EXCLI Journal 2015;14:851-860 – ISSN 1611-2156 Received: April 19, 2015, accepted: May 28, 2015, published: July 27, 2015

Original article:

CYTOCOMPATIBILITY OF A CONDUCTIVE NANOFIBROUS CARBON NANOTUBE/POLY (L-) COMPOSITE SCAFFOLD INTENDED FOR NERVE

Mahboubeh Kabiri1,2,3,*, Saeed Oraee-Yazdani2,4, Masumeh Dodel3,5, Hana Hanaee-Ahvaz2, Sara Soudi2,6, Ehsan Seyedjafari1,3, Mohammad Salehi2,7, Masoud Soleimani8,*

1 Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran 2 Department of Biology, Stem Cell Technology Research Center, Tehran, Iran 3 Department of Nanotechnology and Tissue Engineering, Stem Cell Technology Research Center, Tehran, Iran 4 Functional Neurosurgery Research Center, Shohada Tajrish Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran 5 Department of Textile Engineering, Amirkabir University of Technology, Tehran, Iran, Stem Cell Technology Research Center, Tehran, Iran 6 Department of Immunology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran 7 Department of Biotechnology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 8 Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran * Corresponding authors: Dr Mahboubeh Kabiri, Department of Biotechnology, College of Science, University of Tehran, P.O.Box: 14155-6455, Tehran, Iran, Tel: (98) 21 66491622, Mobile: +98 9122116831; E-mail: [email protected] Dr Masoud Soleimani, Hematology Department, Faculty of Medical Science, Tarbiat Modares University, P.O.Box: 14115-111, Tehran, Iran, Tel: (98) 21 88861065-7; Mobile: +98122875993; E-mail: [email protected] http://dx.doi.org/10.17179/excli2015-282

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/).

ABSTRACT The purpose of this study was to fabricate a conductive aligned nanofibrous substrate and evaluate its suitability and cytocompatibility with neural cells for nerve tissue engineering purposes. In order to reach these goals, we first used to fabricate single-walled carbon-nanotube (SWCNT) incorporated poly(L-Lactic acid) (PLLA) nanofibrous scaffolds and then assessed its cytocompatibility with olfactory ensheathing glial cells (OEC). The plasma treated scaffolds were characterized using scanning electron microscopy and water contact angle. OECs were isolated from of GFP Sprague-Dawley rats and characterized using OEC spe- cific markers via immunocytochemistry and flow cytometery. The cytocompatibility of the conductive aligned nano-featured scaffold was assessed using microscopy and MTT assay. We indicate that doping of PLLA poly- mer with SWCNT can augment the aligned nanosized substrate with conductivity, making it favorable for nerve tissue engineering. Our results demonstrated that SWCNT/PLLA composite scaffold promote the adhesion, growth, survival and proliferation of OEC. Regarding the ideal physical, topographical and electrical properties of the scaffold and the neurotrophic and migratory features of the OECs, we suggest this scaffold and the cell/scaffold construct as a promising platform for cell delivery to neural defects in nerve tissue engineering ap- proaches.

851 EXCLI Journal 2015;14:851-860 – ISSN 1611-2156 Received: April 19, 2015, accepted: May 28, 2015, published: July 27, 2015

Keywords: Nerve tissue engineering, olfactory ensheathing cells, carbon nanotube, composite scaffold, electrospun nanofiber

