Nanoemulsion synthesis of carbonated

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Author’s Accepted Manuscript Nanoemulsion synthesis of carbonated hydroxyapatite nanopowders: effect of variant CO32−/PO43− molar ratios on phase, morphology, and bioactivity Iliya Ezekiel, Shah Rizal Kasim, Yanny Marliana Baba Ismail, Ahmad-Fauzi Mohd Noor

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S0272-8842(18)31000-9 https://doi.org/10.1016/j.ceramint.2018.04.128 CERI18045

To appear in: Ceramics International Received date: 12 February 2018 Revised date: 9 April 2018 Accepted date: 15 April 2018 Cite this article as: Iliya Ezekiel, Shah Rizal Kasim, Yanny Marliana Baba Ismail and Ahmad-Fauzi Mohd Noor, Nanoemulsion synthesis of carbonated hydroxyapatite nanopowders: effect of variant CO32−/PO43− molar ratios on phase, morphology, and bioactivity, Ceramics International, https://doi.org/10.1016/j.ceramint.2018.04.128 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Nanoemulsion synthesis of carbonated hydroxyapatite nanopowders: effect of variant CO32−/PO43− molar ratios on phase, morphology, and bioactivity Iliya Ezekiela, Shah Rizal Kasimb, Yanny Marliana Baba Ismailb, and Ahmad-Fauzi Mohd Noorb* a

School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia, Engineering Campus, 14300 Nibong Tebal, Penang, Malaysia b School of Applied Science and Technology, Department of Ceramics Technology, Federal Polytechnic Auchi P. M. B. 13 Edo State, Nigeria *Corresponding author. Prof. PhD.; Tel.: +60 45996174; fax: +60 45941011; Email: [email protected]

Abstract Bone is a composite material containing carbonated hydroxyapatite (CHA) nanocrystallite and protein matrix. Low-temperature synthesis and assessment of the in vitro response of CHA nanopowders were conducted. Samples of CHA nanopowders were considered for their phase system, chemical compound, and reactions in a simulated body fluid (SBF). The crystallite size and phase for the CO32−/PO43− molar ratios of 0.67 and 1.00 were examined through X-ray diffraction (XRD). Chemical distinctiveness was analyzed by Fourier transform infrared (FTIR) spectroscopy. The compositions of the as-synthesized powders were quantified by X-ray fluorescence, and the carbon contents were estimated by carbon hydrogen nitrogen analysis. The morphology and textural properties were examined by field-emission scanning electron microscopy, transmission electron microscopy (TEM), and Brunauer–Emmett–Teller (BET) analysis. In vitro study was conducted in SBF medium, and the pH was intermittently recorded. Then, the dissolution of Ca2+ and PO43− in SBF was quantified by inductively coupled plasma optical emission spectroscopy. The outcomes of XRD and FTIR confirmed that pure singlephase B-type CHA without any secondary phase was formed. CHA0.67 showed a crystallite size of (Scherrer) 22.7 nm, particle size by TEM (PTEM) of 24.87, and BET ( PSBET ) of 26.48 nm. Meanwhile, CHA1.00 achieved a crystallite and particle size of (Scherrer) 4.67, PTEM of 6.50, and

PSBET of 13.23 nm. Results indicated that increasing the CO32−/PO43− molar ratio decreases the crystallite size and increases the amount of CO32−. Thus, we concluded that the nanoemulsionsynthesized CHA exhibits a crystallite size and chemistry comparable to those of biological hard tissue. Keywords: Carbonated hydroxyapatite, nanoemulsion, nanopowder, bioactivity 1. Introduction A major thrust of biomaterial research is the development of synthetic hydroxyapatite (HA) as the “gold” standard replacement for biological hard tissue. Synthetic HA closely resembles the

