NANO LETTERS
Bone Cell Proliferation on Carbon Nanotubes
2006 Vol. 6, No. 3 562-567
Laura P. Zanello,*,† Bin Zhao,‡ Hui Hu,‡ and Robert C. Haddon*,‡ Department of Biochemistry, UniVersity of California, RiVerside, California 92521, and Center for Nanoscale Science and Engineering, Departments of Chemistry and Chemical & EnVironmental Engineering, UniVersity of California, RiVerside, California 92521 Received September 16, 2005; Revised Manuscript Received January 24, 2006
ABSTRACT We explored the use of carbon nanotubes (CNTs) as suitable scaffold materials for osteoblast proliferation and bone formation. With the aim of controlling cell growth, osteosarcoma ROS 17/2.8 cells were cultured on chemically modified single-walled (SW) and multiwalled (MW) CNTs. CNTs carrying neutral electric charge sustained the highest cell growth and production of plate-shaped crystals. There was a dramatic change in cell morphology in osteoblasts cultured on MWNTs, which correlated with changes in plasma membrane functions.
A major goal in bone bioengineering is to create artificial scaffold materials that have the capacity to sustain bone cell growth and proliferation and increment or replace bone tissue. Bone structure and function depend intimately on the arrangement of cellular and noncellular components at the micro- and nanoscale level.1 These include cell types such as osteoblasts, osteoclasts, and osteocytes embedded in a mineralized extracellular matrix consisting of collagen and a number of noncollagenous proteins.2 Osteoblasts, the bone-forming cells, proliferate on the bone surface and produce and secrete bone matrix proteins. As they progress through stages of cell differentiation under hormonal control, they mineralize the matrix by means of production of hydroxyapatite (HA) crystals. Different scaffold materials have been used as substrates to grow osteoblasts and induce bone formation in vitro. Regeneration of bone by cells in a synthetic extracellular matrix or scaffold is a new approach used in bioengineering aimed at replacing the use of autographs and allographs in bone transplantation.3-5 In the present study, we explore the use of carbon nanotubes (CNTs) as adequate scaffold materials for the growth and proliferation of osteoblasts and subsequent bone formation. Carbon nanotubes are a macromolecular form of carbon6 and are viewed as a class of nanomaterials with high potential for biological applications due in part to their unique mechanical, physical, and chemical properties.7,8 The density of CNTs is similar to that of graphite, which in turn is much * Corresponding authors. E-mail:
[email protected]; Robert.haddon@ ucr.edu. Phone: (951) 827-3159, LPZ; (951) 827-2044, RCH. Fax: (951) 827-3159; (951) 827-4713. † Department of Biochemistry. ‡ Center for Nanoscale Science and Engineering, Departments of Chemistry and Chemical & Environmental Engineering. 10.1021/nl051861e CCC: $33.50 Published on Web 02/16/2006
© 2006 American Chemical Society
lower than other metal bone scaffold materials, including steel and titanium. However, CNTs are the strongest material on earth,8 and their implantation in bone may improve the mechanical properties of damaged bone tissue. From this viewpoint, CNTs may be used not only to stimulate bone regeneration but also to serve a permanent mechanical role. CNTs also have excellent flexibility and elasticity. Having a one-dimensional structure, CNTs are ideal materials for the manufacturing of composite materials. We sought to prime osteoblasts to produce bone in vitro by culturing them on CNTs bearing different chemical modifications that provide net electric charges to the typically noncharged nanomaterial.9-11 Our study focuses on the biocompatibility of functionalized CNTs used as substrates for osteoblast growth and their potential in bone regeneration. To date, few publications report the use of CNTs to grow cells in culture. There are contradictory reports on the biocompatibility of CNTs. On one hand, it has been shown that CNT substrates decreased keratinocyte,12 glial,13 and HEK293 cell survival significantly,14 raising important concerns about the biocompatibility of the nanomaterial. In addition, single-walled (SW) CNTs have been shown to block potassium channel activities in heterologous mammalian cell systems when applied externally to the cell surface,15 which suggests a degree of cytotoxicity. On the other hand, chemically functionalized CNTs have been used successfully as substrates for neuronal growth.16-18 Collagen-CNT composite materials sustained high smooth muscle cell viability,19 and SWNTs in suspension in the culture medium were incorporated into the cell cytoplasm by macrophages and leukemia cells without affecting the cell population growth.20,21 Recently, we reported the mineraliza-
Figure 1. Chemical structure of carbon nanotubes.
