- Nano Express
- Open Access
Hydrothermal Synthesis and Biocompatibility Study of Highly Crystalline Carbonated Hydroxyapatite Nanorods
© Xue et al. 2015
- Received: 8 July 2015
- Accepted: 25 July 2015
- Published: 7 August 2015
Highly crystalline carbonated hydroxyapatite (CHA) nanorods with different carbonate contents were synthesized by a novel hydrothermal method. The crystallinity and chemical structure of synthesized nanorods were studied by Fourier transform infrared spectroscopy (FTIR), X-ray photo-electronic spectroscopy (XPS), X-ray diffraction (XRD), Raman spectroscopy, and transmission electron microscopy (TEM). The biocompatibility of synthesized CHA nanorods was evaluated by cell viability and alkaline phosphatase (ALP) activity of MG-63 cell line. The biocompatibility evaluation results show that these CHA nanorods are biologically active apatites and potentially promising bone-substitute biomaterials for orthopedic application.
- Carbonated hydroxyapatite
- Cell viability
- Alkaline phosphatase activity
- MG-63 cell
Synthesizing materials for preparing HA and CHA nanorods
The hydrothermal method is a typical process which has been widely used in synthesis of inorganic materials for its good repeatability and crystallinity control [15–17]. In this study, we developed a hydrothermal process to synthesize carbonated hydroxyapatite nanorods with different carbonate contents, using ethylene diamine tetraacetic acid (EDTA) and cetyltrimethyl ammonium bromide (CTAB) as templates. The synthesized CHA nanorods were characterized by various analytical measurements to investigate how changes of carbonate levels affect the crystal morphology and structure of CHA nanorods. The effects of synthesized samples on the viability and osteogenic differentiation of the human osteosarcoma MG-63 cells have been measured by an MTT method and alkaline phosphate activity assay [18, 19].
Ca(NO3)2•4H2O, (NH4)2HPO4 and NH4HCO3 were used as a calcium source, phosphorus source, and carbonate source, respectively. Ethylene diamine tetraacetic acid (EDTA) and cetyltrimethyl ammonium bromide (CTAB) served as templates for the CHA nanorods. The phosphorus- and carbonate source solution was added dropwise to a solution of Ca(NO3)2•4H2O, EDTA and CTAB, meanwhile keeping pH at 9~11 by adding ammonium hydroxide solution. After 5-min stirring, the hydroxyapatite suspensions were poured into Teflon-lined stainless steel autoclaves. The autoclaves were placed in an oven for 24 h at 180 °C and then were cooled down to room temperature. The precipitate was washed by deionized water and ethyl alcohol for three times and then dried for 6 h at 80 °C. The details of synthesizing materials for preparing for HA and CHA samples are listed in Table 1.
Transmission Electron Microscope Measurement
Transmission electron microscope (TEM, Tecnai C2 F30 S-Twin, FEI, USA) was carried out to determine particle size and morphology, and selected area electron diffraction (SEAD) was recorded by high-resolution transmission electron microscopy (HRTEM).
Fourier Transform Infrared Spectrometry Measurement
Fourier transform infrared spectrometry (FTIR, ALPHA, Bruker, USA) was used to identify the molecular structure. After sample stage was cleaned up by ethanol wiping, the background was tested from 500 to 3600 cm−1. Finally, the substrate was placed on the diamond sample stage and then the cantilever was dropped onto powder slowly.
X-ray Photo-Electronic Spectroscopy Measurement
The elements composition of the samples were analyzed by X-ray photo-electronic spectroscopy (XPS, ESCALAB250Xi, ThermoFisher Scientific, USA), using a monochromated Al Kα X-ray source.
X-ray Diffraction Measurement
The crystalline phase of the samples was examined by X-ray diffraction (XRD, D8 ADVANCE, Bruker, Germany) with graphite monochromatized Cu Kα radiation operating at 40 kV and 40 mA at room temperature.
Micro-Raman Spectroscopy Measurement
The molecular structure can be further analyzed by Raman spectroscopy (DXR, GX-PT-2412, Thermo, USA) with 532 nm laser as excitation wavelength. The Raman detector was equipped with a charge coupled device (CCD) multichannel detector and Olympus confocal microscope. The laser beam was focused on the sample surface and scanned for a 5-s exposure time for 180 times, meanwhile the powders were measured with extended range grating for 400–4000 cm−1.
Cell Viability and Alkaline Phosphate Activity Assay Measurements
Human osteosarcoma cell line MG-63 cells were cultured in medium containing 10 % of fetal calf serum in a humidified atmosphere of 5 % CO2 at 37 °C, and the medium also contained 100 ug/ml streptomycin and 100 ug/ml penicillin. Then MG-63 cells were seeded in a 96-well cell culture plate with a density of 5 × 103 per well. The next day, cells were treated with samples at the concentration of 0, 20, 40, 60 μg/ml. After 3 days, the cell viability was evaluated by MTT. The MG-63 cells were cultured with samples for 5 days for alkaline phosphate activity assay.
Morphology of CHA Nanorods
FTIR, XPS Spectroscopy, and XRD Pattern of CHA Nanorods
Raman Spectroscopy of CHA Nanorods
Cell Culture and Cell Viability Test
We synthesized highly crystalline carbonated hydroxyapatite nanorods with different carbonate levels by a convenient hydrothermal reaction. As the carbonate content increases, the lengths of nanorods decrease, the widths of nanorods slightly increase, and the crystallinity of CHA nanorods decreases due to lattice defects caused by substitution of CO3 2− ions. The results of biocompatibility and osteogenic differentiation test prove that these CHA nanorods are biological apatites and promising biomaterials in bone-tissue engineering application.
This work was supported by the Jiangsu Province for specially appointed professorship to Prof. P.Z. Zhu, research funds from Yangzhou University, research fund of Liuda Rencai Gaofeng and the support from the Testing Center of Yangzhou University.
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