Roles of fluoride on octacalcium phosphate and apatite formation on

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Roles of fluoride on octacalcium phosphate and apatite formation on amorphous calcium phosphate substrate Mayumi Iijima, and Kazuo Onuma Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b01717 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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Crystal Growth & Design

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Roles of fluoride on octacalcium phosphate and apatite formation on amorphous calcium phosphate substrate Mayumi Iijima and Kazuo Onuma* National Institute of Advanced Industrial Science and Technology Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan

*Corresponding author: Dr. Kazuo Onuma E-mail: [email protected] Tel: +81-29-861-4832 Fax: +81-29-861-6149

Abstract: We examined how the growth of octacalcium phosphate (OCP) and hydroxyapatite (HAp) on an amorphous calcium phosphate substrate is affected by low-dose fluoride (0–2 ppm) in metastable calcium phosphate solution at 37°C. In the absence of fluoride, highly oriented plate-like OCP crystals grow on the substrate while the presence of 0.7–0.8 ppm of fluoride greatly modulated their formation (the crystal sizes were smaller and their orientation was lowered) and it caused tiny plates and needles to appear on the OCP plates. There were two critical fluoride concentrations: (1) at 0.9 ppm, the OCP plates were completely replaced by HAp nanorods; (2) at 1 ppm, the structure of the HAp nanorod assembly changed from an open one to a tightly assembled one. The fluoride suppressed the OCP formation, induced HAp nanorod formation, and promoted tight nanorod assembly. The relationship between the fluoride concentration and the formation of OCP and HAp provides insight into the structural design of an OCP/HAp coating layer and should be useful in the application of fluoride to an OCP/HAp coating on any substrate.

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Roles of fluoride on octacalcium phosphate and apatite formation on amorphous calcium phosphate substrate

Mayumi Iijima and Kazuo Onuma National Institute of Advanced Industrial Science and Technology Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan

ABSTRACT As fluoride is known to affect the biomineralization of calcium phosphates, we examined how the growth at 37°C of octacalcium phosphate (OCP) and hydroxyapatite (HAp) on an amorphous calcium phosphate substrate is affected by low-dose fluoride (0–2 ppm) in metastable calcium phosphate solution. In the absence of fluoride, highly oriented plate-like OCP crystals grow on the substrate. With 0.7–0.8 ppm fluoride, OCP crystal formation was greatly modulated (the crystals were smaller and their orientation was lower); moreover, tiny plates and needles appeared on the OCP plates. There were two critical fluoride concentrations: (1) at 0.9 ppm, the OCP plates were completely


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replaced by HAp nanorods; (2) at 1 ppm, the structure of the HAp nanorod assembly changed from an open one to a tightly assembled one. The fluoride suppressed OCP formation, induced HAp nanorod formation, and promoted tight nanorod assembly. The relationship between the fluoride concentration and the formation of OCP and HAp provides insight into the structural design of an OCP/HAp coating layer and should be useful in the application of fluoride to an OCP/HAp coating on any substrate.

1. INTRODUCTION Hydroxyapatite (Ca10(PO4)6(OH)2; HAp) is biologically important because it is the prototype

of

the

bone

and

tooth

enamel

apatite.

Octacalcium

phosphate

(Ca8(HPO4)2(PO4)4·5H2O; OCP) is also biologically important calcium phosphate, because it is structurally similar to HAp: it is thermodynamically a metastable precursor of HAp and OCP, so it tends to transform into more stable HAp.1 Fluoride affects HAp formation via OCP.2,3 Even a small amount of fluoride plays a crucial role in determining the phase and morphology of the product in calcium phosphate formation.4 Previous studies relevant to tooth enamel formation have shown that the crystal phase and morphology change from ribbon-like OCP to needle-like apatite in the presence of



