BIELEC Monikaq, HUANG An, XIA Yuhao, DAI Honglian1,*

(1. Shenzhen Institute of Wuhan University of Technology, Shenzhen 518000, China; 2. State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan 430070, China)

Abstract: The synthesis of mesoporous β-tricalcium phosphate (β-TCP) powder was performed by using the microemulsion approach, with hexadecyltrimethyl ammonium bromide (CTAB)/cyclohexane/n-octyl alcohol microemulsion system. The influences of different pH values and calcination temperatures on the phase composition of the β-TCP powder were studied. The in vitro proliferation of bone marrow mesenchymal stem cells (BMSCs) in the suspensions of β-TCP powders with meso-structure was studied. The phase composition, mesoporous structure, powder morphology, cell morphology and the optical density (OD) were characterized through X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), Fourier transform infrared (FTIR) spectroscopy, N2 adsorption-desorption isotherms, inverted phase contrast microscopy and Multiskan spectrum, respectively. The mesoporous β-TCP powder with specific surface area of 12.85 m2/g and the average pore size 7.11 nm was obtained through the microemulsion approach (100 g/L CTAB/250 mL/L cyclohexane/250 mL/L n-octyl alcohol) with a controlled pH of 7.0, after calcinating the powder at 800 ℃. It was confirmed that mesoporous β-TCP powder benefits the activity of BMSCs more than the non-mesoporous β-TCP powder.

Key words: β-tricalcium phosphate; mesoporous; microemulsion; bone marrow mesenchymal stem cells

1 Introduction

Mesoporous materials have been attracting intense interest for their applications in the biomedical field, due to their large pore volume, narrow pore size distribution among 2-50 nm, unique surface and large surface area, and a narrow pore size distribution among 2-50 nm, which characteristics conduce to mesoporous materials application in developing new types of catalysts, adsorbents, drug delivery systems, and so on. As the mesoporous structure is also capable of incorporating large numbers of biologically active molecules, is potentially a great material for developing a tissues regeneration system.

Tricalcium Phosphate (TCP, Ca(PO)) is known to have three polymorphs: α,

β

, and α’, that are stable at different temperatures, with the transition from

β

-TCP to α-TCP at 1 180 ℃, and a transition from α-TCP to α’-TCP at 1 470 ℃. Among the three allotropic forms,

β

-TCP is the most suitable to be applied in orthopaedics as a bone filler, due to similarity of its chemical composition with human bone tissue, as well as its good bioactivity and biodegradability and its high dissolution rate in the human biological environment, which promotes bone growth during the progressive degradation. Moreover,

β

-TCP has been shown to exhibit bioresorbability, due to its comparatively high solubility, and osteoconductivity in human bone. Having such beneficial characteristics for biomedical applications,

β

-TCP has become a popular subject of research.Bone regeneration process requires optimal environment for the bone tissue to grow. Porosity of biomaterials is an important characteristic, as it allows growing bone tissues to integrate fully with the material that supports the formation of bone-like apatite on the surface after soaking in simulated body fluids. Bone tissue regeneration, as well as the drug delivery, can be also promoted due to capability of the mesoporous structure to incorporate large numbers of biologically active molecules, including osteogenic agents. Therefore, combining the performance of

β-

TCP with the advantages of mesoporous structure could result in the biomaterial with improved characteristics. However, to the best of our knowledge, in the literature there is little evidence of the mesoporous

β

-TCP research being conducted at the moment. Main challenges in aforementioned research are crystal habit and synthesis difficulties.Here, due to synthesise mesoporous

β

-TCP powder, we applied microemulsion technique. Microemulsions are stable, isotropic mixtures of oil, water and surfactant, frequently in combination with a cosurfactant, serving as the versatile reaction media. Among the wet chemical synthesis methods, microemulsion technique facilitates superior control over morphology and pore size, which is due to the stabilized by surfactant genuine dispersion of 10-100 nm liquid droplets within another immiscible liquid. This method was, to great effect, applied in the process of synthesis of many mesoporous materials, namely: silica, TiO, hydroxyapatite-MgFeOnanocomposite, aluminum oxyhydroxide nanoflakes, ferrihydrite/SiOcomposite, and so on.In this study, microemulsion technique synthesis of the mesoporous

