Enhanced osteoconductivity of sodium-substituted hydroxyapatite by system instability Jung Sang Cho,1 Seung-Hoon Um,2 Dong Su Yoo,3 Yong-Chae Chung,3 Shin Hye Chung,2 Jeong-Cheol Lee,2 Sang-Hoon Rhee1,2 1

Interdisciplinary Program of Bioengineering, College of Engineering, Seoul National University, Seoul 152-742, Korea Department of Dental Biomaterials Science, Dental Research Institute and BK21 Plus, School of Dentistry, Seoul National University, Jongno, Seoul 110-749, Korea 3 Department of Materials Science and Engineering, Hanyang University, Seoul 133-791, Korea 2

Received 17 August 2013; revised 21 October 2013; accepted 16 November 2013 Published online 5 December 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33087 Abstract: The effect of substituting sodium for calcium on enhanced osteoconductivity of hydroxyapatite was newly investigated. Sodium-substituted hydroxyapatite was synthesized by reacting calcium hydroxide and phosphoric acid with sodium nitrate followed by sintering. As a control, pure hydroxyapatite was prepared under identical conditions, but without the addition of sodium nitrate. Substitution of calcium with sodium in hydroxyapatite produced the structural vacancies for carbonate ion from phosphate site and hydrogen ion from hydroxide site of hydroxyapatite after sintering. The total system energy of sodium-substituted hydroxyapatite with structural defects calculated by ab initio methods based on quantum mechanics was much higher than that of hydroxyapatite, suggesting that the sodium-substituted hydroxyapatite was energetically less stable compared with hydroxyapatite. Indeed, sodium-substituted hydroxyapatite exhibited higher dissolution behavior of constituent elements of hydroxyapatite in simulated body fluid (SBF) and Trisbuffered deionized water compared with hydroxyapatite,

which directly affected low-crystalline hydroxyl-carbonate apatite forming capacity by increasing the degree of apatite supersaturation in SBF. Actually, sodium-substituted hydroxyapatite exhibited markedly improved low-crystalline hydroxyl-carbonate apatite forming capacity in SBF and noticeably higher osteoconductivity 4 weeks after implantation in calvarial defects of New Zealand white rabbits compared with hydroxyapatite. In addition, there were no statistically significant differences between hydroxyapatite and sodium-substituted hydroxyapatite on cytotoxicity as determined by BCA assay. Taken together, these results indicate that sodium-substituted hydroxyapatite with structural defects has promising potential for use as a bone grafting material due to its enhanced osteoconductivity compared C 2013 Wiley Periodicals, Inc. J Biomed Mater with hydroxyapatite. V Res Part B: Appl Biomater, 102B: 1046–1062, 2014.

Key Words: sodium, substitution, hydroxyapatite, ab initio, osteoconductivity

How to cite this article: Cho J.S., Um S.-H., Yoo D.S, Chung Y.-C., Chung S.H, Lee J.-C., Rhee S.H. 2014. Enhanced osteoconductivity of sodium-substituted hydroxyapatite by system instability. J Biomed Mater Res Part B: 2014:102B:1046–1062.

INTRODUCTION

Hydroxyapatite has been considered as a representative material for bone grafts due to its similarities with apatite in bone and osteoconductivity. However, a longer time period is reported for substantial bone apposition to occur to the surface of dense hydroxyapatite relative to other bioactive glasses and glass ceramics,1–4 and this disadvantageously increases patient rehabilitation time. Therefore, several methods such as variations of sintering temperature,5 granule size,6,7 introductions of porous structures,8–14 and electrical polarity15–20 have been proposed to improve osteoconductivity of synthetic hydroxyapatite. In addition, foreign ion substitution is also known to enhance osteoconductivity of hydroxyapatite.21–37

For ion substitution, candidate replacement elements are recommendable to be already present in bone apatite at trace levels due to safety purposes. Thus, sodium, magnesium, potassium, strontium, zinc, barium, copper, aluminum, iron, fluorine, chlorine, and silicon38,39 can be considered. Indeed, improved low-crystalline hydroxyl-carbonate apatite forming capacity in SBF and osteoconductivity has already been reported for hydroxyapatite upon partial substitution of phosphate ions with silicate ions.24,26–28,31,40–42 In addition, we have previously reported that substitution of hydroxyl ions in hydroxyapatite with chlorine ions results in increased low-crystalline hydroxyl-carbonate apatite forming capacity in SBF and enhanced osteoconductivity compared with pure hydroxyapatite due to an increase in total system

Correspondence to: S.-H. Rhee (e-mail: [email protected]) Contract grant sponsor: Basic Science Research Program through a National Research Foundation of Korea (NRF), Ministry of Education; contract grant number: 2013R1A1A2004752

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energy.43 While several studies have previously investigated substituting sodium for calcium in hydroxyapatite, they focused primarily on synthesis and characterizations of sodium-substituted carbonate hydroxyapatite rather than sodium-substituted hydroxyapatite itself.44–56 Likewise, there have been no reports examining the low-crystalline hydroxyl-carbonate apatite forming capacity in SBF and osteoconductivity of sodium-substituted hydroxyapatite with an exact structural formula based on defect chemistry. In this study, the effect of sodium-substitution for calcium in hydroxyapatite was investigated for the first time both experimentally and theoretically, with a primary focus on system instability, since this property directly influences low-crystalline hydroxyl-carbonate apatite forming capacity, which in turn dictates apatite osteoconductivity. Sodiumsubstitution was expected to increase system instability of hydroxyapatite due to differences in charge and ionic size between calcium and sodium, which was hypothesized to increase the solubility of sodium-substituted hydroxyapatite. Importantly, increased system instability can be expected to accelerate the release of constituent elements present in hydroxyapatite such as calcium and hydroxyl ions, and thus positively influence its low-crystalline hydroxyl carbonate apatite forming capacity and osteoconductivity. MATERIALS AND METHODS

