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The Surface Structure of Hydroxyapatite Single Crystal and the Accumulation of Arachidic Acid
Journal of Colloid and Interface Science 224, 23–27 (2000) doi:10.1006/jcis.1999.6623, available online at http://www.idealibrary.com on
The Surface Structure of Hydroxyapatite Single Crystal and the Accumulation of Arachidic Acid Kimiyasu Sato, Yasushi Suetsugu, Junzo Tanaka, Sachio Ina,∗ and Hideki Monma∗ National Institute for Research in Inorganic Materials, Tsukuba, Ibaraki 305-0044, Japan; and ∗ Kogakuin University, Hachiouji, Tokyo 192-0015, Japan Received April 12, 1999; accepted November 9, 1999
crucible (10 mmφ × 100 mm) as densely as possible. After weld sealing, the crucible was placed perpendicularly in a HIP furnace. The temperature was raised up to 1400◦ C for 3 h and kept for 1 h for the melting mixture to homogenize. Afterward, the crystal growth was performed by gradually lowering the temperature to 900◦ C at 5◦ C/h. The upper part of the crucible was about 10◦ C lower than the lower part. The crystals grown were chemically treated in an aqueous solution of 10% ammonium chloride at 80◦ C for 1 day to remove the flux and washed by pure water. The yield of crystals was about 65% of the starting materials packed into the crucible. The crystals were colorless and transparent, and their shape was mainly hexagonal, ∼1 to 2 mm in diameter and ∼3 to 5 mm in length. Larger single crystals over 10 mm in length grew partially in the middle of the crucible, and irregular and dendrite crystals grew at the bottom. Details of single crystal preparation are described in Ref. (4). The etching of the single crystal surfaces was carried out at room temperature by soaking in 0.05 N HCl aqueous solution for ∼3 to 30 min. After washing by pure water and drying in a desiccator for several hours, the surfaces of HAp crystals were observed by AFM (Digital Instruments Co., Nanoscope III) with a tapping mode using a resonant frequency of 200 kHz.
Single crystals of hydroxyapatite (HAp) were grown by a flux method using β-tricalcium phosphate and Ca(OH)2 under hot isostatic pressure. After chemical etching by 0.05 N HCl aqueous solution, the HAp crystal surfaces were observed by an atomic force microscope (AFM) and shown dominantly to consist of steps with heights corresponding to a lattice distance d(100) or its 2/3. Arachidic acid films were accumulated on the etched HAp crystal by a Langmuir–Blodgett method. From section analyses by AFM, a distance between the carboxyl groups of arachidic acid and the HAp surface was estimated to be approximately 0.04 nm, sufficiently adjacent for a chemical interaction to take place. °C 2000 Academic Press Key Words: hydroxyapatite; arachidic acid; interfacial interaction; atomic force microscope.
INTRODUCTION
Bone is the typical nanocomposite that mainly consists of inorganic apatite and organic collagen (1). It is therefore important to understand the nature of an inorganic/organic heterointerface for the production of an inorganic/organic composite with bioactive properties similar to bone. In the present study, the single crystal surfaces of hydroxyapatite (HAp) were etched at the atomic level, and arachidic acid with the same functional group as collagen, i.e., carboxyl group (2), was accumulated on the etched HAp surfaces using the Langmuir–Blodgett (LB) method (3). To understand the interaction that occurred between the HAp surface and the carboxyl group, their surface morphologies were observed with an atomic force microscope (AFM).
(2) The Accumulation of LB Thin Film The LB method was applied to accumulate arachidic acid on the HAp crystal surface. First, a chloroform solution of 1 mM arachidic acid, C19 H39 COOH, was prepared and spread on a pure water surface. A fixed surface pressure, typically 25 mN/m, was applied along the water surface to form the monolayer film of arachidic acid. Then, the HAp single crystal prepared in the water was perpendicularly pulled up by controlling the surface pressure by observing a π -A (surface pressure-surface area) curve. The carboxyl head group of arachidic acid adhered to the HAp crystal surface as it was raised up.