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inorganic phase of biological hard tissues and thus has been successfully applied as artificial bone constructs in certain cases; however, the material typically fails to serve as efficiently as natural bone [1]. Carbonated HA (CHA) remains as the most preferred model for replacing bone, dentine, enamel, and calcified tissue [2]. The inorganic component of bone is primarily plate-like (20–80 nm long and 2–5 nm thick) crystalline HA with a small amount of impurities, such as carbonate, which may substitute the phosphate group (forming the B-type substitute) or hydroxyl group (forming the A-type substitute); by contrast, chloride and fluoride may replace the hydroxyl group [3]. The recent research trend is to replicate synthetic HA properties, such as composition stoichiometry, crystal size dimension in nanoscale, and phase as observed in native bone by using ionic (CO32−, Na+, Mg2+, Fe2+, F−, Zn2+, and Si4+) substitutions [4]. The carbonate content in native bone is age dependent and constitutes up to 2–8 wt.% [5]. The synthetic substitution of carbonate ions within the HA structure has been observed for the modification of apatite crystal morphology and its microstructure [6]. Several reports have shown low crystallinity and improved solubility specifically for B-type CHA in bioactivity studies [7-9]. This enhanced solubility of B-type CHA derived from reduced crystallite size relative to that of pure HA reportedly increases bioactivity and improves bone apposition [10]. HA [CaI4CaII6(PO4)6(OH)2] is composed of 10 Ca2+ atoms, of which four are positioned at the CaI site and six are positioned at the CaII site; the four Ca2+atoms are bonded with nine oxygen atoms aligned to the PO43− site, whereas the six Ca2+ atoms are linked to six O2− atoms and a OH− single ion of the PO43− tetrahedron [11]. Methods of synthesizing HA nanopowders have evolved in the last decade into various chemical preparation routes. These preparation methods and their unique advantages significantly affect the precise control of the microstructures. Recent studies categorized the preparation methods as dry, wet, and high-temperature processes; biogenic

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sources; and combination procedures [12]. Dry methods include solid state [13, 14] and mechanochemical methods [15, 16]; wet methods involving chemical precipitation [17, 18], hydrolysis [19], sol–gel [20-22], hydrothermal [23, 24], emulsion (microemulsion and nanoemulsion) [25, 26], and sonochemical processes [27]; and high-temperature processes involving combustion [28] and pyrolysis [29]. The nanoemulsion method enables the acquisition of fine nanoparticles with low crystallinity. Given the small size of droplets of the internal phase, nanoemulsions are used diversely in the transdermal transport of drugs; in applications involving biologically active substances, pharmaceuticals and cosmetics, agrochemicals, paint and varnish; and in petroleum industries [30]. The substitution of CO32− in HA and the effect of this modification on physicochemical properties under calcination regimes and at low temperature have been reported previously [31] [32]. However, the discussion has barely advanced on the influence of variable CO32−/PO43‒ molar ratios on the ion release from nanometric HA synthesized at low temperature. Despite the possible association of cell survival to ion release, ion release is often neglected in bioactivity studies, as reported by Rohanová et al. [33]. In the synthesis of calcium phosphate ceramics, poor biosorption and biodegradation due to sintering can be minimized [34]. Therefore, the aim of this study is to evaluate the variant effect of the molar ratio of carbonate to phosphate [n(CO32−/n(PO43−)] on the phase, morphology, and bioactivity of CHA synthesized at ambient temperatures via nanoemulsion route. The dual (Ca2+; P5+) ion release and pH studies of the intended functional CHA nanopowders are assessed in vitro. A standard molar ratio of HA, [n(Ca2+)/n(PO43−)] = 1.667, was maintained for all variant substitutions, and the substitution process of the synthesized nanopowders was designed to favor a B-type CHA. During synthesis, carbonate ions were incorporated into the HA crystal structure in accordance with the following

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quasi-chemical model: [Ca10 − x/2(PO4)6−x(CO3)x(OH)2] reported by Kovaleva et al. [35], where x = 0.04 and 0.06 mol were qualitatively compared.

2. Experimental procedure 2.1 Material preparation CHA powders were synthesized at an ambient temperature (25 ± 0.01 °C) by the direct pouring nanoemulsion method as adopted elsewhere [36] and in accordance with the following chemical equations: CHA0.67:

10Ca(NO3)2 + 6(NH4)2HPO4 + 4NH4HCO3 → Ca10(PO4)6(CO3)4(OH)2 + (1) 7NH4NO3 + 12HNO3 + H2O + 10NH3,

CHA1.00:

10Ca(NO3)2 + 6(NH4)2HPO4 + 6NH4HCO3 → Ca10(PO4)6(CO3)6(OH)2 + 9.5NH4NO3 + 9HNO3 + 2.5H2O + 10NH3. (2)