Preparations of sodium-substituted hydroxyapatite and pure hydroxyapatite Sodium-substituted hydroxyapatite was synthesized by reacting calcium hydroxide and phosphoric acid with sodium nitrate. The molar ratio of calcium hydroxide, phosphoric acid, and sodium nitrate used for the synthesis was 9.5:6:0.5, respectively. Calcium hydroxide powder was obtained by slaking calcium oxide generated from calcination of calcium carbonate (Wako Pure Chemicals) at 1050 C for 3 h. A 1M calcium hydroxide suspension and 1M phosphoric acid solution were prepared by adding 0.358 mol calcium hydroxide and 0.226 mol phosphoric acid (Wako Pure Chemicals) to 358 mL and 226 mL deionized water, respectively, followed by vigorous stirring. Meanwhile, a solution of 1M sodium nitrate was prepared by dissolving 0.019 mol sodium nitrate (Aldrich) to 19 mL of deionized water. To synthesize sodium-substituted hydroxyapatite, the sodium nitrate solution was mixed with the calcium hydroxide suspension. Next, the phosphoric acid solution was slowly added to the calcium hydroxide suspension containing sodium nitrate at room temperature with vigorous stirring until no other phases except hydroxyapatite could be detected after heat-treating the precipitates at 1100 C for 1 h. After aging for 1 day at room temperature, the suspension was dried in a convection oven at 80 C for 1 day with no filtering process so as to avoid losing water soluble sodium nitrate. The resulting dried mass was then ground and sieved to isolate particles under 75 lm in size. Hereafter, apatite substituted by sodium is referred to as NaAp. As a control, pure hydroxyapatite powder was also synthesized according the same procedures described above

except for the addition of sodium nitrate. Briefly, the synthesized apatite suspension was aged, filtered by rinsing with deionized water, dried, ground, and sieved. Hereafter, pure hydroxyapatite is referred to as OHAp. For low-crystalline hydroxyl carbonate apatite forming capacity and solubility tests, OHAp and NaAp powders were compacted into respective disks with diameters of 10 mm and heights of 2 mm under a pressure of 20 MPa, followed by sintering at 1100 C for 3 h at a heating rate of 5 C/min. For osteoconductivity tests, porous OHAp and NaAp granules were prepared. Polyethylene glycol (PEG; Mw 5 10,000, Aldrich) powder was used as a porogen by generating two different size distributions (75–212 lm and 212–425 lm) by sieving. The two sizes of PEG were mixed mechanically with apatite powders. The mixing ratios among apatite, small sized PEG (75–212 lm), and large sized PEG (212–425 lm) was 60:20:20 (wt %). The resulting mixtures were compacted into disks with a diameter of 50 mm and a height of 30 mm under a pressure of 20 MPa, followed by sintering at 1100 C for 3 h at a heating rate of 5 C/min. The sintered disks were fractured and a granule size range of 425 lm to 600 lm was generated by sieving. Evaluation of low-crystalline hydroxyl-carbonate apatite forming capacity in SBF The low-crystalline hydroxyl-carbonate apatite forming capacity of OHAp and NaAp were assessed in SBF, which was prepared by dissolving reagent grade NaCl, NaHCO3, KCl, K2HPO43H2O, MgCl26H2O, CaCl2, and Na2SO4 in deionized water.57 The solution was buffered at pH 7.25 using tris(hydroxymethyl) aminomethane with 1M HCl at 36.5 C and then sterile-filtered through a presterilized filter unit (Millipore, 0.22 lm). Apatite disks were sterilized under a UV lamp for 0.5 h and then soaked in 30 mL of SBF at 36.5 C for different periods of time. After soaking, the disks were removed from SBF, rinsed gently with deionized water five times, and air-dried under ambient conditions. Characterization All microstructures were observed by a field emission scanning electron microscope (FE-SEM; S-4700, Hitachi). Detailed microstructures of the as-synthesized powders were observed by a transmission electron microscope (TEM; Tecnai F20, FEI) at 200 kV. Microstructural characteristics were determined with image analysis software (Image J, NIH). The shortest and longest diagonals of each crystal were determined, and the shortest diagonal was used to represent crystal size. The average crystal size was determined by measuring at least 500 for each specimen. The crystal phases of each specimen were evaluated with an X-ray diffractometer (XRD; D8 Discover, Bruker) using powder and thin film modes. Determination of the lattice parameters of OHAp and NaAp was performed by Rietveld refinement of XRD data collected from sintered powders (TOPAS v.2.0, Bruker). Refinements were based on the structural data of OHAp using the space group P63/m.58 The least squares refinement method was repeated until a