0.1–2.0 ppm fluoride and that an interlayered structure of OCP and HAP grows in 0.1– 1.0 ppm fluoride.5,6 In an ex vivo remineralization of human enamel, the crystal morphology and crystal organization varied from plate-like loose crystals to densely packed nanocrystal arrays depending on the fluoride concentration and supersaturation level.7,8 The effects of fluoride on the growth and dissolution of HAp are important in dentistry.9,10 A trace amount of fluoride inhibits the progression of caries by adsorbing on enamel surface and by incorporating into HAp crystals, which reduce the rate of demineralization.11,12 Fluoride also greatly promotes HAp formation.13 When HAp is synthesized in a fluoride-containing solution, fluoride is incorporated into the lattice, forming fluoride-containing HAp (F-HAp). The solubility of the F-HAp is lower than that of HAp (i.e., the supersaturation of F-HAp is higher), and the crystallinity of the F-HAp is higher than that of HAP. This results in higher stability of the F-HAp in solution than HAp.14 The higher stability has been ascribed to F− ion substituted OH− on the c-axis, which creates a hydrogen bond between OH− and F−.15 We recently found that highly oriented and tightly assembled HAp nanorods grow on an amorphous calcium phosphate (ACP) substrate if the substrate is immersed in a


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metastable calcium phosphate solution containing 10 ppm fluoride while plate-like µm-size OCP crystals grow with c-axial orientation in the absence of fluoride.16 Cross-sectional characterization of the overgrown layer revealed that, with 10 ppm fluoride, a thin intermediate layer (IL) composed of low-crystalline HAp nanoparticles formed and that HAp nanorods grew on the IL. In contrast, without fluoride, an IL did not form. Instead, small fragmented crystals formed on the substrate, and then plate-like OCP crystals grew on the fragmented crystals. Thus, our study demonstrated for the first time that fluoride affects the overgrowth process and modifies the internal structure of the overgrown layer. Furthermore, it indicated that there are critical fluoride concentrations between 0 and 10 ppm at which the overgrown layer is modified and HAp nanorods form. The present study aimed to find the minimum fluoride concentration at which HAp nanorod assemblies form and to obtain further insight into the effects of fluoride on the overgrowth of OCP and HAp on an ACP substrate. To achieve these aims, we investigated the modification of the overgrown layer on an ACP substrate at fluoride concentrations of 0–2 ppm.



2. EXPERIMENTAL SECTION All solutions were prepared using special grade reagents (Nacalai Tesque, Inc.) and Milli-Q water (total organic carbon: 3 ppb, 18.2 MΩcm; 25°C; Milli-Q, Millipore). 2.1. ACP Substrate preparation. Ice-cold 10 mM Na2HPO4 (99 ml) and 1-M CaCl2·2H2O (1 ml) were mixed with stirring. Immediately after precipitation, the suspension was filtered using a cellulose-acetate filter with 0.22 µm pores (Advance Co.) and rinsed with ice-cold Milli-Q water and 99.5% ethanol (Wako, Ltd., analytical grade). The sediment was immediately frozen at –70°C and then lyophilized and stored in a desiccator at approximately 25% humidity. The product was confirmed to be ACP using a powder X-ray diffractometer. An X-ray diffraction (XRD) profile of the ACP is shown in Fig. S1a, and a scanning electron microscopy (SEM) image of ACP powder is shown in Fig. S1b. About 10 mg of ACP powder was placed in a rectangular mold (2 (width)×10 (length) mm) and pressed at 20 MPa for 10 min at room temperature. The thickness of the compacted material was about 0.3 mm. For calcification, the compacted ACP was cut into sections about 2×2 mm using a surgical knife. SEM images of the surface and a


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cross-section of an ACP substrate are shown in Figs. S1c and S1d. All cross-sectional samples were prepared by cutting the compacted material longitudinally. 2.2.

Calcifying

solution.

Stock

solutions

of

1-M

CaCl2·2H2O,

0.5-M

K2HPO4+KH2PO4 (1:1 molar ratio), 1-M CH3COOH (acetic acid; AcH), 0.1-M CH3COONa·2H2O (sodium acetate; AcNa), and 1000 ppm NaF were made by dissolving appropriate amounts of reagents. Each solution was filtered using a cellulose-acetate filter with 0.22 µm pores (Advance Co.). AcNa (50 mM) was used as a buffering reagent for the CH3COOH. An aliquot of 1-M CaCl2 was added to a phosphate solution containing AcNa to make the concentrations of Ca and PO4 4 mM. The initial pH of the calcifying solution was adjusted to 6.2 (37°C) by adding 1-M AcH. Fluoride-containing solution was prepared by mixing stock NaF solution with this calcium phosphate solution to make solutions with various F– concentrations 0.1–1.0, 1.5, and 2.0 ppm. The supersaturation (σ) of the calcifying solution with respect to OCP and HAp were calculated17 to be 1.27 and 6.35, respectively: σ = (IP/Ksp)1/n – 1 (n = 16 and 18 for OCP and HAp, respectively), where IP is the ionic activity of the solution and Ksp is the solubility product of the material at 37°C.18



