Particle-size-dependent octacalcium phosphate overgrowth on β-tricalcium phosphate substrate in calcium phosphate solution

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Particle-size-dependent octacalcium phosphate overgrowth on β-tricalcium phosphate substrate in calcium phosphate solution Mayumi Iijima, Kazuo Onuma

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Biomaterial Research Group, Division of Health Research, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan

A R T I C L E I N F O

A B S T R A C T

Keywords: Octacalcium phosphate β-tricalcium phosphate Hydroxyapatite Coating

The particle size of ceramics affects their performance in both in vitro and in vivo reactions. We evaluated the effects of the particle size of β-tricalcium phosphate (β-Ca3(PO4)2; β-TCP) on octacalcium phosphate (Ca8(HPO4)2(PO4)4·5H2O; OCP) overgrowth on a β-TCP substrate by using two types of β-TCP substrate, one composed of micrometer-sized particles and one composed of nanometer-sized particles (micro-TCP substrate and nano-TCP substrate), under physiological conditions. When the β-TCP substrate was immersed in a simple calcium phosphate solution, it was quickly covered with OCP. The morphology and size of the OCP crystals, as well as the structure, thickness, and crystal density of the overgrown OCP layer, depended on the β-TCP particle size. When the nano-TCP substrate was immersed, string-like (S) precipitates were initially deposited, and then flake-like (F) crystals formed on them. Plate-like (PL) OCP crystals grew on the flake-like crystals, and a threelayer structure (S-layer/F-layer/PL-layer) was formed, while no such structure was observed on the micro-TCP substrate. Small amounts of tiny OCP crystals and HAp-nanofibers precipitated in the micro-TCP substrate, whereas only HAp-nanofibers were observed in the nano-TCP substrate. These findings will facilitate the structural design of OCP-coating layers on a β-TCP scaffold.

1. Introduction β-tricalcium phosphate (TCP) is a promising bone graft substitute for reconstruction of bone defects [1,2] because of its biocompatibility, osteoconductivity, and resorbability by osteoclastic cells [3–6]. It was developed to overcome the clinical problems of autogenous bone, i.e., its limited availability and difficulties with its general application. βTCP has been applied in the form of granules and three-dimensional (3D) scaffolds [3,7,8] in combination with hydroxyapatite (HAp) [9,10] or organic materials [11,12]. The biocompatibility and degradability of conventional micrometer-sized β-TCP powder have been improved by the use of nanometer-sized β-TCP [13,14]. Octacalcium phosphate (OCP), on the other hand, also has been proven to have promising osteoconductive characteristics. Implanted OCP granules have provided cores for nucleating multiple osteogenic sites [15,16], better enhance bone regeneration than HAp and β-TCP, and are more resorbable than implanted HAp and β-TCP [17,18]. In a comparative study of implantation for 56 days using well grown but non-stoichiometric OCP, a slightly hydrolyzed OCP, a fully hydrolyzed OCP, and β-TCP, the β-TCP had the highest bone formation rate at day 14 while the slightly hydrolyzed OCP had the highest rate at day 56

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because it suppressed early osteoclast activity and reduced inflammation [19]. Since OCP is a metastable phase of HAp and tends to transform into HAp spontaneously [20], it is of particular interest to hypothesize that the intrinsic properties of OCP are responsible for its excellent performance in vivo [15,16]. Although a potential use of OCP granule is as a micro scaffold [21], it is rather difficult to form OCP granules into any shape. In contrast, βTCP granules have been used to form three-dimensional scaffolds. Since both formability and osteoconductivity are important characteristics of materials to be used as a scaffold, the combined usage of β-TCP with its better formability and OCP with its better osteoconductivity would boost the potential of both materials as a bone graft substitute. A practical way to achieve this is coating β-TCP scaffolds with OCP rather than forming scaffolds using a mixture of the two materials. OCP coating is promising for many applications. For example, OCP coating is better than HAp coating at promoting bone formation [22,23]. Coating titania with thin ribbon-like OCP crystals greatly improves its biomineralization ability in simulated body fluid (SBF) [24]. OCP coating enhanced MC3T3-E1 cell proliferation, alkaline phosphatase activity, and extracellular matrix mineralization after 14 days of culturing. Crystalline OCP-coated Ti6A14V disks had a larger volume of

Corresponding author. E-mail address: [email protected] (K. Onuma).

http://dx.doi.org/10.1016/j.ceramint.2017.10.167 Received 15 August 2017; Received in revised form 16 October 2017; Accepted 24 October 2017 0272-8842/ © 2017 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

Please cite this article as: Iijima, M., Ceramics International (2017), http://dx.doi.org/10.1016/j.ceramint.2017.10.167

