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Hydrothermal synthesis and characterization of hydroxyapatite and fluorhydroxyapatite nanosize powders
This article has been downloaded from IOPscience. Please scroll down to see the full text article. 2010 Biomed. Mater. 5 045004 (http://iopscience.iop.org/1748-605X/5/4/045004) View the table of contents for this issue, or go to the journal homepage for more
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IOP PUBLISHING
BIOMEDICAL MATERIALS
doi:10.1088/1748-6041/5/4/045004
Biomed. Mater. 5 (2010) 045004 (7pp)
Hydrothermal synthesis and characterization of hydroxyapatite and fluorhydroxyapatite nano-size powders Leila Montazeri1,2 , Jafar Javadpour1,4 , Mohammad Ali Shokrgozar2,4 , Shahin Bonakdar2 and Sayfoddin Javadian3 1
School of Metallurgy and Materials Engineering, Iran University of Science and Technology, Tehran, Iran 2 National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran 3 Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran E-mail:
[email protected] and
[email protected]
Received 3 March 2010 Accepted for publication 1 June 2010 Published 23 June 2010 Online at stacks.iop.org/BMM/5/045004 Abstract Pure hydroxyapatite (HAp) and fluoride-containing apatite powders (FHAp) were synthesized using a hydrothermal method. The powders were assessed by x-ray diffraction (XRD), Fourier transform infrared (FTIR), scanning electron microscope (SEM) and F-selective electrode. X-ray diffraction results revealed the formation of single phase apatite structure for all the compositions synthesized in this work. However, the addition of a fluoride ion led to a systematic shift in the (300) peak of the XRD pattern as well as modifications in the FTIR spectra. It was found that the efficiency of fluoride ion incorporation decreased with the increase in the fluoride ion content. Fluorine incorporation efficiency was around 60% for most of the FHAp samples prepared in the current study. Smaller and less agglomerated particles were obtained by fluorine substitution. The bioactivity of the powder samples with different fluoride contents was compared by performing cell proliferation, alkaline phosphatase (ALP) and Alizarin red staining assays. Human osteoblast cells were used to assess the cellular responses to the powder samples in this study. Results demonstrated a strong dependence of different cell activities on the level of fluoridation.
a contraction in the a-axis dimension, but with no change in the c-axis dimension [3]. The contraction in the aaxis also brings about a reduction in the volume of the unit cell, so that the chemical and thermal stability of the apatite lattice is enhanced by the virtue of the stronger electrostatic bond between fluoride and the adjacent ions [1]. Due to the favorable effects of fluoride ions on dental caries prevention, they have been used in synthetic dental restorative materials to retard demineralization and activate the re-mineralization of teeth [4]. Moreover, the fluoride ions are known to stimulate the proliferation and differentiation of bone-forming osteoblast cells [5–8]. Thus, fluoridated HAp with the combination of higher stability and biocompatibility is potentially a very attractive biomaterial. However, it is necessary to control the fluoride content in the apatite lattice to achieve the best biological performance. It has been
1. Introduction Hydroxyapatite (HAp) and its family members are of great interest because they are the main components of natural bones and teeth. Biological apatites contain various amounts of anionic and/or cationic substitutions [1, 2]. These substitutions do not alter the basic structural characteristics of HAp but can modify the mechanical properties, solubility and bone bonding capability and improve the overall biological performance of the implant material. Regarding anions, fluorine-substituted HAp has attracted much attention due to the extensive findings of fluoridated HAp in natural bone and dental enamel. Since the fluoride ion is smaller than the hydroxyl ion, the substitution in the HAp lattice results in 4
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Biomed. Mater. 5 (2010) 045004
L Montazeri et al
reported that the high concentration of the fluoride ion can cause reduction in osteoconductivity and other adverse effects such as osteomalacia [9]. In addition, the bioactivity behavior of calcium phosphate particulates depends on their size and morphology. Therefore, these powders have been prepared by different processing methods in order to improve and control both bioactivity and fluoride ion content in the apatite structure [10]. Among the various material processing techniques, hydrothermal is a low energy consuming, environmentally benign fabrication method capable of producing more homogeneous powders with higher degree of crystallinity and a better control over the size and shape [11–13]. Limited information in the literature on the synthesis of substituted HAp powder by this method provided the rational for this study. Thus, the objective of this study was to prepare pure and fluoride ion-containing apatites using a hydrothermal method. Powders produced by this method were characterized by x-ray diffraction (XRD), Fourier transform infrared (FTIR), scanning electron microscope (SEM) and ion-selective electrode (ISE) techniques to investigate the efficiency of fluoride ion substitution in the apatite lattice, its effect on the HAp structure, phase development, particle size and morphology. The effect of fluoride ion substitution on the bioactivity behavior will be studied by performing osteoblast cell proliferation, alkaline phosphatase (ALP) and Alizarin red staining assays.
