Multiwalled Carbon Nanotubes Specifically Inhibit Osteoclast

Osteoblasts also produce osteoprotegerin (OPG), a decoy receptor for RANKL that inhibits the differentiation and functioning of osteoclasts. Several i...
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NANO LETTERS

Multiwalled Carbon Nanotubes Specifically Inhibit Osteoclast Differentiation and Function

2009 Vol. 9, No. 4 1406-1413

Nobuyo Narita,† Yasuhiro Kobayashi,‡ Hiroaki Nakamura,‡ Kazuhiro Maeda,‡ Akihiro Ishihara,‡ Toshihide Mizoguchi,‡ Yuki Usui,† Kaoru Aoki,† Masayuki Simizu,† Hiroyuki Kato,† Hidehiro Ozawa,‡ Nobuyuki Udagawa,‡ Morinobu Endo,§ Naoyuki Takahashi,‡ and Naoto Saito*,| Department of Orthopedic Surgery, Shinshu UniVersity School of Medicine, 3-1-1 Asahi Matsumoto, Nagano 390-8621, Japan, Institute for Oral Science, Department of Biochemistry, Department of Anatomy, Matsumoto Dental UniVersity, 1780 Hiro-oka Gobara, Shiojiri, Nagano 390-0781, Japan, Faculty of Engineering, Shinshu UniVersity, 4-17-1 Wakasato, Nagano 380-8553, Japan, and Department of Applied Physical Therapy, Shinshu UniVersity School of Health Science, 3-1-1 Asahi Matsumoto, Nagano 390-8621, Japan Received October 10, 2008; Revised Manuscript Received February 8, 2009

ABSTRACT Since attention has been paid to the use of multiwalled carbon nanotubes (MWCNTs) as biomaterials in contact with bone, it is critical to understand the reaction of bone cells to MWCNTs. We show that MWCNTs inhibit osteoclastic bone resorption in vivo and that MWCNTs inhibit osteoclastic differentiation and suppressed a transcription factor essential for osteoclastogenesis in vitro. These results suggest that MWCNTs have beneficial effects on bones when they are used as biomaterials.

Introduction. Carbon nanotubes (CNTs) were first reported by Oberlin et al. in 19761 and are extremely valuable and useful materials. CNT research and development was greatly accelerated by Iijima’s report in 1991.2 CNTs possess exceptional mechanical,3-6 thermal,5,7 and electrical properties,8,9 which facilitate their use as reinforcements or additives in various materials such as plastics, metals, and ceramics to improve the properties of the materials and introduce novel functionalities. Consequently, the biomedical applications of CNTs have also been studied extensively.10,11 CNTs also have the potential to be used as scaffold materials in regenerative medicine. When CNTs are used as biomaterials in positions in which they are in direct contact with bone, such as in implants for arthroplasty, bone fracture, or dental treatment and as scaffolds for bone tissue regeneration, the reaction of the bone cells to CNTs is critical for functional maintenance of the biomaterials for a long time in the tissues. * To whom correspondence should be addressed. E-mail: [email protected]. † Department of Orthopedic Surgery, Shinshu University School of Medicine. ‡ Institute for Oral Science, Department of Biochemistry, Department of Anatomy, Matsumoto Dental University. § Faculty of Engineering, Shinshu University. | Department of Applied Physical Therapy, Shinshu University School of Health Science. 10.1021/nl8030746 CCC: $40.75 Published on Web 03/13/2009

 2009 American Chemical Society

Bone is continuously destroyed and re-formed in vertebrates to maintain bone volume and calcium homeostasis. Osteoblasts and osteoclasts are specialized cells responsible for bone formation and resorption, respectively. Osteoclasts are formed from the monocyte/macrophage lineage under the strict control of osteoblasts.12,13 Osteoblasts express two cytokines that are essential for osteoclast differentiation: receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony stimulating factor (M-CSF).14,15 RANKL expression is upregulated by bone-resorbing factors including 1R,25-dihydroxyvitamin D3 [1R,25(OH)2D3]. Osteoclast precursors differentiate into osteoclasts in the presence of RANKL and M-CSF. Mature osteoclasts as well as osteoclast precursors express RANK, which is the RANKL receptor, and RANKL stimulates osteoclast function. Osteoblasts also produce osteoprotegerin (OPG), a decoy receptor for RANKL that inhibits the differentiation and functioning of osteoclasts. Several in vitro studies have shown that CNTs and their nanocomposites such as polylactic acid/CNTs and polycarbonate urethane/CNTs stimulate proliferation, adhesion, and expression of bone-formation-related genes in osteoblasts.16,17 In our previous report, we performed in vivo studies, to demonstrate that implanted MWCNTs had high compatibility with bone tissues and did not induce bone resorption in the