2.3. Substrate calcification. A substrate section was placed at the bottom of a vessel along with 25 ml of the calcifying solution. The vessel was covered with a tight lid and placed in an incubator (37±0.5°C) for 20 hours. The early stage of precipitation was investigated by immersing the substrate for 1, 3, 5, 10, 30, 40, and 60 min and 2 h. All reactions were carried out without stirring. After the reaction terminated, the substrate was removed from the solution, and the solution on the substrate was soaked up with filter paper. The substrate was then quickly rinsed in Mill-Q water, rinsed in 99.5% ethanol (Wako, Ltd., analytical grade), and dried in air. The substrate was stored in a desiccator at approximately at 25% humidity. 2.4. Characterization of overgrown layer. The overgrown layer on the substrate was identified using powder XRD (RINT2000, Rigaku Ltd.) and thin-film in-plane XRD (SmartLab, Rigaku Ltd.). For the powder XRD measurement (monochromated CuKα, 40 kV, 100 mA), the substrate after immersion was set in a quartz sample holder and scanned from 3° to 60° (2θ axis) with a scanning speed of 2°/min. Thin-film in-plane XRD measurements were conducted to measure the diffraction of the lattice planes perpendicular to the substrate surface. A 2×5×0.3 mm substrate was used for the measurements (monochromated CuKα, 45 kV, 200 mA). The substrate after


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immersion was set on a glass slide. It was scanned with a scanning mode of 2θχ/ϕ from 3° to 50° (2θχ) at a scanning speed of 0.1°/min and with an incident angle of 0.2°. The overgrown layer was observed using a field-emission scanning electron microscope (FE-SEM) (5 kV, JSM-7000F, JEOL, Ltd). The samples were coated with Pt (~15 nm thick) prior to observation. To analyze the lattice components in the crystals in the overgrown layer, i.e., PO43−, HPO42−, and H2O, FT-IR spectra were taken using attenuated total reflection (ATR) mode (IRTracer-100, diamond prism, Shimadzu Co.). The ATR FT-IR spectrum of the overgrown layer was obtained in the range 400–4000 1/cm with a resolution of 4 1/cm and with 40 times accumulation. Synthesized OCP, synthesized HAp (Taiheikagaku Co.), and fluorapatite (FAp, Durango, Mexico) were used as references.

3. RESULTS AND DISCUSSION 3.1. 20 hours of overgrowth in fluoride-containing solutions. Powder XRD profiles and thin-film in-plane XRD profiles of overgrown layers obtained for the various fluoride concentrations are shown in Figs. 1 and 2, respectively. In the powder XRD profiles (Fig. 1), the intensities of the (002) and (004) peaks of OCP are strong



while those of the peaks in the a-axis direction are weak. In the thin-film in-plane XRD profiles (Fig. 2), the intensity of the (002) peak is weak while those of the (100), (200), and (010) peaks of OCP and the (100) peak of HAp are strong. The crystals in the overgrown layer were thus identified as OCP or HAp. Furthermore, these peak intensities indicate that the crystals grew on the substrate with c-axis perpendicularity. In a solution containing fluoride, F− ions are incorporated into the HAp lattice, forming F-HAp, and HAp and FAp form a complete solid solution.19 In our previous study, in which an ACP substrate was immersed in calcifying solution containing 10 ppm fluoride, the average fluoride content of the overgrown layer was 2.87±0.28 atomic %.16 In the presence of 1 ppm fluoride, it is possible that less fluoride (one order less) is incorporated into the HAp lattice, forming partially fluoridated HAp. Here, however, HAp was used otherwise specially mentioned. The thin-film in-plane XRD profiles (Fig. 2) clearly show that the intensities of the (100), (200), and (010) peaks of OCP decreased with an increase in the fluoride concentration up to 1 ppm. OCP peaks were not detected at 1.5 ppm and above. On the other hand, the HAp (100) peak was observed at 0.5 ppm and above. The intensity of the (210), (211), (300), and (310) HAp peaks increased with the fluoride concentration.