Figure 2. Quantification of cell growth on CNT types. ROS 17/ 2.8 cell counts were obtained at day 5 in culture and are expressed as percentage of maximum growth achieved on glass (control). Values represent mean cell counts ( SD from n ) 20 fields of observation per treatment; *, p < 0.05 compared with AP-SWNT values. AP-SWNT, SWNT-PEG, and AP-MWNT are electrically neutral (n); SWNT-COOH and SWNT-PABS carry a net negative (-) and zwitterionic (+)(-) electric charge, respectively.
tion of chemically functionalized SWNTs with HA,22 being the first study on the potential use of SWNTs as scaffold for bone growth. For in vivo applications, however, careful consideration of the biocompatibility and toxicity of CNTs is crucial. Bioengineered scaffold materials for the growth of bone have been the focus of intense research.3,5 Ideally, a suitable scaffold material for bone formation should provide the adequate microenvironment for proliferation and differentiation of bone cells as well as formation of a mineralized matrix, therefore acting as a template. In the bioengineered bone, bone formation and remodeling is a dynamic process that involves production and resorption within the scaffold material. Therefore, osteoblasts that grow on CNTs should retain all of the physiological functions found in normal bone. With the purpose of finding the type of CNT that best supports bone formation, we cultured rat osteosarcoma ROS 17/2.8 cells on SWNTs and multiwalled (MW) CNTs, with and without the introduction of chemical modifications. As prepared (AP) CNTs are insoluble in most solvents. Chemical modifications are aimed at increasing their solubility in water and organic solvents. In our study, we used SWNTs and MWNTs covalently functionalized with electrically neutral, zwitterionic, or negatively or positively charged chemical groups (Figure 1).10,16,23 We characterized osteoblast proliferation, cell morphology, and formation of crystals as an indicator of the biocompatibility of CNTs. In addition, we assessed the integrity of the osteoblast plasma membrane, and therefore cell viability, in ROS 17/2.8 cells cultured on CNTs by means of electrical recordings of ion channel activities.24 SWNTs have an average diameter of 1.5 nm, and their length varies from several hundred nanometers to several micrometers. MWNT diameters typically range between 10 and 30 nm. The diameters of SWNTs are close to the size of the triple helix collagen fibers, which makes them ideal candidates as substrates for bone growth. In the present study, AP-SWNTs and nitric acid-treated SWNTs (SWNT-COOH)
were obtained from Carbon Solution Inc. (Riverside, CA), and SWNTs chemically functionalized with poly-(m-aminobenzene sulfonic acid) (SWNT-PABS) and poly ethylene glycol (SWNT-PEG) were obtained by synthetic methods published previously.23 SWNT-COOH, SWNT-PABS, and SWNT-PEG were chosen for the present study on the basis of their net negative, zwitterionic, and neutral electric charge, respectively, at the pH of the experiment. Carbon nanotube coated glass coverslips were prepared as described previously.17 Briefly, CNT samples (100 µg/mL) were sonicated in solvent (water for functionalized CNTs, and 95% ethanol for AP-SWNTs and AP-MWNTs) for about 2 h, and the resulting dispersion was sprayed onto preheated (ca. 80 °C) glass coverslips. Sprayed coverslips were allowed to dry in air and used for cell culture after a sterilization procedure with UV irradiation overnight. Osteoblastic ROS 17/2.8 cells are fully mature osteoblasts that produce mineralized matrix in culture.25-27 ROS 17/2.8 cells (kindly provided by A. W. Norman, University of California-Riverside) were cultured in Ham’s F-12 medium (Sigma) containing 5% fetal bovine serum (Sigma), 5% Serum Plus (JRH Biosciences, Woodland, CA), and 2 mM L-glutamine (Sigma) at 37 °C in a 5% CO2 humidified incubator, as described previously.26 Cells were grown on glass coverslips and CNT-sprayed coverslips in 35-mm plastic culture dishes. The culture medium was changed every 3-4 days. We studied osteoblast proliferation on CNTs in 5-dayold cultures. By day 5, control cells grown on glass reached confluence. Cell growth was calculated on the basis of number of cells per field of observation at a magnification of 200x with an Olympus IX50 inverted microscope. Cell counts were performed with phase contrast and fluorescence microscopy as described in the Supporting Information. We found that the highest cell growth was achieved on APSWNTs at 59 ( 4% of maximal growth obtained on glass, at day 5 (Figure 2). As inferred from Figure 2, the nanomaterials that best supported ROS 17/2.8 cell growth