INTRODUCTION developmental heritage with SC is olfactory ensheathing glial cell (OEC). Normally, their Cell therapy has a promising potential for main role in the olfactory system is to facili- the treatment of neurodegenerative diseases tate and guide olfactory to the hostile or trauma-related injuries that are otherwise very difficult to cure by conventional ap- olfactory bulb throughout the life ( Ramón- proaches. Damages to nervous tissues, in- Cueto and Valverde, 1995). They have cluding spinal cord, brain and peripheral shown to express different neurotrophic fac- nerves, can cause debilitating diseases as tors including glial derived neurotrophic fac- they usually lead to progressive functional tor (GDNF), nerve growth factors (NGF), damages. Current approaches basically rely brain-derived neurotrophic factor (BDNF) on reduction or inhibition of gliosis by using and their receptors (Woodhall et al., 2001), steroids (Hall and Springer, 2004), immune- and can myelinate axons indistinguishably modulatory drugs (Bowes and Yip, 2014) or from myelination by Schwann cells (Barraud et al., 2010). The ex- administration of chondroitinase (Bradbury pressed and produced by OEC could endow et al., 2002), aiming to minimize secondary these cells with regeneration and remy- complications associated with the injury elination potential over several millimeters (Thuret et al., 2006). With the advent of cell- of axons (Imaizumi et al., 1998) and ulti- based therapies, the therapeutic potential of mately restoration of functional synapses different cell types was investigated for (Barnett et al., 2000). Besides, originating nerve regeneration (Hedayatpour et al., from the neural crest, OEC persist in adult 2013). Stem cells from different sources tissues and can be regarded as an abundant such as marrow (BM) (Hafizi et al., and accessible source of autologous cells 2015), blastocysts (ESC, embryonic stem throughout the life (Barraud et al., 2010). cells), fetal tissues and neural stem cells have Regarding the availability of patient specific been investigated frequently, with each dis- OEC and their neurotrophic characteristics, playing varying degrees of regeneration and these cells can be considered as great candi- functional restoration. The transplanted stem dates for cell-based therapy of neural inju- cell derived neural or glial cells/progenitors ries. can partially reestablish the neural connec- A wide range of bioengineered scaffolds tions and result in recovery of function have been developed to assist in the im- (Yazdani et al., 2012). provement of neural regeneration. An ideal Schwann cells (SC), the glial cells that synthetic scaffold must have chemical, me- ensheath and myelinate axons in peripheral chanical, morphological and electrical prop- nervous system (PNS), are of most studied erties mimicking the natural ECM of the de- cell types that can repair injuries of central sired neural tissue. Poly(α-hydroxy esters), nervous system (CNS) owing to their capa- such as poly(L-lactic acid) (PLLA) and its bility to produce neurotropic factors and cell breakdown products are shown to be bio- adhesion molecules (Oudega and Xu, 2006). compatible with neural cells and tissues both SC are available autologously by a biopsy in vitro and in vivo (Schmidt and Leach, from a peripheral nerve in the leg. The re- 2003). Their tunable degradation rate, the leased neurotrophics can induce repair and non-immunogenicity and FDA approval has survival of the damaged neurons and attract made them enormously attractive in tissue endogenous stem or glial cells to the lesion engineering approaches (Potter et al., 2008). site (Ogawa et al., 2002). Another cell type Electrospinning these polymers allows for that shares many characteristics and even

852 EXCLI Journal 2015;14:851-860 – ISSN 1611-2156 Received: April 19, 2015, accepted: May 28, 2015, published: July 27, 2015