870) was used to analyze the HAp and fluoride-substituted apatite powder samples prepared in this study. The FTIR spectra were recorded in the 400–4000 cm−1 wave number range using the KBr pellet technique. The phase analysis of the synthesized powders was carried out using a SIMENS D500 x-ray diffractometer, with Cu Kα radiation at 30 kV, 25 mA at a scanning speed of 1.5◦ min−1 . The morphology and particle size of the powders were examined by a SEM (LeO 1455VP). The powders were platinum coated prior to the SEM study.
2. Materials and methods
2.4. Powder extraction
2.1. Sample preparation
The proliferation and differentiation rates of the osteoblast cells were evaluated using extracted powder prepared according to ISO 1993–5 procedure. 0.1 g of powder samples with different compositions were incubated in 1 ml of culture medium. At the end of 3, 7 and 14 days, the mediums were collected for use in different cellular assays. Fluoride and phosphate ion concentrations in the culture medium were measured by ion chromatography (Metrohm 761). Pure culture medium kept under similar conditions was used as a negative control sample.
2.3. In vitro cell culture The human osteoblast cell line was chosen to evaluate the biological performance of the samples synthesized in this study. The cells were isolated from superior portion of a femoral bone from a 25 year male patient according to previously published protocol [14]. Hematopoietic cells were removed by washing the bone slice several times with sterilized phosphate buffered saline (PBS). Washed bone fragments were incubated at 37 ◦ C in a humidified atmosphere in the presence of 5% CO2 . After 14 days, isolated osteoblast cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; GIBCO, Scotland)/Ham’s F12 culture medium solution supplemented with 10% (v/v) fetal bovine serum (FBS-Seromed, Germany), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin.
Analytical grade Ca(OH)2 and (NH4 )2 HPO4, Merck, Germany and NaF, Labachemie, India, were used as the starting materials in the current study. The first step in the synthesis of pure and substituted HAp powders was the preparation of stock solutions of Ca(OH)2 and (NH4 )2 HPO4 using distilled water. Pure and fluoridated powders with compositions of Ca10 (PO4)6 (OH)2−x Fx (x = 0, 0.4, 0.8, 1.2, 1.6, 2) were coded as HAp, FHAp-1, FHAp-2, FHAp-3, FHAp-4 and FHAp-5, respectively. The different compositions were prepared by initially adding appropriate amounts of NaF solution into a P ion-containing solution. The mixture was then added dropwise into the stirring Ca(OH)2 solution and stirred for 10 min prior to transferring to a stainless autoclave reactor. After 8 h of hydrothermal treatment at 15.2 atm and at a temperature of 200 ◦ C the reacted powders were washed several times with distilled water to remove any residual ions. The powders were then dried at 80 ◦ C for 24 h.
2.5. Cell proliferation The proliferation rate of the osteoblast cells next to different powder extracts was determined by conducting the MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. This test is based on the fact that active cells convert the yellowish MTT to an insoluble purple formazan crystal. The formazan crystals formed are solubilized and the resulting colored solution is quantified using a scanning multiwall spectrophotometer (ELISA reader). Briefly, the cells were placed into a 96-well microtiter plate (Nunc, Denmark) at a density of 1 × 104 cells/well. After 24 h, culture medium in each well was removed and replaced by 90 μl powder extracts plus 10 μl FBS. The medium was discarded after 24 h and 100 μl of 0.5 mg ml−1 MTT (Sigma, USA) solution was added into each well. The cells were then incubated for 5 h at 37 ◦ C. The purple formazan crystals were detected and were dissolved by the addition of 100 μl of isopropanol (Sigma, USA) per well. The plates were incubated at 37 ◦ C
2.2. Characterization The fluoride concentration in the apatite lattice was analyzed using an Elit 101 in a total ionic strength adjustment buffer (TISAB). Samples of 0.2 g from each composition were dissolved in 1 ml of 1 M nitric acid to which 200 ml of deionized water was added. The fluoride content was measured using a solution made by mixing the latter in a 10 ml of buffer solution. Standard solutions made from NaF were used to calibrate the measurement with the same buffer system. FTIR spectroscopy (Thermo Nicolet Nexus 2
Biomed. Mater. 5 (2010) 045004
L Montazeri et al
(300)
The reaction was stopped by adding 20 μl of 1.0 N NaOH to each of the wells. The absorbance of the mixture was read at a 405 nm wavelength using the microplate reader (STAT FAX 2100, USA). The standard p-nitrophenol curve was used to convert the absorbance data to the ALP content. The results were normalized by conducting the simultaneous MTT assay on the cells cultured under the same conditions [15].
FHAp -5
33.12
Intensity (a.u.)
FHAp -4 FHAp -3 FHAp -2
2.7. Alizarin red staining
FHAp -1
20
Alizarin red staining was used to determine the presence of calcific deposition by the osteoblast cells. In this test, 2 × 104 cells were first cultured in a 24-well plate and later were exposed to a medium containing powder extracts and 10% FBS. After 3 days the medium was removed and washed twice with a NaCl solution (0.9%). At this time, the cells were fixed by paraformaldehyde (1%) for 10 min and stained with an Alizarin red solution (2%, pH 4.2) for 45 min at room temperature. The cells were rinsed with the NaCl solution prior to optical microscopy.