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30 min at room temperature to a thickness of ~ 0.3 mm, forming substrates with micrometer-size particles (micro-TCP substrates). About 10 mg of the milled β-TCP powder ranging in size from ~ 10 to ~ 100 nm was similarly molded at 20 MPa for 10 min, forming substrates with nanometer-sized particles (nano-TCP substrates). The surfaces and cross-sections of the micro- and nano-TCP substrates are shown in Fig. S2. The surface reactivity of the two types of β-TCP substrate was examined using ex-situ time-resolved atomic force microscopy (AFM) with a MulitiMode-8 AFM (Bruker AXS GmbH) with a silicon single-crystal cantilever (spring constant = 0.4 Nm–1) used in peak-force tapping mode. Substrate samples were cut into Section ~2 mm × ~2 mm and immersed in standard calcifying solution (pH = 6.2) for 10, 30, or 50 s. A fixed surface area of each calcified sample was continuously observed. Typical scanning conditions were 3 Hz and 512 scan lines. A section profile of the surface was obtained using NanoScope Analysis software (ver. 1.4, Bruker AXS GmbH) after correcting for inclination of the raw image.

newly generated bone formation after implantation in mice [25]. The wet-chemical deposition method was developed to overcome the drawbacks of physical coating techniques. The advantages of this method are that (1) it can use a biomimetic solution [26–29], (2) it enables low-crystalline HAp and/or OCP to be deposited on metallic substrates [28,30–32], and (3) it is useful for coating 3D scaffolds having irregular shapes and size variations. Many of the previous studies of OCP coating on a pre-treated Ti substrate were conducted using a solution with a rather complex composition or a two-step reaction process, e.g., soaking in SBF for 2 h and subsequent soaking in SBF for 48 h to obtain 20–30-μm-long OCP crystals on a substrate [29,32]. Practical applications require that both the solution composition and coating method be simple. It has been shown that a simple calcium phosphate solution can form HAp, HAp/ OCP, and OCP precipitates on Ti at 37 °C [28,33]. Thus, OCP coating should provide β-TCP scaffolds with better biocompatibility and better osteoconductivity. But first, a contradiction must be resolved. In studies comparing the coating of several ceramics (HAp/β-TCP [34], sintered BioglassR, AW glass-ceramics, HAp, α-TCP, β-TCP, and HAp/α-TCP [35]) in SBF at 37 °C, β-TCP was much poorer at inducing calcium phosphate formation both in vitro and in vivo, whereas OCP was deposited on the other ceramics under the same conditions. The β-TCP did not show calcium phosphate deposition even after five days of immersion in SBF and even after implantation in rabbit muscles for six weeks [35]. The present study was designed (1) to examine OCP formation on a β-TCP substrate in a simple calcium phosphate solution, (2) to investigate the structure of the coating layer under various conditions, and (3) to evaluate the effect of the particle size of the β-TCP substrate on the overgrowth of OCP under physiological conditions. For these purposes, micrometer- and nanometer-sized β-TCP particles were used, and time-resolved observations of the early stage of the coating reaction were performed.

2.2. Calcifying solution The calcifying solution was prepared using special grade reagents (Nakalai Tesque, Inc.) and Milli-Q water (total organic carbon: 3 ppb, 18.3 MΩcm (25 °C); Milli-Q, Millipore). Stock solutions of 1-M CaCl2·2H2O, 0.5-M K2HPO4+KH2PO4 (1:1 M ratio), 1-M MgCl2·6H2O, 1-M CH3COOH, 0.1-M CH3COONa·2H2O (AcNa), and 50 mM Tris (trishydroxymethyl-aminomethane)-HCl were made by dissolving appropriate amounts of reagents. Each solution was filtered using a celluloseacetate filter with 0.22-μm pores (Advance Co.). As a standard calcifying solution, 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 5 mM. The initial pH of the standard calcifying solution was adjusted to 6.2 by adding 1-M AcH. To examine the effect of the Ca/PO4 ratio and pH of the standard calcifying solution and co-existing ions on OCP overgrowth, several reactions were conducted in an AcNa-AcH buffered solution with a Ca/ PO4 ratio of 1.33, or 1.67 and a pH of 6.2, in an AcNa-AcH buffered solution with a pH of 5.5, in a 50-mM Tris-HCl buffered solution with and without 142 mM NaCl and 1.5 mM MgCl2 with a pH of 7.4. The ionic concentrations of the calcifying solution are listed in Table 1 along with the degree of supersaturation (σ) with respect to DCPD, β-TCP, OCP, and HAp; σ = (IP/Ksp)1/n (n = 2, 5, 16, and 18 for dicalcium dehydrate (DCPD), β-TCP, OCP, and HAp, respectively). σ was calculated using the ionic concentrations (Table 1) and the Ksp (37 °C) for DCPD, β-TCP, OCP, and HAp [36].

