pubs.acs.org/Langmuir © 2009 American Chemical Society
Bonelike Apatite Formation Utilizing Carbon Nanotubes as Template Lifang Niu, Huiyi Kua, and Daniel H. C. Chua* Department of Materials Science and Engineering, National University of Singapore, 7 Engineering Drive 1, Singapore 117574 Received September 16, 2009. Revised Manuscript Received December 6, 2009 Template-induced hydroxyapatite (HA) has broad prospects in the applied fields of regenerative medicine and bone repair. HA thin coatings have been deposited on vertically aligned multiwalled carbon nanotubes (CNTs) via the high-temperature radio-frequency (rf) magnetron sputtering deposition technique. Simulated body fluid (SBF) solution has been used to soak and incubate the HA/CNTs nanocomposites at 37 °C. SEM, EDS, XRD, and FTIR characterizations revealed bonelike apatite formation on top of HA/CNTs composites. Coating HA material on wellaligned CNT-template provides a way of combining the superior mechanical properties and chemical stability of the CNTs with the excellent biochemical properties of HA.
I. Introduction Hydroxyapatite (HA, Ca10(PO4)6(OH)2) is a form of calcium phosphate that bears close chemical resemblance with the mineral component of bones and teeth.1-3 It promotes tissue adhesion and bone growth by spontaneously forming a biologically active bonelike apatite layer over its surface.4-7 Thus, HA is classified as one of the best biocompatible and bioactive materials, and HA coatings have been found in many biological applications such as dental or skeletal implants and bone repair scaffolds. However, its poor mechanical properties such as brittleness and low wear resistance have limited the use of bulk HA coating in load-bearing and longterm implant applications. To tackle this problem, considerable efforts have been made to improve its fracture toughness and wear resistance by reinforcing HA with bioinert ceramics such as zirconia (ZrO2),8 alumina (Al2O3),9 and bioglass.10 There have also been studies of HA composites reinforced with polymers.11-13 In both cases, high loadings of the less bioactive reinforcing phases are needed in order to achieve the desired mechanical properties. This results in the formation of a less stable interface between the reinforcing phase and the external environment. Carbon nanotubes (CNTs) are ideal candidates as reinforcing materials given their small dimensions, high length/radius ratio, *Corresponding author. E-mail
[email protected]; Tel (65)65168933; Fax (65)67763604. (1) Chen, Y.; Gan, C.; Zhang, T.; Yu, G. Appl. Phys. Lett. 2005, 86, 251905– 251907. (2) Tsui, Y. C.; Doyle, C.; Clyne, T. W. Biomaterials 1998, 19, 2015–2029. (3) Hukovic, M. M.; Tkalcec, E.; Kwokal, A.; Piljac, J. Surf. Coat. Technol. 2003, 165, 40–50. (4) Kokubo, T.; Kim, H. M.; Kawashita, M. Biomaterials 2003, 24, 2161–2175. (5) Siriphannon, P.; Kameshima, Y.; Yasumori, A.; Okada, K.; Hayashi, S. J. Eur. Ceram. Soc. 2002, 22, 511–520. (6) Oyane, A.; Onuma, K.; Ito, A.; Kim, H. M.; Kokubo, T.; Nakamura, T. J. Biomed. Mater. Res., Part A 2003, 64A, 339–348. (7) Leng, Y.; Chen, J. Y.; Qu, S. X. Biomaterials 2003, 24, 2125–2131. (8) Lim, V. J. P.; Khor, K. A.; Fu, L. J. Mater. Process. Technol. 1999, 89, 491– 496. (9) Kong, Y. M.; Bae, C. J.; Lee, S. H.; Kim, H. W.; Kim, H. E. Biomaterials 2005, 26, 509–517. (10) Goller, G.; Demirkiran, H.; Oktar, F. N.; Demirkesen, E. Ceram. Int. 2003, 29, 721–724. (11) Oyane, A.; Kawashita, M.; Kokubo, T.; Minoda, M.; Miyamoto, T.; Nakamura, T. J. Ceram. Soc. Jpn. 2002, 110, 248–254. (12) Roader, R. K.; Sproul, M. M.; Turner, C. H. J. Biomed. Mater. Res., Part A 2003, 67A, 801–812. (13) Bonfield, W.; Grynpas, M. D.; Tully, A. E.; Bowman, J.; Abram, J. Biomaterials 1981, 2, 185–186.