From the wet chemical nanoemulsion method adopted, ammonium bicarbonate (NH4HCO3), calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), and diammonium hydrogen phosphate (NH4)2HPO4 (all ≥ 99% purity; Merck, Darmstadt, Germany) were selected as sources of Ca2+, PO43−, and CO32−, respectively. A standard of 1.67 Ca/P molar ratio, comparable to that of biological bone, was adopted for designing the compositions in Eqs. (1) and (2) [37]. Two samples, namely, CHA0.67 and CHA1.00, were synthesized separately in accordance with Eqs. (1) and (2), where CHA0.67 and CHA1.00 represent [n(CO32−)/n(PO43−)] molar ratios of 0.67 and 1.00, respectively. To prepare CHA0.67 (Table 1), we dissolved the starting amounts of 7.92 and 3.16 g of the precursor reagents (NH4)2HPO4 and NH4HCO3, respectively, in 60 and 40 mL deionized water. The two aqueous solutions were both rigorously stirred for 15 min by magnetic stirring hot plates (FavoritTM, Malaysia) and another 15 min after mixing. Then, 23.62 g of Ca(NO3)2·4H2O was dissolved in 100 mL of acetone solution and poured into the previously 4

prepared aqueous solutions with thorough stirring. The same preparatory procedure was carried out in CHA1.00 but with a change in the amount and volume of NH4HCO3 (4.74 g and 60 mL) given the controlled increase in molarity ratio. The pH of the mixtures was maintained at 11 by adding 2.0 M solution of NaOH and using a Eutech Instruments (EI) pH510 pH meter with an EI glass electrode (Bukit Raja, Klang, Malaysia). Precipitates from the samples were filtered and washed three times with 1,000 mL of deionized water for each cycle for the removal of impurities, derivatives, and ammonia stench. The multiple washing stages ensured a high volumetric dilution of the byproducts (NH4NO3, HNO3, and H2O) and suspension of CHA products. The byproducts were then drained out of the system by vacuum-assisted filtration. Theoretical yields (Yth) of 10 g each for CHA0.67 and CHA1.00 (Table 1) resulted into actual yields (Yat) of 8.32 and 8.50 g, respectively. The percentage of yield [Ypt = (Yat/ Yth)100] for CHA0.67 and CHA1.00 were 83.2 and 85.0% respectively. The resultant white cakes from each sample were dried at 90 °C for 24 h, ground, and passed through a 90 μm mesh. 2.2 Material characterization As-synthesized powders were characterized to ascertain the formation of B-type CHA in the nanoscale and the bioactivity through X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, field-emission scanning electron microscopy (FESEM), X-ray fluorescence (XRF), carbon–hydrogen–nitrogen analysis (CHN), Brunauer–Emmett–Teller (BET) method, transmission electron microscopy (TEM), and inductively coupled plasma optical emission spectroscopy (ICP-OES). We employed XRD in the interval of angles 20° ≤ (2θ) ≤ 60° (Cu Kα radiation; Bruker Advanced X-ray Solution D8, Bremen, Germany) to determine the lattice parameter, crystallite 5

size, and phase of powders. The X’Pert HighScore Plus™ software was used. Inferred patterns were matched with the International Center for Diffraction Data with file number 00-009-0432 designated for standard HA to estimate the phase type and size of crystals. The crystallite size was calculated by XRD through Scherrer’s equation [38] (Eq. (3)):

L

kO , E1 2 cos T

(3)

where L refers to the crystallite size (nm), λ (1.5404 Ǻ) refers to the wavelength of Cu Kα radiation, β) refers to the full width at half maximum (radian), θ is the diffraction angle (degrees), and k is the boarding constant (0.94). In this regard, distinct diffraction peaks of (002), (211), and (300) with high intensities, were selected for evaluation. Rietveld refinement was conducted on the chemical compound to confirm the effect of carbonate vis-à-vis the molar ratio variation of [n(CO32−)/n(PO43−)] on the apatite structure. The types of bonding and chemical distinctiveness were determined by FTIR (Spectrum One; Perkin-Elmer, Waltham, MA, USA) in the 400–4000 cm−1 range with the help of the Spectrum software. The atomic concentration of elements in each composition was quantified by XRF spectrometer (Rix 3000; Rigaku, Tokyo, Japan). The carbonate content was determined by CHN analysis (2400 Series II; Perkin-Elmer, Waltham, MA, USA) by multiplying the weight percentage of carbon content by a factor of 5 [39]. The morphology, particle size, and surface properties of the as-synthesized nanopowders were examined by FESEM (S35VP; Zeiss, Dublin, CA, USA), TEM (Libra 120; Carl Zeiss, Oberkochen, Germany), and BET accordingly. Assessment of nitrogen adsorption–desorption was conducted at approximately 150 °C during the measurement of the BET surface area. The measured volume of gas (N2) adsorbed at a precise pressure for the compositions was conducted 6

after applying liquid nitrogen as coolant. The resultant isotherm adsorption link at Pi/P0 = 0.95 generated the pore volume. From the BET surface area, a derived particle size was estimated using the following formula [40]:

PSBET

6, 000 , SBET ˜ U

(4)

where PSBET is the BET particle size, SBET is the surface area, and ρ is the theoretical density. The sample preparation for TEM was conducted by creating dispersed particles from the acetone solution, 15 min of ultrasonication, and careful release of the mixture on a copper grid. The Image J™ software was employed to confirm the crystallite size of the powders, as determined by TEM. 2.3 Evaluation of in vitro bioactivity The CHA sample bioactivity study was conducted in vitro by using simulated body fluid (SBF) solution prepared in accordance with the recipe established by Kokubo et al. [41]. A ratio of 12.5 mg/mL powder to SBF solution [42] was used, with changes in pH recorded at predetermined sessions. FESEM was employed to evaluate the apatite layer and morphology of the compositions after soaking in SBF solution at 37 °C and pH = 7.4 for 7, 14, and 21 days. The SBF concentration of Ca2+ and P5+ ions in leachates were quantified by ICP-OES (PEO 7300 DV, USA). The leachate was converted into fine aerosol vapor, which allowed for transportation via a nebulizer with an argon stream into a plasmatic burner. The concentrations of ion release of Ca2+ and PO43− were measured at wavelengths of 393.366 and 177.499 nm.

3. Results and discussion

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3.1 Characterization of CHA nanopowders The XRD patterns of the compositions of CHA0.67 and CHA1.00 nanopowders in comparison with commercial HA (Sigma-Aldrich, St. Louis, MO, USA) are shown in Fig. 1. Fig. 1(b) shows the enlarged illustration of the patterns at 25° ≤ (2θ) ≤ 35°, showing a shift of peaks. CHA1.00 was more amorphous than CHA0.67, indicating an early effect of the difference in the CO32−/PO43− ratio. As shown in Fig. 1, by increasing CO32−, the peak reflections at (002) and (300) moved to a lower angle for CHA0.67 and CHA1.00 parameters. Legeros et al. [43] also described the alteration of the peak reflection of (300) to a high angle and a reverse trend for (002) with CO 32− incorporated into the apatite structure. The shift in peaks can be attributed to the strain from planar stress in the apatite structure because of CO32− substitution. Most reports typically described the A-type CHA based on the increase of its a-axis and the decrease of its c-axis lattice constants. By contrast, several reports described pure B-type CHA on the basis of the decrease in its a-axis and the increase in its c-axis lattice constants. However, Kovalena et al. [35] strongly agreed with De Maeyer et al. [44] who reported that the connection between carbonate content and the parameters of the lattice constant is an inconclusive method of establishing CHA types. Ivanova et al. [45] reported that the increase in lattice constant of a in many B-type CHA materials can be attributed to the ionic incorporation around the network O2− tetrahedral site, and possible partial substitution of water at the Ca2+ location. In the present work, the as-synthesized CHA0.67 and CHA1.00 nanopowders increased in their a- and c-axis lattice constants, respectively (Table 2). The increase in a-axis lattice constant may be responsible for the peak reflection at (300) to low 2θ relative to that of the previous assertion by Legeros and coworkers [43]. The absence of the secondary phase and the efficacy of the washing treatment resulted in a nearly stench-free