2. Materials and methods 2.1. Substrate preparation Reagent grade β-TCP powder (Wako, Ltd., analytical grade) was used to form the substrates. Powder X-ray diffraction (XRD) measurements confirmed that the powder was free of other calcium phosphates (Fig. S1(a)). For use in forming substrates with nanometer-sized particles, a portion of the powder was ground using a bead mill (AIMEX Co., Ltd.), dispersed in 99.5% ethanol, and milled with zirconia beads until the particle size reached ~ 100 nm. The milled powder was centrifuged in 99.5% ethanol at 3,000 rpm for 15 min at room temperature. The resulting nanometer-sized particles were centrifuged in 99.5% supernatant ethanol at 15,000 rpm at 4 °C for 15 min. The sediment was lyophilized and stored in a desiccator at –25% humidity. Scanning electron microscopy (SEM) observations revealed that the particles ranged in size from ~10 to ~100 nm. The XRD peaks of the nanometersized β-TCP were broad because of the small particle size (Fig. S1(b)). About 10 mg of the unmilled β-TCP powder ranging in size from 0.5 to 3 µm was placed in a mold (2 × 10 mm) and pressed at 40 MPa for

2.3. Substrate calcification A 25-ml portion of the standard calcifying solution was placed in a plastic vessel and pre-heated in an incubator (37 ± 0.5 °C). A substrate section was then placed at the bottom of the vessel. The vessel was covered with a tight lid and placed in an incubator (37 ± 0.5 °C) for 20 h. The early stage of precipitation was investigated by time-resolved

Table 1 Ionic concentration of calcifying solution and degree of supersaturation (σ) with respect to DCPD, β-TCP, OCP, and HAp. pH

Ca2+

PO4

Ca/PO4

Na+

K+

Cl-

Ac-

Mg2+

σ DCPD

σ β-TCP

σ OCP

σ HAp

5.5 6.2 6.2 6.2 7.4 7.4 7.4

7.50 5.00 4.65 5.00 1.20 2.50 2.50

7.35 4.90 3.50 3.00 1.18 1.00 1.00

1.02 1.02 1.33 1.67 1.02 2.50 2.50

48.4 49.2 49.3 49.4 – 142 142

11.0 7.35 5.25 4.50 1.76 1.50 1.50

15.0 10.0 9.30 10.0 39.0 184 187

58.1 50.0 51.8 51.4 – – –

– – – – – – 1.50

0.80 0.91 0.58 0.52 0.15 −0.0092 −0.0137

0.23 1.88 1.44 1.40 3.49 3.27 3.25

0.47 1.72 1.34 1.30 2.17 1.96 1.95

2.71 7.82 6.68 6.60 15.2 14.8 14.8

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Fig. 1. Time-resolved SEM observations of micro-TCP substrate after immersion in standard calcifying solution for (a) 1 min, (b) 3 min, (c) 10 min, (d) 20 min, (e) 30 min, and (f) 40 min. Note that crystals were deposited directly on β-TCP particles. (g) Thin-film in-plane XRD profiles of substrates after 10-, 30-, 40-, and 60-min immersion. XRD peaks of (100), (200), and (010) of OCP (JCPDF 26–1056) are labeled; characteristic peaks of β-TCP (JCPDF 09-0169) are marked with * in 10-min profile. Arrow in profile of substrate after 30-min immersion indicates shoulder at 4.7°.

Microbeam X-rays were irradiated parallel to the overgrown layer on the substrate by using a collimator with a diameter of 100 µm. The diffraction was recorded on an imaging plate; the digital data on the imaging plate were converted into a 2θ-intensity relationship by using DISPLAY software (Rigaku Ltd.). For the thin-film in-plane XRD measurement (monochromated CuKα, 45 kV, 200 mA), a 2 × 5 × 0.3-mm substrate was used. The substrate after immersion was set on a glass slide and scanned using a scanning mode of 2θχ/φ from 3° to 50° (2θχ) at a scanning speed of 0.1°/min and at an incident angle of 0.2°. The substrates after immersion were 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. The growth rate of OCP crystals in the c-axis direction on the micro- and nano-TCP substrates was estimated by measuring the thickness of the overgrown layer after immersion in the standard calcifying solution for 1, 3, 5, or 20 h. Transmission electron microscopy (TEM) (200 kV, Tecnai Osiris, FEI Co.) and selected area electron diffraction (SAED) measurements of a 200 nm ϕ area were performed. For the TEM measurement, the substrate was embedded in epoxy resin and solidified at 50 °C for 72 h. Thin sections (~ 100 nm) of the resin block were prepared using the focused ion beam (FIB) technique with a Ga ion source (FB-2100, Hitachi Co. Ltd.).