β

-TCP powder was performed through applying CTAB as a surfactant,

n

-octyl alcohol as a cosurfactant, and cyclohexane as an oil phase, in fixed concentrations. As the result of this study, we described the impact of different pH and calcination temperatures on the phase composition of the

β

-TCP powder, the steps of the procedure of microemulsion technique synthesis of mesoporous

β

-TCP powder, and the effect of mesoporous

β

-TCP powder on the proliferation of bone marrow mesenchymal stem cells (BMSCs) in vitro.

2 Experimental

2.1 Materials

CTAB, cyclohexane, n-octyl alcohol, calcium nitrate tetrahydrate (Ca(NO)·4HO), diammonium hydrogen phosphate ((NH)HPO), ammonia solution (NH·HO), anhydrous ethanol, 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 purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Bone marrow mesenchymal stem cells (BMSCs) were derived from SD-type rats. Deionized water was used in this study.

2.2 Preparation of mesoporous β-TCP powder

CTAB (100 g/L), cyclohexane (250 mL/L) and

n

-octyl alcohol (250 mL/L) were dissolved in 40 mL deionized water and stirred for 30 min at 37 ℃, until the microemulsion system was formed. Ca (NO)·4HO solution (20 mL, 0.3 mol/L) was added into the microemulsion system. Subsequently, the (NH)HPOsolution (0.3 mol/L) was added dropwise with stirring at 1 800 rpm, while adjusting the pH with NH·HO solution (1.0 mol/L). The pH values were established as follows: 5.0, 6.0, 7.0, 8.0, and 9.0. The final Ca/P ratio was 1.5. The mixture was stirred continuously for 22 h and, afterwards, aged in the room temperature for 24 h. Then, the mixture was washed 5 times with deionized water and 5 times with ethanol, using centrifugation washing technique. The material was dried at 80 ℃ for 24 h. Finally, obtained powder was calcined at different temperatures (200, 400, 600, 700, and 800 ℃, respectively) for 4 h.

2.3 Cell culture

BMSCs were suspended in α-DMEM solution (2.0×10cells/mL). The suspension was plated into each well of 96-well culture plates (100 μL/well). The plates were incubated for 24 h at 37 ℃ and 5% CO.

2.4 Cell proliferation assay

MTT assay was performed to study the effect of mesoporous

β

-TCP powder on the viability of BMSCs. Mesoporous

β

-TCP powder and non-mesoporous

β

-TCP powder were washed thrice with deionized water and thrice with ethanol. Then, the materials were sterilized in the steam sterilizer and diluted with PBS to obtain three different concentrations. All the suspensions underwent ultrasonic dispersion for 20 min.

β

-TCP suspensions were placed in the wells of 96-well culture plates (20 μL/well) seeded previously with BMSCs. The final concentrations of mesoporous

β

-TCP and non-mesoporous

β

-TCP powder suspensions on the 96-well culture plates were 2, 10, and 20 μg/mL. The wells with no powder suspension added were established as a control group. BMSCs were cultivated for 1, 3, and 5 days (37 ℃, 5% CO) and the medium was replaced on the day 3.

MTT assay was conducted as follows. MTT (20 μL) was added to each well and incubated for 4 h (37 ℃, 5% CO). After MTT was removed, 200 μL DMSO was added to each well to dissolve the optical density of the resulting solution. The final optical density (OD) was measured at a wavelength of 550 nm.

2.5 X-ray diffractometry (XRD)

The structural analysis of mesoporous

β

-TCP powders was conducted by X-ray diffraction (Rigaku D/MAX-RB, Japan). Data was collected for 2

θ

ranging between 5° and 70° using Cu Kα radiation (λ=0.15406 nm).

