11950
J. Phys. Chem. B 2000, 104, 11950-11956
Nucleation of Calcium Phosphate on 11-Mercaptoundecanoic Acid Self-assembled Monolayer in a Pseudophysiological Solution Kazuo Onuma,*,† Ayako Oyane,‡ Tadashi Kokubo,‡ Gabin Treboux,† Noriko Kanzaki,† and Atsuo Ito† National Institute for AdVanced Interdisciplinary Research, Cell Tissue Module Group, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan, and Department of Materials Chemistry, Graduate School of Engineering, Kyoto UniVersity, Yoshida, Kyoto 606-8501, Japan ReceiVed: June 2, 2000; In Final Form: October 2, 2000
Heterogeneous nucleation of calcium phosphate on an 11-mercaptoundecanoic acid self-assembled monolayer (SAM) in a pseudophysiological solution was investigated by in situ and ex situ atomic force microscopy. Calcium phosphate nanodots of 5-10 nm diameter were arranged two-dimensionally in a hexagonal closely packed structure in the initial stage of nucleation (e3 h), reflecting the molecular arrangement of a thiol layer. After this stage, the surface was covered by random nucleated calcium phosphate particles of 20-30 nm diameter. Measurement of the growth rate of a calcium phosphate layer and X-ray photoelectron spectroscopy analysis were performed and possible qualitative models of calcium phosphate growth on a SAM were presented.
Introduction Hydroxyapatite crystals [Ca10(PO4)6(OH)2: HAP], particularly carbonate-containing HAP, are the inorganic components of bone mineral. They are arranged with their [0001] direction parallel to the long axis of collagen, and a minute threedimensional structure of an inorganic-organic composite is formed.1 The formation process of bone should be considered by taking many factors into account such as the threedimensional organization of collagen in relation to the cell biology, the growth kinetics of HAP in a physiological solution, and the inorganic-organic interaction on the interface between collagen and HAP. Landis and co-workers suggested that numerous collagen mutations were related to diseases such as osteogenetic imperfecta by controlling the nucleation of HAP on collagen.2-6 Many reports have been published about the growth kinetics of HAP, including ones from our group, and several mechanisms, such as spiral growth and polynucleation, have been presented.7-15 From the viewpoint of crystal growth, inorganic-organic interaction for the heterogeneous nucleation of HAP is one of the important issues of the process of bone formation.16 Several studies have pointed out the important role played by the carboxylic terminal group for the nucleation of HAP17-19: these studies included the idea that collagen provides a template for the HAP nucleation which might be initiated by associated noncollagenous proteins. Concerning the importance of the carboxylic terminal group of organic substrate, Langmuir-Blodgett (LB) film20-22 and a self-assembled monolayer (SAM) of alkainethiol on a gold substrate23 have been used to investigate the nucleation of calcium phosphate on a nanometric scale. In particular, because alkanethiol on a gold substrate24-31 can be arranged on an atomic scale and is very easy to prepare, * To whom correspondence should be addressed: Dr. Kazuo Onuma, National Institute for Advanced Interdisciplinary Research, Cell Tissue Module Group, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan. E-mail:
[email protected]. † National Institute for Advanced Interdisciplinary Research. ‡ Kyoto University.
it is expected to be suitable as a substrate. Tanahashi and Matsuda attempted to develop a HAP-carboxylic thiol SAM composite in 1997.23 Although they succeeded in confirming HAP nucleation on the thiol surface, they did not discuss the detailed structure of HAP in relation to the molecular structure of thiol on the atomic scale. Because the time scale for observation, that is, a few days, was too long, it was unclear whether HAP actually nucleated on thiol upon recognizing its molecular structure, similar to the biomineralization process, and whether the calcium phosphate that nucleated on the thiol SAM was initially HAP. In the present study, we use in situ and ex situ atomic force microscopy (AFM) to investigate the nucleation of calcium phosphate on 11-mercaptoundecanoic acid SAM, the thiol having a carboxylic terminal group, in a pseudophysiological solution. Special attention is paid to observation during the initial stage of nucleation (1 day), multiple two-dimensional nucleation of calcium phosphate particles, 20-30 nm diameter, is observed. (2) The normal growth rate is time-dependent and discontinuous. The growth rate of the initial stage (1 day) increases with time. These characteristic features suggest that the calcium phosphate grown in the initial stage is different from that in the late stage. Characterization of calcium phosphate was difficult because the thickness of the grown layer was only 10 nm even in the sample grown for 4 days. However, the morphology of the sample grown for 4 days was the same as that of HAP grown on HAP seed crystal. Reflection Fourier transform (FT)-IR with polarized light (RAS FT-IR) measurement was applied to the sample grown for 4 days and the result is shown in Figure 9. The spectrum was obtained by subtracting that of thiol as a background. Although many peaks appear and the signal-tonoise ratio is low, the peaks that correspond to PO43- of HAP (1035 cm-1), the ν4 vibration mode of HPO4 (around 11001150 cm-1), and doublet peaks of PsO bend, which are observed on crystalline HAP (550-600 cm-1), are recognized. Recently, Sato et al.22 succeeded in estimating the calcium phosphate grown on LB film with a carboxylic terminal group, which was the same as our thiol, using transmission FT-IR. They showed that the calcium phosphate grown for 5 days in a solution similar to ours was HAP. Thus, we conclude that the calcium phosphate in the sample grown for 4 days is HAP. We were unable to characterize the calcium phosphate nanodots of the initial stage in the present study. The present solution is supersaturated with respect to octa-calcium phosphate [Ca8H2(PO4)6‚5H2O, OCP] and HAP. The supersaturations for OCP and HAP are 2.6 and 22.0, respectively. From in situ AFM, the growth rate of the calcium phosphate layer, and the higher Ca/P ratio than HAP in the initial stage, it is obvious that the nanodots are not composed of HAP. We also believe that OCP is not the material nucleated in the initial stage. Apart from the lower Ca/P ratio in OCP than in HAP, the structure of OCP resembles that of HAP. Thus, the epitaxial growth of HAP, or the direct structural transformation to HAP is expected. The disordered arrangement of nucleated materials in the late stage is difficult to explain. Based on the discussion mentioned above, we propose possible growth models of calcium phosphate nanodots on a
Nucleation of Calcium Phosphate on HS(CH2)10COOH SAM
J. Phys. Chem. B, Vol. 104, No. 50, 2000 11955
Figure 10. Schematic illustration of the arrangement in the initial stage of growth based on the Ca9(PO4)6 cluster. Black circle indicates the cluster. The cluster combines with the carboxylic group with its c-direction perpendicular to the thiol surface a, and parallel to the thiol surface b. The grown layer resembles that of the (0001) face of HAP crystal in a.