Figure 1. Ectopic bone formation was induced by implantation of collagen/80n-MWCNT or collagen disks impregnated with rhBMP-2. After implantation for 2 weeks, 3 weeks, or 3 months, the implants were recovered. (a) Sections prepared from the implants recovered after 2 weeks were examined by enzymatic histochemical staining for TRAP and then subjected to hematoxylin staining. Red arrows indicate TRAP-positive cells (osteoclasts). The number of TRAP-positive cells was counted in each section. #; p ) 0.038 vs control. (b) BMD of the implants recovered after 3 weeks and 3 months. #; p ) 0.00876 vs control. (c) Soft X-ray images of the ossicle formed after 3 months of implantation. Data are expressed as the mean ( standard deviation. n ) 7 for control group, n ) 8 for 80n-MWCNTs group.

region of implantation in mice.18 To extend these studies, we first examined whether MWCNTs inhibit bone resorption by using an animal model of osteoclastic bone resorption19,20 in vivo and studying the reactions of osteoclasts toward MWCNTs in vitro. There are several major human bone diseases in the context of osteoclastic bone resorption, e.g., osteoporosis, rheumatoid arthritis, metastases of malignant tumors to bone, and osteolysis after artificial joint replacement. If CNTs have any potential in the treatment of bone diseases, they can be used to formulate totally new remedies. Results. We mixed 500 µg of MWCNTs (80n-MWCNTs with an average diameter of 80 nm and length of 8-10 µm) with 2 mg of atelopeptide type I collagen solution and added 5 µg of recombinant human BMP (rhBMP)-2.18 The mixture was freeze-dried and used to prepare pellets of an rhBMP2/collagen/80n-MWCNT composite. As a control, pellets were prepared from an rhBMP-2/collagen composite without 80n-MWCNTs. We implanted the rhBMP-2/collagen/ MWCNT or rhBMP-2/collagen composites in the dorsal musculature of ddy mice and removed the implants after 2 weeks, 3 weeks, or 3 months. The process of new bone formation advances rapidly in this experimental system and was nearly complete at 3 weeks, after which osteoclastic bone resorption could be gradually observed.19,20 Enzymatic histochemical examination at 2 weeks revealed that the number of osteoclasts that appeared in the ectopic bones was significantly lower in the rhBMP-2/collagen/80n-MWCNT group than in the rhBMP-2/collagen group (p ) 0.0380; Figure 1a). The bone mineral density (BMD) is used as an index of the process of ectopic bone resorption, and the BMD Nano Lett., Vol. 9, No. 4, 2009

of the new bone recovered after 3 weeks or 3 months was measured by a bone mineral analyzer. After 3 weeks, the BMD of the rhBMP-2/collagen/80n-MWCNT group was higher than that of the rhBMP-2/collagen group; however, the differences were not significant (p ) 0.492; Figure 1b). After 3 months, the BMD of the rhBMP-2/collagen/80nMWCNT group was significantly higher than that of the rhBMP-2/collagen group (p ) 0.00876; Figure 1b). Soft X-ray images revealed that the radiopacity of the calcified mass in the rhBMP-2/collagen/80n-MWCNT composite recovered after 3 months was higher than that in the rhBMP2/collagen composite without 80n-MWCNTs (Figure 1c). The findings indicated that MWCNTs decreased the number of osteoclasts and inhibited ectopic bone resorption in vivo. We performed an in vitro study to examine the effects of two kinds of MWCNTs (80n-MWCNTs and 150n-MWCNTs; the 150n-MWCNTs had an average diameter of 150 nm and lengths of 8-10 µm21) on the growth of mouse primary osteoblasts and M-CSF-supported BMMφ. We compared these results with those of the effect of CB (conventional carbon material with an average diameter of 80 nm) particles, which served as the control. At concentrations of 5 µg/mL or less, both 80n-MWCNTs and 150n-MWCNTs showed little effect on BMMφ proliferation; however, at 50 µg/mL, significant inhibition was observed (Figure 2a). Using bromodeoxyuridine, which is incorporated into the nuclei of proliferating cells, we confirmed that 5 µg/mL or less of 80n-MWCNTs showed no effect on BMMφ proliferation (see Figure S1 in Supporting Information). At a concentration of 5 µg/mL, neither 80n-MWCNTs nor 150n-MWCNTs 1407