2.6 Fourier-transform infrared (FT-IR) spectroscopy

Fourier transform infrared spectroscopy (Nicolet 6700, USA) was performed to analyse the composition and chemical bonding of mesoporous

β

-TCP. Powders were dried in the oven at 80 ℃ for 6 h and then pressed into transparent disks. FT-IR spectra were analyzed in the frequency range of 400-4 000/cm.

2.7 Field emission scanning electron microscopy (FE-SEM)

Field emission scanning electron microscopy (Zeiss Ultra Plus, Germany) was performed to analyse the surface morphology of the mesoporous

β

-TCP. Powders were placed on a platinum coating plate and sputter-coated with ultra-thin electrically-conducting gold.

2.8 Nitrogen adsorption

Nitrogen adsorption analysis was performed by the Surface Area and Pore Size analyzer (ASAP 2020M, Micromeritics, USA) at 77.3 K. Samples underwent degasseing overnight at 80 ℃. Adsorption and desorption isotherms were recorded in the pressure range of 5.0×10＜

P

/

P

＜ 1.0. The specific surface area and pore size distribution were computed using the Brunauer-Emmett-Teller (BET) equation and the Barrette-Joynere-Halenda (BJH) method.

2.9 Microscopic observation

After the co-culture of materials (mesoporous

β

-TCP powder and non-mesoporous

β

-TCP powder, respectively) and BMSCs for 5 days, the morphologies of cells were observed via inverted phase contrast microscope (CKX31, OLYMPUS, Tokyo, Japan)

2.10 Statistical analysis

All data were expressed as the means ± standard deviation. Statistical differences were confirmed through analysis of variance followed by Student’s

t

-test. A value of

p

＜ 0.05 was assumed statistically significant. Each experiment was performed thrice, at least.

3 Results and discussion

3.1 Effect of different pH values and different calcination temperatures on the phase composition of β-TCP powders

The XRD patterns of the products synthesized at pH values ranging from 5.0 to 9.0 are shown in Fig.1. Curves (c) and (d) show that the products prepared under pH 7.0 and 8.0 had the main (h k l) indices: (1 1 0), (1 0 1 0), (2 1 4), (0 2 1 0), (1 2 8), (2 2 0), (4 0 1 0) and (2 0 2 0) assigned to the formed

β

-TCPs (JCPDS NO. 09-0169).

Fig.1 XRD patterns of β-TCP powder synthesized at different pH values: (a) pH=5.0, (b) pH=6.0, (c) pH=7.0, (d) pH=8.0, and (e) pH=9.0

Curves (a) and (b) indicate that the phase of

β

-calcium pyrophosphate (

β

-CaPO,

β

-CPP) is detected when pH in the microemulsion system is altered from 5.0 to 6.0. As reported in the Ref.[24], the Ca/P value of the initial reagents did not translate directly to Ca/P ratio of the precipitate. Ca/P with the ratio of 1.50 for the initial reagents was decreased down to ＜1.50 under acidic condition.As shown in curve (e), HAP was detected in the product when pH value was in the 9.0. The solubility of HAP was less than

β

-TCP and it had better stability under alkaline condition. Therefore, HAP was more likely to be synthesized when pH was 9.0. HAP was one of the main residues even after the

β

-TCP powder was calcined. The pH therefore had an important influence on the phase composition of the final product. The pH value of 7.0 was selected experimentally to obtain a purified

β

-TCP powder.Fig.2 presents the XRD patterns of the ascalcined

β

-TCP powder and

β

-TCP powder obtained at calcination temperatures ranging from 200 to 800 ℃. At 800 ℃, as shown in curve (f), well defined sharp peaks of

β

-TCP powder were obtained. In the ascalcined and temperature range (200-600 ℃), as shown in curves (a)-(d), the products were mostly amorphous, which remained in agreement with the Ref.[25]. Curves (e) indicated that the phase of HAP was found in the product when the calcination temperature was 700 ℃, which is the phase transition temperature of HAP. So, the calcination temperature of 800 ℃ would have been applied in the following work.