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best fit was obtained between the calculated patterns, which were based on the refined structure models and diffraction patterns. The elemental distributions of calcium, phosphorous, and sodium in the specimens were analyzed with an electron probe microanalyzer (EPMA; JXA-8500F, JEOL). The accelerating voltage was set to 15 kV and the probe current was set to 20 nA using a focused electron beam. Elemental distribution was determined with a qualitative mapping procedure. The measured area was 102 lm 3 102 lm with a step size of 0.4 lm. Functional groups of specimens were analyzed by a Fourier transformed infrared spectroscopy (FTIR; Spectrum 100, Perkin Elmer). For FTIR spectroscopy measurements, specimens were dried for 1 day in a desiccator prior to preparing KBr pellets. The pulverized specimens were subsequently diluted 250-fold with KBr powder and background noise was corrected with data from pure KBr. A total of 128 scans were averaged to yield spectra at a resolution of 4 cm21. The carbonate contents of as-synthesized powders were quantitatively determined with a combustion bulk elemental analyzer (EA Flash 1112, Thermoquest). The atomic concentrations of calcium, phosphorus, and sodium released from specimens into SBF and Tris-buffered deionized water (pH 7.25) after different periods of time were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES; Optima-4300 DV, Perkin Elmer). The pH of SBF and Tris-buffered deionized water as a function of soaking time were measured with a pH meter (DK-20, Horiba). Five samples were tested for each soaking time, and the values were averaged and reported as the mean 6 standard deviation. Ab initio calculation of total system energy For structural optimization of specimens, ab initio calculations were performed using density functional theory with the exchange-correlation energy functional treated by Perdew–Burke–Ernzerhof (PBE) from a generalized gradient approximation59 with a projector augmented wave.60 Using the Vienna ab initio simulation package code,61 the selfconsistent electronic density functional and total energy were calculated with a plane-wave basis that was set to expand to a cutoff energy of 400.0 eV (29.40 Ry). The selfconsistent field calculation was iterated until the total energy difference of the systems between the adjacent iterating steps was less than 1026 eV. All constituent elements in the apatite system were fully relaxed for structural optimization until the maximum Hellmann-Feynman forces62 reached a range of 62.0 meV/Å with an ionic relaxation scheme based on the conjugate gradient method.63 Nonshifted and C-point centered 3 3 3 3 3 and 3 3 3 3 1 k-point grids with the Monkhorst–Pack scheme were used for Brillouin zone sampling.64 A linear tetrahedron method with Bl€ ochl correction for Brillouin zone integration was implemented for the smearing method.65 In vitro cytotoxicity analysis Cytotoxicity analysis with extracts of OHAp and NaAp were performed in compliance with ISO 10993-5 and 12, respec-

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tively. Briefly, OHAp and NaAp were extracted under agitation in deionized water at a ratio of 1 g per 30 mL for 3 days at 37 C. Solvent control and experimental culture medium were prepared by mixing equal volumes of deionized water and extracts with 23 EMEM (Lonza). Subsequently, mouse calvaria-derived preosteoblastic MC3T3-E1 TM cells (CRL-2593 , ATCCV) were seeded into solvent control and experimental culture media a density of 5 3 104 cells/ mL. A blank group was prepared from the solvent control medium, which was centrifuged, pelleted, and stored at 4 C after rinsing with phosphate buffered saline (PBS). Control and test groups were also prepared from each solvent control and experimental culture medium, which were then transferred to 24-well plates (Nunclon surface, Nunc) and cultured at 37 C for 3 days with 5% CO2. Next, cells were collected by centrifugation and the pellets were stored at 4 C after rinsing with PBS. The protein contents of individual groups were measured colorimetrically with a BCA assay kit according to the manufacturer’s instructions (BCA protein assay kit, Thermo). Absorbance was measured at 560 nm with an ELISA microplate reader (Model 550, Bio-Rad). The mean absorbance (A560 nm) and standard deviation of five parallel cultures was calculated and used to assess the percentage of growth inhibition (GI%) as follows: R

  ðA 560nm sampleÞ2ðA 560nm blankÞ G:Ið%Þ5 12 3100 ðA 560nm controlÞ2ðA 560nm blankÞ where A560 nm (sample) was the absorbance of the test extract, A560 nm (blank) was the absorbance of the blank culture (without cells), and A560 nm (control) was the absorbance of the solvent control.66 Data were reported as the mean 6 standard deviation of five parallel groups. Osteoconductivity analysis In vivo testing. New Zealand white male rabbits weighing 2–2.5 kg (eight animals) were used to assess the osteoconductivity of OHAp and NaAp. All animals were kept in single cages and fed a dry diet and water ad libitum. Surgery was performed with general (ketamine; 7.5 mg/kg, Yuhan and Rompun; 3.5 mg/kg, Bayer Korea) and local (2% lidocaine with 1:100,000 epinephrine) anesthesia. For head surgery, the surgical site was carefully shaved and disinfected with iodine and 5% chlorhexidine digluconate. A midline incision was then made from the nasofrontal area to the external occipital protuberance, and a skin periosteal flap was raised to expose the calvarial bone surface. The boundary of the bone defect was outlined using a trephine bur (8 mm in diameter, 3i-Implant Innovations), and the bicortical calvarial bone was removed using a round bur. Both procedures were carried out in the presence of copious saline irrigation. Two defects were prepared for each animal. After defect preparation, one of each of the two grafting materials was placed to fill the respective defects. The periosteum and skin were then repositioned and closed in layers with 4-0 chromic gut sutures (Ethicon). Intramuscular injections of antibiotics were given for 3 days after surgery.

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A total of eight rabbits were euthanized 4 weeks after the surgery. The calvarial bone was collected en bloc and fixed with 10% neutral buffered formalin. Samples were then rinsed with water, dehydrated, and embedded in a super low-viscosity embedding media (Technovit 7200, Exakt Apparatebau) without decalcification. The samples were then sectioned coronally at a thicknesses of 80 lm and polished to 40 lm using a cutting (Secotom-15, Struers) and grinding (Tegrapol-35, Struers) system with a specimen mover plate and thin section holder. All samples were examined under an optical microscope (Eclipse 80i, Nikon) after staining with Sanderson’s Rapid Bone StainTM (Surgipath Medical Industries) and a van Gieson counterstain. This staining combination afforded sufficient contrast to distinguish bone, which stained pink, from osteoid (blue–green), and fibrous tissue (blue–green). Regenerated bone was distinguished from original defects based on histological features, namely, generation of chroma after staining and different morphologies of bone cells and extracellular matrix. Computer-assisted histomorphometric measurements of newly formed bone, implanted granules, and soft tissues were made with image analysis software (Image J, NIH). The ratios of each region were presented as a percentage of the original defect area. Statistical analysis. Statistical analysis was performed using a SPSS v.19.0 software package (SPSS/PC). Data were analyzed by paired Student t-test and represented as the mean 6 standard deviation with a significance level of p < 0.01. Animal research ethics. Approval was obtained for all animal experiments, which were performed according to the guidelines of the Institutional Animal Care and Use Committee of Seoul National University (SNU-120222-1). RESULTS