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In short, the amount of OCP decreased and that of HAp increased with the fluoride concentration. The powder XRD profiles (Fig. 1) clearly show that the intensity of the (002) and (004) OCP peaks decreased between 0 and 0.7 ppm and then increased. On the other hand, the intensity of the peaks in the a-axis direction, such as (112) and (213), increased with the fluoride concentration. This indicates that the addition of fluoride changes the crystal orientation. Fig. 3 shows the relationship between the fluoride concentration and the intensity ratios of the (002) and (112) peaks (I(002)/I(112)) and those of the (002) and (213) peaks (I(002)/I(213)). These ratios conventionally represent the degree of the c-axial orientation of OCP crystals. Both ratios showed the same tendency: they decreased up to 0.7 ppm fluoride and then increased with an increase in the fluoride concentration; the degree of orientation was the lowest at 0.7 ppm. Some of the XRD profiles used to calculate the ratios are shown in Fig. S2. For reference, these ratios of HAp powder in the JCPDS file (No. 9-432) are 0.6 for I(002)/I(112) and 1.0 for I(002)/I(213). The highest ratio (57.3) and even the lowest one (2.9) are indicative of crystal orientation.



Figs. 4 and 5, respectively, show top-view and cross-sectional SEM images of overgrown layers formed using various fluoride concentrations. The major modification caused by the fluoride was a dramatic change in the morphology of the overgrown crystals. Both the top-view and cross-sectional images show that µm-size OCP plates grew when the fluoride concentration was between 0 and 0.8 ppm (Figs. 4(a–e), Figs. 5(a–d)), whereas rod-like crystals grew when the concentration was 0.9 ppm and above (Figs. 4(f–h), Figs. 5(e, f)). There were thus two critical fluoride concentrations: (1) at 0.9 ppm (and above), the OCP plates were completely replaced by HAp nanorods; (2) at 1.0 ppm, the structure of the HAp nanorod assemblies changed from an open one to a tightly assembled one. Higher magnification SEM observation of the cross-sections revealed the modification in the morphology of the OCP plates and in the internal structure of the overgrown layer caused by fluoride. Below the critical concentration, the overgrown layer was comprised of three layers with different textures (Fig. 5): a fragmentary layer (FG-L) on the substrate composed of small crystals (500 nm or less), a secondary layer (S-L) composed of plate-like µm-size crystals with rather low orientation on top of the


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FG-L, and an upper layer (UP-L) composed of long plate-like crystals with orientation higher than that of those in the S-L. To show the overall feature of the overgrown layers obtained for 0–2.0 ppm fluoride, the thicknesses of the FG-L, S-L, and UP-L in an overgrown layer are illustrated in Fig. 6. The effect of fluoride on the thickness was evident at fluoride concentration of 0.7 ppm and higher. The thicknesses of the overgrown layers composed mainly of OCP were greatly reduced for concentrations of 0.7–0.8 ppm. The thickness of the tightly assembled HAp nanorod layer was much less than that of the overgrown layers (about 8–16 µm). This is ascribed to the growth rate of HAp along the c-axis being an estimated few ten nm/h.20 The thickness of HAp nanorod layer at fluoride concentration of 0.9 ppm and higher increased with fluoride concentration, indicating that the fluoride enhanced the nanorod growth. The growth rate of OCP was at least one or two orders higher than that of HAp under the conditions of the previous study. 21 Therefore, the growth of OCP exceeded that of HAp, resulting in eventual OCP overgrowth at 0.7 and 0.8 ppm, where needle like crystals (probably HAp) concomitantly formed in the overgrown layers (see Figs. 7(c, d)). Note that the XRD intensities of the (002) and



(004) peaks were strong at 1.0 and 2.0 ppm fluoride (Fig. 1). This is ascribed to the highly oriented and tightly assembled nanorod array. The thickness of the S-L layer was roughly one-third that of the overgrown layer. For 0.9–2.0 ppm fluoride, no such layer formed; instead, highly oriented HAp nanorods grew on the FG-L. The thickness of the FG-L (1.2–1.6 µm) was not significantly affected by the fluoride. 3.2. Modification of crystal orientation by fluoride. Modification of the crystal orientation in the overgrown layer caused by fluoride can be seen in the SEM images with low magnification (Figs. 4 and 5). The change in the orientation of the plate-like crystals with increasing fluoride concentration is evident in the S-L: the orientation of the crystals decreased as the concentration of fluoride increased up to 0.7 ppm and then increased. This observation is consistent with the change in crystal orientation estimated from the XRD intensity ratios (Fig. 3). Since the thickness of the S-L was almost half that of the overgrown layer, the S-L would have contributed to XRD intensity. Thus, the intensity ratios reflect the degree of crystal orientation in the S-L. The inconsistency between the top-view image of the 1.5 ppm sample and the degree of orientation estimated from the XRD intensity is explained as follows.