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Figure 3. Morphology of ROS 17/2.8 cells cultured on AP-SWNTs (A-C), AP-MWNTs (D-F), and control cultures on glass cover slips (G-I), as seen with SEM. (A) Osteoblast colony on AP-SWNTs. (B) A flat cell body of a single cell extends over almost the entire field of observation; the cell nucleus protrudes in the center. (C) Tape-like cytoplasmic prolongations (arrow) extend from the flat body of a ROS 17/2.8 cell (a portion of it shown at the left upper corner of the picture) on an evenly distributed AP-SWNT substrate. (D) Osteoblast colony on AP-MWNTs. The nanotubes aggregate unevenly in areas of the glass surface (notice bundles on CNTs on the right). (E) Image of a single ROS 17/2.8 cell on AP-MWNTs. A round single-cell body extends thin neurite-like cytoplasmic prolongations (arrow) that reach the nanotube bundles. (F) SEM micrographs at higher magnification show a detail of long threadlike cytoplasmic prolongations (arrow) that extend from the round body of a single ROS 17/2.8 cell (partially seen on the left, upper corner), interweave with, and reach individual AP-MWNTs. (G) ROS 17/2.8 cell colony cultured on glass. (H) Image of a single cell obtained at higher magnification. (I) Detail of a portion of the cell cytoplasm (covering the left upper half of the picture) in contact with the glass surface; no cytoplasmic prolongations are observed.
were AP-SWNTs and SWNT-PEG (57 ( 4%), which are electrically neutral CNTs. In third place and fourth place, SWNT-COOH and SWNT-PABS supported 29 ( 8% and 20 ( 4% of cell proliferation relative to the control on glass, respectively. Lowest growth achieved on AP-MWNTs may be in part due to loss of MWNTs into the solution because of a decreased attachment of this CNT type to the glass, as reported before.17 In addition, we found that nonfunctionalized AP-MWNTs sustained a significantly higher cell growth than functionalized MWNTs, with values below 15% (data not shown). In all three cases with highest cell growth, CNT types carry no net electrical charge, and are designated as “neutral” throughout this paper. However, cell counts obtained from negatively charged SWNT-COOH and zwitterionic SWNT-PABS were significantly lower, indicating that the presence of electric charges does not favor osteoblast growth and proliferation in culture. In all cases, however, osteoblasts stained positive for alkaline phosphase, an indicator of the osteoblast phenotype, after 7 days in culture. In addition, Von Kossa staining showed the formation of mineralization nodules after 2 weeks in culture (data not shown). 564
Next, we studied ROS 17/2.8 cell morphology in CNT cultures with scanning electron microscopy (SEM). Osteoblasts express different morphology when cultured on smooth versus rough substrates. It has been found that as surface microroughness increases, osteoblast differentiation increases, and proliferation decreases, depending on the type and density of the points of contact between the cytoplasm and the surface.28,29 Regardless of the presence or absence of net electric charges, the nanomaterials used in our study offer a variety of topographies, including rough, continuous SWNT surfaces, and three-dimensional MWNT bundles, in comparison with the smoothness of glass. For SEM analysis, cells were grown for 5 days and fixed in 4% formalin (Sigma) in 0.1 M sodium cacodylate buffer, for 1 h at room temperature. Coverslips were then washed three times with 0.1 M sodium cacodylate. Samples were incubated with 1% osmium tetroxide in the same buffer, for 1 h at room temperature. Dehydration was performed in an ethanol series (30%, 50%, 70%, 80%, 95%, and 100%), and samples were critical point dried. Samples were then covered by a thin gold/palladium layer and observed on a Philips SEM XL-30 microscope. As shown in Figure 3A-C, ROS 17/2.8 cell bodies grew Nano Lett., Vol. 6, No. 3, 2006
Figure 4. Cubic (A) and plate-shaped (B) crystals produced by osteoblastic ROS 17/2.8 cells cultured on glass (A), and AP-SWNTs (B) for 10 days. Crystals tend to disperse in A but associate in clusters in B.