The microstructures of apatite powders synthesized by reacting calcium hydroxide and phosphoric acid (a) without and (b) with sodium nitrate were observed by TEM (Figure 1). The crystal shapes present in the two specimens were similar to each other, and the average crystal sizes in the apatite synthesized without sodium nitrate were 13.8 6 3.3 nm in width and 37.2 6 10.7 nm in length while those of the apatite synthesized with sodium nitrate were 14.8 6 2.0 nm in width and 42.7 6 12.6 nm in length. To assess the formation of apatite after synthesis, phase analysis of the powders synthesized without and with sodium nitrate was performed by continuous mode XRD (Figure 2). For both samples, only a low-crystalline apatite phase was detected. To evaluate the incorporation of sodium into the hydroxyapatite structure during synthesis, functional groups of apatite powders synthesized without and with sodium nitrate were measured by FTIR (Figure 3). The asymmetrical stretching (t3) and bending (t4) modes of PO4 were detected at 1087 and 1039 cm21 for t3 and 601 and 571

FIGURE 1. TEM images of as-synthesized (a) hydroxyapatite (OHAp) and (b) sodium-substituted hydroxyapatite (NaAp) powders.

cm21 for t4.39 Symmetric stretching modes of PO4 were also observed at 962 cm21 for t1 and 469 cm21 for t2.39 Stretching and liberation modes of OH were detected at 3572 and 630 cm21, respectively. The bands present at 1633 and 3443 cm21 were assigned to H2O bending66,67 and an OH envelope,39 respectively. Likewise, bending and stretching modes of CO3 were observed at 1454 cm21 for t368–70 and 1422 cm21 for t1,70,71 respectively, while a bending mode for t2 was also observed at 874 cm21.68,72 The magnitudes of CO3 bands observed with apatite synthesized with sodium nitrate were much larger than those observed for apatite synthesized without sodium nitrate, which was consistent with the results of combustion bulk elemental analysis. The detected amount of CO2 present in apatite powder synthesized without sodium nitrate was

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FIGURE 2. XRD patterns of as-synthesized hydroxyapatite (OHAp) : and sodium-substituted hydroxyapatite (NaAp) powders. hydroxyapatite.

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1.03 6 0.02 wt % while that of apatite powder synthesized with sodium nitrate was 2.16 6 0.04 wt %. Surface microstructures of OHAp [Figure 4(a)] and NaAp [Figure 4(b)] disks after sintering at 1100 C for 3 h were observed using FE-SEM. The grain shapes were similar to each other; however, the grain size of NaAp was approximately three times larger than that of OHAp. Specifically, the average grain sizes of OHAp and NaAp were 0.32 6 0.14 lm and 0.97 6 0.38 lm, respectively. The sintered density of OHAp was about 98%, while that of NaAp was about 95%. Phase analysis for OHAp and NaAp disks after sintering at 1100 C for 3 h was obtained using continuous mode

FIGURE 3. FTIR spectra of as-synthesized hydroxyapatite (OHAp) and sodium-substituted hydroxyapatite (NaAp) powders.

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FIGURE 4. FE-SEM images of (a) hydroxyapatite (OHAp) and (b) sodium-substituted hydroxyapatite (NaAp) disks after sintering at 1100 C for 3 h.

XRD (Figure 5). Both of the specimens exhibited pure hydroxyapatite structures. To evaluate change of hydroxyapatite structure after sintering, functional groups of OHAp and NaAp disks after sintering at 1100 C for 3 h were evaluated by FTIR (Figure 6). In all specimens, the asymmetrical stretching (t3) and bending (t4) modes of PO4 were detected at 1087 and 1039 cm21 for t3, and 601 and 571 cm21 for t4, respectively.39 In addition, the symmetric stretching modes t1 and t2 of PO4 were observed at 962 and 469 cm21, respectively.39 Stretching and liberation modes of OH were detected at 3572 and 630 cm21.39 Interestingly, three new bands were observed at 3555, 677, and 433 cm21 in the NaAp specimen, originating from OOHO, HO:OH, and Ca3AO configurations.73 Microstructures observed by FE-SEM and corresponding elemental maps measured by EPMA of sodium, calcium, and phosphorous distributed in (a) OHAp and (b) NaAp disks sintered at 1100 C for 3 h are shown in Figure 7, and the colored scale bars indicate the relative concentrations of each element. Only calcium and phosphorous were detected in the grains of OHAp disk [Figure 7(a)], while sodium was not detected. In contrast, sodium, in addition to calcium and phosphorous, was homogeneously distributed on every apatite grain from NaAp disks [Figure 7(b)].

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FIGURE 5. XRD patterns obtained from hydroxyapatite (OHAp) and sodium-substituted hydroxyapatite (NaAp) disks after sintering at 1100 C for 3 h. : hydroxyapatite.

