen® ) nerve conduits and stem cells for peripheral nerve gap repair, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.029
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Q4 Fig. 1. Adult stem cell multi-potency. ASC obtained from fat (A) and bone marrow-derived MSC (E) of adult rats were successfully differentiated into three cell lineages: adipogenic (Oil Red O staining of fat droplets, B and F), chondrogenic (toluidine blue staining of proteoglycans, C and G) and osteogenic (Alizarin Red S staining for calcium deposition, D and H). Scale bar = 100 m. Lower row: differentiated Schwann cell like ASC (dASC) and primary Schwann cells (SC) immunostained for S100 (green), GFAP (red) and DAPI (blue). Scale bar = 20 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Immunostaining on frozen sections using primary antibodies rabbit anti-S100 and rabbit anti-PGP 9.5 (both 1:500, Dako, UK) was performed as previously described [11]. The sections were examined under fluorescence microscope. Using a micro-grid, the regenerating front directed toward the distal end was measured in each section [11,24]. Axonal regeneration distance (PGP 9.5) and S100 positive cell distribution inside the conduit were evaluated. The four highest regeneration values for each conduit were selected and the average of these was recognized as representative of the maximal length of the regeneration cone.
3.2. Cell seeding and conduit properties Prior to in vivo experiments, we tested the NeuraGen® conduit and its capacity to retain cells within. Two conduits were seeded with either dASC or SC, and then fixed after 48 h. Cell retention was qualitatively judged with scanning electron microscopy. A good cell distribution was found throughout the conduits and there was a satisfactory adhesion of the cells to collagen walls (Fig. 2). During surgery the collagen conduits were easy to handle and micro-surgical suture was uneventful. 3.3. Postoperative complications and autotomy
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2.8. Data analysis One-way analysis of variance (ANOVA) with Bonferroni multiple comparison test was used to statistically analyze data (GraphPad Prism). Significance was determined as *p < 0.05.
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All animals survived; none of the animals developed infections or surgical complications. As expected, at the time of wound reopening after 2 weeks in vivo, collagen conduits showed no signs of resorbtion. Hematoma, infection, scarring or excess inflammatory reactions were not detected. Lower limb autotomy was present in 1 out of 20 animals (5%). A supplementary animal was operated to re-establish the numbers per group. 3.4. Axon regeneration and Schwann cells infiltration
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ASC obtained from fat and bone marrow-derived MSC of adult rats showed a flattened fibroblast-like morphology and both showed multi-lineage differentiation potential (Fig. 1). MSC and ASC also differentiated into a SC-like phenotype as previously described [11,14]. The stem cells showed obvious morphological changes and exhibited the spindle-like shape typical of SC; a significant number of the cells co-expressed the SC markers (S100, GFAP, and p75) (Fig. 1, lower row).
Axonal regeneration distance was assessed proximally by PGP 9.5 immunohistochemistry (Fig. 3). Immunohistochemistry on longitudinal sections showed the growing patterns of the regenerating nerves. The growth regeneration front profile was generally better defined in the NeuraGen® + SC group when compared to the other experimental groups (empty, dASC, dMSC), in which there was occasional mis-directional growth. Moreover, SC displayed a particular affinity to type I collagen, with two lateral sprouts along the collagen walls accompanying the central growth
Please cite this article in press as: P.G. di Summa, et al., Collagen (NeuraGen® ) nerve conduits and stem cells for peripheral nerve gap repair, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.029
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Fig. 2. Scanning electron micrographs of the collagen tube at increasing magnifications. Longitudinal sections of collagen tube, seeded with SC (A–C) and dASC (D–F) exhibiting a network aspect and semi-permeability. Scale bars: A, D =200 m; B, C, E, F = 20 m.
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regeneration cone (Fig. 3). No statistically significant differences were noticed in proximal axonal sprouting (Fig. 4). By using alternate sections stained with PGP 9.5 (axons) and S100 (Schwann cells) a clear interposed pattern between regenerating axons and Schwann cells sprouting at the proximal stump was observed (Figs. 3 and 4). Statistical analysis showed that the collagen conduits seeded with SC significantly improved SC infiltration at the distal nerve stump compared with the empty conduit (NeuraGen® +SC: 5.18 ± 0.29 mm, empty NeuraGen® 3.85 ± 0.26 mm, *p < 0.05, all values mean ± SEM). No significant difference was noticed between the SC group and stem cells groups (dASC, dMSC) with either type of antibody staining.