the generation of aligned fibers with diame- nous suspension, and then combined with ters in the nano-meter range that are suitable DMF and PLLA in the same proportions in directed axonal outgrowth through provi- stated above. The final concentration of sion of appropriate contact guidance (Chew SWCNT in solution was equivalent to 3 % of et al., 2006; Kabiri et al., 2012; Potter et al., the PLLA mass. The polymer solution was 2008). Furthermore, the electrospun nano- ultra-sonicated and stir homogenized over- fibrous sheets have the capacity to be rolled night before electrospinning. A syringe and packed within a defined volume, provid- pump was used to feed the solution through ing enough substrate for cell transplantation an extension tube ended in a blunted 21- (Goto et al., 2010). gauge needle. A voltage potential of 25 kV We hypothesized that further functional- was applied between the needle and the col- izing of the PLLA nanofibers with an elec- lector. The nanofiber jet was collected on a trically conductive compound can aid to stainless steel cylinder rotating at 2400 RPM mimic the inherently conductive nature of at a fixed distance of 15 cm from the spin- the nerve tissues. Electrically conductive ma- neret tip. Oxygen plasma surface treatment terials such as polypyrrole, polyaniline and was performed using a low frequency plasma carbon nanotubes (CNT) have been effec- generator set on 40 kHz (Diener Electronics). tively used in drug delivery and biosensor The hydrophobic/hydrophilic nature of the applications and for the fabrication of nerve nanofiber scaffolds before and after plasma guidance conduits in nerve tissue engineer- treatment was evaluated by measuring the ing (Chronakis et al., 2006; MacDonald et contact angle of water droplets using the ses- al., 2008; Schmidt et al., 1997; Voge et al., sile drop method (G10 contact angle goni- 2008; Zhang et al., 2007). The resultant con- ometer, Kruss). ductive composite would inherit both the The morphology of the nanofibrous scaf- physical properties of polymeric materials folds and the surface characteristics of cell and the electrical characteristics of the con- seeded scaffolds were evaluated by SEM ductive material needed for specific applica- (Philips Xl-30). The scaffold or cell/scaffold tions such as nerve and cardiac tissue engi- constructs were washed thoroughly with neering. PBS and fixed with 2.5 % glutaraldehyde so- In the present study we made a conduc- lution for two hours. The dehydrated sam- tive nano-featured substrate of single walled ples were sputter coated with gold for 90 s carbon nanotubes (SWCNT) and PLLA and and deposited onto SEM stubs. The electro- assessed its biocompatibility with well char- spun nanofibers were imaged and the fiber acterized OEC, aiming to use the composite diameter was measured from 10 random im- system as a nerve guidance conduit and a ages using Image J software (NIH). cell delivery platform to the nerve lesion for nerve tissue engineering purposes. OEC isolation and culture OEC were obtained from olfactory bulbs MATERIALS AND METHODS of adult GFP rats following a procedure ap- proved by the Animal Care and Ethics of Scaffold fabrication and characterization Electrospinning (Nanoazma, Iran) was Stem Cell Technology research center, Iran. used to construct SWCNT/PLLA aligned The olfactory bulb was dissected, finely minced and digested in collagenase/dispase nanofibrous scaffolds. PLLA (MW=157000, Sigma-Aldrich) was dissolved in a solvent II solution as previously described in details mixture of chloroform and N,N-dimethyl- (Salehi et al., 2009). After enzyme deactiva- formamide (DMF) (8.5:1.5, v/v) to have a fi- tion the tissue was mechanically triturated nal concentration of 3.5 % w/v. SWCNT and cells were spun down with centrifuga- (Plasmachem) nanoparticles were first well tion. The cell pellet was cultured in DMEM/ dispersed in chloroform to form a homoge- F12 (Gibco) supplemented with 10 % fetal

853 EXCLI Journal 2015;14:851-860 – ISSN 1611-2156 Received: April 19, 2015, accepted: May 28, 2015, published: July 27, 2015

bovine serum (FBS, Sigma), 2 mM L- were stained with anti-P75 (Sigma) and anti- glutamine (Gibco), 100 IU/ml penicillin and S100 (Chemicon) antibodies. PE-conjugated 100 µg/ml streptomycin (Gibco). Cultures secondary antibody (Abcam) was used to la- were incubated in 5 % CO2 incubator for 18 bel the cells. Paraformaldehyde (PFA) fixed hours. Next the supernatant was cultured for cells were analyzed on a FACS Calibur cy- another 36 hours in a new uncoated flask. tometer (Becton Dickinson, San Jose, CA) The supernatant was then used to feed poly- and analyzed with Win MDI 2.8 software. L-lysine (Sigma) coated flasks, knowing most of the OEC have not attached to the Immunofluerescence assay previous uncoated flasks. Cells were cultured A monolayer culture of OEC was fixed on poly-l-lysine coated plates until appropri- with 4 % PFA, and permeabilized with 0.1 % ate cell number was achieved. Triton X-100 for intracellular antigen stain- ing, and blocked with 5 % goat serum. Cells Cell seeding and proliferation assay were then incubated at 4 °C overnight with Scaffolds were sterilized in 70 % ethanol anit-P75 primary antibody. Cells were then for 2 hours and then rinsed copiously with incubated with PE-conjugated secondary an- PBS prior to use in culture. The biocompati- tibody for 3 hours at room temperature. The bility of composite scaffolds was assessed by cells nuclei were stained with 4',6- examining cell proliferation. Cell prolifera- diamidino-2-phenylindole dihydrochloride tion was measured on day 1, 7, 14 and 21 (DAPI; 1:1000) and visualized by fluores- from a starting cell seeding number of 5000 cent microscope (TE2000-S; Nikon-Eclipse, cells on the SWCNT/PLLA scaffolds, or in a Tokyo, Japan). standard poly-l-lysine coated tissue culture polystyrene (TCP) well control. Relative cell Statistical analysis number was quantified using the MTT (me- In all the experiments of this original re- thyl thiazolyl diphenyl tetrazolium bromide) search article the statistical analysis was per- assay, as per manufacturer’s instructions and formed using the unpaired Student’s t test, compared to 2-dimensional (2D) TCP con- where results were considered significant trols. Briefly, the MTT assay was performed when p < 0.05. All experiments were done in by incubating cells at various time points triplicate, unless otherwise stated and data with MTT (1 mg/ml) solution for 3 hours. expressed as the mean ± standard deviation. MTT absorbance was measured spectropho- tometrically at 570 nm and the absorbance RESULTS was assumed to be directly proportional to OEC characterization the number of viable cells. OEC were isolated from olfactory bulb To investigate cell morphology of the of GFP Sprague -Dawley rats. The isolated OEC on aligned nanofibers, cell-scaffold cells were phenotypically characterized by constructs were fixed using 2.5 % glutaral- flow-cytometery and immune-fluerescence dehyde solution. The specimens were then assay before use. The morphology of the undergone dehydration using series of grad- cells is shown in Figures 1A and B. OEC ed ethanol solutions. The dried samples were populations are composed of adherent cells then sputter coated with gold and observed with heterogeneous morphologies, but most- using SEM. ly display spindle or polygonal shape. P75 is Flow cytometery analysis a neurothrophin receptor (also known as The enriched cell population was im- P75NTR, or CD271) expressed on the cell munostained with cell specific antibodies to surface of OEC which is normally used for determine purity of the culture. In brief, cells both OEC identification and enrichment. The were permeabilized with 0.5 % Triton X-100 results of this study indicated that the en- and blocked with 1 % BSA. Next the cells riched OEC population was immunopositive