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Microstructures and detailed microstructures (inset photographs) of OHAp disks after soaking in SBF for various time intervals were observed by FE-SEM (Figure 8). No obvious microstructural changes were observed for OHAp during the testing period. Microstructures and detailed microstructures (inset photographs) of NaAp disks after soaking in SBF for various time intervals were observed by FE-SEM (Figure 9). Small apatite crystals present on NaAp grain surfaces, discerned by acicular-shaped small white particles from the detailed microstructure images, were newly observed after only 3 h of soaking in SBF [Figure 9(a)]. The numbers and sizes of small apatite crystals increased with time, and clearly observed even under low magnification after 6 h of soaking [Figure 9(b)]. After 9 h [Figure 9(c)], the entire NaAp disk surface was covered by numerous acicular-shaped new apatite crystals. Consistent with these observations, the newly formed apatite layer gradually thickened with increasing duration of the testing period. Changes in the atomic concentrations of (a) calcium, (b) phosphorous, and (c) sodium, as well as (d) pH, and (e) ionic activity product (IAP) of apatite in SBF in which OHAp and NaAp disks were soaked as a function of time are shown in Figure 10. There were no discernible changes after soaking OHAp disks in SBF. Soaking of OHAp disks caused no change in the pH or concentration of sodium while only a minor decrease in the concentrations of calcium and phosphorous. In contrast, soaking NaAp disks increased the concentrations of calcium and pH of SBF after 3 and 9 h, respectively, but decreased quickly thereafter. Likewise, the concentration of phosphorous in SBF was slightly decreased up to 6 h, and decreased quickly there-

FIGURE 6. FTIR spectra of hydroxyapatite (OHAp) and sodiumsubstituted hydroxyapatite (NaAp) disks after sintering at 1100 C for 3 h.

after. Conversely, the sodium concentration of SBF in which NaAp disks had been soaked increased very little during the testing period. The IAP of apatite in SBF were calculated for OHAp and NaAp disks based on the release of calcium and phosphorous, as well as changes in pH data over time [Figure 10(e)]. For the NaAp specimen, IAPs reached a maximum value after 6 h of soaking, after which they decreased rapidly. Conversely, there was very little decrease in IAP for OHAp during the observation period. Phase analyses for (a) OHAp and (b) NaAp disks after soaking in SBF for 1 week were performed using TF-XRD with an incident angle of 1 (Figure 11). Only high crystalline hydroxyapatite phase was observed on OHAp disk before and after soaking [Figure 11(a)]. However, after soaking NaAp disks, high crystalline hydroxyapatite changed to low-crystalline hydroxyapatite as evidenced by decreased intensity and peak broadening [Figure 11(b)]. Changes in the atomic concentrations of (a) calcium, (b) phosphorous, and (c) sodium, as well as (d) pH of Tribuffered deionized water after soaking OHAp and NaAp disks for various time intervals are shown in Figure 12. For the NaAp specimen, the concentrations of calcium and sodium, as well as pH, increased gradually with soaking time, while the concentration of phosphorous was not changed at all. In contrast, no changes in ion concentrations were observed for OHAp during the testing period. The in vitro cytotoxicities of OHAp and NaAp were analyzed by cell growth inhibition testing. Specifically, changes in the rates of proliferation of cells were assessed by measuring total protein contents of preosteoblastic MC3T3-E1 cells cultured with extracts of OHAp and NaAp, and were compared to solvent control media. The percent change in

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FIGURE 7. FE-SEM images and corresponding elemental maps measured by EPMA of calcium, phosphorous, and sodium distributed in (a) hydroxyapatite (OHAp) and (b) sodium-substituted hydroxyapatite (NaAp) disks after sintering at 1100 C for 3 h. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 8. FE-SEM images (50003) of pure hydroxyapatite (OHAp) disks after soaking in SBF for (a) 3 h, (b) 6 h, (c) 9 h, (d) 1 day, (e) 3 days, and (f) 7 days (inset micrographs are detailed microstructures, 20,0003).

growth inhibition of OHAp and NaAp were 1.41 6 0.12% and 1.83 6 0.13%, respectively, the difference of which was not statistically significant (p > 0.05), indicating that the cytotoxicities of OHAp and NaAp were similar. The osteoconductivities of OHAp and NaAp granules were also evaluated using a calvarial defect model in New Zealand white rabbits. Figure 13 shows the optical microscopic images of whole defects [Figure 13(a,b)] and more detailed images [Figure 13(c,d)] 4 weeks after surgery for each group. In the OHAp-treated defects [Figure 13(a,c)], the majority of grafted granules (black color) were surrounded by soft tissues (pale blue color), and small amounts of new bone formation (pink color) were observed. In contrast, in the NaAp-treated defects [Figure 13(b,d)], formation of thick and sound new bone

(pink color) was observed around grafted granules. The results of histomorphometric analyses are shown in Table I. The average occupied portion of OHAp was 48.3 6 4.5%, while that of NaAp was 46.1 6 3.9%, the difference of which was not statistically significant between the two groups (p > 0.01). Conversely, the average areas occupied by newly formed bone were 5.8 6 1.8% for the OHAp-treated defects and 51.2 6 5.3% for NaAp-treated defects, while the average areas of soft tissue were 45.9 6 4.9% and 2.7 6 1.2%, respectively; the areas occupied by new bone as well as average areas of soft tissue were significantly different between the two groups (p < 0.01). The atomic structures of OHAp [Ca10(PO4)6(OH)2] were optimized by atom position, cell volume, and cell shape.

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FIGURE 9. FE-SEM images (50003) of sodium-substituted hydroxyapatite disks (NaAp) after soaking in SBF for (a) 3 h, (b) 6 h, (c) 9 h, (d) 1 day, (e) 3 days, and (f) 7 days (inset micrographs are detailed microstructures, 20,0003).

Meanwhile, 1 3 1 3 2 NaAp supercells with the structural formula shown in Scheme 1 were derived by combining the initial amount of sodium and FTIR results (Figures 3 and 6), where one sodium-substitution, and vacancies for one carbonate ion (doubly positively charged) and two hydrogen molecules (a negatively charged) were optimized by atom position, cell volume, and cell shape. The hydroxyl ion arrangements of NaAp were set to a “tail to tail” configuration of HO:OH along the [0 0 0 1] axis based on FTIR results (Figure 6) and a previous study.73 The atomic structures of OHAp unit cell [Figure 14(a)] and 1 3 1 3 2 sodium-substituted supercells [Figure 14(b)] in the crystal plane (1 0 0) with full ionic relaxation are illustrated in Figure 14. Table II summarizes the structural parameters in comparison with experimental data (ab initio and Rietveld) and previously