Langmuir 2010, 26(6), 4069–4073
high strength, and stiffness. By incorporating CNTs into certain form of matrix, their outstanding properties can be best exploited. There have been considerable interests and investigation work in the application of CNTs in composite materials.14-17 Because of the lack of systematic studies on the biological and toxicological properties of CNTs, there have also been discrepancies in biocompatibility and toxicity reports.18 But recent studies suggest that CNTs in composites bear minimal probability of being released into the body and have no noticeable detrimental effects.1,19 There have been several studies reporting on the incorporation of CNTs into HA to improve its mechanical properties for loadbearing applications. Methods used to disperse CNTs in a matrix include physical blending1,20,21 and in situ formation.22-24 Physical blending, such as ball milling and dry mixing, tend to produce inhomogeneous dispersal due to CNTs’ high aspect ratio. While reported in situ techniques produce more homogeneous composites, they usually involve complicated wet chemical procedures. It is also noted that the orientation of the reinforcement was not addressed in both methods. Thus, the objective of this study is to incorporate CNTs into HA with a uniform dispersal of the CNT reinforcing phase. In practice, we have deposited HA seeds on vertically aligned multiwalled CNTs via the high-temperature radio-frequency (rf) magnetron sputtering deposition technique. We report the process of bonelike apatite formation in the matrix of the HA/CNTs composites when incubated in simulated body fluid (14) Peigney, A. Nat. Mater. 2003, 2, 15–16. (15) Balazsi, C.; Konya, Z.; Weber, F.; Biro, L. P.; Arato, P. Mater. Sci. Eng., C 2003, 23, 1133–1137. (16) Peigney, A.; Laurent, C.; Flahaut, E.; Rousset, A. Ceram. Int. 2000, 26, 677–683. (17) Peigney, A.; Flahaut, E.; Laurent, C.; Chastel, F.; Rousset, A. Chem. Phys. Lett. 2002, 352, 20–25. (18) Smart, S. K.; Cassady, A. I.; Lu, G. Q.; Martin, D. J. Carbon 2006, 44, 1034–1047. (19) Li, J.; Liao, H.; Hermansson, L. Biomaterials 1996, 17, 1787–1790. (20) Kealley, C.; Elcombe, M.; van Riessen, A.; Ben-Nissan, B. Key Eng. Mater. 2006, 309-311, 61–64. (21) Kealley, C.; Ben-Nissan, B.; van Riessen, A.; Elcombe, M. Key Eng. Mater. 2006, 309-311, 597–600. (22) Zhao, L.; Gao, L. Carbon 2004, 42, 423–460. (23) Wei, Q.; Yang, X. P.; Chen, G. Q.; Tang, J. T.; Deng, X. L. New Carbon Mater. 2005, 20, 164–170. (24) Aryal, S.; Remant Bahadur, K. C.; Dharmaraj, N.; Kim, K. W.; Kim, H. Y. Scr. Mater. 2002, 54, 131–135.
Published on Web 12/18/2009
DOI: 10.1021/la9034722
4069
Article
Niu et al.
(SBF) solution. Coating HA material onto well-aligned CNTtemplate is a way of combining the superior mechanical properties and chemical stability of the CNTs with the excellent biochemical properties of HA. Conventional physical and in situ chemical deposition techniques typically yield planar flat surfaces. This methodology, on the contrary, produces a consistent surface topography of nanometer-scale corrugation reinforced by a large area of aligned CNTs blanket over the substrate. The as-deposited HA encapsulate the CNTs’ tips and form many HA islands. The presence of these HA islands not only serve as nucleation sites for further spontaneous and rapid apatite formation but also greatly reduce the chance of stress accumulation, thereby lowering the chance of film delamination. The bioactivity of this hybrid HA-CNTs matrix, which can be correlated to the rate of bonelike apatite formation, is then assessed in vitro by the SBF incubation.