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complete reaction, in line with the findings of Krajewski et al. [39]. The as-synthesized nanopowders were observed to be nanocrystalline, with CHA1.00 exhibiting a more notable decrease in crystallite size than that of CHA0.67. According to McElderry et al., this substantial decrease in crystallite size results from the low resolution of the reflection peak that is caused by the perturbed lattice when the carbonate content is increased [46]. In comparison to XRD studies of heat treated carbonated HA reported elsewhere [47], our compositions are devoid of the typical single or multiple phases such as CaO/ α or β‒TCP associated with calcinated schedules. Furthermore, CHA0.67 and CHA1.00 displayed nanometric dimensions (Scherrer’s; 4.67 and 22.7 nm) lower than that of heat treated carbonated HA. The lower crystallite diminution in carbonated HA has been attributed to the atomic disorder and strained lattices (high energy state) created by non-growth crystallite conditions [48]. An extensive work on calcined carbonated HA by Garskaite et al [31] showed that the increase in crystallite size directly correlates to crystallization kinetics of such as crystallization temperature and heating rate. Person et al. [49] previously expounded on the interpretation of phase information from the peak parameters of CHA. However, the peak heights for commercial HA, CHA0.67, and CHA1.00 were compared by extracting the peak data directly from the peak list of XRD (Fig. 2). The results indicated a large increase in peak height for CHA0.67 relative to those of commercial HA and CHA1.00 nanopowders. By contrast, the broadening of peaks and decrease in height generated by the formation of a poorly crystalline phase were observed during carbonate substitution in CHA1.00 nanopowders with high carbonate content. The increase in carbonate content generally creates a short-range CO32− substitution [39], which leads to phase transition into a poorly crystalline or amorphous phase, as observed in CHA1.00. 9

The FTIR results shown in Fig. 3 and Table 3 strongly support the XRD results obtained (Fig. 1) from the nanopowders. The B-type CO32− substitution for CHA0.67 and CHA1.00 was confirmed with the band originating from the stretching vibrations of CO32− ions at 870–875, 1,410–1,430, 1,450–1,470, and 1,640 cm−1 [50]. The as-synthesized CHA was completely devoid of the PO43− spectral band spread at the v2 bending mode (878–888 cm−1), which is an A- or AB-type marker, as reported by Fleet et al. [51]. The PO43− spectral band for the as-synthesized B-type CHA was detected at approximately 550–570 (v4) and 1,020–1,120 cm−1 (v3) [52]. The broad band at approximately 1,600–1,700 and 3,200–3,600 cm−1 in all the synthesized samples was possibly due to water adsorption [53]. The commercial HA bands showed different structural bands with the visible absence of CO32− associated with CHA0.67 and CHA1.00. XRF spectroscopy, energy-dispersive X-ray spectroscopy (EDS), and CHN analysis were conducted to assess the elemental compositions and stoichiometry of the as-synthesized nanopowders (Table 4). EDS and CHN showed a trend of increased CO32− for CHA1.00 with respect to that of CHA0.67. By contrast, XRF analysis displayed an increment in Ca/P relative to that of HA (1.67); this result indicates the CO32− substitution into the PO43− site. These findings strongly support our FTIR analysis. The morphologies of the CHA0.67 and CHA1.00 nanopowders were investigated by FESEM (Fig. 3). The FESEM micrograph shows a fibrous particle with similar agglomerated morphologies for both compositions. EDS analysis (Fig. 4) revealed that the elemental composition of both CHA nanopowders confirmed the presence of phosphorus, calcium, and carbon, and that the high carbonate content of CHA1.00 reflects the high carbonate substitution in the structure. The TEM images in Fig. 5 illustrate that the CHA0.67 nanopowder exhibits a uniform size of rodlike crystals because of the highly crystalline phase similar to that of commercial HA. This 10

outcome is consistent with the rod-like crystalline HA reported by Sadat-Shojai et al. [12]. By contrast, CHA1.00 displayed regular spherical crystals with 20.4% smaller average size than that of the CHA0.67 crystals. Such significant reduction in crystallite size with the poorly crystalline phase of CHA1.00 was investigated and determined by Smolen et al. [54] to be closely similar to the stoichiometry and nanosize particle of human bone. In a study on CaP particles, Uskoković et al. stated that the nanosphere and rod-like crystals exhibit viable non-toxic responses to cells at concentrations up to 5 mg/mL of RAW 264.7 culture because of the nanometric dimension among other factors [55]. The nanosphere and rod-like crystals in our work agrees with this finding and thus show potential for safe cell-versus-crystal interactions. BET was employed to evaluate the textural properties, such as surface area (SBET) and derived BET particle size ( PSBET ), of the synthesized nanopowders. In Table 4, the BET results of the surface area of both compositions agree with those of the low-temperature synthesis reported elsewhere [56] with high surface area and small particle sizes. CHA1.00 exhibited a relatively large surface area (143.93 m2/g) and hence, a small particle size ( PSBET ) of 13.23 nm. By contrast, CHA0.67 exhibited a relatively small surface area (72.1 m2/g) and consequently, a large particle size ( PSBET ) of 26.42 nm. The high surface area and nanoscale particle size (less than 100 nm) of non-calcined CaP bioceramics have been shown to improve cell grafts at the cell–implant interface or protein adsorption and can thus enhance bone growth kinetics [34] [56]. Therefore, the combined effect of high surface area and smaller particle size shown in this work by TEM and BET may provide a potential pathway in dental restoration, bone filler, and drug release applications.