observation of the substrate after immersion in the calcifying solution for 1, 3, 5, 10, 20, 30, 40, or 60 min. After the reaction terminated, the substrate was removed from the solution, and the solution remaining on the substrate was soaked up with filter paper. The substrate was then quickly rinsed in Mill-Q water. After the water was removed, the substrate was rinsed in 99.5% ethanol (Wako, Ltd., analytical grade) and dried in air. The substrate was stored in a desiccator at –25% humidity. 2.4. Characterization of overgrown crystals and substrates The precipitate on the substrate was identified using powder XRD (RINT2000, Rigaku Ltd.), microbeam XRD (RAPID, 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) at a scanning speed of 2°/min. Since OCP crystals grow with the c-axis perpendicular to the substrate, the powder XRD patterns showed intense (002) and (004) peaks and did not show (100), (200), or (010) peaks. The diffraction of the lattice planes perpendicular to the substrate surface was measured using microbeam XRD (RAPID, Rigaku Ltd.) and thin-film in-plane XRD (SmartLab, Rigaku Ltd.). For the microbeam XRD measurement (CuKα, 50 kV, 30 mA), a cross-section sample (0.2–0.4-mm thick) cut perpendicular to the substrate was prepared and fixed on a borosilicate-glass capillary (WJM-Glas/Müller GmbH Ltd.). 3

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particles in the nano-TCP substrate, or precipitation did not occur in either surface before 1 min. Sequential AFM images of micro- and nanoTCP substrates after 10-, 30-, and 50-s immersion are shown in Fig. S3(a) and S3(b).

3.1.2. Overgrowth on micro-TCP substrate Time-resolved SEM and thin-film in-plane XRD measurements of substrates in the early stage (after 1-, 3-, 10-, 20-, 30-, and 40-min immersion) are shown in Fig. 1. After 1-min immersion (Fig. 1(a)), there were sparse island-like aggregates of string-like precipitates, which gradually grew into small flakes (3, 10 min; Fig. 1(b), (c)). These flakes increased in size (20, 30 min; Fig. 1(d), (e)) and grew into platelike crystals that fully covered the substrate surface after 40 min (Fig. 1(f)). Several irregularly shaped crystals grew parallel to the substrate (* in Fig. 1(e) and (f)). In the thin-film in-plane XRD profiles of the 10- and 30-min immersion substrates (Fig. 1(g)), the observed peaks are ascribed to the βTCP substrate; there are no peaks corresponding to an overgrown layer although there is a shoulder peak at 4.7° (2θ) (indicated by arrow) in the 30-min profile. The XRD peaks at 4.7° and 9.4–9.7° (2θ) in the 40and 60-min profiles correspond to the (100), (200), or (010) peaks of OCP (JCPDF 26-1056). This indicates that the plate-like crystals evident in Fig. 1(e) and (f) were OCP. The intensity of the β-TCP XRD peaks (* in the 10-min profile in Fig. 1(g)) decreased as the OCP overgrowth increased. The cross-sectional SEM image of the substrate after 30-min

Fig. 2. Cross-sectional SEM image of micro-TCP substrate after 30-min immersion. Note that at this stage of overgrowth, a shoulder peak of OCP was observed in the thin-film XRD profile (Fig. 1(g)). Plate-like crystals that grew into the substrate are indicated by arrows, and small crystals that formed inside the substrate are encircled.

3. Results 3.1. Early stage of overgrowth in standard calcifying solution 3.1.1. Surface reactivity of micro- and nano-TCP substrates The ex-situ time-resolved AFM observations after 10-, 30-, or 50-s immersion did not reveal any changes in the surfaces of the two types of substrate. This indicates that dissolution of the TCP particles, which might occur due to the change in solubility caused by atomizing the

Fig. 3. Time-resolved SEM observations of nano-TCP substrate after immersion in standard calcifying solution for (a) 1 min, (b) 3 min, (c) 5 min, (d) 10 min, (e) 20 min, and (f) 40 min (g) Thin-film in-plane XRD profiles of substrates after 5-, 30-, 40-, and 60-min immersion. XRD peaks of (100), (200), and (010) of OCP (JCPDF 26-1056) are labeled; characteristic peaks of β-TCP (JCPDF 09–0169) are marked with * in 5-min profile.

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Fig. 4. SEM images of micro-TCP substrate after 60-min immersion. (a) Top-view and (b) cross-sectional view. Plate-like crystals (PL) grew on thin layer of flake-like crystals (F), under which had formed a thin layer of strings (S) on the substrate, thereby forming an overgrowth layer with a three-layer structure (S-layer/F-layer/PL-layer). Dotted lines are guides for the eye.

immersion shown in Fig. 2 shows that plate-like crystals grew directly on the micro-TCP particles and that small plate-like crystals grew into the substrate (arrows in Fig. 2). Small crystals formed on and between the β-TCP particles (encircled in Fig. 2). Fig. 5. (a) Powder XRD and (b) thin-film in-plane XRD profiles of micro-TCP substrate after 20-h immersion in standard calcifying solution with Ca/PO4 ratio of 1.0.