Fig.2 XRD patterns of the as-calcined β-TCP powder and the β-TCP powder obtained at different calcination temperatures for 4 h: (a) as-calcined, (b) 200, (c) 400, (d) 600, (e) 700, and (f) 800 ℃

3.2 FT-IR analysis of β-TCP powder

Fig.3 shows the typical FT-IR spectra of the

β

-TCP powder. It could be seen that the characteristic peak at 3 432 cmbelongs to a bending mode of HO. The peaks at 1 120, 1 074, and 1 043 cmwere respectively attributed to phosphate stretching vibrational modes of νin PO. The peaks at 975 and 945 cmwere respectively attributed to the phosphate bending vibrational modes of

ν

in PO. The bands at 608 and 551 cmwere assigned to the

ν

vibration of PO

Fig.3 FTIR spectra of the β-TCP powder prepared by the microemulsion technique

ions. The bands at 432 cmcorresponded to the

ν

vibration of POions. The tendency of the spectra corresponded greatly with the standard spectra of

β

-TCP.

3.3 FE-SEM analysis of the surface morphology of β-TCP powder

The surface morphology and crystal structure of mesoporous

β

-TCP powder synthesized by microemulsion technique was investigated by FE-SEM and HRTEM, as given in Fig.4. The particles of the sample seen in the picture were worm-like, irregular and aggregation partially. However, the presence of disordered, non-uniformity, and relatively continuous mesostructure was apparent. In the process of synthesis of

β

-TCP powder by microemulsion technique, the concentrations of surfactant, co-surfactant and oil phase, the stability of the microemulsion, and calcination temperature could all affect the surface

Fig.4 FE-SEM image and HRTEM lattice micrographs of the β-TCP powder synthesized by the microemulsion technique

morphology. The FE-SEM of the sample heated to 800 ℃ has shown the presence of dense wormhole-like mesopores should be resulting from the densification of the mesopore walls at higher temperature and consequent pore shrinkage.

3.4 N2 adsorption/desorption and BJH pore size distribution of mesoporous β-TCP powder

Nadsorption/desorption isotherms of the

β

-TCP powder synthesized by the microemulsion technique are shown in the Fig.5. The isotherms exhibited a type IV isotherm for mesoporous materials with H3-type hysteresis loop, indicating the presence of non-uniform mesopores. However, the adsorption/desorption isotherms at lower relative pressure (＜ 0.6

P

/

P

) had no better curve fitting, suggesting the existence of amounts of micropores in the

β

-TCP powder.

Fig.5 N2 adsorption/desorption isotherms of the mesoporous β-TCP powder

The pore size distribution of mesoporous

β

-TCP sample presented in Fig.6 revealed a broader range of pore size distributions in the formed

β-

TCP mainly below 35 nm with combined microporous and mesoporous structures. The average pore size in the

β

-TCP powder measured by the BJH method was was determined as 7.11 nm and the specific surface area was calculated as 12.85 m/g.

Fig.6 The BJH pore size distribution of the mesoporous β-TCP powder

3.5 The formation of mesoporous -TCPs

A credible mechanism for the formation of the mesoporous

β

-TCP powder by the microemulsion technique is presented in Fig.7. At first, microemulsion was formulated by adding surfactant (CTAB), oil (cyclohexane), and cosurfactant (

n-

octyl alcohol) into an aqueous solution and CTAB formed a hexagonal micelle in the microemulsion. After the addition of an aqueous Casolution to the microemulsion, the CTAB-Cacluster, which consisted of many Caspecies bound to the surface of the micelle, was formed. After an aqueous POsolution was added, a rod-like CTAB-calcium phosphate cluster was formed because of the electrostatic interactions betweenand Cain the CTAB-Camicroemulsion. The micelle acted as a nucleating point for the growth of calcium phosphate crystals at adequate aging temperature and time. The mesoporous

β

-TCP powder was obtained after the removal of CTAB by calcining at 800 ℃.

