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Vol. 8. Issue 3.
Pages 2481-3388 (May - June 2019)
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Vol. 8. Issue 3.
Pages 2481-3388 (May - June 2019)
Original Article
DOI: 10.1016/j.jmrt.2019.02.025
Open Access
Synthesis and characterization of mesoporous hydroxyapatite powder by microemulsion technique
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An Huang, Honglian Dai
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daihonglian@whut.edu.cn

Corresponding author.
, Xiaopei Wu, Zheng Zhao, Yanzeng Wu
State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan 430070, China
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Abstract

The mesoporous hydroxyapatite (HAP) powder was synthesized by using the microemulsion (hexadecyltrimethyl ammonium bromide (CTAB)/cyclohexane/n-octyl alcohol) system. The optimal ratios of concentrations of CTAB, cyclohexane and n-octyl alcohol in the microemulsion on morphology of the HAP were studied. The effects of pHs and calcination temperatures on the phase composition of the HAP powder were discussed. The phase composition, morphology, mesoporous structure and the optical density (OD) were characterized and confirmed by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FESEM), N2 adsorption–desorption isotherms and Multiskan Spectrum, respectively. We further investigated the effect of the HAP powder with meso-structure on the proliferation of bone marrow mesenchymal stem cells (BMSCs) and MC3T3 in vitro, and in vitro study of drug release of mesoporous HAP powder. Through the optimized microemulsion system (CTAB 25g/L/cyclohexane 125mL/L/n-octyl alcohol 250mL/L) at pH of 11.0, the mesoporous HAP was formed. The specific surface area of the HAP powder product is 13.62m2/g with an average pore size of 19.56nm after calcinating at 850°C.

Keywords:
Hydroxyapatite
Mesoporous
Microemulsion
Cells
pH
Calcinating
Full Text
1Introduction

HAP (Ca10(PO4)6(OH)2) has been utilized for biomedical materials reconstruction of damaged bones and tooth zones for decades for its unique properties such as biocompatibility, osteo-reconstruction conductivity and bone integration [1–5]. The biological performance of HAP has been known for the flexibility of its structure to the surrounding environment [6,7]. HAP ceramic coating with a designated microstructure can be extensively used for many applications. In the field of biomedical ceramics, pore-size modification technique has thus been important for HAP productions. The smaller pore-sized and uniformed HAP ceramics are more desirable, that has broader applications for its flexibility, effective crack dispersion and biocompatability compared to the non-uniformed aperture HAP and the macroporous HAP [8,9].

Mesoporous material has gained great attentions in the field of biomaterials in recent years [9,10], mesoporous HAP have exhibited a higher drug-loading capacity and boosted drug release property [11]. Compared to other traditional biomaterials, the mesoporous coating materials also have displayed a higher bone forming ability and an easier induction of apatite in vitro [12,13]. A doped HAP nanocrystals through the surfactant-free method have potential applications in blue phototherapy and photodynamic therapy [14]. One new promising approach for a multimodal contrast agent is the use of doped HAp as a ceramic nanoparticle-sized contrast agent or marker for biomedical luminescence imaging [15]. On the other hand, the HAp nanorods with excellent luminescent properties could be applied to live cell imaging [16]. Fluoridated HAP nanoparticles for cell-imaging [17]. Since mesoporous HAP have proven to be a desirable coating material with good biocompatibility [18–21], it would be worthwhile to explore new ways to optimize the methodology for uniformed and better properties of HAPs.

Mesoporous HAP can be synthesized by various techniques including sol–gel technology [18], hard-templating method [22], gas–liquid chemical precipitation method [23] and soft-templating technique [24–26]. Among them, soft-templating technique is the most commonly used. The soft-templating technique is based on the self-assembly of surfactant molecules into micelles where soluble HAP precursors are condensed. However, the undesired grain growth and disorderly aperture formation are the problems after the thermal treatment in HAP process [22]. Therefore, a new method of improvement in this study for the preparation of a uniformed and mesoporous HAP.