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reported results.74,75 The lattice parameters of OHAp and NaAp calculated by PBE were in good agreement with results obtained by the Rietveld method and those reported earlier,74,75 within an error of about 1–2%. The lattice parameters a and c of NaAp decreased when sodium was substituted for calcium, which was identical to the results obtained with Rietveld refinement. The total energy differences between OHAp and NaAp were calculated with DE defined as equation (1): DE5Etot ðNaApÞ22 3 Etot ðOHApÞ 1 nlPO 4 1 nlCa 1nlH2 2nlNa (1) where Etot ðNaApÞ and Etot ðOHApÞ are the total energies of the corresponding systems, and lPO4 ; lCa ; lH2 ; and lNa are OSTEOCONDUCTIVITY OF SODIUM-SUBSTITUTED HYDROXYAPATITE

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FIGURE 10. Changes in atomic concentrations of (a) calcium, (b) phosphorous, and (c) sodium, as well as changes in (d) pH, and (e) ionic activity products (IAP) of apatite in SBF after soaking hydroxyapatite (OHAp) and sodium-substituted hydroxyapatite (NaAp) disks for various time intervals.

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FIGURE 11. TF-XRD measurements of (a) hydroxyapatite (OHAp) and (b) sodium-substituted hydroxyapatite (NaAp) disks before and after soaking in SBF for 1 week. : hydroxyapatite.

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the chemical potentials of the corresponding systems, and n is the number of corresponding systems, respectively. The calculated total energy difference between OHAp and NaAp was 116.85 eV, which suggested that the structure of NaAp was much more energetically unstable than that of OHAp. Table III summarizes the binding energies between apatite structures and the constituent elements of apatite. The binding energy was defined as equation (2) Ebind 5 Etot ðstructureÞ2Etot ðstructure 2elementsÞ1lelements (2) where Etot ðstructureÞ; Etot ðstructure 2elementsÞ; and lelements are the total energies and chemical potentials of the corresponding systems, respectively. The binding energies varied with the atomic positions in the crystal structures, but the differences were very small. Thus, only average binding energies are reported in Table III. The binding energies of calcium and phosphate were decreased by sodium substitution, suggesting that the bond between apatite structure and constituent elements of apatite became energetically unstable. In addition, the higher total system energy and energetically unstable binding of constituent elements of NaAp than those of OHAp were in good concordance with the increasing dissolution behaviors of NaAp in SBF and Tris-buffered deionized water (Figures 10 and 12).

DISCUSSION

In this study, we investigated the effect of substituting sodium for calcium in hydroxyapatite on low-crystalline hydroxyl-carbonate apatite forming capacity in SBF and osteoconductivity, and analyzed the results in the context of

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system instability. When apatite powder was synthesized by the reaction of calcium hydroxide, phosphoric acid, and sodium nitrate, sodium-substituted low-crystalline hydroxyl carbonate apatite was produced (Figures 2 and 3). It has been already reported that as the amount of sodium (x in Scheme 2) substituting calcium increased, the same amount of carbonate ions was introduced into the phosphate sites to balance the total net charge (Scheme 3).48 The substitution of sodium for calcium was confirmed indirectly by the elevated amount of carbonate ions present in sodium-substituted low-crystalline hydroxyl carbonate apatite after synthesis compared with the amount of carbonate ions in low-crystalline hydroxyl carbonate apatite (Figure 3). These results were corroborated by the detection of CO2 gas from hydroxyl carbonate apatite and sodiumsubstituted hydroxyl carbonate apatite during combustion bulk elemental analysis. Specifically, the amount of CO2 gas detected from the hydroxyl carbonate apatite powder was 1.03 6 0.02 wt % while that of sodium-substituted hydroxyl carbonate apatite powder was 2.16 6 0.04 wt %. After sintering products at 1100 C for 3 h, sodiumsubstituted hydroxyapatite lacking carbonate ions were generated, which was indirectly confirmed by XRD (Figure 5), FTIR (Figure 6), and EPMA (Figure 7) analyses. The peak positions of pure hydroxyapatite and sodium-substituted hydroxyapatite were almost identical. Thus, XRD measurements were not able to provide direct evidence for the substitution of sodium for calcium in hydroxyapatite. FTIR results (Figure 6) also provided the indirect evidence for the substitution of sodium for calcium in hydroxyapatite structures. Specifically, during sintering, carbonate ions were released from products as CO2 gas by thermal decomposition, which resulted in carbonate ion sites with doubly

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FIGURE 12. Changes in atomic concentrations of (a) calcium, (b) phosphorous, (c) sodium, as well as (d) pH in Tris-buffered deionized water after soaking hydroxyapatite (OHAp) and sodium-substituted hydroxyapatite (NaAp) disks for various time intervals.

positively charged vacancies (Scheme 4). To balance the total net charge (Scheme 5), loss of hydrogen from hydroxyl group occurred simultaneously with the formation of a doubly positively charged vacancy in the carbonate site, producing a negatively charged two vacancies at hydrogen site as shown in Scheme 3. Consequently, this would have produced a “tail to tail” configuration of (HO:OH) in an OH chain.73 Indeed, three new bands (OOHO, HO:OH, and Ca3AO configurations)73 originating from a single oxygen appeared after sintering due to loss of a hydrogen in a OH chain (Figure 6). Elemental mapping of pure OHAp and NaAp by EPMA (Figure 7) corroborated XRD and FTIR results; sodium was observed uniformly throughout the apatite grain surfaces of NaAp disks [Figure 7(b)] but not in OHAp disks [Figure 7(a)]. Together, these results clearly demonstrated successful sodium-substitution of the calcium site of hydroxyapatite after sintering. However, calcium oxide was also formed along with NaAp when the amount of added sodium (x) in the structural formula Ca 102x Na x ðPO 4 Þ62x ðCO 3 Þx ðOHÞ2 exceeded 0.5 (data not shown). Thus, there is an appropriate concentration range for generating single-phase NaAp.