854 EXCLI Journal 2015;14:851-860 – ISSN 1611-2156 Received: April 19, 2015, accepted: May 28, 2015, published: July 27, 2015

for P75 (Figures 1C, D). The flow-cyto- onto the scaffolds. We have previously metric results revealed the purity of the cells shown that addition of 3 % w/w CNT to the to be ~78 % (Figure 2A). Given that OEC PLLA fibers can dramatically enhance its are in fact a glial cell type (also known as ol- electrical conductive properties with minimal factory ensheathing ) residing the olfac- CNT agglomeration in the fiber constructs tory system, expression of glial markers such and no cytotoxic effects on cellular behavior as glial fibrillary acidic protein and S100 can (Kabiri et al., 2012). The composite scaf- be used for immunostaining of these cells folds used in this study were shown to have (Kawaja et al., 2009). Although S100 is ba- nanometer diameter fibers, well aligned ori- sically known as Schwann cells’ marker, but entation and high conductivity, totally mak- OEC and Schwann cells share many pheno- ing them a suitable substratum for nerve tis- typic and antigenic characteristics. Previous sue engineering applications. The attachment reports also show a close relationship in growth behavior and antigenic markers of OEC and Schwann cells (Ramón-Cueto and Avila, 1998). The isolated cell population used in this study was 63 % immunoreactive to S100 marker (Figure 2B).

Effect of guidance cues on the alignment of OEC The nanostructure of SWCNT doped PLLA scaffold is shown in Figure 3A. These nanofibers have a narrow range of diameter distribution with an average of ~430nm.We observed an excellent increase in hydro- philicity of the nanofibers after modification Figure 1: Characterization of isolated OEC. (A) in vitro morphology of isolated cells with light mi- with oxygen plasma, with a reduction in wa- croscopy; (B) GFP expressing OEC: The cells ter contact angle from 137 to a non-detec- show a bipolar or three polar characteristic most- table amount. Indeed such surface modifica- ly connected with each other; (C) P75 positive tions are commonly used to enhance protein OEC; (D) Nuclei stained with DAPI; Scale bars adsorption and subsequent cell attachment 10µm.

Figure 2: Characterization of isolated OEC. In vitro expression of OEC markers as quantified by flow cytometery (A) P75 and (B) S100 markers.

855 EXCLI Journal 2015;14:851-860 – ISSN 1611-2156 Received: April 19, 2015, accepted: May 28, 2015, published: July 27, 2015

Figure 3: Effect of guidance cues on the alignement of OEC. (A) Aligned SWCNT/PLLA nanofibers used as the substratum for OEC, scale bar= 10µm; (B) OEC grown on culture plates showing random orientation, magnification 100x; (C) SEM micrographs of OEC aligned on nanofiber SWCNT/PLLA scaffolds, scale bar= 2µm; (D) Fluorescence image of aligned OEC grown on SWCNT/PLLA nano- fibrous scaffolds, magnification 100x.