EXPERIMENTALS AND METHODS
(1) Crystal Growth of HAp and Surface Etching Single crystals of HAp were grown by a flux method using β-tricalcium phosphate (β-TCP; Ca3 (PO4 )2 ) and Ca(OH)2 under a hot isostatic pressure (HIP) of 50 MPa. The high pressure was needed because Ca(OH)2 decomposes into CaO and water above 580◦ C. The pressure medium was the Ar gas. Starting material was the mixture of β-TCP and Ca(OH)2 , whose ratio was 2 : 3 by weight, which was molded into cylindrical pellets 8 mm in diameter. The pellets were then packed into a platinum
RESULTS AND DISCUSSION
(1) The Surface Morphology of Etched Single Crystal Figure 1 shows the AFM images of a HAp single crystal before chemical etching. The surfaces observed proved to be (h00) by 23
0021-9797/00 $35.00
C 2000 by Academic Press Copyright ° All rights of reproduction in any form reserved.
24
SATO ET AL.
FIG. 1. The surface of as-grown HAp single crystal as observed by AFM: (a) directly after the crystal growth, (b) HAp surface left in the air for 8 days.
the X-ray diffraction analysis. Directly after the crystal growth, a step/terrace structure was observed on the surface, suggesting that the HAp crystal grew by a layer-by-layer mechanism. However, the step/terrace structure disappeared upon leaving the crystal in the air for 8 days. This indicates that, in the vicinity of the HAp surface, some ions moved from the inside to the surface and then a secondary chemical phase formed. According to an Auger electron spectroscopic experiment by Suetsugu et al. (5), calcium carbonate formed on the surface by reacting with carbon dioxide in the atmosphere, as calcium ions could move near the surface even at room temperature. We surveyed etching conditions for the HAp surface with HCl, acetic acid, etc. It was found that 0.05 N HCl aqueous solution was most effective for the atomic scale etching of the HAp surfaces. Figure 2 shows the etched surfaces as a function of soaking time. Figure 2a shows the surface after 3 min of etching. Terraces about 100 nm in width were observed and steps ran approximately parallel to the c axis. The variation of surface height (i.e., section analysis) was also shown in Fig. 2; the step heights were typically 0.60–0.65 or 0.90–0.94 nm. Figure 2b shows the surface after 10 min of etching. In this case, the terrace width became narrower than 100 nm, indicating that the dissolution had been progressing. Since the step height did not change in comparison to that in Fig. 2a, it is considered that the dissolution occurred more selectively along the horizontal direction of the terrace than along the perpendicular direction. Figure 2c shows the surface etched for 20 min. The dissolution of the terraces progressed further and the step became rectilinear. With respect to Fig. 2d, narrowness of the terrace width prevents precise section analysis. The step height of Figs. 2c and 2d, however, almost did not change in comparison with Figs. 2a and 2b. As a result of detailed section analysis, the step height √ corresponded to 2/3 of d(100) or d(100) itself (0.82 nm = 3/2a0 ). In the HAp crystal structure, ionic density changes along the
[210] axis at intervals of 1/3 and 2/3 of d(100) as shown in Fig. 3(6). The layers with higher ionic density correspond to steps observed by AFM. Therefore, the fundamental step height of the HAp crystal consisted of the 1/3 or 2/3 of d(100). However, the step height of 1/3 of d(100) was seldom observed in
FIG. 4. The AFM image and section analysis of arachidic acid accumulated on a HAp single crystal.
ACCUMULATION OF ARACHIDIC ACID ON THE HAp SURFACE
25
FIG. 2. AFM image and section analysis of HAp surfaces. The surfaces were etched for (a) 3 min, (b) 10 min, (c) 20 min, and (d) 30 min by 0.05 N HCl aqueous solution.
26
SATO ET AL.
