Synthesis and Characterization of Strontium Fluor-Hydroxyapatite Nanoparticles For Dental Applications

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Synthesis and characterization of

Strontium Fluor-Hydroxyapatite
nanoparticles for dental applications
Mehrdad Khakbiz
Microchemical Journal
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Synthesis and characterization of Strontium Fluor- Hydroxyapatite

Alireza Rajabnejadkeleshteri , Armin Kamyar , Mehrdad Khakbiz ,

Zahra Lotfi bakalani , Hamideh Basiri
PII: S0026-265X(19)32274-X
DOI: https://doi.org/10.1016/j.microc.2019.104485
Reference: MICROC 104485
To appear in: Microchemical Journal
Received date: 24 August 2019

Revised date: 28 November 2019
Accepted date: 29 November 2019
Please cite this article as: Alireza Rajabnejadkeleshteri , Armin Kamyar , Mehrdad Khakbiz ,
Zahra Lotfi bakalani , Hamideh Basiri , Synthesis and characterization of Strontium Fluor-
Hydroxyapatite nanoparticles for dental applications, Microchemical Journal (2019), doi:
https://doi.org/10.1016/j.microc.2019.104485
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Highlights:
• F-SrHA has been synthesized with combination of precipitation and pH-cycle methods.
• The crystal size of F-SrHA is smaller than HA
• The amount of fluorine in F-10%SrHA is 2.56 wt%
• The presence of strontium and fluorine in the structure of HA increased cell proliferation
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Synthesis and characterization of Strontium Fluor- Hydroxyapatite
Alireza Rajabnejadkeleshteria, Armin Kamyara, Mehrdad Khakbiza*,

Zahra Lotfi bakalania, Hamideh Basiria
a
Division of Biomedical Engineering, Faculty of New Sciences and Technologies,
University of Tehran, North Karegar Ave., PO Box 14395-1561, Tehran, Iran.
*
Corresponding author: Mehrdad Khakbiz (khakbiz@ut.ac.ir ) , Faculty of New Sciences
and Technologies, University of Tehran
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Abstract
Being biocompatible, bioactive, having low solubility in a wet environment and showing
similarities to mineral part of hard tissue in human’s body, hydroxyapatite has so many
applications in dentistry and related hard tissue concerns. Fluoride ion being found in bone
and enamel can be joined to hydroxyapatite and reduce solubility and also hold
biocompatibility relatively like hydroxyapatite. Playing main role in bone regeneration,
strontium can be used partially or totally to treat biomaterials and increase biocompatibility
and bioactivity. In this study hydroxyapatite and strontium hydroxyapatite were synthesized
by chemical precipitation of different percentages of strontium by which substitutes calcium.
Additionally, Fluoride was incorporated into the apatite structure, using pH-cycling method
and producing Strontium- Fluoride hydroxyapatite. Diammonium phosphate, calcium nitrate,
strontium nitrate and sodium fluoride were starting materials and solution of sodium
hydroxide and nitric acid were used to control pH. Accordingly, synthesized nano-powders
characterized by XRD, FTIR, F-selective electrode, inductively coupled plasma spectroscopy
and SEM. Subsequently, biological assessment of fluoride’s and strontium’s influence on the
proliferation and differentiation of MG63 cell line was conducted by MTT and ALP tests.
Results of XRD and FTIR showed that nano-crystals of HA, SrHA, FHA and F-SrHA were
successfully achieved by this method. SEM images revealed that prepared crystalline
powders morphology was nano-size and spherical. Biocompatibility also approved by MTT
tests. Also, since the process of calcification of hard tissues (bones and teeth) is effected by
alkaline phosphatase enzyme (ALP), the influence of both alone and combined fluoride and
strontium on cell differentiation was investigated. Consequently, results represent the ability
of these elements to enhance remineralization of tooth enamel.
Keywords:
Strontium, fluoride, hydroxyapatite, pH-cycling, precipitation, biological assessment
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1. Introduction