of OEC onto the nanofibrous scaffolds pro- Effect of SWCNT/PLLA substrate on OEC vides a qualitative assessment of the bio- proliferation compatibility of the composite fibers. When For determining OEC proliferation (ex- cultured on aligned nanofibers, OEC prolif- pressed by absorbance values), cells were erate and follow the orientation of the fibers; seeded on the scaffolds and subjected to in contrast, on tissue culture plates cells they MTT assay at different time points (1, 7, 4, tend to grow in a random manner (Figures 21 days). The assay indicated that the prolif- 3B-D). As shown in SEM micrographs, OEC eration of scaffold seeded OEC was just tightly attached and got aligned with fibers. slightly better after 7 days of culture com- Our results indicate that the aligned nano- pared to TCP plate cultured cells. However, fiber composites can serve as contact guid- after 14 and 21 days, there was no statistical ance to direct cell alignment. This strategy difference between cell viability and prolif- can have promising implications in guided eration on SWCNT/PLLA scaffolds and peripheral nerve regeneration. TCP plates (Figure 4). This suggests that SWCNT/PLLA fibers can show OEC sup- portive capacity just as TCP surfaces. Col- lectively, the results from Figures 3 and 4 suggest good biocompatibility of OEC with CNT/PLLA materials in vitro.

856 EXCLI Journal 2015;14:851-860 – ISSN 1611-2156 Received: April 19, 2015, accepted: May 28, 2015, published: July 27, 2015

cell to promote repair of the injured spinal cord. In most cell transplantation approaches, a cell suspension of several hundred thousand is directly injected via fine needles or glass capillaries. However, cell viability and effi- cacy of engraftments is usually very low (Paul et al., 2009). In recent years, OEC have gained enormous acceptance as a potential source of therapeutic cell in neural lesions and they have shown to promote the survival of injured neurons and functional recovery Figure 4: OEC proliferation estimated by MTT (Silva et al., 2014). These cells have a dual assay method at four different time points (aver- nature of astroglial cells and Schwann cells age ± standard deviation (SD)). Cells were cul- (Vincent et al., 2005). Use of OEC is espe- tured on the composite scaffolds or TCP plates cially attractive given the potential for autol- with the same surface area. The results have ogous transplantation, the ability for in vitro been reproduced in two independent studies. Each bar is the average of three replicates; error expansion and excellent neurotrophic and bars indicate standard deviations. The asterisks migratory characteristics ( Ramón-Cueto and imply statistical difference with the first time point Valverde, 1995). Numerous studies of autol- (p < 0.05). ‡ implies significant difference with ogous OEC transplantation to the patients TCP surface at the same time point. with SCI have proved these cells to be feasi- ble and safe with no evidence of any adverse effects (Mackay-Sim et al., 2008). Particular- DISCUSSION ly, of significant importance for the treat- Damages to the nervous system leave a ment of SCI is to stimulate the proliferative significant impact on the quality of life. activity of endogenous neuro progeni- Amongst neural repair strategies, cell thera- tor/stem cells along with the migration of py has gained increasing attention due to its resident glial cells to the site of injury that potential to replace lost neurons or neurogli- could be best addressed using OEC’s migra- as, reduce cysts and cavities and ultimately tory and neurotrophic potential. The trans- creating a permissive environment for axon plantation of OEC for SCI is interesting from regeneration. Clearly, cell therapy has great a translational perspective; however, scaf- potential for repairing damages to the nerv- folds as cell delivery vehicles can be em- ous system, however regarding the multifac- ployed to improve cell viability after trans- torial and multiphasic pathophysiology of plantation (Bensaïd et al., 2003; Malafaya et the brain and spinal cord injury, it needs to al., 2007). OEC engraftment on a biocompat- be combined with other interventions to ible scaffold not only guarantees direct cell maximize tissue regeneration. One strategy delivery to the site of injury but can provide is to use scaffolds to aid replacing lost tissue, the lesion site with accumulated neural inhibiting gliosis, enhancing synaptic plastic- growth factors and establishment of a gradi- ity and provision of contact guidance for the ent towards the graft (Kemp et al., 2008). newly sprouted axons. The promises of bio- Besides, such scaffold constructs can provide engineering approaches utilizing combined transplanted cells with a suitable microenvi- cellular therapy strategies and biomaterial ronment to maintain their functional pheno- design and fabrication, provoked us to make type, morphology, cell signaling and guiding a conductive nanofibrous scaffold ideal for potential while avoiding or diminishing im- nerve tissue engineering and investigate its munological rejection of the transplanted cytocompatibility with a promising type of cells (Wang et al., 2007).