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3.2 In vitro bioactivity in SBF Apatite formation results from the interaction between a rich-Ca2+, which is a product of cationic/anionic intra-reaction within an immersed sample and PO43‒ in the SBF media [57]. In another explanation, bone-like apatite layer may be a precipitate formed by the supersaturated dissolution of immersed HA releasing Ca2+ and PO43‒ in SBF solution [47]. The ion release and apatite-forming capabilities of CHA0.67 and CHA1.00 nanopowders under calibrated pH conditions were assessed in vitro by using SBF media. Notable formation of apatite deposit on the nanopowders appeared at approximately 7 days of immersion in the SBF solution (Fig. 6). Both CHA compositions showed complete cladding of cauliflower-like apatite on the surface from 7 days to 21 days. This morphology was previously reported as a direct consequence of bone-like apatite deposited from SBF media [58]. The apatite layer thickens relatively as the soaking time increases and becomes more apparent in CHA1.00 and thereby confirming the bioactivity of both nanopowders. To avoid supersaturation, we used a dilution factor of 10 in 1 M HNO3 in the ion release analysis; hence, the values in Fig. 7 were multiplied by 10 in this discussion. The Ca2+ and P5+ released into the SBF for CHA0.67 increased as the immersion period was extended and confirmed the dissolution of Ca2+ and P5+ (Fig. 7). The early dissolution of bioceramics exerted a major influence on the interaction between implant material and bone [42]. A similar trend was observed for CHA1.00 as the release of Ca and P ions increased with prolonged soaking time. The Ca2+ release rate for CHA1.00 of 1.15 mMol to the SBF solution was relatively higher than that of CHA0.67 (0.40 mMol) after 21 days of soaking. The P5+ release rate for CHA1.00 was slightly lower than that of CHA0.67 in the early soaking period but showed a close value in releasing 1.71 12

and 1.76 mMol after 21 days of immersion. An increased functional P–O group in HA was recounted to favor protein adsorption because of anionic surface charge and electrophoretic mobility mechanism [56]. The ion release studies (Fig. 8), showed the mean (SD) pH values in CHA0.67 to be 7.3±0.28, 7.2±0.28, and 6.9±0.28 for 7, 14, and 21 days, respectively. Similarly, the pH values of 7.4±0.28, 7.0±0.28, and 6.5±0.42 were recorded in the same sequence of days for CHA1.00. The pH of the SBF solution for 7–21 days for CHA1.00 increased in the first 7 days of soaking and decreased thereafter alongside CHA0.67 to the end of the soaking period. Gheitanchi et al. opined that the decline in pH was linked to the consumption of hydroxyl ion because of the relocation of calcium and phosphate alongside hydroxyl ions toward the surface of the material [58]. The healthy pH range in humans is from 6.0 to 7.50, with the serum venous and arterial pH tightly regulated from 7.35 to 7.45 [59]. Osteoclastic bone resorption also importantly operates preferably in a pH less than the neutral region (< 7.0) because of the decarboxylation of proteins [60]. The materials synthesized and examined in this work exhibited an acceptable pH range (CHA0.67: 6.9±0.28 to 7.3±0.28 and CHA1.00: 6.5±0.42 to 7.4±0.28) for functional nanopowder applications.

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4. Conclusion B-type CHA nanopowders with variant CO32− to PO43− molar ratios were successfully synthesized via the nanoemulsion route at low temperatures and then characterized. The assumption that CO32− substitutes for PO43−, as described in the chemical formulations of Ca9.98(PO4)5.96(CO3)0.04(OH)2 for CHA0.67 and Ca9.97(PO4)5.94(CO3)0.06(OH)2 for CHA1.00, was well validated. The increase in CO32−/PO43− molar ratio resulted in the rise in surface area and refinement of particle size; however, no significant change in morphology was observed. Structural information showed a significant change in phase from highly crystalline to amorphous phase with increasing carbonate content. In vitro bioactivity tests of the powders indicated a rapid formation of apatite layer and relatively improved bioactivity values in CHA1.00; such enhancement included high Ca2+ release (1.15 mMol) possibly because of increased carbonate content (7.80 wt.%) and smaller nanometric values. Notwithstanding, both materials (CHA0.67 and CHA1.00) showed excellent biofunctional qualities, such as pH, CO32− content, and nanodimensions, compatible with human body applications. The overall results highlight that this improved nanosized bioactive synthetic CHA may offer promising bone tissue engineering and drug delivery system applications. Acknowledgments The authors sincerely acknowledge the financial support provided by the USM Graduate Assistant Scheme and the support from the Fundamental Research Grant Scheme, FRGS MOHE (6071736).

