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Carbon nanotubes functionalized with fibroblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation

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IOP PUBLISHING Nanotechnology 24 (2013) 435101 (8pp)

NANOTECHNOLOGY

doi:10.1088/0957-4484/24/43/435101

Carbon nanotubes functionalized with broblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation
Eri Hirata1 , C ecilia M enard-Moyon2 , Enrica Venturelli2 , Hiroko Takita1 , 1 Fumio Watari , Alberto Bianco2 and Atsuro Yokoyama1
Graduate School of Dental Medicine, Hokkaido University, Kita 13, Nishi 7, Kita-ku, Sapporo 060-8586, Japan 2 CNRS, Institut de Biologie Mol eculaire et Cellulaire, Laboratoire dImmunopathologie et Chimie Th erapeutique, F-67000 Strasbourg, France E-mail: erieri@den.hokudai.ac.jp
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Received 26 June 2013, in nal form 13 August 2013 Published 27 September 2013 Online at stacks.iop.org/Nano/24/435101 Abstract Multi-walled carbon nanotubes (MWCNTs) were functionalized with broblast growth factor (FGF) and the advantages of their use as scaffolds for bone augmentation were evaluated in vitro and in vivo. The activity of FGF was assessed by measuring the effect on the proliferation of rat bone marrow stromal cells (RBMSCs). The presence of FGF enhanced the proliferation of RBMSCs and the FGF covalently conjugated to the nanotubes (FGFCNT) showed the same effect as FGF alone. In addition, FGFCNT coated sponges were implanted between the parietal bone and the periosteum of rats and the formation of new bone was investigated. At day 14 after implantation, a larger amount of newly formed bone was clearly observed in most pores of FGFCNT coated sponges. These ndings indicated that MWCNTs accelerated new bone formation in response to FGF, as well as the integration of particles into new bone during its formation. Scaffolds coated with FGFCNT could be considered as promising novel substituting materials for bone regeneration in future tissue engineering applications. (Some gures may appear in colour only in the online journal)

1. Introduction
Carbon nanotubes (CNTs) are endowed with interesting mechanical [1], chemical [2] and physical properties [3, 4]. Because of their unique properties, CNTs have attracted a great deal of attention for applications as the components of advanced biomaterials [57]. For instance, CNTs were used as reinforcements or additives in various materials, such as collagen [8], chitosan [9] and polymers [10, 11] to improve the properties of the materials and introduce novel functionalities. In addition, CNTs can be easily modied with proteins in order to make possible not only an enhancement of the biocompatibility but also to induce new characteristics through chemical functionalization [1215].
0957-4484/13/435101+08$33.00 1

These ndings encourage the possible use of CNTs for biomedical applications, either as carriers of therapeutic molecules or as immune modulator systems [16, 17]. We have previously reported strong cell adhesion to a CNT-coated polycarbonate membrane with a dense meshwork nanostructure of CNTs [18]. On the basis of this result, we decided to use CNTs as a coating substance and developed a 3D collagen scaffold coated with MWCNTs (MWCNT-coated sponge) [19]. The cells adhered well to the inner surface of the CNT-coated collagen sponges during the seeding process due to CNTs that entrapped the cells on the surface [20]. We also showed that poly (L-lactide) (PLLA) improved the surface wettability and cell proliferation once coated with CNTs [21]. In addition,
c 2013 IOP Publishing Ltd Printed in the UK & the USA