Microemulsion system/technique has been recently applied in many fields such as oil production, coating, solvent extraction, cleansing agents, enzyme-catalyzed reaction, preparation of nano-particles, and porous or mesoporous materials. Therefore, it is a key to have an optimized microemulsion system to be made for uniformed and mesoporous HAP. Microemulsion system contains a surfactant phase, an oil phase and a water phase. Microemulsion system is an isotropic oil–water mixture characterized with low viscosity. It is therefore, transparent or semitransparent depending on the environmental temperature. It is thermo dynamically stabile. The size range of the droplets in the system is between 5 and 20nm in diameter [27]. Advantages of the system include its anti-aggregation property, excellent stability and self-controlled formation of apertures. The microemulsion technique has been employed to prepare silicon materials with mesoporous structure [28], mesoporous aluminum oxyhydroxide nano flakes [29], and other mesoporous materials such as, TiO2, ZnO, CdS, carbon foams, polyvinyl benzenes and so on. We reported here the preparation of a microemulsion system used for the synthesis of a uniformed and mesoporous HAP powder.

In this study, the mesoporous HAP was synthesized by a microemulsion system using CTAB as a surfactant, n-octyl alcohol as a cosurfactant, cyclohexane as oil phase. The optimization of the microemulsion composition was reported. The influential factors for the microemulsion system to produce mesoporous HAP such as pHs and calcination temperatures on the phase composition of the HAP powder were studied. The combinations of CTAB, cyclohexane and n-octyl alcohol in the microemulsion system on the morphology of higher purity meso-structural HAP with improved uniform surface and the effect of mesoporous HAP powder on the proliferation of BMSCs and MC3T3 and in vitro study of drug release properties of the mesoporous HAP powder were researched and discussed.

2Materials and methods2.1Materials

Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), diammonium hydrogen phosphate ((NH4)2HPO4), ammonia solution (NH3·H2O), Anhydrous ethanol, CTAB, cyclohexane and n-octyl alcohol were of analytical reagent grade and phosphate buffered saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and α-Dulbecco's modified Eagle's medium (α-DMEM) were acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bone marrow mesenchymal stem cells (BMSCs, derived from SD-type rates). MC3T3 (Mouse embryo osteoblast cells). Deionized water was used throughout the study.

2.2Preparation of mesoporous HAP powder

The mesoporous HAP was synthesized as the follows. CTAB (with concentrations of 5.0g/L, 25g/L and 125g/L, respectively), cyclohexane (25mL/L, 125mL/L and 625mL/L, respectively) and n-octyl alcohol (125.5mL/L, 250mL/L and 500mL/L, respectively) were dissolved in 40mL deionized water and stirred for 30min at 37°C to make a preparation system respectively. Ca(NO3)2·4H2O solution (20mL, 0.3mol/L) was added to make a pre-microemulsion system. Then, the (NH4)2HPO4 solution (0.3mol/L) was added dropwise to the mixed solution under vigorously stirring force (1800rpm) simultaneously, Ca/P solution with the ratio of 1.67 and NH3·H2O solution (1.0mol/L) was added to adjust the pH to (5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0, respectively) to make the final microemulsion system. After being continuingly stirred for 6h, and then aged for 24h at room temperature, the mixture of the microemulsion system was washed with deionized water 3–5 times and ethanol 3–5 times in sequence, precipitated HAP particle preparations were filtered from the mixture and then dried at 80°C for 24h. Finally, the dried preparations were calcined to form HAP powders at different temperatures (200, 400, 600, 700, 750, 800 and 850°C for 4h, respectively).

2.3Cell culture

The cell suspension (2.0×104cells/mL) was obtained by mixing the BMSCs or MC3T3 with α-DMEM solution and then plated into each well of 96-well culture plates (100μL/well). The plates were incubated for 24h at 37°C in a humidified incubator of 5% CO2.

2.4Cell proliferation assay

The effect of mesoporous HAP powder on the viability of BMSCs was measured using MTT assay. The raw material (100mg mesoporous HAP powder) were cleaned carefully with deionized water 3 times and ethanol 3 times in sequence and sterilization treatment by steam sterilizer. The materials were diluted by PBS for five different concentrations respectively and then ultrasonic dispersion about 20min. The materials suspension were obtained and added to the each well of 96-well culture plates (20μL/well) containing the cell suspension. The final five concentrations of materials were 0.002, 0.01, 0.02, 0.06 and 0.1μg/mL. The wells without materials samples were used as a control group. All cells were cultivated at 37°C in 5% CO2 incubator for 1, 3, 5 and 7 days. The culture medium was replaced on the third day. MTT (20μL) was added to each well and incubated for 4h at 37°C. The MTT was removed and 200μL DMSO was added to each well to dissolve the formazan crystals. The optical density (OD) was measured at a wavelength of 490nm.