Figure 2. MWCNTs inhibited osteoclast formation in cocultures of osteoblasts and bone marrow cells. (a) Effects of 80n-MWCNTs, 150n-MWCNTs, and CB on the proliferation of BMMφ and primary osteoblasts. BMMφ (2.5 × 104 cells/well) were cultured in the presence of M-CSF (25 ng/mL) in 96-well plates. Osteoblasts (1 × 104 cells/well) were also cultured. After culturing for 1 day, various doses of 80n-MWCNTs, 150n-MWCNTs, or CB were added to the cultures. Cell proliferation was estimated by assaying cell viability using an Alamar Blue assay kit. Media samples were collected at 24, 48, and 72 h after exposure to 80n-MWCNTs, 150n-MWCNTs, or CB, which were centrifuged at 11000g to remove 80n-MWCNTs, 150n-MWCNTs, and CB. The fluorescence intensity of the supernatant was measured. n ) 4. (b, c) Osteoblasts and bone marrow cells were cocultured with or without 80n-MWCNTs, 150n-MWCNTs, or CB in the presence of 1R,25(OH)2D3 (10-8 M). After culturing for 6 days, the cells were fixed and stained for TRAP. TRAP-positive cells containing more than three nuclei were regarded as osteoclasts and counted. n ) 4. #; p < 0.001 vs control. (b). Red giant cells represent TRAP-positive osteoclasts. Red arrows indicate CB incorporated into osteoclasts (c). (d) The expression levels of RANKL, OPG, and M-CSF mRNAs in osteoblasts were unaffected by 80n-MWCNTs, 150n-MWCNTs, or CB. Osteoblasts were cultured for 24 h with 80nMWCNTs, 150n-MWCNTs, or CB in the presence of 1R,25(OH)2D3 (10-8 M). The expression levels of RANKL, OPG, M-CSF, and GAPDH mRNAs were determined by semiquantitative RT-PCR. The ratios relative to the GAPDH were determined by densitometry using NIH image software.

induced apoptotic cell death in BMMφ as evidenced by DNA laddering and annexin V staining (data not shown). Osteoblast growth was unaffected by the addition of 80nMWCNTs, 150n-MWCNTs, or CB at concentrations of up to 50 µg/mL (Figure 2a). The effects of MWCNTs and CB, 1408

at concentrations of 0.5 and 5 µg/mL, on osteoclast formation were examined in cocultures of mouse osteoblasts and bone marrow cells (Figure 1, pars b and c). Treatment of the cocultures with 1R,25(OH)2D3 (10-8 M) induced the formation of tartrate-resistant acid phosphatase (TRAP, a marker Nano Lett., Vol. 9, No. 4, 2009