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osteoblasts on the CNT-coated sponges displayed osteogenic activity earlier than on the non-coated sponges [22]. These ndings demonstrated that CNT-coatings can be effective for the adhesion and differentiation of osteoblasts by improving the surface morphology of the scaffolds. Bone mesenchymal stem cells (BMSCs) derived from bone marrow are envisaged for applications in the eld of regenerative medicine due to existing multipotent progenitors. These cells expand to terminally differentiate into osteoblasts, chondrocytes, adipocytes, tenocytes and neural cells [23]. Indeed, stem cells or progenitor cells could be isolated from almost every tissue of the body [24, 25]. BMSCs in particular have been explored to create bone tissue using biomaterial-based approaches [26, 27]. However, BMSCs still do not proliferate and differentiate well compared to rat primary osteoblasts [28]. It is known that broblast growth factor (FGF) increases the proliferation of BMSCs [29] and accelerates bone regeneration [30]. It has been reported that FGF contained in a collagen gel increased new bone formation [31]. FGF needs to be delivered locally in a precisely controlled manner, thus assuring a sustained release from the surface of biomaterials such as medical implants and tissue scaffolds [32]. Kwan et al suggested that controlled FGF secretion can signicantly increase bone formation by adipose stem cells in vivo [33]. CNTs could be used as a support for the controlled release of FGF following its conjugation to the nanotubes. The unique properties of CNT-coating could be applied to bone tissue regeneration. In this study, CNTs were functionalized with broblast growth factor and the use of the conjugates as scaffolds for bone augmentation was evaluated.

2.1.1. Preparation of FGF/CNT covalent conjugate. A suspension of MWCNT-COOH (10 mg) in anhydrous DMF (6 ml) was sonicated in a water bath (Transsonics Digitals Elma, 20 W, 40 kHz) for 10 min. N ethyl-N -(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC HCl) (100 mg) and N -hydroxysuccinimide (NHS) (50 mg) were added. The mixture was sonicated for 10 min and stirred for two days. The suspension was ltered over a polytetrauoroethylene (PTFE) membrane (0.45 m pore size, Millipore R ). The solid recovered on the lter was dispersed in DMF (100 ml), sonicated for 5 min in a water bath, and ltered again over a PTFE membrane. This sequence was repeated twice with DMF and once with dichloromethane. The resulting solid was dried under vacuum for 5 h. The NHS-ester MWCNTs (9.5 mg) were added to a solution of FGF-2 (950 g) in 9.5 ml of PBS. The suspension was sonicated for 30 s in a cold water bath (<10 C) and then shaken at 4 C for 22 h. The suspension was centrifuged (4500 rpm, 5 min, 4 C). The precipitate was redispersed in PBS, sonicated for 10 s in a cold water bath, and centrifuged again. This sequence was repeated 3 times. The precipitate was dispersed in PBS and dialyzed against PBS using Spectra/Por R membrane dialysis with MWCO of 300 000 Da for two days at 4 C. This sample is dened as Conjugate throughout the paper. 2.1.2. Preparation of FGF/CNT non-covalent mixture. The FGF/CNT non-covalent mixture was prepared in the same conditions as the Conjugate but without the activation step using NHS. This sample is dened as Mix throughout the paper. 2.2. Cell proliferation assay RBMSCs obtained from the femora of syngenetic male Fisher 344 rats were isolated and cultured using the method described by Maniatopoulos et al [34]. The cells were cultured in basic medium: -modied minimum essential medium ( -MEM: Sigma, France) containing 15% fetal bovine serum (FBS; MP Biomedicals, France) and 1% penicillin/streptomycin (PenStrep; Gibco, France) supplemented with 50 g ml1 ascorbic acid, 5 l ml1 L-glutamine, 10 mM -glycerophosphate and 62 mg g1 dexamethasone. RBMSCs were subcultured to reach 70% conuence and released by trypsin. RBMSCs were seeded on 96-well culture plates at a density of 1.5 105 cells/well in 200 l of the basic medium and incubated for 4 h. After initial attachment, the culture medium was replaced by a medium including low serum medium ( -MEM supplemented with 2% FBS and 1% PenStrep) and the cells were cultured for 24 h. The medium was replaced by the low serum medium containing each suspension (PBS, FGF, Control CNTs, Mix and Conjugate). Control CNTs, Mix or Conjugate were dispersed in PBS at 1 g ml1 concentration by ultrasonication. According to the TGA result, 70 g of FGF was estimated as linked to 1 mg of CNTs. Each suspension was added to the medium at a nal concentration of 300 ng ml1 . FGF alone was used as control at the
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2. Experimental details
2.1. Functionalization of multi-walled CNTs (MWCNTs) and characterization Carboxylated MWCNTs were purchased from NanoLab (USA) (MWCNT-COOH, purity > 95%, 30 15 nm in diameter, 15 m in length, Lot PD30L1-5-CO2 H# 2010-03-01). Basic broblast growth factor (FGF-2) was purchased from Kaken Pharmaceutical (Japan). All reagents and solvents were purchased from different commercial suppliers and used as received. Thermogravimetric analyses (TGA) were performed using a TGA Q500 TA instrument with a ramp of 10 C min1 under N2 using a ow rate of 60 ml min1 from 100 to 800 C. Transmission electron microscopy (TEM) was performed on a Hitachi H600 microscope with an accelerating voltage of 75 kV. To prepare the TEM grids, the suspensions of CNTs in phosphate buffered saline (PBS) were dialyzed against MilliQ water for two days using a Spectra/Por R dialysis membrane with a molecular weight cutoff (MWCO) of 300 kDa. The dispersions of CNTs were then deposited on carbon-coated copper TEM grids (FormvarCarbon lm on 300 square mesh copper grids from Electron Microscopy Sciences) and air-dried.