The low-crystalline hydroxyl carbonate apatite-forming capacity in SBF was remarkably enhanced in NaAp as compared with OHAp (Figures 8 and 9). The induction time for the formation of low-crystalline hydroxyl carbonate apatite with a discernible acicular shape was only 3 h on NaAp disks [Figure 9(a)], while low-crystalline hydroxyl carbonate apatite did not form on OHAp disks during the testing period. However, low-crystalline hydroxyl carbonate apatite also formed on the surface of OHAp after one week of soaking (data not shown). The late forming rate of low-crystalline hydroxyl carbonate apatite on OHAp in this study compared to other reports must originate from the different initial pH of SBF. The pH used in this study was 7.25 but others were almost 7.4.27,28,76–83 The calculated initial ionic activity product of SBF with pH 7.25 is 10296.6 while that of SBF with pH 7.4 is 10295.2. It means the initial ionic activity product of SBF used in this experiment was more than 10 times lower than the SBF with pH 7.4. This corroborates low forming rate of hydroxyl carbonate apatite on OHAp in this experiment compared to other reports. Conversely, a thick apatite layer formed on NaAp disk after soaking in SBF for 9 h. This increase in crystal formation was also confirmed by a

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FIGURE 13. Optical microscopic images of whole defects and more detailed images of porous hydroxyapatite [OHAp, (a) original magnification 403 and (c) original magnification 1003] and sodium-substituted hydroxyapatite granules [NaAp, (b) original magnification 403 and (d) original magnification 1003] at 4 weeks after implantation into calvarial defects of New Zealand white rabbits. (Stained with Sanderson’s Rapid Bone StainTM and a van Gieson counterstain). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

decrease in peak height of NaAp observed by TF-XRD [Figure 11(b)], which was due to the thick, newly formed lowcrystalline hydroxyl carbonate apatite layer preventing detection of underneath NaAp phase. In contrast, there was no change in OHAp peak heights for OHAp disk before and after soaking in SBF, which supported the observation that lowcrystalline hydroxyl carbonate apatite was not formed on the surface of OHAp disks. Together, these results demonstrated that the low-crystalline hydroxyl carbonate apatite-forming capacity of NaAp in SBF was superior to that of OHAp. ICP-AES evaluation of SBF with soaking time (Figure 10) provided an explanation of the formation behavior of lowTABLE I. Histomorphometric Analyses of Hydroxyapatite (OHAp) and Sodium-Substituted Hydroxyapatite (NaAp) Granules 4 Weeks After Implantation in Calvarial Defects of New Zealand White Rabbits

Group

Implanted Granules (%)

Regenerated Bone (%)

Soft Tissue (%)

OHAp NaAp

48.3 6 4.5 46.1 6 3.9

5.8 6 1.8 51.2 6 5.3a

45.9 6 4.9 2.7 6 1.2a

a Significant difference between the two groups after 4 weeks (p < 0.01).

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crystalline hydroxyl carbonate apatite on the surface of NaAp disks. Specifically, for NaAp, the increased calcium [Figure 10(a)] and hydroxyl ions [Figure 10(d)] in SBF after 3 and 9 h of soaking, respectively, indicated that the ions were rapidly released from NaAp disks. The subsequent decreases in concentration of calcium and hydroxyl ions with phosphorous ions implied that these ions were consumed by the new formation of low-crystalline hydroxyl carbonate apatite crystals, because calcium, phosphorous, and hydroxyl ions are the constituent elements of apatite. It should be noted that the ionic activity product of apatite in SBF also reached a maximum value after 9 h of soaking of NaAp disks, and decreased rapidly thereafter [Figure 10(e)]. Indeed, the maximum ionic activity product of apatite after 9 h of soaking was about 10295.9 for NaAp, which was significantly high to provoke nucleation and growth of apatite crystals in SBF, considering that the solubility product of apatite in aqueous solution is only 5.5 3 102118 at 37 C.84

SCHEME 1. The structural formula of 1 æ 1 æ 2 sodium-substituted hydroxyapatite supercells where one sodium-substitution, and vacancies for doubly positively charged one carbonate ion and a negatively charged two hydrogen molecules.

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FIGURE 14. Crystal structures of (a) hexagonal unit cell of hydroxyapatite (space group P63/m) consisting of 10 calcium atoms, 6 phosphorous atoms, 26 oxygen atoms, and 2 hydrogen atoms, (b) 1 3 1 3 2 supercells of sodium-substituted hydroxyapatite replacing one calcium with one sodium, one carbonate ion vacancy, and two hydrogen molecules vacancies from the hydroxyapatite structure. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

TABLE II. Comparisons of Structural Parameters a (A˚) and c (A˚) Calculated by PBE and Rietveld Methods with Reported Values ˚) a (A

c (A˚)

c/a

SCHEME 2. The structural formula of sodium-substituted low crystalline hydroxyl carbonate apatite, where x is the amount of sodium and carbonate ions substituting calcium and phosphate ions, respectively.

9.354 9.421 9.424 9.432 9.331 9.403

6.837 6.881 6.879 6.881 6.830 6.871

0.730 0.730 0.730 0.730 0.730 0.731

SCHEME 3. The substitution mechanism for sodium and B-type carbonate ions in hydroxyapatite.

Taken together, these results indicated that the rapid release of calcium and hydroxyl ions from NaAp disks increased the degree of apatite supersaturation in SBF, and consequently induced the formation of low-crystalline hydroxyl carbonate apatite on NaAp disks. Meanwhile, there was no change in sodium concentration during the testing period, which was

SCHEME 4. The structural formula of sodium-substituted hydroxyapatite after sintering with the new formation of doubly positively charged vacancy in the carbonate site and negatively charged two vacancies at hydrogen site.