II. Experimental Method In this study, an rf (∼13.56 MHz) plasma-enhanced chemical vapor deposition (PECVD) system was employed to grow aligned multiwalled CNTs normal to silicon (100) substrate by catalytic decomposition of C2H2 using iron nanoparticles as catalysts. First, a 8 nm thick layer of iron catalyst was sputtered on Si substrate by RF magnetron sputtering. The iron-coated substrates were then brought in the PECVD chamber, where the iron coatings were heat-treated at 700 °C in H2 plasma (rf power 100 W) for 10 min to promote the formation of catalyst particles. After which, a gaseous mixture of C2H2 (flow rate 15 sccm) and H2 (flow rate 60 sccm) was introduced into the PECVD system at substrate temperature of ∼700 °C and chamber temperature of ∼400 °C. Deposition was performed for 10 min, at a working pressure of 1.2 Torr and rf power of 100 W. The as-grown multiwalled CNTs were found to have an average length of 14 μm. Two sets of samples were deposited: (1) HA film on Si substrate and (2) HA film on vertically aligned CNT-template. The target material used was a commercial high-density HA plate of purity 99.9%. The samples were fixed on a rotating substrate holder. Prior to deposition, the chamber was evacuated to a base pressure of below 2 10-6 Torr, and the sample stage was heated up to 500 °C. The process pressure was adjusted to 10 10-3 Torr using pure argon gas flow of 20 sccm. The rf power was varied from 40 to 140 W. Deposition was conducted for 30 min. The SBF solution used for in vitro incubation was prepared by sequentially dissolving appropriate quantities of NaCl, NaHCO3, KCl, K2HPO4 3 3H2O, MgCl 3 6H2O, CaCl2, and Na2SO4 in deionized (DI) water and buffering with tris(hydroxymethyl)aminomethane ((CH2OH)3CNH2). The final pH value was adjusted to 7.4 by titrating with 1 M HCl. Details of its composition were reported elsewhere.25 The deposited samples were cut into rectangular pieces of 5 5 mm, immersed in SBF solution, and then maintained at 37 °C in an incubator throughout all the tests. After incubation for varying periods, the specimens were collected, gently washed with DI water, and dried for further characterizations. A Bruker D8 X-ray diffractometer (XRD) was used to analyze the phase compositions of the samples. Generation of X-rays were carried out using a Cu KR tube (λ = 0.154 nm) at 40 kV and 40 mA. The specimen surface was scanned at step size of 0.02° with scan speed of 0.4 s per step. A Phillips XL 30-FEG scanning electron microscope (SEM) with an attached energy dispersion X-ray spectroscopy (EDS) was used to characterize surface morphology and elemental compositions. A low accelerating voltage of 5 kV was used for the secondary electron imaging (SEI) of microstructures from sample surfaces. FTIR spectra were collected over the range of 4000-500 cm-1 using a Perkin-Elmer System 2000 Fourier transform infrared spectrometer. (25) Kokubo, T.; Takadama, H. Biomaterials 2006, 27, 2907–2915.
4070 DOI: 10.1021/la9034722
Figure 1. SEM images of HA/CNTs composites deposited at rf powers of (a, b) pristine, (c, d) 40 W, (e, f) 90 W, and (g, h) 140 W.
III. Experimental Results Microstructural Features of the As-Grown Films. The morphologies of CNTs with rf magnetron sputtered HA coatings prepared at different rf powers were studied using SEM as shown in Figure 1a-h. According to the top view images, HA/CNTs composites are uniformly thick and appear microstructurally homogeneous at low magnifications. The HA/CNTs sample produced at power of 40 W shows little or no difference as compared with the pristine CNT sample. This may indicate that the deposition rate of HA at 40 W is too low such that the coating is too thin to be visible under SEM. As the rf power increases, the top portions of the CNTs become much thicker, suggesting that the produced HA coating was likely to have adhered onto the tips of CNTs. Elemental analysis by EDS is presented in Figure 2a. Because of the less compact nature of the vertically aligned HA/ CNTs composites grown on Si substrate, the X-ray escaping from Si element is readily detected. The energy-dispersive spectrum confirms the presence of Ca, P, and O specimens. Quantitative analyses show that the Ca/P atomic ratio of the HA coatings varies between 1.7 and 2.1. The SEM image of HA film directly deposited on Si substrate at 100 W is also shown in Figure 3 for comparison purposes. The HA-coated Si substrate shows increased surface microcracks and delaminations, suggesting the presence of residual stresses during the film growth process. It is believed that during deposition, while the grains grow, the poor thermal conductivity of the ceramic layer results in random heat accumulation and thus local Langmuir 2010, 26(6), 4069–4073
Niu et al.