32.86
HAp
25
30
35 40 45 2 Theta (degrees)
50
55
60
Figure 1. XRD patterns of hydrothermally synthesized powders.
for 15 min prior to absorbance measurements. The optical density (OD) of formazan in the solution was measured at 545 nm using a multiwall microplate reader (STAT FAX 2100, USA). The results were normalized with respect to the control sample.
2.8. Statistical analysis The Student t-test was used for the analysis of statistical difference on the results for the in vitro cellular assays, and p < 0.05 was considered significant.
2.6. Alkaline phosphatase activity The functional activity of the cells was examined by measuring the ALP enzyme activity. The isolated osteoblast cells were placed into a 96-well plate (2 × 103 cells/well) containing 80% powder extract, 10% culture medium and 10% FBS. After 7 days, the medium was removed and the cells were washed twice with Hank’s buffer salt solution (HBSS; Gibco, USA). Afterward, 70 μl of cell lysis buffer (10 mM Tris, 2 mM MgCl2 , 0.05% triton, pH 8.5) was added to each well. The cells were incubated at 37 ◦ C for 30 min followed by 24 h at 4 ◦ C. 100 μl of p-nitrophenol phosphate (Sigma, USA) was added into each well and they were incubated for 1 h at 37 ◦ C.
3. Results and discussion 3.1. Sample characterization The results of determination of fluoride ion concentrations in various samples are presented in table 1. It should be noted that the measurements of the F ion content using an F-selective electrode were done on as hydrothermally synthesized powders without further calcinations. As indicated in this table, the efficiency of fluoride ion incorporation for the
Figure 2. FTIR spectra of hydrothermally synthesized powders. 3
Biomed. Mater. 5 (2010) 045004
L Montazeri et al
(a)
(b)
Figure 3. SEM micrographs of powder samples: (a) HAP; (b) FHAp -5.
Figure 4. The osteoblast cell viability results for various extracted powder samples.
F-added to the initial formulation (mol F mol−1 apatite)
F content determined by a fluoride-selective electrode (mol F mol−1 apatite)
FHAp-1 FHAp-2 FHAp-3 FHAp-4 FHAp-5
0.4 0.8 1.2 1.6 2
0.37 0.5 0.67 1.09 1.42
p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.
HAp and FHAp-5 powders occurred at 2θ angles of 32.86◦ and 33.12◦ , respectively. This shift has been related to the a-axis contraction caused by smaller size of F ions compared to OH ions. The a-axis contraction changes linearly with increasing fluoride content. Another point to note is the presence of sharper peaks in the XRD patterns of the fluorinated samples. This feature may be due to increased crystallinity of fluorinated powders as compared to pure HAp. Figure 2 shows the FTIR spectra results for the pure HAp and powders with different percentages of fluoride ion substitution. For the pure HAp powder, a single band at 3573 cm−1 is observed which is associated with the OH stretching vibration mode in the apatite lattice [3]. With the increase in the fluoride ion concentration the intensity of this characteristic band decreases and a new band appears at around 3544 cm−1 . The appearance of this new band is an indication for the fluoride–hydroxyl substitution in the lattice [18]. As shown in figure 2, broadening of this band occurs with a further increase in the fluoride ion content in the structure, a feature which has been related to different configurations of fluoride and hydroxyl ions along the c-axis of the lattice [18]. Another major change in the FTIR spectra with the introduction of F ions occurs around 631 cm−1 vibrational mode. This mode of vibration has been assigned to the OH liberation band in the lattice [18]. The disappearance of this band with the increase
Table 1. Effect of the fluoride content on the efficiency of fluorine incorporation. Sample code
∗
sample FHAp-1 is around 90%, whereas for other samples the efficiency reduces to about 60%. These results are interesting, because similar F ion incorporation efficiency has been reported on the fluoridated HAp powder samples prepared by a pH cycling method after calcinations at 1200– 1400 ◦ C [5]. The XRD patterns of the as prepared powders are shown in figure 1. As can be seen, the substitution of fluorine did not significantly affect the diffraction patterns of the as prepared powders. All patterns reveal a single phase crystalline apatite structure. The most obvious difference is the slight shift of the (3 0 0) peak to the right-hand side in the fluoridated samples which indicates the substitution of OH with F ions in the apatite structure [6, 16, 17]. For example, the (3 0 0) reflections from 4
Biomed. Mater. 5 (2010) 045004
L Montazeri et al
Figure 5. Alkaline phosphatase (ALP) activity of the osteoblast cell, for powder extraction periods of 3, 7 and 14 days. 0.01.
(a)
∗
p < 0.05, ∗∗ p