857 EXCLI Journal 2015;14:851-860 – ISSN 1611-2156 Received: April 19, 2015, accepted: May 28, 2015, published: July 27, 2015

Having a high aspect ratio, electrospun delivery platform, it was necessary to test its nanofibers can provide more available sur- biocompatibility with the desired cell type face area for the cells to grow as compared to beforehand. As such, the biological charac- TCP plates; this in turn enables having more teristics and proliferation capability of rat confluent population (Figures 2B, D) which OEC were assessed in advance of imple- means transplanting greater number of cells menting in vivo studies. The viability and/or into the defect site. Besides, the capacity of proliferation of the cells, measured by MTT such small diameter electrospun sheets to be assay, were similar and even significantly rolled and packed into a cylindrical shape, higher on SWCNT/PLLA nanofibers after not only provides an aligned nanosized sub- seven days, as compared to TCP surfaces stratum to direct axons via topographical (Figure 2). There was an increasing trend in cues, but also offers ample substrate to de- cell number with time up to 14 days which liver repair promoting cells to the site of in- then tended into the plateau phase likely be- jury (Li and Shi, 2007). In a similar study, cause of contact inhibition effects in conflu- Goto et al. (2010) showed the cytocompati- ent cultures. These results indicate that the bility of a rolled sheet of scaffold plasma treated electrospun SWCNT/PLLA with SCs and proved the efficiency of the scaffolds are as suitable of a substrate for construct to promote axonal outgrowth. OEC as is the widely used poly-lysine coated Electrical stimulation has previously TCP plates. We concluded that the been shown to guide axon orientation and di- SWCNT/PLLA nanofibers have no cytotoxic rect extension (Zhang et al., 2007), or inhibitory effects on OEC survival and outlining the importance of the conductive proliferation and can be used as a conductive substrate in enhanced nerve regeneration. nerve conduit material to facilitate nerve cell Aligned nanofibers of SWCNT-doped PLLA proliferation and axonal extension. composites have a conductivity of 6 mS/cm We further proved the regenerative po- which is in sharp contrast to non-conductive tential of this cell/scaffold construct in an in PLLA nanofibers (10-8–10-9 mS/cm) (Kabiri vivo study confirming peripheral nerve re- et al., 2012). Doping of PLLA with a con- generation capacity in sciatic nerves in rats ductive substance not only endows the scaf- (Kabiri et al., 2015). Herein we introduced fold with conductive properties but can lead an aligned nanofibrous composite scaffold to relative reduction in fiber diameter, likely with structural and electrical features suitable due to higher electrostatic repulsive force to for nerve tissue engineering and showed its surface tension ratio in the fluid jet of the cytocompatibility with OEC as a well proved conductive polymer mixture. cell type with neuroprotective characteristics Only a few studies have addressed the for the repair of nerve and spinal cord inju- interactions of neural or glial cells with nan- ries. We believe this combined cell-scaffold ofibrous composite substrates (Yang et al., strategy exhibits superior promoting effects 2005). We had previously shown that as compared with either intervention on its CNT/PLLA nanofibrous scaffolds can both own. support cell adhesion and proliferation and promote neural differentiation of embryonic CONCLUSION stem cells (Kabiri et al., 2012). The compo- OEC are considered one of the most site scaffold was indeed able to direct the promising cell candidates for the treatment alignment of sprouted cells from the embry- of nerve injuries. They can directly partici- oid bodies. In the present study we observed pate in axonal extension and myelination and similar directionality and contact guidance secrete large number of neurotrophic factors with OEC seeded on SWCNT/PLLA scaf- for neural differentiation and maturation. On folds. Since this scaffold was destined to the other hand the topographical, mechanical serve as a nerve guidance conduit and a cell and electrical cues to direct axonal and neu-