flat on AP-SWNTs, similar to their growth on glass (Figure 3G-I). Typical cell diameters for flat ROS 17/2.8 osteoblasts was of the order of 40 µm, which resembled osteoblasts found on the surface of natural bone. However, cell bodies were spherical when cultured on AP-MWNTs. They developed long threadlike cytoplasmic prolongations, as seen in Figure 3D-F. Round cell bodies on MWNTs measured approximately 15 µm in diameter. This cell morphology resembles that of osteocytes, the fully differentiated osteoblasts embedded in the bone matrix. Osteocytes connect and communicate with neighbor cells by means of thin cytoplasmic prolongations that run across the mineralized matrix. Interestingly, a similar neurite-like growth pattern was described for neuronal cultures on AP- and functionalized MWNTs.17 SEM observations performed at high magnification revealed the morphology of physical contacts between cell and CNT materials, as shown in Figure 3C, F, and I. Spherical osteoblasts on AP-MWNTs grew long threadlike cytoplasmic prolongations that reached the nanotube bundles as a way to anchor to a discontinuous, three-dimensional substrate (Figure 3F). These thin pseudopods had diameters in the range of 10-20 nm, close in size to MWNT diameters. Alternatively, flat osteoblasts on AP-SWNTs grew shorter, tape-like cytoplasmic prolongations that adhered to the more evenly distributed layer of nanotubes (Figure 3C). Nanometer-scale cytoplasmic prolongations were not observed in ROS 17/2.8 cells grown on glass (Figure 3I). Next, we investigated if the processes of bone production were initiated in CNT cultures by observing crystal formation with SEM. We found that ROS 17/2.8 cells cultured on glass produced cubic crystals after the first week in culture (Figure 4A). These crystals (100-500 nm in length) dispersed at random in intercellular spaces, suggesting that they were not a case of nonspecific ectopic mineralization. On the contrary, ROS 17/2.8 cells cultured on SWNTs produced plate-shaped crystals (100-1000 nm in length, and approximately 20 nm thick) similar in shape to HA crystals found in woven bone,30 which aggregated in clusters outside the cells (Figure 4B). Although plate-shaped crystals aggregated on the nanomaterials in a disordered fashion, our results indicate that CNTs constitute a suitable substrate for deposition of a mineralized matrix. To assess the viability of osteoblasts in CNT cultures, we studied cell membrane functionality in ROS 17/2.8 cells Nano Lett., Vol. 6, No. 3, 2006
Figure 5. Voltage-gated chloride currents recorded from ROS 17/ 2.8 cells cultured on AP-SWNTs (open circles) and AP-MWNTs (full circles). Current-to-voltage relations obtained from a series of depolarizing 400-ms voltage steps between -60 and 80 mV. Current amplitudes (pA) are calculated relative to the individual cell capacitance value (pF). Data points correspond to mean values ( SD from n ) 4 individual cells per treatment. In MWNTs, SD values are smaller than the symbols. No significant differences in Cl- current amplitudes were found between cells cultured on APSWNTs and AP-MWNTs (p < 0.05). Raw current traces (bottom panel) show noninactivating outward currents obtained at high depolarizing potentials. Holding potential ) -50 mV. Vm: membrane voltage.
grown on neutrally charged AP-SWNTs and AP-MWNTs. Osteoblasts are secretory cells. Fusion of secretory vesicles to the plasma membrane and further release of the products of secretion into the medium depend strongly on the functioning of osteoblast ion channels and their response to mechanical and chemical stimuli.31,32 Manipulation of ion channel activities in bone may be an effective way to amplify bone matrix production.33,34 Osteoblasts express a variety of voltage-gated ion channels.35-37 We characterized previously the activity of steroid hormone-sensitive calcium and chloride channels in ROS 17/2.8 cells.26,27 In the present work, whole-cell patchclamp experiments were performed essentially as described previously.26 Chloride and calcium channel activities were recorded in the presence of 150 mM TEA-Cl, 3.1 mM KCl, 20 mM BaCl2, 1 mM MgCl2, 4 mM NaHCO3, 10 mM Hepes, 20 mM sucrose, pH 7.4 (adjusted with TEA-OH) in the bath, and 150 mM Cs-methansulfonate, 15 mM NaCl, 4 mM MgCl2, 5 mM EGTA, 10 mM Hepes, pH 7.4 (adjusted with