enzyme of osteoclasts)-positive osteoclasts (red cells). Addition of 5 µg/mL of 80n-MWCNTs or 150n-MWCNTs to the coculture significantly inhibited osteoclast formation induced by 1R,25(OH)2D3. CB showed no effect on osteoclast formation in the coculture. Interestingly, TRAP-positive osteoclasts containing CB particles were often observed but those containing MWCNTs were rarely detected in the cocultures (Figure 2c, red arrows in lower panels). This suggests that precursor cells, which have incorporated MWCNTs into the cell body, cannot differentiate into osteoclasts. The RT-PCR analysis showed that treatment of osteoblasts with 1R,25(OH)2D3 (10-8 M) induced RANKL mRNA expression and suppressed OPG mRNA expression (Figure 2d). Osteoblasts constitutively expressed MCSF mRNA, regardless of the presence or absence of 1R,25(OH)2D3. The expression of RANKL, OPG, and MCSF mRNAs in osteoblasts treated with 1R,25(OH)2D3 was unaffected by 5 µg/mL of 80n-MWCNTs, 150n-MWCNTs, or CB (Figure 2d). These results suggest that MWCNTs act on osteoclast precursor cells but not on osteoblasts to inhibit osteoclast formation in the coculture. Osteoclasts were also formed on day 3 in BMMφ cultures treated with RANKL (100 ng/mL) and M-CSF (25 ng/mL) in the absence of osteoblasts. Unexpectedly, when MWCNTs and RANKL were simultaneously added to the BMMφ culture, RANKL-induced osteoclast formation was not inhibited by the addition of MWCNTs up to a concentration of 5 µg/mL (Figure 3a). However, we found that pretreatment of BMMφ with MWCNTs but not with CB at a concentration of 5 µg/mL for 24 h strongly inhibited osteoclastic differentiation (Figure 3b). Pretreatment for 1 h with 5 µg/mL MWCNTs failed to inhibit osteoclast formation. Both 80nMWCNTs and CB were incorporated into BMMφ after incubation for 24 h but not after 1 h (Figure 3c). This suggests that BMMφ in which MWCNTs were incorporated could not differentiate into osteoclasts. In the coculture, MWCNTs added on day 1 could effectively but not completely suppress osteoclast formation induced by 1R,25(OH)2D3 (Figure 2, parts b and c). These results also suggest that some osteoclast precursors incorporated MWCNTs before they were exposed to RANKL expressed by osteoblasts in the coculture. Next, we examined the effects of MWCNTs on the intracellular signals that control osteoclast differentiation. The interaction of RANKL and RANK stimulated NF-κB activation and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in osteoclast precursors22 and finally induced NFATc1, which is a key transcription factor in osteoclast differentiation.23 Western blot analysis showed that RANKL stimulated IκBR degradation and p38 MAPK phosphorylation in BMMφ treated with or without 80n-MWCNTs or CB (Figure 3d). RANKL upregulated the expression of the NFATc1 protein in BMMφ. Interestingly, pretreatment with 80n-MWCNTs even for 24 h failed to suppress NFATc1 induction in BMMφ (Figure 3e). These results were unexpected because NFATc1 overexpression in osteoclast precursors induced their differentiation into osteoclasts even in the absence of RANKL stimulation. Next, we examined the RANKL-induced nuclear translocaNano Lett., Vol. 9, No. 4, 2009

tion of NFATc1 and NF-κB p65 in BMMφ that had been pretreated with 80n-MWCNTs or CB (Figure 4). The nuclear localization of NFATc1 was detected in less than 10% of the BMMφ treated with M-CSF alone (Figure 4a). Treatment of BMMφ with RANKL increased NFATc1 nuclear localization by approximately 20%. The RANKL-induced nuclear localization of NFATc1 was completely suppressed by the pretreatment of BMMφ with 80n-MWCNTs for 24 h but not for 1 h. CB showed no effect on the RANKL-induced nuclear localization of NFATc1 in BMMφ. NF-κB p65 was translocated into the nuclei of BMMφ within 30 min in response to RANKL (Figure 4b). The RANKL-induced nuclear localization of NF-κB p65 was unaffected by both 80n-MWCNTs and CB. These results suggest that 80nMWCNTs incorporated into BMMφ may specifically inhibit the transcriptional activity of NFATc1 in osteoclast precursors. When osteoclasts were cultured for 36 h on dentine slices in the presence of osteoblasts, osteoclasts formed resorption pits on the slices (Figure 5a). Addition of either MWCNTs or CB at a concentration of 5 µg/mL or less to the pit-forming activity assay did not influence osteoclast function. However, the pit-forming activity of osteoclasts was significantly inhibited by the addition of 50 µg/mL 80nMWCNTs or 150n-MWCNTs (Figure 5a). In contrast, CB at a concentration of 50 µg/mL failed to inhibit osteoclast function. Previous studies have shown that purified osteoclasts died rapidly due to apoptosis, and RANKL promoted the survival of osteoclasts. Addition of 50 µg/mL 80nMWCNTs or 150n-MWCNTs suppressed the RANKLsupported survival of purified osteoclasts (Figure 5b). The two MWCNTs (80n-MWCNTs and 150n-MWCNTs) were observed as black traces in areas where MWCNTs-containing osteoclasts died (Figure 5b, arrows in right panels). There was no effect of 50 µg/mL CB on RANKL-supported osteoclast survival. Electron microscopic analysis confirmed that 80n-MWCNTs were incorporated into osteoclasts (Figure 5c). Some 80n-MWCNT fibers were observed in the vicinity of the mitochondria in osteoclasts (Figure 5c, left). These results suggest that MWCNTs but not CB incorporated into osteoclasts specifically suppressed the function of osteoclasts through unique unknown mechanisms. Discussion. By means of in vivo experiments using an animal model,19,20 we showed that MWCNTs inhibit bone resorption. The number of osteoclasts in ectopic bones was significantly lower in the rhBMP-2/collagen/MWCNT group than in the rhBMP-2/collagen group in the early time when both bone formation and resorption were the most active. In the later time when both bone formation and resorption became stable, TRAP-positive osteoclasts could not be detected in both groups (data not shown) probably because the number of osteoclasts was below the limit of test sensitivity. Attenuation of minerals from ectopic bones 3 months after implantation was clearly less pronounced in the rhBMP-2/collagen/MWCNT group than in the rhBMP2/collagen group. Thus, the suppression of osteoclastic bone resorption in the BMP-induced ectopic bone was believed to be due to the influence of MWCNTs. 1409