4. Discussion The potential advantages of a nerve conduit over autologous nerve sensory graft in nerve repair include reduction of collateral sprouting, no donor site morbidity, and operational time saving, especially in multiple nerve injuries [17]. However, nerve conduits have limited benefit in treating long gaps. Cell transplantation into the conduits has been studied as a possible solution to improve the conductive microenvironment of the hollow nerve guides. Electron microscope images after 48 h in vitro incubation showed effective cells retaining into the conduits; this was particularly evident when seeding with Schwann cells, suggesting an effective adhesion to
Fig. 3. PGP 9.5 staining shows axonal regeneration into the collagen conduits either empty or seeded with different regenerative cells. (A) Empty collagen conduit. (B) Collagen conduit + dMSC. (C) Collagen conduit + dASC. (D) Collagen conduit + SC. Right column: detail of SC infiltration into the collagen conduit, suggesting a particular affinity of transplanted SC to the collagen walls. Nerve stumps regrowth from left to right in all images. Scale bar = 1 mm.
Please cite this article in press as: P.G. di Summa, et al., Collagen (NeuraGen® ) nerve conduits and stem cells for peripheral nerve gap repair, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.029
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Fig. 4. S100 staining showing SC cell migration across the collagen conduits either empty or seeded with different regenerative cells. (A) Empty collagen conduit. (B) Collagen conduit + dMSC. (C) Collagen conduit + dASC. (D) Collagen conduit + SC. Nerve stumps regrowth from left to right in all images. Scale bar = 1 mm. Lower row: quantification of regeneration distances measured with PGP 9.5 and S100 cell staining (proximal and distal infiltration, respectively). Values are mean distances (mm) ± SEM (*p < 0.05).
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collagen, consistent with previous literature showing good Schwann cells affinity to collagen substrates [23]. All conduits (empty or cell-seeded) supported early axonal regeneration. No signs of conduit resorbtion were noticed at 2 weeks consistent with the expected complete degradation period of 4 years [7]. Empty NeuraGen® conduits displayed a particularly good performance with over 5 mm of average axonal regeneration after 2 weeks, which compares favorably with other experimental conduits [11,18]. In all groups axonal regeneration distances overlapped SC migration patterns, suggesting that regenerating axons in the proximal stump were associated to Schwann cells, confirming the “SC-axon partnership” during nerve regrowth, which has been previously described as a key sign of effective early regeneration [4]. However, transplanted cells did not significantly improve the regeneration distances. At first glance, the generally similar proximal regeneration patterns (Fig. 3) suggest that the important local cells infiltration, allowed by the semi-permeable conduit, could have concealed the effect of delivered cells. Semi-permeability of
collagen material is one of the main characteristics of NeuraGen® guides [16] and has been identified as an important feature to promote nerve regeneration by allowing the beneficial passage into the lumen of bioactive factors and local cells such as proliferating macrophages, Schwann cells and fibroblasts, which are involved in the first phases of nerve regeneration [5]. Although previous experiments revealed initial beneficial effects of transplanted regenerative cells [11,13], it may be that longer time points are required to detect significant improvements, as shown previously when using Schwann cells [18]. What was interesting was the significantly better SC infiltration at the distal stump in the NeuraGen® conduits seeded with SC. We cannot rule out the possibility that the transplanted SC migrated from the walls of the conduit to the distal stump but the pattern of staining is consistent with an infiltration of endogenous SC. SC are the main actors of this regeneration trigger serving as scaffolds for regenerating axons by expressing surface adhesion molecules, and attracting them while proliferating and releasing neurotrophic factors [4].