3.1.3. Overgrowth on nano-TCP substrate Time-resolved SEM and thin-film in-plane XRD measurements of substrates in early stage (after 1-, 3-, 5-, 10-, 20-, and 40-min immersion) are shown in Fig. 3. After 1-min immersion (Fig. 3(a)), there were particles with a size of 10–20 nm on the substrate. These particles fused into strings (3 min; Fig. 3(b)), which subsequently fused lengthwise (5 min; Fig. 3(c)) and formed flakes (10 min; Fig. 3(d)). These flakes were distributed homogenously on the substrate and covered the whole surface. With longer immersion, the flakes grew (20. 40 min; Fig. 3(e), (f)). The SEM image in Fig. 4(a) shows that, after 1 h, plate-like crystals homogeneously covered the substrate. The cross-sectional view in Fig. 4(b) shows that the plate-like crystals (PL in Fig. 4(b)) grew on a thin layer of flake-like crystals (F), under which a thin layer of strings (S) had formed (corresponding to Fig. 3(a)–(f)). The overgrown layer thus formed a three-layer structure: string (S)-layer / flake (F)-layer / plate (PL)-layer. In the thin-film in-plane XRD profiles of the 5-, 30-, 40-, and 60-min immersion substrates (Fig. 3(g)), the broad peaks at 26° (2θ) and 31° (2θ) increased with time. The shoulder peak at 4.7° (2θ) (indicated by the arrow) and the small peak at 9.7° (2θ) in the 40-min profile became more pronounced in the 60-min profile. The (100), (200), and (010) peaks of OCP indicated that the plate-like crystals in Fig. 3(f) and Fig. 4 were OCP. The peaks marked with * in the 5-min profile are ascribed to the β-TCP substrate.

Table 2 Thickness of overgrown layer on micro- and nano-TCP substrates after 20-h immersion in calcifying solution for three Ca/PO4 ratios and pH of 6.2. Ca/PO4

1.0

1.33

1.67

micro-TCP substrate nano-TCP substrate

75 µm 66 µm

57 µm 42 µm

52 µm 31 µm

3.2. Substrate immersion in standard calcifying solution for 20 h 3.2.1. Overgrown layer formation After 1-h immersion of both substrates in the standard calcifying solution, the OCP crystals were 4–6 µm long. As the immersion period increased, the crystals grew in the c-axis direction. On the whole, the thickness on both substrates increased in the same manner; 3 h was enough to form a 10-μm-thick layer; the layer was 15–20 µm thick after 5 h and 66–70 µm thick after 20 h (Table S1). The growth rate of OCP estimated from the sectional SEM images is ~ 1 µm/h (Fig. 4(b)). The powder XRD profiles of both substrates after 3-, 5-, and 20-h immersion exhibited an increase in the intensities of the (002) and (004) peaks of OCP (Fig. S4), corresponding to the increase in the thickness of the overgrown layer. Fig. 5(a) and (b), respectively, show powder XRD and thin-film inplane XRD profiles of the micro-TCP substrate after 20-h immersion in the standard calcifying solution. In the powder XRD profile (Fig. 5(a)), the (002) and (004) peaks are strong, while the other reflections are very weak. In the thin-film in-plane XRD profile (Fig. 5(b)), the peaks in 5

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Fig. 6. 2θ-intensity converted microbeam XRD profile of (a) micro-TCP substrate and (b) nano-TCP substrate after 20-h immersion in standard calcifying solution. XRD peaks of OCP (JCPDF 26-1056) are shown in middle plot in (a), those of HAp (JCPDF 9-0432) are shown in middle plot in (b), and those of β-TCP (JCPDF 09-0169) are shown in bottom plots.

TEM image in Fig. 7(c1) shows that the d spaces were 0.815 and 0.345 nm (blue and magenta lines, respectively, in Fig. 7(c2)). Thus, HAp nanofibers also precipitated inside the substrate. Microbeam XRD measurement of the nano-TCP substrate indicated the precipitation of HAp (Fig. 6(b)). TEM observation revealed that fine fibers surrounded the nanoparticles (Fig. 8a and (b)), which was not revealed by SEM. The texture of the substrate resembled that of the micro-TCP substrate (Fig. 7(a)) while there were more fibers than in the micro-TCP substrate. The SAED image of the circled area in Fig. 8(b) shows the pattern of TCP (Fig. 8(c)). The HR-TEM image of a fiber in Fig. 8(d) shows spacing of 0.346 nm in the fiber axis direction. These observations show that HAp nanofibers formed in the nano-TCP substrate during 20-h immersion,

the a-axis direction, (100), (200), (010), are strong, while the peaks in the c-axis direction, (002), is weak. This difference is due to the c-axial orientation of the OCP crystals on the substrate. Combined measurement of both XRDs enabled the oriented overgrown crystals to be identified as OCP. The thickness of the overgrown layer depended on the calcifying solution composition, whereas all the components were identified as OCP by the XRD measurements (data not shown). Table 2 shows the thickness of the overgrown layer, which was measured using the SEM images, after 20-h immersion in a solution with a Ca/PO4 ratio of 1.0, 1.33, or 1.67. For each ratio, the overgrown layer was thicker on the micro-TCP substrate than on the nano-TCP substrate.