FIG. 3. The surface state of the (h00) terraces of HAp single crystal. Positively charged layer, [Ca8 (PO4 )4 ]4+ ; negatively charged layer, [Ca(PO4 )2 (OH)2 ]4− .
our experiments; this was, for example, due to the charge neutrality between adjacent terraces or the structural instability of step edge for 1/3 step height. (2) The Adsorption State of Arachidic Acid Arachidic acid was accumulated by the LB method on the HAp crystal surface that was etched for 3 min with HCl solution. Figure 4 shows the AFM image of the accumulated surface. Though the monolayer of arachidic acid formed a planar triangular lattice on the water surface at the application of surface pressure, the AFM image in Fig. 4 did not reflect such arrangement on the water but the LB film obtained was inhomogeneous as a whole. According to the section analyses, the thickness of the built-up film was 5.1 to 5.4 nm which was approximately equal to twice the molecule length of arachidic acid, i.e., 2 × 2.7 nm (3, 7). This result suggested the formation of a bilayer film of arachidic acid in which the hydrophilic carboxyl
groups of arachidic acids faced the HAp surface as shown in Fig. 5. In general, arachidic acids are roughly perpendicularly accumulated on a substrate but slightly lean toward the HAp single crystal surface. For example, the leaning angle is about α = 20◦ for arachidic acids on a hydrophobic substrate (8). As shown in Fig. 5, we defined here d1 as a distance between the HAp surface and the carboxyl groups and d2 as an accumulation distance between arachidic acids. The distance between the two facing terminal methyl groups in the bilayer structure is 0.3 nm (9). When we suppose that α = 20◦ and d2 = 0.3 nm, the distance between the terminal carboxyl group and the HAp surface was estimated at 0.04 nm or less on the basis of the thickness of the dyad film determined, i.e., ∼5.1–5.4 nm. The length estimated for d1 is sufficiently short for a chemical interaction to occur between the carboxyl head group of arachidic acid and the HAp surface. As shown in Fig. 3, it is conjectured from Fig. 2 that the terraces parallel to the (h00) planes have charges of +4 or −4 per unit area. Even if any types of lattice relaxation take place in the terraces to reduce the surface charges, the total surface charges probably differ from terrace to terrace. The hydrophilic carboxyl groups of arachidic acids attract positively charged terraces but repulse negatively charged terraces. It is thought that such attractive or repulsive interactions are related to the inhomogeneous accumulation of arachidic acids on the HAp surfaces shown in Fig. 4. CONCLUSIONS
FIG. 5. The schematic accumulation state of arachidic acid on a HAp surface.
HAp single crystals were grown by a flux method, and the interaction of inorganic and organic substances was examined. When etched by 0.05 N HCl aqueous solution, the step height of HAp surfaces corresponded to 2/3 of d(100) or d(100) itself and the direction of the steps was almost parallel to the c axis. Arachidic acids were accumulated onto the HAp crystal surfaces by a Langumuir–Boldgett method. The hydrophilic carboxyl group of arachidic acid was sufficiently adjacent to the
ACCUMULATION OF ARACHIDIC ACID ON THE HAp SURFACE
HAp surface, probably less than 0.04 nm; it was thought that a chemical interaction took place between the charged HAp surface and the hydrophilic carboxyl group of arachidic acid. REFERENCES 1. Bloom, W., and Fawcett, D. W., in “A Textbook of Histology,” p. 194. Chapman & Hall, New York, 1994. 2. Piez, K. A., in “Biochemistry of Collagen” (G. N. Ramachandran and A. H. Reddi, Eds.), p. 1. Plenum, New York, 1976.
27
3. Petty, M. C., in “Langmuir–Blodgett Films” (G. G. Roberts, Ed.), p. 133. Plenum, New York, 1990. 4. Suetsugu, Y., Takahashi, Y., Cho, S.-B., Okamura, F. P., and Tanaka, J., “Key Engineering Materials,” Vols. 132–136, pp. 2037, 1997. 5. Suetsugu, Y., Hirota, K., Fujii, K., and Tanaka, J., J. Mater. Sci. 31, 4541 (1996). 6. Kay, M. I., Young, R. A., and Posner, A. S., Nature 204, 1050 (1964). 7. Petty, M. C., “Langmuir–Boldgett Films: An Introduction.” Cambridge Univ. Press, Cambridge, UK, 1996. 8. Sato, K. et al., in preparation. 9. Carlino, S., and Hudson, M. J., J. Mater. Chem. 5(9), 1433 (1995).