2.5X-ray diffractometry (XRD)

The structural analysis of mesoporous HAP powder was performed by X-ray diffraction (Rigaku D/MAX-RB, Japan). Data were collected for 2θ ranging between 5° and 70° using Cu Kα radiation (λ=0.15406nm).

2.6Field emission scanning electron microscopy (FE-SEM)

The surface morphology of the mesoporous HAP powder was visualized by a field emission scanning electron microscope (Zeiss Ultra Plus, Germany). HAP samples were placed on a platinum coating plate. Then the HAP powder was made conductively by sputtering a thin layer of gold onto the powder surface for the emission scanning under FE-SEM.

2.7Transmission electron microscope (TEM)

High-resolution transmission electron microscopy (HRTEM) images were taken digitally using a JEOL 2010 microscope operated at 200kV and fitted with a low-background Gatan double tilt holder and a Si (Li) X-ray detector. Before this study, the mesoporous HAP powder was ultrasonically dispersed in ethanol for 5min with a drop of suspension deposited on holey carbon-coated copper grids.

2.8Fourier-transform infrared (FT-IR) spectroscopy

Fourier transform infrared spectroscope (Nicolet 6700, USA) was also used to determine the composites and chemical bonding of HAP. Samples were oven-dried at 80°C for 6h before being compacted into transparent disks. All FT-IR spectra were recorded in the frequency range of 400–4000cm−1.

2.9Nitrogen adsorption

Nitrogen adsorption analysis was performed at 77.3K by the Surface Area and Pore Size analyzer (ASAP 2020M, Micromeritics, USA). Samples were degassed over night at 80°C. Adsorption and desorption isotherms were recorded in the relative pressure range of 5.0×10−2<P/Po<1.0. The specific surface area and pore size distribution were calculated by the Brunauer–Emmett–Teller (BET) equation and the Barrette–Joynere–Halenda (BJH) method.

2.10Dynamic light scattering

The hydrodynamic size distribution of the mesoporous HAP powder was performed by the dynamic light scattering. Before this study, the mesoporous HAP powder (0.1g) was dispersed in deionized water (100mL) and Ultrasonic processing for 30min.

2.11Statistical analysis

Each experiment was performed at least in triplicate. All data were expressed as the means±standard deviation and statistical differences were determined by analysis of variance followed by Student's t-test. A value of P<0.05 was considered statistically significant.

2.12In vitro study of drug release

To investigate the influence of the mesoporous structure on drug release properties, firstly 0.20g alendronate sodium-loaded mesoporous HAP were soaked in 15mL PBS (pH 7.4) at 37°C. At different interval, then 5mL PBS solution was taken-off and replaced with 5mL of fresh PBS respectively. The amount of alendronate sodium released in the PBS was then assayed by UV-Vis at the wavelength of 266nm.

3Results and discussions3.1Influences of pHs and calcination temperatures on the phase composition of the HAP powder

The XRD patterns of the product synthesized at pHs from 5.0 to 11.0 were shown in Fig. 1. (f) and (g) show that the product prepared under pH 10.0 and 11.0 had the main (hkl) indices: (002), (211), (112), (300), (202), (310), (222), (213), (321) and (004) assigned to the formed HAP powder (JCPDS no. 09-0432).

Fig. 1.

XRD patterns of HAP powder synthesized at different pHs: (a) pH=5.0, (b) pH=6.0, (c) pH=7.0, (d) pH=8.0, (e) pH=9.0, (f) pH=10.0 and (g) pH=11.0.

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As shown in (c)–(e), β-tricalcium phosphate (β-Ca3(PO4)2, β-TCP) was detected in the product when pH in the microemulsion system was changed from 7.0 to 9.0. As reported in the literature [30], Ca/P ratio of 1.67 at the beginning would be dropped down to 1.50 at the end. Under our experimental conditions, when pH was changed from 7.0 to 9.0. β-TCP is one of the main residues formed after the HAP was calcined.