Figure 3. MWCNTs incorporated into BMMφ inhibited the differentiation of BMMφ into osteoclasts. (a) BMMφ were cultured for 3 days with RANKL (100 ng/mL) and M-CSF (25 ng/mL) in the presence or absence of 80n-MWCNTs, 150n-MWCNTs, or CB. Cells were fixed and stained for TRAP. n ) 4. (b) BMMφ were treated with or without 80n-MWCNTs, 150n-MWCNTs, or CB for 24 or 1 h. BMMφ were further cultured for 3 days in the presence of RANKL (100 ng/mL) and M-CSF (25 ng/mL), fixed, and stained for TRAP. n ) 4. #; p < 0.001 vs control. (c) Phase-contrast microscopy of BMMφ treated with 80n-MWCNTs or CB. BMMφ were incubated for 24 h with 80n-MWCNTs or CB, fixed, and observed under a phase-contrast microscope. (d, e) Neither 80n-MWCNTs nor CB attenuated RANKLinduced signals in BMMφ. BMMφ were cultured with or without 80n-MWCNTs or CB under the same conditions as those in the experiments in (b). BMMφ were then treated for 20 min with RANKL (100 ng/mL) to activate NF-κB and p38 MAPK (d). BMMφ were treated with RANKL (100 ng/mL) for 24 h to induce NFATc1 (e). Cell lysates were subjected to SDS-PAGE, followed by immunoblotting with specific antibodies.

Next, by conducting in vitro experiments, we showed that MWCNTs inhibit osteoclast differentiation and function through a unique mechanism. Previous studies have shown that single-walled CNTs, which were functionalized via oxidation/amidation processes, induced the production of inflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor-R (TNF-R) in mouse macrophages.24 MWCNTs were also reported to induce TNF-R production 1410

in rat peritoneal macrophages.25 However, in our supplemental study, even at a concentration of 50 µg/mL, neither 80n-MWCNTs nor 150n-MWCNTs could induce IL-6 or TNF-R production in BMMφ cultures (Figure S2 in Supporting Information). In addition, 80n-MWCNTs and 150nMWCNTs at a concentration of 50 µg/mL failed to inhibit alkaline phosphatase activity in primary osteoblast cultures (Figure S3 in Supporting Information). These results suggest Nano Lett., Vol. 9, No. 4, 2009