Article
Figure 4. XRD spectra of (a) as-deposited HA/CNTs (140 W, 500 °C, 30 min) and (b) after SBF incubation of 20 days. Increase in the intensity of HA peaks is observed.
Figure 2. EDS spectra of (a) as-deposited HA/CNTs (at 140 W) and (b) after SBF incubation of 20 days. Figure 5. FTIR spectra of (a) as-deposited HA/CNTs (140 W, 500 °C, 30 min) and (b) after SBF incubation of 20 days.
Figure 3. SEM image of HA film directly deposited on Si substrate.
temperature increase of the substrate. As the HA films undergo thermal contraction during cooling down, cracks are generated. HA/CNTs composite, on the other hand, exhibits no transverse cracks due to the template function of CNTs, which effectively scatters heat and dissipates the accumulated stresses into the individual nanosized tips. The existence of CNT-template increases the overall surface roughness of the HA coating, giving rise to more exposed sites and atoms for further apatite formation during SBF incubation. Surface Characterization by XRD and FTIR. Figure 4a shows the typical XRD pattern of the as-sputtered HA on CNTtemplate prepared at 140 W. The coated apatite is characterized by broad diffraction peaks owing to small crystallites and defective structure. Figure 4b presents the pattern of HA/CNTs specimen with SBF incubation of 20 days. Compared to the diffraction pattern of preimmersion HA/CNTs specimen, the Langmuir 2010, 26(6), 4069–4073
diffraction indicates an increased intensity of HA pattern and the presence of carbonated HA. It clearly shows that the Ca- and P-rich layer consists of a carbonate-containing hydroxyapatite with disordered structure and thus poor crystallinity. For those CNTs specimens with no HA coating but with SBF incubation, no specific XRD reflection lines were observed. FTIR was used to detect the presence of functional groups of phosphate and carbonate, so as to provide more information on the apatite formed on the HA/CNTs composite after incubation in SBF. The FTIR spectra of as-deposited HA/CNTs (140 W, 500 °C, 30 min) and HA/CNTs with SBF incubation of 20 days are shown in Figure 5. The characteristic absorption bands of phosphate are clearly resolved at 1035 cm-1. The presence of peaks at about 630 cm-1 is due to the OH- vibrations, which can be found in apatite structure. For the HA/CNTs specimen soaked in SBF solution for 20 days, carbonate substitution can be seen by the two absorption bands between 1410 and 1540 cm-1 and at ∼880 cm-1, which correspond to the CO32vibrational modes. Combinding the findings from XRD and FTIR, it can be confirmed that the formed layer is carbonatecontaining apatite, which has similar composition and structure to bone apatite. SEM and EDS Analyses of the SBF Immersion Behavior. Figure 6 shows SEM images of top and cross sections of HA/CNTs specimens soaked in SBF for varying periods. It can be seen from Figure 6a that after HA deposition the tips of CNTs were homogenously covered by a layer of material, with an increase in tube diameter from ∼15 to ∼30 nm. From the cross section view it can be seen that although the top portion of CNTs DOI: 10.1021/la9034722
4071
Article
Niu et al.
The EDS analysis of the HA/CNTs composites with 20 days of SBF incubation is shown in Figure 2b. The intensities of characteristic Ca and P peaks after soaking are normalized against those before soaking. It can be seen that Ca and P concentrations at the surface of HA/CNTs are much enhanced compared to those of the HA/CNTs composites with no SBF incubation. Because of the formation of HA layer on the CNT-template, the intensity of X-ray emission from Si substrate is greatly depressed. For those non-HA-coated CNT specimens, no appreciable changes of the elemental concentration can be seen along soaking time. These results are consistent with those of SEM observations described above.