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Figure 1. Preparation of Conjugate (A) and Mix (B).

same concentration as in Mix and Conjugate suspensions. At day 1 and 3 after culturing in the presence of the different preparations, the relative number of viable cells was determined by Cell Proliferation Reagent WST-1 (Roche, Indianapolis, IN, USA). BMSCs were seeded into 96-well plates at a density of 1 103 cells/well in 200 l of the basic medium for 4 h and then the medium was replaced by the low serum medium. After 22 h, the medium was replaced with the low serum medium including MAPK inhibitor 20 M SU5402 (Merck KGaA, Darmstadt, Germany). Stock solution of SU5402 was prepared in DMSO at 10 mM concentration. The stock solution was added to the culture medium at 1:500. An equal concentration of DMSO was used as control. The cells were cultured for 2 h. Then, the cells were added with low serum medium including FGF or Conjugate at a nal concentration of FGF 300 ng ml1 and further cultured for 24 h. The cell proliferation activity was evaluated with the WST-1 test. 2.3. Animal experiment The surface of a collagen sponge was coated with MWCNTs according to a previously reported method [22]. Control CNTs, Mix, and Conjugate were dispersed in PBS at a 1 g ml1 concentration by ultrasonication. Collagen sponge honeycombs (AteloCell; 9 mm in diameter, 2 mm in thickness, Koken Co., Japan) were soaked in each MWCNT suspension for 5 min and rinsed with PBS to remove the excess of nanotubes. After coating, each sponge was observed by scanning electron microscopy (SEM; S4800 Hitach, Japan) and used for animal experiments. Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, Hokkaido University. Eleven-week-old Wistar rats (320360 g) were put under general anesthesia by an intraperitoneal injection of Somnopentyl (sodium pentobarbital, 50 mg kg1 body weight). Pockets were formed between the parietal bone and the periosteum. The materials were implanted in the pockets. At week two after surgery, rats were sacriced and materials were harvested with the surrounding tissue, then xed using 10% neutral buffered
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Figure 2. TGA of MWCNT-COOH, Conjugate, and Mix under N2 .

formaldehyde. The hard tissue was decalcied with ethylene diamine tetraacetic acid (EDTA). Specimens were stained by hematoxylin and eosin (HE). They were examined for tissue responses including inammation, osteogenesis and absorption of the material.

3. Result and discussion

The FGF-2 protein was covalently bound to MWCNT-COOH by amidation reaction. For this purpose, the carboxylic functions were rst activated with NHS in the presence of EDC (gure 1(A)). The NHS-ester groups were then coupled to the amine functions of FGF, affording Conjugate. As control, we prepared a non-covalent conjugate through the adsorption of FGF on MWCNT-COOH (Mix) (gure 1(B)). This sample was obtained under the same conditions as Conjugate, but without the activation step using NHS. Both covalent and non-covalent FGF/CNTs were characterized by TGA and TEM. TGA was performed under an inert atmosphere to assess the level of functionalization. The weight losses at 500 C for MWCNT-COOH, Conjugate, and Mix were 8.7%, 15.7%, and 15.5%, respectively (gure 2).