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Figure captions Fig. 1. Phase properties of CHA nanopowders: (a) XRD patterns of commercial HA, CHA0.67, and CHA1.00, and (b) redrawn XRD patterns at 25° ≤ 2θ ≤ 35°, confirming a shift in peaks. Fig. 2. Selected peak height of commercial HA, CHA0.67, and CHA1.00: elucidating phase information. Fig. 3. FTIR spectrum: (a) commercial HA and as-synthesized (b) CHA0.67 and (c) CHA1.00. Fig. 4. FESEM morphology and EDS confirmation of chemical constituent: (a) CHA0.67 and (b) CHA1.00. Fig. 5. TEM micrographs: structural properties of (a) CHA0.67 and (b) CHA1.00 nanopowders. Fig. 6. FESEM micrographs of as-synthesized nanopowders: (a) CHA0.67 and (e) CHA1.00. In vitro bioactivity evaluation after soaking: CHA0.67 in (b) 7, (c) 14, and (d) 21 days; CHA1.00 in (f) 7, (g) 14, and (h) 21 days. Fig. 7. Released ions (Ca and P) to the SBF solution after immersion: (a) CHA0.67 and (b) CHA1.00. Fig. 8. pH during the soaking period for CHA0.67 and CHA1.00 in SBF media.

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Table captions Table 1 Precursor parameters of the ambient temperature synthesis of CHA nanopowders Molarity and media of precursors [Ca2+] [PO43−] [CO32−] Sample Acetone solution Water solution Water solution 0.11 M Ca(NO3)2 0.06 M (NH4)2HPO4 0.04 M NH4HCO3 CHA0.67 0.11 M Ca(NO3)2 0.06 M (NH4)2HPO4 0.06 M NH4HCO3 CHA1.00 a b c Yth; Theoretical yield, Yat; actual yield, Ypt; percentage yield

Synthesis yield Yth Yat Ypt a b (g) (g) (%)c 10.0 8.32 83.2 10.0 8.50 85.0

Table 2 Crystal structure of the reference HA and CHA as-synthesized nanopowders Lattice constant (Å) Sample

a=b

c/a ratio

Crystallite/particle size (nm) LScherrera

c

PTEMb

PSBET c

9.4180 6.8840 0.7309 HA (09-0432) 9.4311 6.8862 0.7302 22.7 24.87 26.48 CHA0.67 9.4728 6.9110 0.7296 4.67 6.50 13.23 CHA1.00 a b c LScherrer; Scherrer calculation, PTEM; TEM average particle size, PSBET ; BET average particle size

Table 3 Functional groups and wavelengths of HA and as-synthesized CHA nanopowders Absorption location (cm‒1) Functional group CHA0.67 CHA1.00 HAӂ ‒ 3593 3593 3602 (OH ) 1640 1640 1639 (OH‒ ) 1461 1462 ‒ (CO32‒) v3 3‒ 1078 1080 1117 (PO4 ) v3 872 871 ‒ (CO32‒) v2 568 568 569 (PO43‒) v4 ӂ Commercial HA from Sigma-Aldrich

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Reference 3200-3600 [53] 1600-1700 [53] 1450-1470 [50] 1020-1120 [52] 870-875 [50] 550-570 [52]

Table 4 Chemical and BET textural properties of the as-synthesized nanopowders Samples

Phase Formula

[n(CO32‒)/n(PO43‒)]

CHA0.67 CHA1.00

Ca9.98(PO4)5.96(CO3)0.04(OH)2.00 Ca9.97(PO4)5.94(CO3)0.06(OH)2.00

0.67 1.00

a

CO32‒ (wt. %) a 3.10 7.80

(Ca/P)b 1.89 1.88

CO32‒ content; CHN analysis, b Ca/P; XRF analysis, c (SBET); BET specific surface area

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SBET (m2/g) c 72.10 143.93

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