Figure 6. SEM images of (a) as-deposited HA/CNTs, (b) after SBF incubation of 10 days, (c) after SBF incubation of 16 days, (d) after SBF incubation of 20 days, and (e) after SBF incubation of 35 days.
was getting thicker, the CNTs underbrush remained uncoated. As can be seen in Figure 6b, the specimen soaked for 10 days showed the homogeneous growth of the coating layer and had a tube diameter of 100 nm. It is worth noting that the HA layer started to grow from CNTs’ tips down into CNTs’ underbrush. Once the specimen had been soaked for 16 days, the layer got thicker and the individual HA-coated tubes started to fuse with the neighboring ones. Fragmental particles were also observed on the surface. After being soaked for 20 days, the top surface looked less corrugated due to the fusion of adjacent HA globules to from a continuous film. Cross-section image reveals that the HA film continued to spread into the deep side of vertically aligned CNTs underbrush and filled the free space between individual carbon tubes. The amount and thickness of the formed HA layer increase as the soaking time increases. As shown in Figure 6e, it took about 35 days of incubation to form a concrete, homogeneous apatite layer along the entire length of the CNTs. As a comparison, the CNTs specimens with no HA coating showed no material growth at all during varying incubation periods. 4072 DOI: 10.1021/la9034722
IV. Discussion There are several deposition methods of HA, which include sputtering,26,27 plasma-spraying,28 pulsed laser deposition,29 and ion-beam deposition.30 Different experimental conditions can significantly influence the characteristics of the deposited HA films. In this work, since the vertically aligned CNTs were used as template, the use of oxygen or water vapor gaseous environment was avoided to prevent potential CNT oxidation. Previous attempts of depositions at substrate temperatures lower than 400 °C had resulted in amorphous films. To obtain crystalline HA, rf magnetron sputtering deposition was performed at substrate temperature of 500 °C. The samples underwent in situ postheat-treatment in vacuum chamber at 500 °C for 2 h and were slowly cooled down to room temperature at 10 °C/min. It is believed that such in situ annealing helps in the formation of a crystalline film. The mechanisms of the bonelike apatite formation on bioactive materials through in vitro incubation in SBF solution have been reported by a number of groups. Khor et al. reported that a dissolution/precipitation process dominates the apatite formation of thermally sprayed amorphous calcium phosphate coatings.31 Kokudo et al.’s work reveals that the negative charges of surface functional groups induce the accumulation of solution ions and facilitate the transformation into bonelike apatite.4 The presence of OH- and PO43- ions at the surface of HA makes it negatively charged. Upon incubation in SBF solution, the negatively charged surface electrostatically combines with the positively charged Ca2þ ions from the solution and forms Ca-rich calcium phosphate. As more Ca2þ ions approach the surface, the surface acquires a net positive charge and starts to combine with the negatively charged PO43- ions to form metastable amorphous calcium phosphate, which gradually transforms into stable bonelike apatite. CNTs have various potential applications due to their excellent electrical, mechanical, and chemical properties.32-34 To better explore the use of their high surface area, there is often a need to attach functional groups to them, either by chemical or by physical ways. In this work, well-aligned CNTs with selfstanding geometry were used as template to obtain HA/CNTs (26) van Dijk, K.; Schaeken, H. G.; Wolke, J. G. C. J. Biomed. Mater. Res. 1995, 29, 269–276. (27) Wolke, J. G. C.; van der Waerden, J. P. C. M.; de Groot, K.; Jansen, J. A. Biomaterials 1997, 18, 483–488. (28) Lu, Y.; Xiao, G.; Li, S.; Sun, R.; Li, M. Appl. Surf. Sci. 2006, 252, 2412– 2421. (29) Cotell, C. M. Appl. Surf. Sci. 1993, 69, 140–148. (30) Choi, J. M.; Lee, I. S.; Kim, H. E. Biomaterials 2001, 20, 469–473. (31) Khor, K. A.; Li, H.; Cheang, P. Biomaterials 2003, 24, 769–775. (32) Treacy, M. M. J.; Ebbesen, T. W.; Gibson, J. M. Nature 1996, 381, 678–680. (33) Dai, H.; Hafner, J. H.; Rinzler, A. G.; Colbert, D. T.; Smalley, R. Nature 1996, 384, 147–150. (34) Ebbesen, T. W.; Lezec, H. J.; Hiura, H.; Bennett, J. W.; Ghaemi, H. F.; Thio, T. Nature 1996, 382, 54–56.