The Surface Structure of Hydroxyapatite Single Crystal and the Accumulation of Arachidic Acid Kimiyasu Sato, Yasushi Suetsugu, Junzo Tanaka, Sachio Ina,∗ and Hideki Monma∗ National Institute for Research in Inorganic Materials, Tsukuba, Ibaraki 305-0044, Japan; and ∗ Kogakuin University, Hachiouji, Tokyo 192-0015, Japan Received April 12, 1999; accepted November 9, 1999
crucible (10 mmφ × 100 mm) as densely as possible. After weld sealing, the crucible was placed perpendicularly in a HIP furnace. The temperature was raised up to 1400◦ C for 3 h and kept for 1 h for the melting mixture to homogenize. Afterward, the crystal growth was performed by gradually lowering the temperature to 900◦ C at 5◦ C/h. The upper part of the crucible was about 10◦ C lower than the lower part. The crystals grown were chemically treated in an aqueous solution of 10% ammonium chloride at 80◦ C for 1 day to remove the flux and washed by pure water. The yield of crystals was about 65% of the starting materials packed into the crucible. The crystals were colorless and transparent, and their shape was mainly hexagonal, ∼1 to 2 mm in diameter and ∼3 to 5 mm in length. Larger single crystals over 10 mm in length grew partially in the middle of the crucible, and irregular and dendrite crystals grew at the bottom. Details of single crystal preparation are described in Ref. (4). The etching of the single crystal surfaces was carried out at room temperature by soaking in 0.05 N HCl aqueous solution for ∼3 to 30 min. After washing by pure water and drying in a desiccator for several hours, the surfaces of HAp crystals were observed by AFM (Digital Instruments Co., Nanoscope III) with a tapping mode using a resonant frequency of 200 kHz.
Single crystals of hydroxyapatite (HAp) were grown by a flux method using β-tricalcium phosphate and Ca(OH)2 under hot isostatic pressure. After chemical etching by 0.05 N HCl aqueous solution, the HAp crystal surfaces were observed by an atomic force microscope (AFM) and shown dominantly to consist of steps with heights corresponding to a lattice distance d(100) or its 2/3. Arachidic acid films were accumulated on the etched HAp crystal by a Langmuir–Blodgett method. From section analyses by AFM, a distance between the carboxyl groups of arachidic acid and the HAp surface was estimated to be approximately 0.04 nm, sufficiently adjacent for a chemical interaction to take place. °C 2000 Academic Press Key Words: hydroxyapatite; arachidic acid; interfacial interaction; atomic force microscope.
INTRODUCTION
Bone is the typical nanocomposite that mainly consists of inorganic apatite and organic collagen (1). It is therefore important to understand the nature of an inorganic/organic heterointerface for the production of an inorganic/organic composite with bioactive properties similar to bone. In the present study, the single crystal surfaces of hydroxyapatite (HAp) were etched at the atomic level, and arachidic acid with the same functional group as collagen, i.e., carboxyl group (2), was accumulated on the etched HAp surfaces using the Langmuir–Blodgett (LB) method (3). To understand the interaction that occurred between the HAp surface and the carboxyl group, their surface morphologies were observed with an atomic force microscope (AFM).
(2) The Accumulation of LB Thin Film The LB method was applied to accumulate arachidic acid on the HAp crystal surface. First, a chloroform solution of 1 mM arachidic acid, C19 H39 COOH, was prepared and spread on a pure water surface. A fixed surface pressure, typically 25 mN/m, was applied along the water surface to form the monolayer film of arachidic acid. Then, the HAp single crystal prepared in the water was perpendicularly pulled up by controlling the surface pressure by observing a π -A (surface pressure-surface area) curve. The carboxyl head group of arachidic acid adhered to the HAp crystal surface as it was raised up.