858 EXCLI Journal 2015;14:851-860 – ISSN 1611-2156 Received: April 19, 2015, accepted: May 28, 2015, published: July 27, 2015

ral network connection can be provided by a Bradbury EJ, Moon LD, Popat RJ, King VR, Bennett well-designed scaffold. In the present study GS, Patel PN, et al. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature. we first isolated and characterized OECs 2002;416:636-40. from GFP rats and then investigated contact guidance and in vitro cytocompatibility of Chew SY, Wen Y, Dzenis Y, Leong KW. The role of conductive SWCNT/PLLA aligned nano- electrospinning in the emerging field of nano- fibrous scaffolds with OEC in order to assess medicine. Curr Pharm Des. 2006;12:4751-70. the scaffold suitability as a nerve guidance Chronakis IS, Grapenson S, Jakob A. Conductive conduit for nerve tissue engineering. We polypyrrole nanofibers via electrospinning: electrical showed that OEC well adhere and proliferate and morphological properties. Polymer. 2006;47: on these scaffolds and get aligned along the 1597-1603. fibers direction. By adding neurotrophic ef- Goto E, Mukozawa M, Mori H, Hara M. A rolled fects of the OEC to the electrical and topo- sheet of collagen with cultured Schwann cells: graphical contact guidance cues of the model of nerve conduit to enhance neurite growth. J SWCNT/PLLA scaffold we can promote ax- Biosci Bioeng. 2010;109:512-8. onal outgrowth and glial migration from the Hafizi M, Atashi A, Bakhshandeh B, Kabiri M, Nadri nerve into the graft and finally better nerve S, Hajihosseini R, et al. MicroRNAs as markers for regeneration. neurally committed CD133+/CD34+ stem cells derived from human umbilical cord blood. Biochem Acknowledgement Genet. 2013;51:175-88. This study was completely supported by Hall ED, Springer JE. Neuroprotection and acute stem cell technology research center and spinal cord injury: a reappraisal. NeuroRx. 2004;1: university of Tehran. There is no conflict of 80-100. interest and no benefit of any kind will be Hedayatpour A, Ragerdi I, Pasbakhsh P, Kafami L, received either directly or indirectly by the Atlasi N, Mahabadi VP, et al. Promotion of remye- author(s). lination by adipose trans- plantation in a cuprizone model of multiple sclerosis. Conflict of interest Cell J. 2013;15:142. The authors declare that they have no Imaizumi T, Lankford KL, Waxman SG, Greer CA, conflict of interest. Kocsis JD. Transplanted olfactory ensheathing cells remyelinate and enhance axonal conduction in the REFRENCES demyelinated dorsal columns of the rat spinal cord. J Neurosci. 1998; 18:6176-85. Barnett SC, Alexander CL, Iwashita Y, Gilson JM, Crowther J, Clark L, et al. Identification of a human Kabiri M, Soleimani M, Shabani I, Futrega K, olfactory ensheathing cell that can effect transplant- Ghaemi N, Ahvaz HH, et al. Neural differentiation of mediated remyelination of demyelinated CNS axons. mouse embryonic stem cells on conductive nanofiber Brain. 2000;123:1581-8. scaffolds. Biotechnol Lett. 2012;34: 1357-65.

Barraud P, Seferiadis AA, Tyson LD, Zwart MF, Kabiri M, Oraee-Yazdani S, Shafiee A, Hanaee- Szabo-Rogers HL, Ruhrberg C, et al. Neural crest Ahvaz H, Dodel M, Vaseei M, et al. Neuroregenerat- origin of olfactory ensheathing glia. Proc Natl Acad ive effects of olfactory ensheathing cells transplanted Sci. 2010;107:21040-5. in a multi-layered conductive nanofibrous conduit in peripheral nerve repair in rats. J Biomed Sci. 2015; Bensaïd W, Triffitt J, Blanchat C, Oudina K, Sedel L, 22:35. Petite H. A biodegradable fibrin scaffold for mesenchymal stem cell transplantation. Biomaterials. Kawaja MD, Boyd JG, Smithson LJ, Jahed A, 2003;24:2497-2502. Doucette R. Technical strategies to isolate olfactory ensheathing cells for intraspinal implantation. J Bowes AL, Yip PK. Modulating inflammatory cell Neurotraum. 2009;26:155-77. responses to spinal cord injury: all in good time. J Neurotraum. 2014;31:1753-66. Kemp SW, Walsh SK, Midha R. Growth factor and stem cell enhanced conduits in peripheral nerve regeneration and repair. Neurol Res. 2008;30:1030-8.