3.2.2. Precipitates in the substrates SEM observation of the micro-TCP substrate showed that tiny platelike crystals grew on and between the particles after 30-min immersion (Fig. 2). As the immersion period was increased, the number of these crystals increased. A cross-sectional view of the micro-TCP substrate after 20-h immersion is shown in Fig. S5. Microbeam XRD focused on the central part of the substrate (Fig. 6(a)) revealed OCP formation in the substrate. TEM observation showed tiny thin plates about 500 nm long and less than 100 nm thick (magenta arrows in Fig. 7(a)). It also revealed fine fibers deposited around rather small β-TCP particles with sizes less than 500 nm (blue arrows in Fig. 7(a)). The SAED pattern of the fibers (encircled in Fig. 7(a)) gave the diffraction spots corresponding to the (002) and (004) planes of HAp (Fig. 7(b)). The width of the fibers was about 10 nm (Fig. 7(c1)). The fast Fourier transformation (FFT) of the HR-

3.3. Substrate immersion in pH 7.4 calcifying solution for 20 h In the Tris-HCl buffered solution with pH 7.4 (Table 1), plate-like crystals grew on both substrates (Fig. 9). The crystals were identified as OCP (Fig. S6) from the thin-film in-plane XRD measurement. Crosssectional SEM observations of both substrate types (Fig. 9(a2), (a3), (b2), and (b3)) showed that OCP crystals grew directly on the micrometer-sized particles while on the nano-TCP substrate, OCP crystals in micrometer-size grew on the thin layer of flake-like crystals (F-layer), under which strings (S-layer) had formed, constructing the aforementioned three-layer structure (Fig. 9(b3)) and that small crystals grew inside the micro-TCP substrate (Fig. 9(a3)). In the presence of NaCl (142 mM), OCP crystal growth was suppressed on both substrates (data not shown). 6

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Fig. 7. TEM observation and SAED measurement of micro-TCP substrate after 20-h immersion in standard calcifying solution. (a) Bright field image. Blue and magenta arrows, respectively, indicate HAp nanofibers and tiny plate-like OCP crystals. (b) SAED pattern of aggregate of fibers in region circled in (a). (c1) HR-TEM image of a fiber, and (c2) FFT of (c1). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

4. Discussion

OCP crystal growth was greatly suppressed in Tris-HCl buffered solution with NaCl or NaCl+Mg (Table 1). In the presence of NaCl, small plate-like OCP crystals formed sparsely on the micro-TCP substrate (Fig. 10(a1)), while small flakes on the nano-TCP substrate were connected (Fig. 10(b1)). Mg, in coexistence with NaCl at pH 7.4, synergistically disturbed the formation of OCP plates on the substrate. In the presence of NaCl and Mg, sparse island-like aggregates of small flakes formed on the micro-TCP substrate (Fig. 10(a2)) while a wall-like structure formed on the nano-TCP substrate (Fig. 10(b2)).

The present study demonstrated that both micro-and nano-TCP substrates were coated with OCP in a simple calcium phosphate solution within a short period, even in a solution with NaCl at pH 7.4 and in the presence of Mg, which is a well-known growth inhibitor of OCP [37]. Overgrown layers that formed both in an acidic and in a basic solution had characteristic long plate-like OCP crystals with high crystallinity while overgrown layer that formed in a basic solution in the presence of NaCl and Mg had flake-like OCP crystals with XRD profiles inferior to the profiles of those grown in an acidic solution. A previous study showed that slightly hydrolyzed OCP has a higher bone formation rate than well grown OCP and fully hydrolyzed OCP [19]. With the present protocol to precipitate OCP, it is possible to control the morphology and crystallinity of the OCP simply by changing the solution composition. Varying the particle size of the β-TCP had great effect on the early stage of overgrowth and precipitates inside the substrates, as explained below. The initial precipitate on the micro-TCP substrate was string-like materials, while that on the nano-TCP substrate was nanometer-scale particles, which subsequently changed into flake-like crystals. We deduced the phase of the initial precipitates on both substrates from characterization of the substrates after 20-h immersion in the standard calcifying solution. That is, the microbeam XRD patterns and HR-TEM

3.4. Substrate immersion in pH 5.5 calcifying solution for 20 h Micrometer-sized plate-like dicalcium phosphate dehydrate (DCPD) grew on both substrates when immersed in calcifying solution with a pH of 5.5 for 20 h (Fig. S7(a) and (b)). The plates that formed on the micro-TCP substrate were much larger than those that formed on the nano-TCP substrate, and fewer plates formed on the micro-TCP substrate. The formation of strings (after 3 min: Fig. S7(c)) and small flakelike crystals (after 5 and 10 min: Fig. S7(e) and (f)) proceeded simultaneously with the DCPD formation on the nano-TCP substrate. After 20 h, these flakes had grown into small plates with lengths of about 500 nm. Although the XRD measurements showed only peaks of DCPD (Fig. S7(g)), formation of OCP was suggested.