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Although the stems of the apatite nanorods were highly oriented and tightly assembled, their tips were thinner than the stems and thus were slightly separated. The tips became thinner and the crystal density became lower with an increase in fluoride concentration (Figs. 5(e), (f); Figs. 4(f)–(h)). Thus, the top part of the nanorods would have bent and lost their orientation once they were removed from the reaction solution. 3.3. Modification of crystal morphology by fluoride. High-resolution SEM observation of the FG-L clearly revealed the morphological changes in the component crystals caused by fluoride (Fig. 7). Without fluoride, the FG-L was composed of regularly arranged small plates with a length of about 500 nm (Fig. 7(a)). With a slight increase in the fluoride concentration, the crystals in the FG-L changed into irregular flakes at 0.3 ppm and then into slightly elongated irregular flakes with better orientation at 0.5 ppm (Fig. 7(b)). Small needle-like precipitates were observed on these irregular flakes (white dashed arrows in Fig. 7(b)). The needle-like crystals became evident at 0.7 and 0.8 ppm (white arrows in Figs. 7(c) and (d)). At 0.8 ppm, the FG-L had an abundance of such crystals, which were partly assembled side-by-side and well arranged. Subsequent growth of larger plates of OCP crystals started from the FG-L (Figs. 7(a)–(d)). Therefore, the orientation of the crystals in the FG-L affected that of



the crystals in the S-L. This means that fluoride controlled the modification of crystal orientation. The thin-film in-plane XRD (Fig. 2) indicated concomitant formation of apatite at 0.3 ppm and above. The modification evidenced in the SEM images is the formation of a string-like texture on the (100) face of the OCP crystals (indicated by black arrows in Figs. 7(b)–(d)). A string-like texture also formed on fragmented crystals in the FG-L at 0.5 ppm (Fig. 7(b), black arrows). At 0.7 and 0.8 ppm, small needle-like crystals formed on the substrate in the FG-L (indicated by white arrows in Figs. 7 (c) and (d)). Needle-like precipitates on the irregular flakes and needle-like crystals on the substrate in the FG-L became abundant at higher fluoride concentrations. In the XRD measurement, the intensity of apatite increased with the fluoride concentration (Fig. 2). Taken together, the XRD and SEM results indicate that these needle-like crystals were apatite. A similar string-like texture on OCP plates has been observed in other experimental systems in the presence of 0.5 ppm fluoride.6

3.4. ATR FT-IR analysis. Fig. 8 shows ATR FT-IR spectra of samples with various fluoride concentrations 20 h after immersion. Synthesized OCP and OCP grew on the


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substrate without fluoride (labeled “no F” in the figure) showed the same IR absorption bands as the samples with fluoride. The bands are assigned to the lattice components: PO43−, HPO42−, and H2O.22,23 The 0.7 ppm sample, which had a plate-like morphology and XRD peaks corresponding to both OCP and apatite, exhibited a spectrum similar to that for the “no F” OCP sample, with the exception of the band at 524.6 1/cm. This band, assigned to P–O bending of HPO42−, was observed for the “no F” sample but not for the 0.7 ppm sample. The band assigned to γ

OH

(HPO4)23 was observed at 915 1/cm for the

0.7 ppm sample but not for the 1 and 2 ppm samples. Thus, a very small amount of HPO42− may have been contained in the 0.7 ppm sample. This is consistent with the XRD results (Fig. 2). Moreover, the 0.7 ppm sample showed the band assigned to the P–O stretch of PO43− at 1022 1/cm while the “no F” sample showed the band at 1011 1/cm. Interestingly, the position of this band again shifted to 1011 1/cm for the 1 ppm sample and then returned to 1021 1/cm for the 2 ppm sample although both samples showed similar needle-like morphology and XRD profiles of apatite. The unexpected modulation in the absorption band of the P–O stretch of PO43− can, in part, be ascribed to the stoichiometry of the samples and the fluoride incorporated into the lattice.