Thermodynamically, hydroxyapatite (Ca10(PO4)6(OH)2, HA) is the most lasting crystalline
phase of calcium phosphate salts in body fluid. As it is the principal mineral constituent of
tooth and other calcified tissues [1, 2], synthetic hydroxyapatite holds exceptional bioactive
characteristics such as osteoconductivity and cytocompatibility due to its maximum
correspondence to the mineral portion of bone tissue [3]. Nevertheless, synthetic HA which is
stoichiometric, decomposes into distinct phases like tricalcium phosphate (TCP; Ca3 (PO4)2)
at high sintering temperatures. This phase impurity resulted from inadequate thermal
stability, often leads to unfavorable dissolution behavior in vivo [4]. Therefore, the perennial
efficacy of hydroxyapatite is disputable, as it has relatively high solubility when exposed to
body fluid [5, 6].
Across the wide range of HA structures, HA nanoparticles, with suitable stoichiometry and
purity, have attracted enormous interest in biomedical applications [7]. HA nanoparticles
have ultrafine structure and high surface reactivity similar to the mineral found in bones [8].
As the biological HAs are plate-like crystals that have nanoscale thickness, it is found that
synthetic HA nanoparticles paralleling natural hard tissue minerals is the best bioceramic to
use for bone substitution and regeneration [2, 9]. In addition, demineralization may be
hindered when particle size decrease to nano-size level [10]. Moreover, nanoscale
hydroxyapatite indicates developed densification and sintering properties because of its high
surface energy and, therefore, problems like micro-cracks, can be prevented [11, 12]. The
better cell proliferation and differentiation in HA nanoparticles may be due to better surface
functional characteristics compared to micro-size particles [9, 13, 14].
The non-stoichiometric HA can be synthesized with incorporation of various ionic
substitutions into the crystal lattice [15]. Reports authenticate that substituting ions exist in
body’s hard tissues such as strontium (Sr) and fluorine (F) into HA can result in improvement
of biomaterial characteristics, such as the crystallinity and dissolution rate under
physiological circumstances. Consequently, this improvement may impact bioactivity, thus
drawing more attention in research community [16-19].
Fluorine is a substantial trace element among hard tissues, which can advance HA
crystallization and mineralization in bone formation process [20]. Fluorine-doped HA
Ca10(PO4)6(OH)2-xFx (FHA) is reported to have low solubility and high bioactivity and
biocompatibility in compare to hydroxyapatite. Furthermore, the doping of fluorine in
hydroxyapatite increases the differentiation and proliferation of osteoblasts and consequently
induces bone regeneration [21, 22].
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Strontium is one of the most significant cations in hard tissues, stimulating cell growth and
preventing bone resorption as well as confronting osteoporosis[23, 24]. Strontium have the
capability to improve gene expression in osteoblastic cells and the ALP activity of
mesenchymal stromal cells (MSCs) as well as prevent the osteoclasts’ differentiation [25,
26]. Sr-substituted hydroxyapatite (SrHA) demonstrates osteoinduction as well as high
solubility [27, 28]. Thus, it is rational to conclude that incorporation of fluorine and strontium
into HA could be desirable for the advancement of the bioactivity.
There are various techniques of synthesizing ion-substituted hydroxyapatite nanoparticles,
such as pH-cycling [29], precipitation [30] and sol-gel [31] methods. The pH-cycling
technique was first presented by Duff [29] to prevent high temperature function and
utilization of volatile fluorine reagent. In our study, we have synthesized HA nanoparticles
and Sr-HA nanoparticles with the precipitation technique [30]. Then, F-Sr-HA was
synthesized by PH-cycling method. Finally, the resulting powders were characterized by X-
ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron
microscope (SEM) and F-selective electrode.
2. Materials and methods