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Figure 3. TEM of MWCNT-COOH (a), Conjugate (b), and Mix (c). Photograph of suspensions of MWCNT-COOH, Conjugate, and Mix in water (50 g ml1 ).

Figure 4. BMSC proliferation at day 1 and 3 after exposure to PBS (vehicle), FGF, Control CNTs (MWCNT-COOH), Mix and Conjugate ( p < 0.05, n = 5). Figure 5. BMSC proliferation at day 1 in the presence of the FGF receptor inhibitor SU5402 (10 M) added in the culture medium 2 h before FGF stimulation ( p < 0.05, n = 5) (DMSO: dimethylsulfoxide).

The weight loss difference (7%) between MWCNT-COOH and Conjugate or Mix allowed an assessment of the amount of protein in the nanotube samples, which corresponds to about 70 g mg1 of construct for both Conjugate and Mix. The nanotubes were then observed by TEM. Figure 3 shows the morphology of the starting MWCNT-COOH (gure 3(a)), MWCNT-COOH with covalently linked FGF (Conjugate, gure 3(b)), and MWCNT-COOH with adsorbed FGF (Mix, gure 3(c)). The TEM images show that the conditions used for the conjugation of FGF did not affect the morphology of the nanotubes. A difference in terms of dispersibility in water was observed (gure 3(d)). Indeed, the aqueous suspension of Conjugate was more stable in comparison with that of MWNT-COOH and Mix. The different conjugates were used to assess the proliferation capacity of BMSCs. In the presence of FGF (FGF alone, Mix and Conjugate), the BMSCs proliferated better than without the protein (PBS and CNTs) (gure 4). Among FGF alone, Mix and Conjugate, there were no signicant differences. In a previous study, the risk of denaturation of bone morphogenetic proteins during their covalent bonding to CNTs was reported [15]. Indeed, immobilization of enzymes and proteins onto nanomaterials can sometimes affect their secondary structures and often decrease their activity [35]. This seems not to be the case for our substrates (Mix and Conjugate) as there is no signicant difference in their activity, although a slight decrease was observed for the nanotube-based conjugates. This might be
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attributed to the accessibility of the protein once linked to the tubes. FGF intracellular signaling is mainly mediated by the mitogen activated protein kinase (MAPK) pathway. In a previous report, it was shown that FGF-2 increased the proliferation of bone marrow-derived mesenchymal stem cells through extracellular signal regulated kinase (ERK) of the MAPK pathway during BMSC proliferation [36]. It is known that SU5402 inhibits ERK and reduces the proliferation of BMSCs [37]. We have observed that SU5402 treatment decreased cell proliferation in the presence of Conjugate (gure 5). This seems to prove that the FGF covalently linked to CNTs regulated the proliferation of BMSCs via the MAPK pathway. Based on these results, the activity of FGF was almost completely maintained and acted through FGF signaling after the covalent modication. After the in vitro study, we decided to investigate the tissue responses, especially osteogenesis of the scaffolds coated with the FGF-functionalized CNTs implanted in bone, in order to understand their potential for bone tissue regeneration. We have previously reported that new bone formed around the MWCNT-coated sponges more than around the uncoated sponge after implantation in the femur [22]. For this purpose, collagen sponges coated with Conjugate and Mix were prepared following the approach that was previously reported [19]. The materials were then

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Figure 6. SEM images of each sponge surface. An upper part of a collagen sponge (a); the surface of a collagen sponge (b); CNT-coated sponge (c) and (d), Mix-coated sponge (e) and (f); Conjugate-coated sponge (g) and (h). Images (d), (f) and (h) are magnied views of (c), (e) and (g), respectively.