carboxylic thiol SAM based on both the ionic species and the calcium phosphate cluster. First, the model based on ionic species is presented. In the previous study,23 a Ca ion was considered to combine with two neighboring -COO- groups in the initial stage as COO-sCa2+s COO-. Here, we assume that this mechanism rarely occurs for two reasons: (1) The distance between two -COO- groups is 0.5 nm, whereas the diameter of the Ca ion is only 0.2 nm. (2) Even if binding occurs as a result of distortion of the -COOgroups, the charge of the Ca ion is canceled by the two COOgroups, thus, PO43-, a negative counterion, cannot reach the surface, and the growth of calcium phosphate does not proceed. For the growth of calcium phosphate to proceed, one Ca ion should combine with one -COO- group, as COO-sCa2+s, leaving a positive charge to attract PO43-. The binding continues until the bond, (COO-sCa2+)sPO43-sCa2+, is formed. In this case, the average Ca/P ratio of the layer grown at the initial stage is 2.0, which is higher than that of HAP, and the structure of the initial layer does not resemble that of HAP. The size of calcium phosphate on COO- is about 0.8 nm if linear binding between ions occurs. This size is much smaller than that of observed nanodots, 5-10 nm diameter. However, we should point out that the observed size of nanodots is obvious because the tip radius of the AFM probe is also 5-10 nm. The actual size of nanodots is much smaller than 5 nm, and it is possible that a 1 nm nanodot or group of nanodots was observed as a 5 nm cluster. Second, we discuss the model based on the calcium phosphate cluster. In previous studies using in situ AFM, optical interferometry and dynamic light scattering, we demonstrated that calcium phosphate clusters exist in the pseudophysiological solution.36,37 The most probable cluster is Ca9(PO4)6.38 When the cluster combines with -COO- groups with its c-direction perpendicular to the thiol surface, as shown in Figure 10a, the structure of the initial layer quite resembles that of the (0001) face of HAP crystal although the higher Ca/P ratio than that of HAP can be achieved by coexistence of the cluster and free calcium ions. This contradicts the observed morphological change between the initial and the late stages, and the discontinuity of the growth rate. However, when the cluster
combines with its c-direction parallel to the thiol surface, as shown in Figure 10b, the structure of the initial layer does not resemble that of HAP. In this model, the cluster is stabilized by combining with two -COO- groups. The c-direction of the cluster is inclined about 7° from [110]. Coexistence of Ca ions with the cluster is also necessary to maintain a higher Ca/P ratio than HAP. Conclusion Regularly arranged nanodots composed of calcium phosphate were formed on the carboxylic thiol SAM. The arrangement of nanodots reflects the structure of the thiol surface in the initial stage of growth, however, it transforms to a disordered state in the late stage of growth. The growth rate and the morphology of the grown layer indicate that the calcium phosphate nucleated in the initial stage is different from that in the late stage. Acknowledgment. One of the authors (K.O.) would like to thank Dr. T. Ishida of JRCAT for his helpful discussion on thiol SAM. References and Notes (1) Landis, W. J. Connect. Tissue Res. 1996, 34, 239. (2) Landis, W. J.; Paine, M. C.; Hodgens, K. J.; Glimcher, M. J. J. Ultrastruct. Mol. Struct. Res. 1986, 95, 142. (3) Landis, W. J. Bone 1995, 16, 533. (4) Fratzl, P.; Paris, O.; Klaushofer, K.; Landis, W. J. J. Clin. InVest. 1996, 97, 396. (5) Camacho, N. P.; Landis, W. J.; Boskey, A. L. Connect. Tissue. Res. 1996, 35, 259. (6) Misof, K.; Landis, W. J.; Klaushofer, K.; Fratzl, P. J. Clin. InVest. 1997, 100, 40. (7) Koutsoukos, P. G.; Amjad, Z.; Tomson, M. B.; Nancollas, G. H. J. Am. Chem. Soc. 1980, 102, 1553. (8) Nancollas, G. H.; Koutsoukos, P. G. Prog. Cryst. Growth Charact. 1980, 3, 77. (9) Moreno, E. C.; Zahradnik, R. T.; Glazman, A.; Hwu, R. Calcif. Tissue Res. 1977, 24, 47. (10) Moreno, E. C.; Varughese, K. J. Cryst. Growth 1981, 53, 20. (11) Arends, J.; Christoffersen, J.; Christoffersen, M. R.; Eckert, H.; Fowler, B. O.; Heughebaert, J. C.; Nancollas, G. H.; Yesinowskl, J. P.; Zawacki, S. J. J. Cryst. Growth 1987, 84, 515. (12) Christoffersen, M. R.; Christoffersen, J. J. Cryst. Growth 1992, 121, 608.
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