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Fig. 8. TEM observation and SAED measurement of nano-TCP substrate after 20-h immersion in standard calcifying solution. (a) Bright field image. White, blue, and magenta arrows, respectively, indicate nano-TCP particle, HAp nanofibers and tiny plate of OCP. (b) Magnified image of area around a nano-TCP particle surrounded by fibers, (c) SAED pattern of circled area, and (d) HRTEM image of a fiber. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

precipitating phase is the size of the β-TCP particles. The factors that determine the precipitating phase are the structural relationship between OCP–β-TCP and HAp–β-TCP and their interfacial tension (γSL). Fig. 11 shows the structural relationship between HAp and β-TCP and that between OCP and β-TCP for two cases: (001)OCP // (110)β-TCP and (001)HAp // (110)β-TCP. The diagrams were drawn using VESTA software. Because the (110) plane of β-TCP has both a small Miller index and a large interplanar spacing, it could be the plane most likely to develop. Since the β-TCP microcrystals and nanoparticles were randomly packed in the substrate surface, we assumed that the (110) plane mainly appears in the substrate surface. Since most of the HAp nanofibers and plate-like OCP grew perpendicular to the surface of the β-TCP crystal, their c-axs were assumed to be perpendicular to the β-TCP crystal. Thus, the lattice miss-match between the (001)OCP plane and (110)β-TCP plane, and that between the (001)HAp plane and (110)β-TCP plane were calculated using the unit cell dimensions of OCP [39], HAp [40], and β-TCP [41]. The mismatch between the a-axis of OCP and the c-axis of β-TCP was 5.35% (corresponding to upper image in Fig. 11) and that between the b-axis of OCP and the c-axis of β-TCP was 1.82%. On the other hand, the mismatch between the a-axis of HAP and the c-axis of β-TCP was 0.535% (corresponding to lower image in Fig. 11). The lattice mismatch in HAp is much smaller than that in OCP, suggesting that HAp is better suited than OCP for being deposited on β-TCP. The γSL values of OCP, β-TCP, and HAp are 2.9, 3.8, and 9.3 mJ/m2, respectively [36]. This indicates that the energetic affinity between OCP and β-TCP is higher than that between β-TCP and HAp, meaning that OCP growth on β-TCP is a natural consequence. Atomizing the micro-TCP crystals would increase the interfacial tension so that it approached that of HAp. As a result, the energetic affinity between HAp and β-TCP would increase, making it easier to deposit HAp on a nanoTCP substrate. It should be noted that our ex-situ time-resolved AFM observations (Fig. S3(a) and (b)) revealed that both types of surface

images indicated that the major precipitate inside the nano-TCP substrate after immersion was nanometer-scale HAp needles (Figs. 6(b) and 8). On the other hand, inside the micro-TCP substrate, the major precipitate was OCP (Fig. 6(a)), and the HAp was not detected by microbeam XRD measurement. However, the HR-TEM in Fig. 8 reveals a small number of HAp nanofibers among the β-TCP particles. Moreover, HAp nanofibers were always localized around particles with a size of less than 500 nm (Fig. 7). From these observations, we concluded that the initial precipitate on the nano-TCP substrate was HAp while that on the micro-TCP was OCP although the final growth phases were OCP in both cases. Our results strongly suggest that nano-TCP particles induce HAp. The growth rate of HAp along the c-axis was estimated to be a few tens nm/h in a previous investigation [38]. The growth rate of OCP was at least one or two orders higher than that of HAp under the conditions of the present study. Therefore, on the nano-TCP substrate, even though the first precipitate was HAp, the growth of OCP exceeded that of HAp, resulting in eventual OCP overgrowth on the nano-TCP substrate. The predominant crystals that formed inside the substrate were plate-like OCP (micro-TCP substrate) and HAp nanofibers (nano-TCP substrate) (Figs. 7 and 8). A notable finding is that HAp nanofibers surrounded the small particles (~ 500 nm or less) in the micro-TCP substrate. This demonstrates that the deposition of HAp nanofibers is closely related with the nanometer-sized TCP particles. In other words, HAp nanofibers formed directly, and its formation was induced by the nano-TCP particles rather than being transformed from OCP crystals. This is supported by the finding that their morphology and sizes were obviously different (Fig. 7(a)). The general consensus is that, when HAp forms via phase transformation from OCP crystals, the original morphology of the OCP plates is preserved as a pseudomorph [21]. A pseudomorphic relationship was not observed between the HAp nanofibers and plate-like OCP, indicating that the HAp nanofibers and platelike OCP formed independently and that the factor controlling the 8