implanted between the parietal bone and the periosteum of rats in order to investigate the tissue responses by using onlay grafting [38]. Onlay grafting, in which materials are implanted directly on the bone, is a better procedure to evaluate materials for ridge augmentation, because the materials implanted on the bone will receive pressure from the periosteum, mucosa, or skin [39]. In this study, onlay grafting is considered the best procedure to evaluate carbon nanotubes for bone formation in order to assess the osteogenesis and resorption of the materials. The collagen sponge had pores longitudinally connected from the top to the bottom (gure 6(a)) and a smooth surface (gure 6(b)). After coating, the surfaces of the sponges were homogeneously covered by MWCNT-COOH (gures 6(c) and (d)), Mix (gures 6(e) and (f)) and Conjugate (gures 6(g) and (h)).
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At day 14 after implantation into rats, all untreated sponges disappeared and nebulous bone-like tissue was observed (gures 7(a) and (b)). This result suggested that the uncoated sponges were absorbed before the formation of a sufcient amount of bone. CNT-coated sponges were still present while their shape became at (gure 7(c)). The bone was formed in some of the pores close to the host parietal bone and the pores of the upper regions were lled by scar granulation tissues (gure 7(d)). Mix-coated sponges were also attened and some of the pores were again lled by bone tissues (gures 7(e) and (f)). Bone tissues were clearly observed in most pores of Conjugate-coated sponge (gures 7(g) and (h)). Some of the CNTs of the Mix-, Conjugate- and CNT-coated sponge were directly integrated in newly formed bone. These ndings indicated that the

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Figure 7. Histology at day 14 after implantation. FGF-treated sponge (a) and (b). CNT-coated sponge (c) and (d). Mix-coated sponge (e) and (f). Conjugate-coated sponge (g) and (h). Images (b), (d), (f) and (h) are magnied views of (a), (c), (e) and (g), respectively. CB: calvarial bone, white asterisk: new bone, white arrow head: collagen sponge, white arrow: CNTs. Scale bars correspond to 500 (left column) and 100 m (right column), respectively.

MWCNT-coated sponge had favorable biocompatibility with bone. Moreover, the FGF on the MWCNTs accelerated new bone formation. We have previously reported that CNT-coating maintained the shape of collagen sponges and enhanced bone formation according to the pore morphology [22]. CNTs were more likely adsorbed on the collagen surface of Conjugate-coated sponges (gures 7(g) and (h)) in comparison to Mix-coated sponges (gures 7(e) and (f)). The FGF on the Conjugate promoted bone formation because CNTs may retain the pore morphology. It was reported that newly regenerated bone results from the action of FGF-2 in facilitating nutrition by increased vascularization [40] and
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the promotion of the proliferation of BMSCs [41]. Slowly degraded Conjugate-coated sponge might release FGF for a longer time period and lead to a prolonged vascularization. In view of these results, functionalization of CNTs with FGF could be developed as a useful coating substance to create bio-resorbable materials such as collagen, -tricalcium phosphate and polymers.

4. Conclusions
Functionalized carbon nanotubes have potential applications in drug, vaccine and gene delivery because they can be

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well dispersed and further derivatized with active molecules, making them compatible with biological systems [12]. Covalent conjugation of FGF tailors CNTs with the appropriate function in the proliferation of BMSCs. In this study, we applied CNTs conjugated with FGF as a coating substance of a collagen sponge. At day 14 after implantation, a larger amount of newly formed bone was observed around the sponges coated with functionalized carbon nanotubes. Scaffolds coated with these conjugates could be considered as promising novel substituting materials for bone regeneration in future applications in the eld of tissue engineering.

Acknowledgments
EH wishes to thank the Japan Society for the Promotion of Science (JSPS) for a Research Fellowship for Young Scientists (ID No. 24006076), Iketani Science and Technology Foundation (ID No. 0241039) and Marubun Research Promotion Foundation. AY wishes to thank the Ministry of Education, Science, Culture and Sport of Japan for a Grant-in Aid for Basic Research B (ID No. 22390361). AB wishes to thank JSPS for the Invitation Fellowship for Research in Japan (ID No. S-12072). This work was supported by CNRS. The authors are indebted to Alessia Battigelli for TGA experiments. TEM images were recorded at the RIO Microscopy Facility Platform of Esplanade Campus (Strasbourg, France).

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