(a) and (b) indicates that calcium pyrophosphate (Ca2P2O7, CPP) was formed in the product when pH was adjusted between 5.0 and 6.0, pH in the microemulsion system therefore played an important role in the phase composition of the final product. To obtain a high purity of HAP powder, pH of 11.0 was therefore selected under our experimental conditions.

Fig. 2 presents the XRD patterns of the as-calcined HAP powder and HAP powder obtained at the calcination temperatures ranging from 200 to 850°C. All HAP powder produced under such conditions exhibited a typical phase of the high purity without residues such as β-TCP and CPP. The characteristic peaks of the purer HAP power were narrower and sharper when the calcination temperatures increased from 200 to 850°C, indicating that higher temperature facilitates the crystallization in the system. Therefore, the calcination temperature of 850°C would be optimized for next work.

Fig. 2.

XRD patterns of the as-calcined HAP powder and the HAP powder obtained at different calcination temperatures for 4h: (a) as-calcined, (b) 200°C, (c) 400°C, (d) 600°C, (e) 700°C, (f) 750°C, (g) 800°C and (h) 850°C.

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3.2Influences of concentrations of CTAB, cyclohexane and n-octyl alcohol in the microemulsion system on the surface morphology of mesoporous HAP powder

The concentrations of CTAB, cyclohexane and octyl alcohol are known to be important for the formation of microemulsion.

Fig. 3 shows the surface morphology of HAP powder prepared at different CTAB concentrations (5.0g/L, 25.0g/L and 125g/L, respectively). As shown in Fig. 3b, the HAP powder had worm-like particles at the CTAB concentration of 25.0g/L and exhibited relatively smooth, continuous and well-ordered mesoporous morphology. In contrast, the HAP powder with a great numbers of disordered and aggregated macroporous particles was observed at the concentrations of 5.0g/L and 125g/L (Fig. 3a and c). It was seemingly that the uniformity of a mesoporous HAP powder was affected remarkably by the concentration of CTAB in the microemulsion system at extremities. Organic substances such as surfactants may have interacted with inorganic substances in the microemulsion system by intensified electrostatic forces to form an orderly arrangement structure through a cooperative formation mechanism [31,32]. As an amphiphillic molecule, CTAB would be existent in the interface of the dispersed aqueous phase and the continuous organic phase [33]. The mesoporous structure of HAP is considered to be formed with the removal of the intermediate assembly parts of charged surfactant molecules through high temperature treatment [34]. In our experimental conditions, the CTAB concentration of 25.0g/L was optimal for the formation of uniformed surface morphology for a mesoporous HAP.

Fig. 3.

FE-SEM images of the surface morphology of the HAP powder synthesized at different CTAB concentrations: (a) 5.0g/L, (b) 25.0g/L and (c) 125g/L.

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As shown in Fig. 4b, the HAP powder formed in the experimental condition exhibited uniform worm-like morphology and mesoporous structure. The egg-shaped particles of HAP were with width of about 40nm and length of about 100nm when the concentration of cyclohexane was at 125mL/L. Compared with the mesoporous HAP powder obtained in cyclohexane at 125mL/L, the HAP synthesized under the cyclohexane concentrations of 25mL/L and 625mL/L displayed larger grains and pores respectively due to the agglomerations of small grains and under different cyclohexane concentrations, as shown in Fig. 4a and c. It was shown that cyclohexane, as oil phase, played an important role in the microemulsion system. Since water and oil phase separation is in nature, a transparent and homogeneous microemulsion formation will not be the case when the cyclohexane concentration was at low or high extremes, consequentially leading to the formation of more disorderly and macroporous structure of HAP particles. The mesoporous structure should therefore be prepared under the cyclohexane concentration of 125mL/L.

Fig. 4.

FE-SEM images of the surface morphology of the HAP powder synthesized at different cyclohexane concentrations: (a) 25mL/L, (b) 125mL/L and (c) 625mL/L.

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Fig. 5 presents the surface morphology of the HAP synthesized under three n-octyl alcohol concentrations respectively (125.5mL/L, 250mL/L and 500mL/L). The existence of pores was observed in the FE-SEM images of the HAP powder prepared under the n-octyl alcohol concentrations of 125.5mL/L and 500mL/L (Fig. 5a and c). In contrast, an orderly uniformed mesoporous HAP powder were synthesized under the n-octyl alcohol concentration of 250mL/L as shown in Fig. 5b. n-Octyl alcohol as a cosurfactant in the microemulsion system may have reduced interfacial tensions and adjusted the hydrophilic and lipophilic forces in the emulsion, making a finer microemulsion in the system possible. In contrast, low or high concentrations of n-octyl alcohol interfered the stability of the microemulsion system. The optimized concentration of n-octyl alcohol in the system of the study was 250mL/L.