3.5. Early stage of overgrowth in fluoride-containing solution. Fig. 9 shows time-resolved SEM observation of precipitates after 1, 3, 5, 10, 30, and 60 min of immersion in solution with 0, 0.7, and 1.0 ppm fluoride. To identify the initial precipitates, thin-film in-plane XRD and ATR FT-IR measurements were conducted. Subsequent SEM observation showed that without fluoride, the precipitates were in the initial stage of flake formation within 1–10 min. A "root of tree" structure was observed at 5 min (white arrows), and then this structure changed into flakes over time.16 With 0.7 and 1.0 ppm fluoride, nanoparticles precipitated after 1 min; the amount increased for 3–5 min, and flakes with uneven edges formed after 10 min. Without fluoride, flakes with even edges formed after 30 min while with 0.7 and 1.0 ppm fluoride, irregularly shaped small flakes formed after 30 min. Thus, acceleration of the initial stage of crystal growth due to fluoride was observed. After 60 min immersion without fluoride and with 0.7 ppm fluoride, plate-like crystals formed, while with 1 ppm fluoride, small plates with a string-like texture formed on the surface, indicating the effect of fluoride. Time-resolved thin-film in-plane XRD measurement of 0.7 and 1.0 ppm samples are shown in Fig. 10. The XRD profiles for 10–60 min precipitates for both samples showed similar broad peaks at 26° and 32° (2θχ). These peaks, corresponding to the


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(002) and (211) peaks of HAp, indicate the formation of an apatite-like structure. The improvement in the peak profile with time indicates maturation of this phase, however the (100) peaks of both OCP and HAp were not observed even after 60 min. The 20 h immersion sample showed (100) peaks of OCP at 4.7° (Fig. 2). Since the intensity of the (100) peak of OCP is the strongest, this peak is generally used as the criterion for the existence of OCP. If the intensity ratio of the (100) and (211) peaks for the 1 h product is assumed to be the same as that for the 20 h product, the (100) peak should be observed with an intensity almost the same as that of the (211) peak. However, it was not observed throughout the 1 h reaction (Fig. 10). This suggests that the product in the early stage is immature HAp or immature OCP without regular periodicity in the [100] direction, presumably due to a missing HPO4–H2O portion.24 In the ATR FT-IR spectra of the 0.7 ppm samples after 1 and 20 h immersion (Fig. S3), an absorption band of HPO4 at 915 1/cm, which is an indication of an ideal OCP structure,23 was not observed. This is consistent with the XRD results. The 915 1/cm absorption band was observed only for the “no F” and 0.7 ppm samples after 20 h (Fig. 7). Taken together, the results of the thin-film in-plane XRD measurement and ATR



FT-IR spectra for the 0.7 and 1.0 ppm samples indicate that immature HAp or immature OCP without regular periodicity in the a-axis direction is likely to form. 3.6. Effect of fluoride on HAp nanorod formation. The dramatic change in the crystals in the overgrown layer between 0.8 and 0.9 ppm is indicative of the fluoride effect. Although µm-sized plate-like crystals were observed at 0.8 ppm, the XRD indicated that the main component was HAp and that the OCP content was much less than that of HAp (Fig. 2). The needle-like crystals in the FG-L and the needle-like precipitates on the OCP plates were likely HAp. It is also likely that there was partially transformed HAp (more strictly, F-HAp) in the plate-like crystals.5,24 At 0.9 ppm, HAp nanorods grew. It is likely that in this amount of fluoride, partially fluoridated HAp precipitates and grows preferentially because incorporated fluoride contributes to a decrease in Ksp and stabilizes the F-HAp.8,9 The lowest Ksp value (6.55×10−63) was obtained for the solid state phase of Ca5(PO4)3OH0.43F0.57, while the Ksp values of FAp and HAp at 37°C were determined to be 3.19×10−61 and 7.36×10−60, respectively.8 Besides the thermodynamic and structural contributions of fluoride incorporated into HAp to its growth, we speculate that fluoride in the solution changed the stability of the calcium phosphate clusters as a growth unit when ACP transformed into OCP or HAp,