Please cite this article in press as: P.G. di Summa, et al., Collagen (NeuraGen® ) nerve conduits and stem cells for peripheral nerve gap repair, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.029
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Thus, the improved distal SC infiltration may secondarily enhance proximal regeneration at later time points. The amplified SC infiltration at the distal stump is consistent with the sharper growth cone pattern in the NeuraGen® + SC group, when compared with the other experimental groups. Transplanted SC seemed to have a preferential affinity to the collagen walls (Figs. 3 and 4), building connections to the distal stump along the collagen walls. This goes in line with previous literature describing expression of a number of integrin and non-integrin receptors on the surface of SC which interact with the extracellular matrix (ECM) molecules, including collagen fibrils, during axon regrowth [6]. The preferential affinity of SC for collagen may also explain the increased distal cell infiltration, which could contribute to enhanced neurotrophic factor release by the cells [1]. It would be interesting to know if the distal cell infiltration would provide benefits in cases of longer, critical distance gap injuries. In these situations, it is hypothesized that with prolonged denervation the distal stump is progressively less supportive to receiving the axons which eventually cross the gap. By transplanting Schwann cells in the conduits they might enhance endogenous distal Schwann cell survival, proliferation, migration and alignment to provide a more effective pathway for the regenerating axons. The signals transmitted between the transplanted cells and endogenous cells need to be elucidated to further optimize the use of the more clinically relevant stem cells. The stem cells did not play a significant role in term of axonal regeneration in this study, despite showing effective in vitro differentiation toward a Schwann cell phenotype. These initial findings are in contrast with previous reports of our research group and others [7,9,12] using various experimental conduits. A few studies have described the application of SC cells and MSC to collagen guides [19,23], but to our knowledge no literature exists on the association of ASC with collagen nerve guides. Recently, Ladak and coworkers seeded NeuraGen® with SC and MSC to cross a 12 mm gap after total sciatic nerve transection in rats [15]. The authors reported a significant increase in retrogradely labeled motoneurons in the NC groups seeded with dMSC and SC. However, compared with empty conduits no functional improvements were shown in SC or dMSC groups [15]. This may suggest that, even if collagen remains one of the most versatile and effective conduits for peripheral nerve repair, it requires further modifications to allow it to be coupled with therapeutic cells [15,23]. Future studies will have to consider the use of multiple approaches to guarantee the best conduit design to combine ideal mechanical properties with the optimal cell performance. We previously reported the positive effect of extracellular matrix molecules such as laminin and fibronectin on neurotrophic activity and cell viability of dASC [10]. Surface functionalization by laminin could improve the intraluminal guidance and directionality [4,22] and at the same time help preserve the neurotrophic effect of the SC-like differentiated stem cells, thereby supporting regeneration across nerve gaps.
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This study confirms the good performance of commercially available collagen tubes for peripheral nerve repair, with substantial axonal regeneration at 2 weeks post implantation. The supportive environment of the conduits appears to be enhanced by addition of Schwann cells. Whether significant differences can be observed among the various cell types at longer time points will be assessed in future studies. This preliminary report suggests that tissue engineering refinements such as extracellular matrix coatings of the collagen scaffolds might be needed to enhance the effect