Fig. 5.

FE-SEM images of the surface morphology of HAP powder synthesized under different octyl alcohol concentrations: (a) 125.5mL/L, (b) 250mL/L and (c) 500mL/L.

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3.3TEM images of the mesoporous HAP powder

As shown in Fig. 6, the mn-HAP (mesoporous nano-hydroxyapatite) powder obtained at without calcination and after calcination. Certain agglomeration of HA particles were observed. Long nanorods with mesoporous structure were seen in Fig. 6a and b, the increase in particle size with temperature takes place by aggregation of these agglomerated particles to form secondary particle, this has been schematically represented in Fig. 6b. The mesoporous structure in Fig. 6a was clearer than that in Fig. 6b. The pores were formed by the removal of the intermediate assemblies of the charged surfactant molecules. By adjusting the heating rate at 5–10°C/min, the porous structure did not collapse, but form a mesoporous structure.

Fig. 6.

TEM images of the mesoporous HAP powder obtained at (a) without calcination and (b) after calcination.

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3.4FT-IR analysis of HAP powder

Fig. 7 shows typical FT-IR spectra of the uniformed mesoporous HAP powder. The characteristic peaks were between 3572 and 632cm−1. It is in a range of vibrational mode of structural OH groups of mesoporous HAP powder. The peaks at 1090, 1044 and 962cm−1 were respectively contributed to phosphate stretching vibrational modes of ν3, ν3, and ν1 in PO43−. The peaks at 602, 571 and 474cm−1 were respectively contributed to the phosphate bending vibrational modes of ν4, ν4, and ν2 in PO43−. The broader peak at 3431cm−1 and the narrower peak at 1629cm–1 were due to H2O bending mode and adsorbed water, respectively. The peaks for the carbonate anions were also observed at 1458, 1412 and 1381cm−1, respectively. It might be due to released CO2 from mixing, stirring and reaction processes [35].

Fig. 7.

FTIR spectra of the HAP powder prepared by the microemulsion technique.

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3.5N2 adsorption/desorption and BJH pore size distribution of mesoporous HAP powder

Fig. 8 shows the N2 adsorption/desorption isotherms of the HAP synthesized by the microemulsion technique. A typical type IV isotherm with H1-type hysteresis loop is due to capillary condensation, indicating the presence of a mesoporous structure. The area of hysteresis loop was narrow. The adsorption branch was nearly parallel with the desorption one. These two phenomenons suggested the existence of microporous structure in the formed HAP. Additionally, the isotherms displayed a sharp inflection in the range of 0.96–0.99 P/Po, suggesting the HAP powder contains a broader pore size distribution of the mesoporous structure. The pore size distribution of the sample mesoporous HAP presented in Fig. 9 reveals a broader range of pore size distributions in the formed HAP mainly below 25nm with combined microporous and mesoporous structures. The broader range of pore size distributions might be produced by collapses of partial mesoporous structure after intensified heating treatment, higher temperature could contribute to particle grow than deagglomeration because of the lack of any obstacle for particle growth, subsequently, the tighter packing and agglomeration of HAP powder lead to the disappearance of mesopores [18]. The average pore size was 19.56nm measured by the BJH method and the specific surface area calculated was 13.62m2/g in the HAP powder.

Fig. 8.

N2 adsorption/desorption isotherms of the mesoporous HAP powder.

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Fig. 9.

The BJH pore size distribution of the mesoporous HAP powder.

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3.6The formation of mesoporous HAP

Fig. 10 shows the forming process of the mesoporous HAP by the microemulsion technique. A microemulsion was formulated by adding CTAB (surfactant), cyclohexane (oil) and n-octyl alcohol (cosurfactant) into an aqueous solution. Initially, CTAB was formed a hexagonal micelle [36]. Ca2+ ions were interacted with CTAB molecules in the micelles to form hexagonal CTAB-Ca2+ Clusters in the microemulsion. PO43− could have electrostatically interacted with Ca2+ in the CTAB-Ca2+ microemulsion to be precipitated on the external surface of the hexagonal CTAB-Ca2+ Clusters to form rod-like CTAB-HAP Clusters. The mesoporous structures were then retained after the removal of CTAB by calcining at a high temperature [37].