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and this stability change controlled the final growth phase. Previous investigations showed that the phase transformation of ACP into HAp (and probably into OCP) proceeds via incorporation and rearrangement of calcium phosphate clusters as essential units in pseudo-physiological solutions.25,26 Ab initio calculation of the energetic stability of the calcium phosphate cluster showed that there are many isomers with different structures but with similar free energies.27 Fluoride might reduce the cluster stability needed for OCP growth and increase the stability needed for HAp growth. The F-HAp nanorods, which stood apart from each other at 0.9 ppm, were tightly assembled side by side at 1 ppm and above. A possible explanation is that fluoride above 1 ppm enhances nucleation and growth of F-HAp on the side face of already formed F-HAp. Fig. 11 shows SEM images of a cross-section of the overgrown layer obtained for 1.0 ppm fluoride. In the top part of the layer (Fig. 11(a)), there were many small crystals in the early stage of growth on the side faces of the nanorods, as indicated by the white arrows. Inspection of the fractured nanorods (Fig. 11(b)) revealed that each nanorod was composed of several tiny nanorods with thicknesses of 10–20 nm (encircled). Similar HAp nanorod assemblies have been detected in simulated body fluid containing 1 mM fluoride in a supersaturation-dependent manner,28 and the



assembly of nano HAp crystallites has been explained as self-epitaxial nucleation mediated self-assembly.29–31 In our experimental system, crystal formation occurred on an ACP substrate, and the calcifying solution was a simple calcium phosphate solution containing AcNa 50 mM, so a lower fluoride concentration might be effective. 3.7. Implications for OCP-HAp coating. The internal structure of the OCP-HAp overgrown layer, i.e., the morphology, orientation, and phase of the component crystals, was effectively and variously modified by fluoride. The layer thickness was controlled by simply changing the immersion period. The current results show that the physical properties of the coating layer, such as the thickness and topography, affect the in vivo performance of the coated material. The effect of the thickness of a HAp coating on the formation of new bone and on osteoblastic activity has been examined for the 1–200 µm thickness range32–34 and for the lower thickness range of 200 nm to 2 µm.35–36 It is notable that a thinner coating improved the initial stage of osteoinduction.36 The morphologies and sizes of the component crystals of the overgrown layer as well as their density are related to the topography of the coating layer, which is known to affect osteoblastic activity.37–39 The effect of OCP40,41, HAp42–44, and TCP45–47 crystalline or granule morphology and size on bone regenerative capacity has been demonstrated.


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Since the optimal thickness and surface structure of the coating layer differs case by case, the effects of varying the thickness and surface structure of the OCP coating layer on its in vitro and in vivo performance are yet to be investigated. The various surface structures identified in our study are applicable to the structural design of coating layers.

4. CONCLUSION This study has shown that a low level of fluoride significantly modulates the overgrowth of OCP and HAp on an ACP substrate. Fluoride modifies the morphology, orientation, and assembly of the component crystals, resulting in the modification of the internal structure of the OCP-HAp overgrown layer. At one critical fluoride concentration (0.9 ppm and above), plate-like OCP crystals were completely replaced by HAp nanorods. At another critical concentration (1 ppm and above), HAp nanorods assembled tightly side by side. These findings should be useful in the application of fluoride to calcium phosphate coating on any substrate and indicate the potential effectiveness of controlling the internal structure of an OCP coating layer.



Notes The authors declare no competing financial interests. Author contributions K. O. conceived and designed the study. M. I. and K. O. equally contributed to performing the experiments and preparing the article. Appendix A: Supporting information Supplemental data associated with this article can be found in the online version. Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS KAKENHI C; 16K04954). The FE-SEM observations and thin-film in-plane XRD measurements were, respectively, supported by the National Institute of Materials Science (NIMS) microstructural characterization platform and the University of Tokyo advanced characterization nanotechnology platform through the "Nanotechnology Platform" program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. We thank Shimadzu Corporation for performing the ATR FT-IR spectroscopy of the overgrown layers and Taiheikagaku Corporation for kindly providing the hydroxyapatite sample. We thank Dr.


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Atsuo Ito of the National Institute of Advanced Industrial Science and Technology (AIST) for calculating the supersaturation.