of the delivered stem cell, improving cell-material interactions and, as a result, regeneration across nerve gaps. References [1] S.J. Armstrong, M. Wiberg, G. Terenghi, P.J. Kingham, ECM molecules mediate both Schwann cell proliferation and activation to enhance neurite outgrowth, Tissue Eng. 13 (2007) 2863–2870. [2] J.H. Bell, J.W. Haycock, Next generation nerve guides: materials, fabrication, Q3 growth factors, and cell delivery, Tissue Eng. Part B: Rev. (2011). [3] J. Caddick, P.J. Kingham, N.J. Gardiner, M. Wiberg, G. Terenghi, Phenotypic and functional characteristics of mesenchymal stem cells differentiated along a Schwann cell lineage, Glia 54 (2006) 840–849. [4] Y.Y. Chen, D. McDonald, C. Cheng, B. Magnowski, J. Durand, D.W. Zochodne, Axon and Schwann cell partnership during nerve regrowth, J. Neuropathol. Exp. Neurol. 64 (2005) 613–622. [5] C. Cheng, D.W. Zochodne, In vivo proliferation, migration and phenotypic changes of Schwann cells in the presence of myelinated fibers, Neuroscience 115 (2002) 321–329. [6] M.A. Chernousov, D.J. Carey, Schwann cell extracellular matrix molecules and their receptors, Histol. Histopathol. 15 (2000) 593–601. [7] W. Daly, L. Yao, D. Zeugolis, A. Windebank, A. Pandit, A biomaterials approach to peripheral nerve regeneration: bridging the peripheral nerve gap and enhancing functional recovery, J. R. Soc. Interface 9 (2012) 202–221. [8] D.N. Deal, J.W. Griffin, M.V. Hogan, Nerve conduits for nerve repair or reconstruction, J. Am. Acad. Orthop. Surg. 20 (2012) 63–68. [9] P.G. di Summa, D.F. Kalbermatten, E. Pralong, W. Raffoul, P.J. Kingham, G. Terenghi, Long-term in vivo regeneration of peripheral nerves through bioengineered nerve grafts, Neuroscience 181 (2011) 278–291. [10] P.G. di Summa, D.F. Kalbermatten, W. Raffoul, G. Terenghi, P.J. Kingham, Extracellular matrix molecules enhance the neurotrophic effect of Schwann cell-like differentiated adipose-derived stem cells and increase cell survival under stress conditions, Tissue Eng. Part A 19 (2013) 368–379. [11] P.G. di Summa, P.J. Kingham, W. Raffoul, M. Wiberg, G. Terenghi, D.F. Kalbermatten, Adipose-derived stem cells enhance peripheral nerve regeneration, J. Plast. Reconstr. Aesthet. Surg.: JPRAS 63 (2010) 1544–1552. [12] J.H. Gu, Y.H. Ji, E.S. Dhong, D.H. Kim, E.S. Yoon, Transplantation of adipose derived stem cells for peripheral nerve regeneration in sciatic nerve defects of the rat, Curr. Stem Cell Res. Ther. 7 (2012) 347–355. [13] D.F. Kalbermatten, P.J. Kingham, D. Mahay, C. Mantovani, J. Pettersson, W. Raffoul, H. Balcin, G. Pierer, G. Terenghi, Fibrin matrix for suspension of regenerative cells in an artificial nerve conduit, J. Plast. Reconstr. Aesthet. Surg. 61 (2008) 669–675. [14] P.J. Kingham, D.F. Kalbermatten, D. Mahay, S.J. Armstrong, M. Wiberg, G. Terenghi, Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro, Exp. Neurol. 207 (2007) 267–274. [15] A. Ladak, J. Olson, E.E. Tredget, T. Gordon, Differentiation of mesenchymal stem cells to support peripheral nerve regeneration in a rat model, Exp. Neurol. 228 (2011) 242–252. [16] S.T. Li, S.J. Archibald, C. Krarup, R.D. Madison, Peripheral nerve repair with collagen conduits, Clin. Mater. 9 (1992) 195–200. [17] M.F. Meek, J.H. Coert, US Food and Drug Administration/Conformit Europeapproved absorbable nerve conduits for clinical repair of peripheral and cranial nerves, Ann. Plast. Surg. 60 (2008) 110–116. [18] A. Mosahebi, M. Wiberg, G. Terenghi, Addition of fibronectin to alginate matrix improves peripheral nerve regeneration in tissue-engineered conduits, Tissue Eng. 9 (2003) 209–218. [19] F.R. Pereira Lopes, L. Camargo de Moura Campos, J. Dias Correa Jr., A. Balduino, S. Lora, F. Langone, R. Borojevic, A.M. Blanco Martinez, Bone marrow stromal cells and resorbable collagen guidance tubes enhance sciatic nerve regeneration in mice, Exp. Neurol. 198 (2006) 457–468. [20] L.A. Pfister, M. Papaloizos, H.P. Merkle, B. Gander, Nerve conduits and growth factor delivery in peripheral nerve repair, J. Peripher. Nerv. Syst. 12 (2007) 65–82. [21] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143– 147. [22] N. Rangappa, A. Romero, K.D. Nelson, R.C. Eberhart, G.M. Smith, Laminincoated poly(l-lactide) filaments induce robust neurite growth while providing directional orientation, J. Biomed. Mater. Res. 51 (2000) 625– 634. [23] F. Stang, H. Fansa, G. Wolf, M. Reppin, G. Keilhoff, Structural parameters of collagen nerve grafts influence peripheral nerve regeneration, Biomaterials 26 (2005) 3083–3091. [24] M. Tohill, C. Mantovani, M. Wiberg, G. Terenghi, Rat bone marrow mesenchymal stem cells express glial markers and stimulate nerve regeneration, Neurosci. Lett. 362 (2004) 200–203.
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