Figure 4. MWCNTs inhibited the nuclear translocation of NFATc1 but not that of NF-κB p65 in BMMφ. (a) Nuclear translocation of NFATc1 in BMMφ. BMMφ were pretreated with 80n-MWCNTs or CB for 24 or 1 h and then stimulated with RANKL for 24 h. NFATc1 (green) was detected by immunostaining with anti-NFATc1 antibodies. Cells were counterstained with PI (red) to label nuclei. The percentage of cells that possessed NFATc1-positive nuclei (means ( standard deviation) is indicated in each panel. n ) 6. #; p < 0.001 vs control. (b) Nuclear translocation of NF-κB p65 in BMMφ. BMMφ were treated for 24 h with or without 80n-MWCNTs and CB and stimulated with RANKL and M-CSF for 30 min. The cells were immunostained with anti-NF-κB p65 antibodies (green) and counterstained with PI (red). The percentage of cells that possessed NF-κB p65-positive nuclei (mean ( standard deviation) is indicated in each panel. n ) 6.

that MWCNTs specifically affect cells of the osteoclastic lineage. We showed the interesting result that BMMφ with incorporated MWCNTs could not differentiate into osteoclasts, while those with CB could differentiate. We also demonstrated that MWCNTs that had incorporated into BMMφ selectively inhibited the nuclear localization of NFATc1, a key transcription factor for osteoclast lineage cells. These results may explain why MWCNTs specifically affect osteoclast differentiation. CNTs have been reported to interact with organelles and bind to some proteins and DNA.26-29 In addition to the above properties, MWCNTs but not CB possess many holes in their structures that may specifically trap NFATc1 in the cytosol of osteoclast precursors. Interestingly, it was also demonstrated that MWCNTs are incorporated into mature osteoclasts and inhibited the latter’s functions. In this study, we concentrated Nano Lett., Vol. 9, No. 4, 2009

on determining the effects of CNTs on osteoclasts along with the underlying mechanisms. CNTs and their nanocomposites have been reported to enhance proliferation of osteoblasts.30,31 Thus, the effects of CNTs on bone modeling, in which osteoblasts and osteoclasts are coupled, remain to be investigated in future studies. The experiments reported here provide information on new inhibitory mechanisms of MWCNTs in bone resorption. With MWCNTs included in implant materials in contact with bone, osteolysis around the implants can be prevented, which is a critical problem in orthopedic or dental implant treatment. In addition, when using MWCNTs as the scaffold material for bone tissue regeneration, the inhibitory effect on osteoclasts may promote bone formation or maintain bone quantity. There are many diseases that arise from bone resorption and/or bone destruction due to osteoclasts. Osteoclastic bone resorption leads to 1411

Figure 5. MWCNTs inhibited the pit-forming activity and survival of osteoclasts. (a) Osteoclast preparations were cultured for 36 h on dentine slices with or without 80n-MWCNTs, 150n-MWCNTs, or CB. The dentine slices were then recovered and were stained with Mayer’s hematoxylin to visualize the resorption pits. The red region represents the resorption pits formed on the dentine slices (right panel). The number of the resorption pits was counted. n ) 5. #; p < 0.01 vs control. (b) Purified osteoclasts were cultured for 36 h with or without 80n-MWCNTs, 150n-MWCNTs, or CB in the presence of RANKL (100 ng/mL). TRAP-positive cells containing more than three nuclei were counted as surviving osteoclasts. n ) 4. #; p < 0.001 vs control. (c) TEM images of osteoclasts containing incorporated 80n-MWCNTs. Osteoclasts were cultured for 24 h on dentine slices in the presence of 80n-MWCNTs. Cells were then fixed and processed for transmission electron microscopy analysis. Arrowheads indicate 80n-MWCNTs. Key: OC, osteoclast; NC, nuclei; Mt, mitochondria.

osteoporosis and osteolysis. Osteoclastic bone destruction occurs in rheumatoid arthritis and metastases of malignant tumors to the bone. The osteoclast inhibitory effect of MWCNTs may be applied for the treatment of such diseases. In conclusion, this is the first report in which the mechanistic basis of the effect of CNTs on bone tissues and bone cells, namely, osteoclasts, has been explored. CNTs are representative of nanosize materials. There are many questions on the interactions between nanosize materials and cells and/or tissues. We believe that elucidation of such 1412