NaOH) in the pipet. Patch-clamp recordings24 were performed with a Heka EPC-9 amplifier (ALA Scientific Instruments Inc., Westbury, NY). Patch pipets of about 2 MΩ were fabricated with a DMZ Universal micropipet puller from Drummond capillaries (Drummond Scientific Co., Broomall, PA), coated with Sylgard elastomer (Dow Corning Corp., Midland, MI) to reduce capacitative transients, and fire-polished. Giga seals progressed rapidly and stably. Figure 5 shows current-to-voltage relations and raw current traces of a voltage-gated, outward rectifying chloride current that activates at depolarizing potentials in ROS 17/2.8 cells cultured on AP-SWNTs and AP-MWNTs. This Cl- current 565
Figure 6. HVA calcium currents obtained from ROS 17/2.8 osteoblasts cultured on CNTs. Right panel: current-to-voltage relations obtained from a series of depolarizing 200-ms-long voltage steps applied to cells cultured on AP-SWNTs and AP-MWNTs (open and full circles, respectively). Values correspond to averages ( SD obtained from n ) 4 individual cells per treatment (*, p < 0.05). Current amplitudes (pA) were calculated relative to the individual cell capacitance value (pF). Holding potential ) -50 mV. Left, upper panel: inward Ca2+ current traces obtained from an osteoblast cultured on AP-SWNTs. Left, bottom panel: calcium current potentiation evoked by the L-type Ca2+ channel agonist S(-) BayK 4688 applied to cells cultured on AP-SWNTs. Full squares show current-to-voltage relations obtained before the addition of the agonist. Open circles correspond to values in the presence of Bay K after 2 min obtained from the same cell. Open diamonds show complete blockade of Ca2+ currents by cadmium applied to the bath at the end of the experiment.
resembles a hormone and volume-sensitive anionic current that we described earlier in the same cells.26 Chloride current amplitude was reduced by 200 µM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, Sigma), a specific Clchannel blocker applied to the bath (data not shown). We did not find any statistically significant differences in Clcurrent amplitudes and voltage sensitivity in ROS 17/2.8 cells grown on AP-SWNTs and AP-MWNTs. However, current amplitudes measured from cells on AP-MWNTs at membrane potentials over 40 mV were slightly larger than those recorded from AP-SWNT cultures, as shown in the same figure. High-threshold, voltage-activated (HVA) calcium currents were recorded from ROS 17/2.8 cells cultured on APSWNTs and AP-MWNTs, as shown in Figure 6. We found that HVA Ca2+ current amplitudes obtained at 20 mV, the membrane voltage value for maximal activation, were ca. twofold larger in osteoblasts cultured on AP-MWNTs than AP-SWNTs. This Ca2+ current was enhanced significantly by 0.5 µM S(-) BayK 8644 (Sigma, Figure 6, left panel), a dihydropyridine agonist specific for L-type Ca2+ channels. Calcium currents were completely blocked by 500 µM Cd2+ added to the bath at the end of the experiment (not shown). Inward Ca2+ currents recorded in AP-SWNT and AP-MWNT cultures resembled an L-type Ca2+ channel involved in exocytosis of bone materials described in ROS 17/2.8 cells cultured on plastic dishes.27,38 Large Ca2+ current amplitudes obtained in AP-MWNTs correlate with the changes in cell morphology found in this nanomaterial substrate (see Figure 3). This may be explained by a differential expression of 566
L-type Ca2+ channel quantities in ROS 17/2.8 cells grown on different CNT types and/or different osteoblast differentiation stages.39 Normal plasma membrane electrical functions are necessary for osteoblasts to maintain exocytotic activities, and therefore bone formation. Here we prove that electrical activities of the osteoblast membrane are maintained, and Ca2+ channel functions enhanced, in cells grown on neutral CNTs, confirming a degree of biocompatibility of APSWNTs and AP-MWNTs. Ours is the first description of electrical activities of ion channels in a cell type cultured on CNTs. Treatment of bone defects in humans including those associated with the removal of tumors, trauma, and abnormal bone development faces important limitations. Current therapies such as autographs, allographs, and metal prostheses do not generally favor bone regeneration itself. Instead, they replace the lost bone by an artificial material. One novel aspect of modern