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FIGURE CAPTIONS Figure 1

Powder XRD profiles of overgrown layers obtained for various fluoride

concentrations. Due to c-axial orientation of crystals, (002) and (004) peaks were intensified. Figure 2

Thin-film in-plane XRD profiles of overgrown layers obtained for various

fluoride concentrations. Diffraction plane indices attributed to OCP are shown in blue, and those to HAp are shown in brown. Figure 3

Relationships between intensity ratios of (002) and (112) peaks (I(002)/I(112))

and of (002) and (213) peaks (I(002)/I(213)) and fluoride concentration. Figure 4

Top-view SEM images of ACP substrate (a) in absence of fluoride and in

presence of (b) 0.3, (c) 0.5, (d) 0.7, (e) 0.8, (f) 0.9, (g) 1.0, and (h) 1.5 ppm fluoride after 20 h immersion. Small plate-like crystals (indicated by arrows) are evident on OCP plates in (b), (c), (d), and (e). Note drastic change in morphology between (e) and (f). Figure 5

Cross-sectional SEM images of overgrown layers obtained after 20 hours (a)

without fluoride and with (b) 0.5, (c) 0.7, (d) 0.8, (e) 0.9, and (f) 1.0 ppm fluoride. Note drastic change in morphology between 0.8 and 0.9 ppm. In (a), (b), (c), and (d), overgrown layers clearly contain three layers with different textures. These layers are



referred to as FG-L (fragmentary layer), S-L (secondary layer), and UP-L (upper layer). In (a), FG-L is too thin to be observed while it is evident in (b), (c), and (d). Dashed lines in (e) and (f) show approximate boundaries between substrate and FG-L and between FG-L and S-L or UP-L. Figure 6

Thicknesses of FG-L, S-L, and UP-L in the overgrown layer obtained after

20 hours in the presence of 0–2.0 ppm fluoride. Standard deviation for total thickness of the overgrown layers is indicated by magenta line on top of each bar. Deviations for 0.9 ppm and higher were negligible and are thus omitted. Thickness measurements were performed at a minimum of ten positions, and average and standard deviations of data were calculated for each fluoride concentration. Figure 7

High-resolution SEM images of overgrown layer near boundary of substrate

and overgrown layer (a) without fluoride and with (b) 0.5, (c) 0.7, (d) 0.8, (e) 0.9 and (f) 1.0 ppm fluoride. Fragmentary layer (FG-L) and S-L (secondary layer) are indicated except in (e) and (f), where S-L layer is not evident. Specific string-like texture caused by fluoride on OCP plates is indicated by black arrows in (b), (c), and (d). Needle-like precipitates on fragmented crystals are indicated by white dashed arrows in (b). Small


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needle-like crystals observed around top of FG-L in (c) and (d) are indicated by white arrows. Figure 8

ATR FT-IR spectra of overgrown layers on ACP substrate obtained after 20

h for fluoride concentrations of 0, 0.7, 1.0, and 2.0 ppm. For reference, spectra of synthesized OCP, synthesized low-crystalline HAp, and (001) face of mineral FAp are also shown. Figure 9

Time-resolved SEM images of overgrown layers without fluoride and with

0.7 and 1.0 ppm fluoride. White arrows show root of tree locations. Time-resolved thin-film in-plane XRD measurement of precipitates with

Figure 10

(a) 0.7 and (b) 1.0 ppm fluoride. To enable comparison of product intensities for the various times, the axis scales in (a) and (b) were set equal. The diffraction indices superimposed in each figure, (002) and (211), correspond to those of HAp, but this does not necessary mean that the grown material was HAp. Immature OCP has the same peak pattern. Figure 11

Cross-sectional SEM images of HAp nanorod assembly obtained after 20 h

in presence of 1.0 ppm fluoride. (a) Top part of overgrown layer. Crystals in early stage of growth on side face of nanorods are indicated by white arrows. (b) Higher



magnification image. Fractured nanorods (encircled) show that each nanorod was composed of several tiny nanorods with thickness of 10–20nm.


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For Table of Contents Use Only

Roles of fluoride on octacalcium phosphate and apatite formation on amorphous calcium phosphate substrate Mayumi Iijima and Kazuo Onuma* National Institute of Advanced Industrial Science and Technology Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan

*Corresponding author: Dr. Kazuo Onuma

Synopsis Relationship between fluoride concentration and grown materials on ACP substrate. Critical concentration less than 0.9 ppm of fluoride resulted OCP growth but it was inhibited with concentration. Above 1 ppm, tightly assembled HAp nanorods like as tooth enamel forms on the substrate.



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Roles of fluoride on octacalcium phosphate and apatite formation on

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