EXPERIMENTALS AND METHODS
(1) Crystal Growth of HAp and Surface Etching Single crystals of HAp were grown by a flux method using β-tricalcium phosphate (β-TCP; Ca3 (PO4 )2 ) and Ca(OH)2 under a hot isostatic pressure (HIP) of 50 MPa. The high pressure was needed because Ca(OH)2 decomposes into CaO and water above 580◦ C. The pressure medium was the Ar gas. Starting material was the mixture of β-TCP and Ca(OH)2 , whose ratio was 2 : 3 by weight, which was molded into cylindrical pellets 8 mm in diameter. The pellets were then packed into a platinum
RESULTS AND DISCUSSION
(1) The Surface Morphology of Etched Single Crystal Figure 1 shows the AFM images of a HAp single crystal before chemical etching. The surfaces observed proved to be (h00) by 23
0021-9797/00 $35.00
C 2000 by Academic Press Copyright ° All rights of reproduction in any form reserved.
24
SATO ET AL.
FIG. 1. The surface of as-grown HAp single crystal as observed by AFM: (a) directly after the crystal growth, (b) HAp surface left in the air for 8 days.
the X-ray diffraction analysis. Directly after the crystal growth, a step/terrace structure was observed on the surface, suggesting that the HAp crystal grew by a layer-by-layer mechanism. However, the step/terrace structure disappeared upon leaving the crystal in the air for 8 days. This indicates that, in the vicinity of the HAp surface, some ions moved from the inside to the surface and then a secondary chemical phase formed. According to an Auger electron spectroscopic experiment by Suetsugu et al. (5), calcium carbonate formed on the surface by reacting with carbon dioxide in the atmosphere, as calcium ions could move near the surface even at room temperature. We surveyed etching conditions for the HAp surface with HCl, acetic acid, etc. It was found that 0.05 N HCl aqueous solution was most effective for the atomic scale etching of the HAp surfaces. Figure 2 shows the etched surfaces as a function of soaking time. Figure 2a shows the surface after 3 min of etching. Terraces about 100 nm in width were observed and steps ran approximately parallel to the c axis. The variation of surface height (i.e., section analysis) was also shown in Fig. 2; the step heights were typically 0.60–0.65 or 0.90–0.94 nm. Figure 2b shows the surface after 10 min of etching. In this case, the terrace width became narrower than 100 nm, indicating that the dissolution had been progressing. Since the step height did not change in comparison to that in Fig. 2a, it is considered that the dissolution occurred more selectively along the horizontal direction of the terrace than along the perpendicular direction. Figure 2c shows the surface etched for 20 min. The dissolution of the terraces progressed further and the step became rectilinear. With respect to Fig. 2d, narrowness of the terrace width prevents precise section analysis. The step height of Figs. 2c and 2d, however, almost did not change in comparison with Figs. 2a and 2b. As a result of detailed section analysis, the step height √ corresponded to 2/3 of d(100) or d(100) itself (0.82 nm = 3/2a0 ). In the HAp crystal structure, ionic density changes along the
[210] axis at intervals of 1/3 and 2/3 of d(100) as shown in Fig. 3(6). The layers with higher ionic density correspond to steps observed by AFM. Therefore, the fundamental step height of the HAp crystal consisted of the 1/3 or 2/3 of d(100). However, the step height of 1/3 of d(100) was seldom observed in
FIG. 4. The AFM image and section analysis of arachidic acid accumulated on a HAp single crystal.
ACCUMULATION OF ARACHIDIC ACID ON THE HAp SURFACE
25
FIG. 2. AFM image and section analysis of HAp surfaces. The surfaces were etched for (a) 3 min, (b) 10 min, (c) 20 min, and (d) 30 min by 0.05 N HCl aqueous solution.
26
SATO ET AL.