Fig. 10.

The flow chart of the formation of the mesoporous HAP powder by the microemulsion technique.

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3.7Effects of the mesoporous HAP powder on the proliferation of BMSCs and MC3T3

The hydrodynamic size distribution of mesoporous HAP powder via the microemulsion processing route, as shown in Fig. 11, was in the range of sub-micrometers, with an average particle size of about 1.5μm. Drying and calcination resulted in the formation of agglomerates of HAP powder and a skewed distribution which centered to larger particle size [38].

Fig. 11.

The hydrodynamic size distribution of mesoporous HAP after calcination.

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The MTT assays were performed to evaluate the effects of the mesoporous HAP powder on the proliferation of BMSCs and MC3T3, as shown in Figs. 12 and 13. It was found that the OD values measured at 490nm increased when the culture period of BMSCs and MC3T3 under different concentrations (0.002, 0.01, 0.02 and 0.06μg/mL) of materials were prolonged from 1 to 7 days. The presence of 0.1μg/mL mesoporous HAP powder induced a significant decrease in cell proliferation in relation to the control group after 3, 5 and 7 days in culture (P<0.05), as shown in Fig. 12. The presence of 0.01μg/mL, 0.02μg/mL and 0.06μg/mL mesoporous HAP powder induced a significant decrease in cell proliferation in relation to the control group at 7 day in culture (P<0.05), as shown in Fig. 13. These results suggested that the material under lower concentrations could allow cell attachment and proliferation, but cytotoxic at a higher concentration. This property makes the mn-HAP powder useful in many fields, such as biomedical applications [39], induced emission dye-based fluorescent organic nanoparticles via emulsion polymerization and their cell imaging applications [40].

Fig. 12.

Effects of different concentrations of mesoporous HAP powder on the BMSCs proliferation under different culture time. Each bar represents the mean±standard deviation (n=3). *P<0.05, when compared with control.

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Fig. 13.

Effects of different concentrations of mesoporous HAP powder on the MC3T3 proliferation under different culture time. Each bar represents the mean±standard deviation (n=3). *P<0.05, when compared with control.

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3.8In vitro study of drug release properties of the mesoporous HAP powder

Fig. 14 indicates the cumulative alendronate sodium release rate from synthetic sample in PBS (pH 7.4). Obviously, the release kinetics of mesoporous HAP powder include the initial fast release, which was induced by the breakage of hydrogen bonding on the surface of crystal, and the following sustained release mainly attributed to the various surface property and mesoporous structure [41]. However, due to the time limits, it is not possible to study the sustained release law of drugs in detail, but our study on drug release in a short time shows that the mesoporous structure of apatite has potential applications in the field of drug release. Also, the mesoporous HAP powder has a potential application of surface modification and biomedical applications [42].

Fig. 14.

The cumulative alendronate sodium release rate of mesoporous HAP powder.

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4Conclusions

The mesoporous HAP powder was prepared by the microemulsion technique. After calcination at 850°C for 4h, a high-purity mesoporous HAP powder with uniformed and well-ordered morphology was obtained in the microemulsion system of CTAB 25g/L, cyclohexane 125mL/L and n-octyl alcohol 250mL/L at pH 11.0. The N2 adsorption/desorption and BET studies had proved the uniformed and mesoporous structure in the HAP. The specific surface area was 13.62m2/g. The mesoporous structure had a broader pore size distribution with an average pore size of 19.56nm. The MTT assays demonstrated that the material's biocompatibility under lower concentrations (0.002, 0.01, 0.02 and 0.06μg/mL), but cytotoxic at a higher concentration (0.1μg/mL). The HAP product produced under optimized conditions from the study meets the standard of homology and micromeso-structure of HAP. It has potentials for applications of bone tissue crafts and drug delivery, as well as coating material for the future studies.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (No. 2018YFB1105500, 2016YFC1101605 and 2016YFB1101302), the National Natural Science Foundation of China (No. 51772233), and the Science and Technology Project of Wuhan (No. 2018010401011273).

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