tissue engineering is the attempt to create tissue replacement by culturing bone cells on synthetic 3D scaffolds3,5 or live prosthesis. The synthetic scaffold material can be either biodegradable, disappearing as the new bone grows, or nonbiodegradable, such as CNTs. In the last case, the nonbiodegradable material behaves as an inert matrix on which cells proliferate and deposit new live matrix, which must become functional, normal bone. Our study demonstrates promise for the use of CNTs in bone regeneration. However, more studies are needed to address how the body will interact with nonbiodegradable CNTs, more specifically the reaction of the immune system. We demonstrate in this paper that neutrally charged CNTs sustain osteoblast proliferation and bone-forming functions. We also demonstrate that cell shape, and possibly cell differentiation, can be controlled by the use of SWNTs or MWNTs, which suggests the possibility of combining nanomaterials. Although we cannot conclude from our observations that the surface charge of nanomaterials alone is responsible for our results obtained for osteoblast growth, our work opens up a promising chapter in the study of biological applications of CNTs. Hydrophilicity and phobicity, topography, and presence of impurities in AP- and functionalized CNTs are key factors to be addressed in future studies. Our results provide insight into the understanding of the degree of biocompatibility between live cells and CNTs, and the real possibilities for CNTs to be used as an alternative material for the treatment of bone pathologies that lead to bone loss, with the potential for the regrowth of normal bone. We found that osteoblasts grow and produce mineralized bone when cultured on electrically neutral CNTs. This growth is diminished by chemical modifications that introduce a net electric charge to the CNTs. In addition, we verified that ROS 17/2.8 cells retain electrical properties necessary for adequate secretion of bone materials when cultured on CNTs. These results suggest that electrically neutral CNTs can be considered as potential filling materials for the treatment of injured bone. We conclude that CNTs sustain osteoblast growth and bone formation, and thus represent a potential technological Nano Lett., Vol. 6, No. 3, 2006
advance in the field of bone bioengineering. CNTs show promising biocompatibility with osteoblast cells, and they appear to modulate the cell phenotype. Application of CNTs to bone therapy may lead to the development of new bonegraft materials and techniques. Acknowledgment. This work was supported by the USPHS grant DK-071115-01 to L.P.Z., and DOD/DARPA/ DMEA under Award DMEA90-02-2-0216 to R.C.H. Carbon Solutions, Inc. acknowledges NSF SBIR Phase I and Phase II Awards DMI-0110221 from the Division of Design, Manufacture and Industrial Innovation, a DARPA Phase I SBIR administered by the U.S. Army Aviation and Missile Command (Award W31P4Q-04-C-R171) and a AFOSR STTR Phase I Award (Contract no. FA09550-04-C-0122). Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Taton, T. A. Nature 2001, 412, 491. (2) Mann, S.; Weiner, S. J. Struct. Biol. 1999, 126, 179. (3) Ma, P. X.; Zhang, R.; Xiao, G.; Franceschi, R. J. Biomed. Mater. Res. 2001, 54, 284. (4) Shin, H.; Zygourakis, K.; Farach-Carson, M. C.; Yaszemski, M. J.; Mikos, A. G. J. Biomed. Mater. Res. A. 2004, 69, 535. (5) Shea, L. D.; Wang, D.; Franceschi, R. T.; Mooney, D. J. Tissue Eng. 2000, 6, 605. (6) Iijima, S. Nature 1991, 354, 56. (7) Ajayan, P. M. Chem. ReV. 1999, 99, 1787. (8) Yu, M. F.; Files, B. S.; Arepalli, S.; Ruoff, R. S. Phys. ReV. Lett. 2000, 84, 5552. (9) Sinnott, S. B. J. Nanosci. Nanotechnol. 2002, 2, 113. (10) Chen, Y.; Haddon, R. C.; Fang, S.; Rao, A. M.; Eklund, P. C.; Lee, W. H.; Dickey, E. C.; Grulke, E. A.; Pendergrass, J. C.; Chavan, A.; Haley, B. E.; Smalley, R. E. J. Mater. Res. 1998, 13, 2423. (11) Niyogi, S.; Hamon, M. A.; Hu, H.; Zhao, B.; Bhowmik, P.; Sen, R.; Itkis, M. E.; Haddon, R. C. Acc. Chem. Res. 2002, 35, 1105. (12) Shvedova, A. A.; Castranova, V.; Kisin, E. R.; Schwegler-Berry, D.; Murray, A. R.; Gandelsman, V. Z.; Maynard, A.; Baron, P. J. Toxicol. EnViron. Health A 2003, 66, 1909. (13) McKenzie, J. L.; Waid, M. C.; Shi, R.; Webster, T. J. Biomaterials 2004, 25, 1309.
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