FIG. 3. The surface state of the (h00) terraces of HAp single crystal. Positively charged layer, [Ca8 (PO4 )4 ]4+ ; negatively charged layer, [Ca(PO4 )2 (OH)2 ]4− .
our experiments; this was, for example, due to the charge neutrality between adjacent terraces or the structural instability of step edge for 1/3 step height. (2) The Adsorption State of Arachidic Acid Arachidic acid was accumulated by the LB method on the HAp crystal surface that was etched for 3 min with HCl solution. Figure 4 shows the AFM image of the accumulated surface. Though the monolayer of arachidic acid formed a planar triangular lattice on the water surface at the application of surface pressure, the AFM image in Fig. 4 did not reflect such arrangement on the water but the LB film obtained was inhomogeneous as a whole. According to the section analyses, the thickness of the built-up film was 5.1 to 5.4 nm which was approximately equal to twice the molecule length of arachidic acid, i.e., 2 × 2.7 nm (3, 7). This result suggested the formation of a bilayer film of arachidic acid in which the hydrophilic carboxyl
groups of arachidic acids faced the HAp surface as shown in Fig. 5. In general, arachidic acids are roughly perpendicularly accumulated on a substrate but slightly lean toward the HAp single crystal surface. For example, the leaning angle is about α = 20◦ for arachidic acids on a hydrophobic substrate (8). As shown in Fig. 5, we defined here d1 as a distance between the HAp surface and the carboxyl groups and d2 as an accumulation distance between arachidic acids. The distance between the two facing terminal methyl groups in the bilayer structure is 0.3 nm (9). When we suppose that α = 20◦ and d2 = 0.3 nm, the distance between the terminal carboxyl group and the HAp surface was estimated at 0.04 nm or less on the basis of the thickness of the dyad film determined, i.e., ∼5.1–5.4 nm. The length estimated for d1 is sufficiently short for a chemical interaction to occur between the carboxyl head group of arachidic acid and the HAp surface. As shown in Fig. 3, it is conjectured from Fig. 2 that the terraces parallel to the (h00) planes have charges of +4 or −4 per unit area. Even if any types of lattice relaxation take place in the terraces to reduce the surface charges, the total surface charges probably differ from terrace to terrace. The hydrophilic carboxyl groups of arachidic acids attract positively charged terraces but repulse negatively charged terraces. It is thought that such attractive or repulsive interactions are related to the inhomogeneous accumulation of arachidic acids on the HAp surfaces shown in Fig. 4. CONCLUSIONS
FIG. 5. The schematic accumulation state of arachidic acid on a HAp surface.
HAp single crystals were grown by a flux method, and the interaction of inorganic and organic substances was examined. When etched by 0.05 N HCl aqueous solution, the step height of HAp surfaces corresponded to 2/3 of d(100) or d(100) itself and the direction of the steps was almost parallel to the c axis. Arachidic acids were accumulated onto the HAp crystal surfaces by a Langumuir–Boldgett method. The hydrophilic carboxyl group of arachidic acid was sufficiently adjacent to the
ACCUMULATION OF ARACHIDIC ACID ON THE HAp SURFACE
HAp surface, probably less than 0.04 nm; it was thought that a chemical interaction took place between the charged HAp surface and the hydrophilic carboxyl group of arachidic acid. REFERENCES 1. Bloom, W., and Fawcett, D. W., in “A Textbook of Histology,” p. 194. Chapman & Hall, New York, 1994. 2. Piez, K. A., in “Biochemistry of Collagen” (G. N. Ramachandran and A. H. Reddi, Eds.), p. 1. Plenum, New York, 1976.
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3. Petty, M. C., in “Langmuir–Blodgett Films” (G. G. Roberts, Ed.), p. 133. Plenum, New York, 1990. 4. Suetsugu, Y., Takahashi, Y., Cho, S.-B., Okamura, F. P., and Tanaka, J., “Key Engineering Materials,” Vols. 132–136, pp. 2037, 1997. 5. Suetsugu, Y., Hirota, K., Fujii, K., and Tanaka, J., J. Mater. Sci. 31, 4541 (1996). 6. Kay, M. I., Young, R. A., and Posner, A. S., Nature 204, 1050 (1964). 7. Petty, M. C., “Langmuir–Boldgett Films: An Introduction.” Cambridge Univ. Press, Cambridge, UK, 1996. 8. Sato, K. et al., in preparation. 9. Carlino, S., and Hudson, M. J., J. Mater. Chem. 5(9), 1433 (1995).