Langmuir 2010, 26(6), 4069–4073
Niu et al.
nanocomposites. Brunauer-Emmer-Teller (BET) surface areas for the as-grown CNT arrays by means of N2 adsorption measurement at -196 °C is determined to be 54 m2/g. The presence of CNTs blanket underneath HA coating produces a homogeneous nanometer-scale corrugated topograph and larger surface area of HA layer as compared to as-deposited HA film with no CNT-template. Additionally, HA/CNTs composites show no obvious surface microcracks, indicating the capability of overcoming thermal expansion inconsistency and inner stress dissipation. The as-grown HA/CNTs composites contain numerous nanometer-sized HA islands, which also serve as nucleation sites when soaked in SBF solution. Furthermore, the difference in hydrophilicity between HA and CNTs may lead to the formation of solution-air interface at the phase boundary between HA and CNTs, thus resulting in an increase in local ion concentrations. This would favor the consumption of solution ions and promote successive deposition of apatite layers. The large number of HA nanosites and the incresed local ion concentrations are believed to be the main driving forces of spontaneous, rapid apatite formation. With the spatial support of the CNTs template, the growth of apatite extends not only planarly but also vertically into the CNTs underbrush. The HA/CNTs composites are eventually woven into a concrete apatite pallet braced by the inner freestanding CNTs. Additionally, the usage of aligned CNTs as template guarantees a uniform dispersal of the reinforcing phase into the ultimate HA film and thus imparts homogeneous properties to the overall composite. Aligned fibers provide the material with antibrittle property by preventing crack propagation.35,36 It is expected that (35) Teo, E. H. T.; Yung, W. K. P.; Chua, D. H. C.; Tay, B. K. Adv. Mater. 2007, 19, 2941–2945. (36) White, A. A.; Best, S. M.; Kinloch, I. A. Int. J. Appl. Ceram. Technol. 2007, 4, 1–13.
Langmuir 2010, 26(6), 4069–4073
Article
the HA films reinforced with CNTs improve the stress distribution of the composite film through load transfer from the HA to the CNT network. The correlation between mechanical properties of HA/CNTs composites and the length and density of CNTs is still under investigation.
V. Conclusion In summary, HA films were coated onto CNTs templates by using PECVD and RF magnetron sputtering deposition. Upon subsequent incubation in SBF solution, the HA-coated tips work as functional structures for apatite nucleation, with nanosized calcium phosphate precipitating onto the individual HA/CNTs composites and gradually growing into a three-dimensional bonelike apatite fabric. Results of the SEM and EDS analyses described above show that HA/CNTs nanocomposites form a Ca- and P-rich thin layer on its surface by means of SBF incubation, which is absent in bare CNT-template. XRD and FTIR results show that the Ca- and P-rich layer consists of small crystallites of carbonate-containing hydroxyapatite. The compositional and structural characteristics of the surface apatite are similar to those of the natural bone apatite. Results of SEM observation at different SBF soaking days show that the surface apatite layer was formed successively. Coating HA material on well-aligned CNT-template can be one way of combining the superior mechanical properties and chemical stability of the CNTs with the biochemical properties of HA. This new method of apatite formation may provide an alternative way for the design and preparation of bioactive materials with improved mechanical properties and tailored microstructure and macrodimension control. Acknowledgment. The authors acknowledge NUS WBS R-284-000-046-101 for funding this work.
DOI: 10.1021/la9034722
4073