interactions can lead to the development of new treatments for many diseases in the near future. Acknowledgment. This study was supported by CLUSTER (second stage) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a Grant-inAid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Nos. 18390557, 17390497, 19390476, 18390495, and 18201021), and by the Research and Development of Nanodevices for Practical Utilization of Nanotechnology of the New Energy Nano Lett., Vol. 9, No. 4, 2009

and Industrial Technology Development Organization, Japan (No. 07001418). Supporting Information Available: Description of materials and methods and figures showing MWCT effects on alkaline phosphatase activity in osteoblasts, on production of IL-6 and TNF-R in BMMφ, and on proliferation of BMMφ. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Oberlin, A.; Endo, M.; Koyama, T. J. Cryst. Growth 1976, 32, 335. (2) Iijima, S. Nature 1991, 354, 56. (3) Treacy, M. M. J.; Ebbesen, T. W.; Gibson, J. M. Nature 1996, 381, 678. (4) Salvetat, J. P.; et al. Phys. ReV. Lett. 1999, 82, 944. (5) Ruoff, R. S.; Lorents, D. C. Carbon 1995, 33, 925. (6) Miyagawa, H.; Misra, M.; Mohanty, A. K. J. Nanosci. Nanotechnol. 2005, 5, 1593. (7) Berber, S.; Kwon, Y. K.; Tomanek, D. Phys. ReV. Lett. 2000, 84, 4613. (8) Ebbesen, T. W.; et al. Nature 1996, 382, 54. (9) Dai, H.; Wang, E. W.; Lieber, C. M. Science 1996, 272, 523. (10) Zanello, L. P.; Zhao, B.; Hu, H.; Haddon, R. C. Nano Lett. 2006, 6, 562. (11) Saito, N.; et al. Curr. Med. Chem. 2008, 15, 523. (12) Suda, T.; et al. Endocr. ReV. 1999, 20, 345. (13) Chambers, T. J. J. Pathol. 2000, 192, 4. (14) Teitelbaum, S. L.; Ross, F. P. Nat. ReV. Genet. 2003, 4, 638.

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(15) Karsenty, G.; Wagner, E. F. DeV. Cell 2002, 2, 389. (16) Supronowicz, P. R.; et al. J. Biomed. Mater. Res. 2002, 59, 499. (17) Price, R. L.; Waid, M. C.; Haberstroh, K. M.; Webster, T. J. Biomaterials 2003, 24, 1877. (18) Usui, Y.; et al. Small 2008, 4, 240. (19) Saito, N.; et al. Nat. Biotechnol. 2001, 19, 332. (20) Nakamura, M.; et al. Endocrinology 2003, 144, 5441. (21) Endo, M.; et al. Carbon 2001, 39, 1287. (22) Matsumoto, M.; Sudo, T.; Saito, T.; Osada, H.; Tsujimoto, M. J. Biol. Chem. 2000, 275, 31155. (23) Takayanagi, H.; et al. DeV. Cell 2002, 3, 889. (24) Dumortier, H.; et al. Nano Lett. 2006, 6, 1522. (25) Muller, J.; et al. Toxicol. Appl. Pharmacol. 2005, 207, 221. (26) Meng, S.; Maragakis, P.; Papaloukas, C.; Kaxiras, E. Nano Lett. 2007, 7, 45. (27) Kam, N. W. S.; O’Connell, M.; Wisdom, J. A.; Dai, H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11600. (28) Nepal, D.; Geckeler, K. E. Small 2007, 3, 1259. (29) Park, K. H.; Chhowalla, M.; Iqbal, Z.; Sesti, F. J. Biol. Chem. 2003, 278, 50212. (30) Giannona, S.; Firkowska, I.; Rojas-Chapana, J.; Giersig, M. J Nanosci. Nanotechnol. 2007, 7, 1679. (31) Balani, K.; et al. Biomaterials 2007, 28, 618. (32) Mizuno, A.; et al. Biochem. Biophys. Res. Commun. 1998, 247, 610. (33) Suda, T.; Jimi, E.; Nakamura, I.; Takahashi, N. Methods Enzymol. 1997, 282, 223. (34) Nakamichi, Y.; et al. J. Immunol. 2007, 178, 192. (35) Komarova, S. V.; Pereverzev, A.; Shum, J. W.; Sims, S. M.; Dixon, S. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 2643.

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