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Fig. 9. SEM images of (a) micro-TCP substrate and (b) nano-TCP substrate after 20-h immersion in Tris-HCl buffer solution with pH of 7.4 and Ca = PO4 1.3 mM. Top view (a1, b1), and cross-sectional view (a2, b2). (a3) and (b3) are higher magnifications of (a2) and (b2). (a3) shows plate-like crystals (PL) that grew on TCP particles; many small crystals grew inside the substrate. (b3) shows plate-like crystals (PL) on the flake-layer (F) on top of the string layer, forming a three-layer structure.

Fig. 10. SEM images of micro-TCP substrate (a1, b1) and nanoTCP substrate (a2, b2) after 20-h immersion in Tris-HCl buffer solution with pH of 7.4, Ca 2.5 mM, PO4 1 mM. (a1, a2) with 142 mM NaCl and (b1, b2) with 142 mM NaCl and 1.5 mM Mg.

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Fig. 11. Structural relationship between HAp and βTCP (lower) and that between OCP and β-TCP (upper). Lower case: (001)HAP // (110)β-TCP; upper case: (001)OCP // (110) β-TCP. a-axis and c-axis directions of OCP and HAp are indicated. a-axis of OCP, a-axis of HAp, and c-axis of β-TCP are in horizontal direction. For visual aid, Ca of HAp and P of β-TCP is connected by blue lines and Ca of OCP and P of β-TCP is connected by magenta lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

were unreactive during the first 50 s of immersion, judging from their precipitation inducing reaction and dissolution behavior, which might caused by atomizing the TCP crystals. Therefore, the initial precipitates observed after 1-min immersion would reflect the property of the substrate surface. Thus, the phase of the precipitate was determined by the particle size due to the particle size dependency of the γSL. Along with the affinity, the smaller lattice mismatch between HAp and β-TCP (0.535%) than between OCP and β-TCP (5.35% and 1.82%) led to HAp deposition on the nano-TCP particles. In each calcifying solution used, the growth phase was OCP regardless of the type of substrate (Figs. 1(g), 3(g), S3(a), and S3(b)). Particularly, for the nano-TCP substrate, the initial deposits were HAp and OCP was lately nucleated; however, the growth phase was OCP. This is ascribed to the fact that OCP grows much faster than HAp. In our previous study [42], we evaluated the overgrowth of apatite and OCP on an amorphous calcium phosphate (ACP) substrate. In the absence of fluoride, a two-layer structure formed on the substrate, i.e., a thin layer of irregularly shaped flakes and a thick layer of plate-like OCP crystals. For the ACP substrate, a “root of tree” similar to the strings formed on the nano-TCP substrate formed between 3 and 5 min. It changed to irregularly shaped flakes between 5 and 10 min and then grew into plate-like OCP between 30 min and 1 h. For the nano-TCP substrate, the strings were HAp nanofibers, so they remained as formed without dissolving or changing into flakes during 20-h immersion. As a result, a three-layer structure formed. There is a general consensus that the physical properties of the coating layer, i.e., its thickness and topography, affect the in vivo performance of the coated material. The effect of the thickness of a plasma-sprayed HAp coating on the formation of new bone has been examined for the 1–200-μm thickness range [43–45] while the effect of

a thinner calcium phosphate coating on osteoblastic activity has been investigated for the 200-nm to 2-μm thickness range [46–48]. The thin coating was found to improve the initial stage of osteoinduction [46]. The morphologies and sizes of the component crystals of the overgrown layer as well as its crystal density affect the topography of the coating layer, which is known to affect osteoblastic activity [49–51]. However, the current information regarding the optimal coating thickness range and surface roughness is insufficient. The effect 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. 5. Conclusion Our protocol for forming OCP on a β-TCP substrate enables fast OCP crystal overgrowth in solution with a simple composition under nearly physiological conditions. The morphology and size of the OCP crystals, as well as the structure, thickness, and crystal density of the overgrown OCP layer, depended on the β-TCP particle size. The growth phase was OCP regardless of the type of substrate while the initial precipitate and precipitates inside the substrate depended on the β-TCP particle size. Nano-sized particles induced HAp nanofibers. Factors that determine the precipitating phase could be their interfacial tension (γSL) and the structural relationship between OCP and β-TCP and between HAp and β-TCP. On the nano-TCP substrate, plate-like OCP crystals grew on a previously formed thin layer of flake-like crystals that had a layer of string-like precipitates under it, resulting in a three-layer structure. In contrast, no such structure was formed on the micro-TCP substrate. These findings are applicable to the structural design of OCP-coating 10

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layers, which should boost the potential of β-TCP scaffolds as a bone graft substitute.

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