859 EXCLI Journal 2015;14:851-860 – ISSN 1611-2156 Received: April 19, 2015, accepted: May 28, 2015, published: July 27, 2015

Li J, Shi R. Fabrication of patterned multi-walled Schmidt C, Shastri V, Vacanti J, Langer R. Stimul- poly-l-lactic acid conduits for nerve regeneration. J ation of neurite outgrowth using an electrically con- Neurosci Meth. 2007;165:257-64. ducting polymer. Proc Nat Acad Sci USA. 1997;94: 8948-53. MacDonald RA, Voge CM, Kariolis M, Stegemann JP. Carbon nanotubes increase the electrical Silva NA, Sousa N, Reis RL, Salgado AJ. From conductivity of -seeded collagen . basics to clinical: a comprehensive review on spinal 2008;4:1583-92. cord injury. Progr Neurobiol. 2014;114:25-57.

Mackay-Sim A, Feron F, Cochrane J, Bassing- Thuret S, Moon LD, Gage FH. Therapeutic inter- thwaighte L, Bayliss C, Davies W, et al. Autologous ventions after spinal cord injury. Nat Rev Neurosci. olfactory ensheathing cell transplantation in human 2006;7:628-43. paraplegia: a 3-year clinical trial. Brain. 2008;131: 2376-86. Vincent AJ, Taylor JM, Choi‐Lundberg DL, West AK, Chuah MI. Genetic expression profile of Malafaya PB, Silva GA, Reis RL. Natural–origin olfactory ensheathing cells is distinct from that of polymers as carriers and scaffolds for biomolecules Schwann cells and . Glia. 2005;51:132-47. and cell delivery in tissue engineering applications. Adv Drug Deliv. Rev. 2007;59:207-33. Voge CM, Kariolis M, MacDonald RA, Stegemann JP. Directional conductivity in SWNT-collagen-fibrin Ogawa Y, Sawamoto K, Miyata T, Miyao S, composite biomaterials through strain-induced matrix Watanabe M, Nakamura M, et al. Transplantation of alignment. J Biomed Mater Res A. 2008;86:269-77. in vitro‐expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal Wang B, Han J, Gao Y, Xiao Z, Chen B, Wang X, et cord contusion injury in adult rats. J Neurosci Res. al. The differentiation of rat adipose-derived stem 2002;69:925-33. cells into OEC-like cells on collagen scaffolds by co- culturing with OECs. Neurosci Lett. 2007;421:191-6. Oudega M, Xu X-M. transplantation for repair of the adult spinal cord. J Neurotraum. 2006; Woodhall E, West AK, Chuah MI. Cultured olfactory 23:453-67. ensheathing cells express , brain- derived neurotrophic factor, glia cell line-derived Paul C, Samdani AF, Betz RR, Fischer I, Neuhuber B. neurotrophic factor and their receptors. Mol Brain Grafting of human bone marrow stromal cells into Res. 2001;88:203-13. spinal cord injury: a comparison of delivery methods. Spine. 2009;34:328. Yang F, Murugan R, Wang S, Ramakrishna S. Electrospinning of nano/micro scale poly (L-lactic Potter W, Kalil RE, Kao WJ. acid) aligned fibers and their potential in neural tissue systems for neural progenitor cell-based therapy. engineering. Biomaterials. 2005;26:2603-10. Front Biosci. 2008;13:806-21. Yazdani SO, Pedram M, Hafizi M, Kabiri M, Ramón-Cueto A, Avila J. Olfactory ensheathing glia: Soleimani M, Dehghan M-M, et al. A comparison properties and function. Brain Res Bull. 1998;46:175- between neurally induced bone marrow derived 87. mesenchymal stem cells and olfactory ensheathing glial cells to repair spinal cord injuries in rat. Tissue Ramón-Cueto A, Valverde F. Olfactory bulb Cell. 2012;44:205-13. ensheathing glia: a unique cell type with axonal growth-promoting properties. Glia. 1995;14:163-73. Zhang Z, Rouabhia M, Wang Z, Roberge C, Shi G, Roche P, et al. Electrically conductive biodegradable Salehi M, Pasbakhsh P, Soleimani M, Abbasi M, polymer composite for nerve regeneration: electricity- Hasanzadeh G, Modaresi MH, et al. Repair of spinal stimulated neurite outgrowth and axon regeneration. cord injury by co-transplantation of embryonic stem Artif Organs. 2007;31:13-22. cell-derived motor neuron and olfactory ensheathing cell. Iran Biomed J. 2009; 13:125-35. Schmidt CE, Leach JB. Neural tissue engineering: strategies for repair and regeneration. Annu Rev Biomed Eng. 2003;5:293-347.

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