OHAp (ab initio) OHAp (Rietveld) Ref. 74 Ref. 45 NaAp (ab initio) NaAp (Rietveld)

TABLE III. Comparisons of Binding Energies Between Structures and Constituent Elements of Apatites Structure OHAp

NaAp

Elements

Binding Energy (eV)

Ca PO4 OH Ca PO4 OH Na

212.01 217.06 26.35 210.01 215.89 26.53 24.94

attributed to the very small amount of sodium that was released (0.16 mM after 7 days in Tris-buffered deionized water, Figure 12) compared with the initial amount of sodium in SBF (140 mM).

SCHEME 5. Total net charge balance mechanism of sodium-substituted hydroxyapatite for loss of carbonate and hydrogen ions during sintering.

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In contrast, there were only slight changes in the constituent elements of apatite during the testing period of OHAp. The increased IAPs of apatite in SBF were similarly negligible for OHAp disks [Figure 10(e)]. Taken together, these observations support a minor forming capacity of low-crystalline hydroxyl carbonate apatite on OHAp (Figure 8). The exact dissolution behaviors of OHAp and NaAp were assessed by soaking pure OHAp and NaAp disks in Trisbuffered deionized water (pH 7.25), which did not have a capacity to induce new apatite formation because it did not contain any constituent elements of apatite except hydroxyl ions. Specifically, when NaAp disks were soaked in Trisbuffered deionized water, calcium [Figure 12(a)], sodium [Figure 12(c)], and hydroxyl ions [Figure 12(d)] were released quickly in a time-dependent manner. In contrast, when OHAp disks were soaked in Tris-buffered deionized water, the release of constituent elements of apatite was negligible. The dissolution behaviors of OHAp and NaAp in SBF and Tris-buffered deionized water could be explained in terms of total system and binding energies between apatite structures and the constituent elements of apatite. Specifically, when sodium was substituted for calcium in OHAp, the total system energy of NaAp was notably increased compared with that of OHAp (116.85 eV). Likewise, the higher total system energy of NaAp corresponded with the higher solubility of NaAp compared with that of OHAp. The calculated binding energies between apatite structures and the constituent elements of apatite (Table III) were consistent with the ICP-AES results obtained with SBF and Tris-buffered deionized water. Specifically, the binding energies between apatite structures and constituent elements of apatite (calcium and phosphate) decreased with sodium substitution (Table III), indicating that the dissolution of each of the constituent elements of apatite became easier with substitution of calcium with sodium in hydroxyapatite. The smallest and highest binding energies of sodium and phosphate ions demonstrate that these elements were the easiest and the hardest to dissolve out from the apatite structure, respectively. Indeed, sodium ion was released quickly whereas phosphate ion was not dissolved out from NaAp disk into Tris-buffered deionized water within the testing period (Figure 12). Meanwhile, the dissolution of hydroxyl ions from NaAp was higher than that from OHAp (Figures 10 and 12) even though binding energy of hydroxyl ions of OHAp was lower than that of NaAp. When sodium ions, which were substituted for triangular calcium sites39,50 (Figure 5), were dissolved out first due to its lowest binding energy (Table III), binding between hydroxyl ions and hydroxyapatite structure became unstable, which provoked the quick dissolution of hydroxyl ions. Indeed, the release of calcium, phosphorous, and hydroxyl ions was increased with NaAp compared with pure OHAp (Figures 10 and 12). NaAp with enhanced the low-crystalline hydroxyl carbonate apatite-forming capacity in SBF exhibited significantly higher osteoconductivity compared with OHAp (Figure 13, Table I). Specifically, formation of thick and sound bone was observed on the surfaces of NaAp granules without intervening fibrous tissues. Conversely, the degree of new bone-forming capacity in OHAp granules was low. These

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FIGURE 15. Schematic diagram of the channel region of a 1 3 1 3 2 supercell of sodium-substituted hydroxyapatite. [Color figure can be viewed in the online issue, which is available at wileyonline library.com.]

results strongly suggested that NaAp, which exhibited excellent the low-crystalline hydroxyl carbonate apatite-forming capacity in SBF, also had good osteoconductivity, which has been already reported elsewhere.1,79,85–90 Conversely, the low osteoconductivity of OHAp granules might originate from their dense surface structure6–8,91 (Figure 8) due to no forming capacity of hydroxyl carbonate apatite even though they had a few macro-pores (Figure 13). Collectively, the results of this study indicate that NaAp has enhanced osteoconductivity compared with OHAp, but a similar degree of cytotoxicity. The improved low-crystalline hydroxyl carbonate apatite-forming capacity in SBF and osteoconductivity of NaAp was attributed primarily to improved solubility, which could be explained by a higher system energy achieved by sodium-substitution for calcium in hydroxyapatite. Further, our results suggest that NaAp demonstrates encouraging potential as a material for bone grafts due to its improved osteoconductivity compared with OHAp. CONCLUSIONS

Here, we evaluated for the first time, the effect of substituting sodium for calcium in hydroxyapatite. Sodium-substituted hydroxyapatite was successfully synthesized by a precipitation method followed by sintering. Substitution of sodium for calcium in hydroxyapatite resulted in enhanced low-crystalline hydroxyl carbonate apatite-forming capacity in SBF compared with pure hydroxyapatite, which could be explained by a higher system energy achieved by sodium-substitution for calcium. In addition, sodium-substituted hydroxyapatite exhibited excellent osteoconductivity compared with pure hydroxyapatite in calvarial defects of New Zealand white rabbits 4 weeks after surgery. Taken together, our results indicate that sodiumsubstituted hydroxyapatite demonstrates encouraging potential as a material for bone grafts due to its enhanced osteoconductivity compared with hydroxyapatite.

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OSTEOCONDUCTIVITY OF SODIUM-SUBSTITUTED HYDROXYAPATITE