Fig.7 The flow chart of the formation of the mesoporous β-TCP powder by the microemulsion technique

3.6 Effects of the mesoporous β-TCP powder on the morphology and proliferation of BMSCs

Effects of different concentrations of two materials (mesoporous

β

-TCP powder and nonmesoporous

β

-TCP powder) on the morphology of BMSCs and the control group are shown in Fig.8. The Primary BMSCs were spindle-shaped and adherent to the dish (Fig.8 C). All cells on dish spread well and exhibited spindle-like morphology in the experimental groups, indicating nontoxicity of two materials. However, the cells viability was different under the different concentrations of materials. The cells on dish spread and grew better at the mesoporous

β

-TCP powder concentrations of 2 and 10 μg/mL (Fig.8 A1 and Fig.8 A2) when compared with the concentration of 20 μg/mL (Fig.8 A3). As expected, the cells containing the mesoporous

β

-TCP powder spread and grew better compared to the non-mesoporous

β

-TCP powder, suggesting that the effect of mesoporous

β

-TCP powder on the activity of BMSCs was better than that of the non-mesoporous

β

-TCP powder, demonstrating good cytocompatibility of the mesoporous

β

-TCP powder.

Fig.8 Effects of different concentrations of two materials on the morphology of BMSCs: (A) mesoporous β-TCP powder (A1:2, A2:10, and A3:20 μg/mL), (B) non-mesoporous β-TCP powder (B1:2, B2:10, and B3:20 μg/mL), and (C) the control group

To further confirm the biocompatibility of the mesoporous

β

-TCP powder, the MTT assay was performed to evaluate the effects of the mesoporous

β

-TCP powder and non-mesoporous

β

-TCP on the proliferation of BMSCs, as shown in Fig.9. It was found that the OD values measured at 550 nm increased when the culture period of BMSCs under different concentrations (2, 10, and 20 μg/mL) of materials was prolonged from 1 day to 5 days. This result could be translated into a significant increase in the cells proliferation. The OD values of mesoporous

β

-TCP powder samples were higher than those of non-mesoporous

β

-TCP powder samples, comparing identical concentrations, after 1 day, 3, and 5 days in culture. In addition, the presence of 20 μg/mL mesoporous

β

-TCP powder induced a significant increase in cell proliferation in relation to the nonmesoporous

β

-TCP powder after 1 and 3 days in culture (

P

＜0.01). After 3 days in culture, the OD value of BMSCs containing the mesoporous

β

-TCP powder of 2 μg/mL was significantly higher than that the OD value of the non-mesoporous

β

-TCP powder sample (

P

＜0.05). The proliferation of BMSCs co-cultured with the 10 μg/mL of mesoporous

β

-TCP powder was further increased (

P

＜0.05) compared to the non-mesoporous

β

-TCP powder cultured on dish for 3 and 5 days. These results suggested that the effect of mesoporous

β

-TCP powder on the proliferation of BMSCs was better than that of the non-mesoporous

β

-TCP powder.

Fig.9 Effects of different concentrations of two materials on the BMSCs proliferation under different culture time: (A) mesoporous β-TCP powder and (B) non-mesoporous β-TCP powder. Each bar represents the mean ± standard deviation (n = 3). *P＜0.05, ** P＜0.01 when compared with nonmesoporous β-TCP powder

4 Conclusions

In this study, high-purity mesoporous

β

-TCP powder with non-uniform and disordered morphology was synthesized through micro emulsion technique. The material has a broader pore size distribution, an average pore size of 7.11 nm and a specific surface area of 12.85 m/g. The mesoporous

β

-TCP powder has shown better effect on the proliferation of BMSCs than that of the non-mesoporous

β

-TCP powder.