2.1 Synthesis of HA
Hydroxyapatite powder (Ca10(PO4)6(OH)2) was synthesized by precipitation technique using

Ca(NO3)2.4H2O (98.5%, Merck) and (NH4)2HPO4 (99%, Merck) as starting materials and
NaOH solution (1M; 97%, Merck) for pH adjustment. A solution of Ca(NO3)2.4H2O (23.251
g in 177 ml distilled water) was vigorously stirred. A suspension of (NH4)2HPO4 (7.891 g in
124 ml distilled water) was added dropwise to the calcium nitrate solution. The pH of
calcium nitrate solution in all experiments was kept 11 by NaOH solution.
The precipitated hydroxyapatite powder has been removed from solution by centrifugation
(3000 rpm) and washed three times by distilled water. The product was dried at 37◦C for 24h
and then calcined at 800 ◦C for 1 h [15, 32].
2.2 Synthesis of SrHA
Strontium hydroxyapatites (Ca10-xSrx(PO4)6(OH)2) was synthesized by precipitation technique

using Ca(NO3)2.4H2O (98.5%, Merck), (NH4)2HPO4 (99%, Merck) and Sr(NO3)2 (99%,
Merck) as starting materials and NaOH solution (1M; 97%, Merck) for pH adjustment. In
this study, the 5 mol% SrHA (with 5 mol% Ca2+ replaced by Sr2+) was termed 5%SrHA and
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the 10 mol% SrHA (with 10 mol% Ca2+ replaced by Sr2+) was termed 10%SrHA in the
following. For 5%SrHA, a solution of Ca(NO3)2.4H2O (23.823 g) and Sr(NO3)2 (1.029 g)
in 177 ml distilled water was vigorously stirred. A suspension of (NH4)2HPO4 (7.707 g in 124
ml distilled water) was added dropwise to the calcium nitrate solution. For 10%SrHA, a
solution of Ca(NO3)2.4H2O (20.202 g) and Sr(NO3)2 (2.011 g) in 177 ml distilled water was
vigorously stirred. A suspension of (NH4)2HPO4 (6.531 g in 124 ml distilled water) was
added dropwise to the calcium nitrate solution. The pH of calcium nitrate solution in all
experiments was kept 11 by NaOH solution. The precipitated hydroxyapatite powder has
been removed from solution by centrifugation (3000 rpm) and washed three times by distilled
water. The product was dried at 37◦C for 24h and then calcined at 800 ◦C for 1 h [15, 33].
2.3 Synthesis of FHA
Fluoride hydroxyapatite (Ca10(PO4)6(OH)2-2xF2x) was synthesized by pH cycling technique. 5

g of the hydroxyapatite created before was suspended in solution of sodium fluoride (0.43g
NaF (98.5%, Merck) in 0.5 L distilled water) in order to prepare fluorine substituted
hydroxyapatite (FHA). The solution was equilibrated at pH 7 overnight. The pH of the
solution was then brought down to 4 by slowly dropping HNO3 (1M; 65%, Merck). After 30
min, the pH of the solution was brought up to 7 again by adding 1 M NaOH (1 N standard
solution, Acros). The pH cycling process was repeated three times. The precipitate was then
aged for one day at room temperature. After the pH cycling, the precipitate was subsequently
washed five times with deionized water to eliminate sodium residues remaining in the
solution. The precipitate was then dried at 80 ◦C for 24 h and ground into fine powders [34,
35].
2.4 Synthesis of F-SrHA
Strontium Fluoro-hydroxyapatite (Ca10-xSrx(PO4)6(OH)2-2yF2y) was synthesized by pH cycling

technique. 5 and 10 g of the SrHA powders produced above was suspended in the sodium
fluoride solution (0.43g NaF (98.5%, Merck) in 0.5 L distilled water) in order to prepare F-
5SrHA and F-10SrHA. The solution was equilibrated at pH 7 overnight. The pH of the
solution was then brought down to 4 by slowly dropping HNO3 (1M; 65%, Merck). After 30
min, the pH of each solution was brought up to 7 again by adding NaOH (1M; 97%, Merck).
The pH cycling process was repeated three times. The precipitates were then aged for one day
at room temperature. After the pH cycling, the precipitates were subsequently washed five
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times with deionized water to eliminate sodium residues remaining in the solution. The
precipitate was then dried at 80 ◦C for 24 h and ground into fine powders. The flowchart of
all synthesis processes are demonstrated in Fig. 1. In addition, schematic illustration of
synthesized samples is shown in Fig. S2.
2.5 Characterization techniques (XRD, FTIR and SEM)
X-ray diffraction pattern (XRD) was recorded using inel-EQUIN 3000X diffractometer. The
measurement was performed by using Cu-Kα radiation (ƛ=1.5406 A) at 40 kV and 20 mA
with 2Ɵ range of 4◦ to 70◦. The samples were scanned at a rate of 2 min-1. Fourier Transform
Infrared (FTIR) spectra were recorded on a Thermo Fisher Scientific (iS10) FT-IR
Spectrometer instrument using the conventional KBr pellet technique (sample to KBr ratio
1:100) in the range 400-4000 cm-1. The morphology of samples was observed by scanning
electron microscope (SEM) (EM3200, KYKY). In order to provide sufficient electrical
conductivity, samples were coated by a thin layer of gold before scanning with sputter coater
(SBC12, KYKY).
2.6 Chemical analysis of different samples

The chemical composition of the hydroxyapatite powders and substituted samples were
reported by ICP (Inductively Coupled Plasma spectroscopy) on a AGILENT ICP-7900
spectrometer. Samples were initially submitted with litium metaborate to alkaline fusion in a
graphite crucible. The following fusion cake was dissolved with nitric acid. Diorite specimen
was utilized for calibration. Then, the compositions have been easily gained.
2.7 F-selective electrode

Fluorine concentration measurement was conducted using F-selective electrode (Orion 94-
09) in a total ionic strength adjustment buffer (TISAB). 10 mg of each sample was dissolved
in 20 ml of 0.2 M HCL and then diluted by 10 ml deionized water and 40 mg trisodium
citrate 0.2 M. The fluorine content was determined by mixing 10 ml of such solution and 10
ml of the buffer. Standard solutions made from NaF were used to calibrate the measurement
in the same buffer solution.
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2.8 MTT assay
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-lium bromide) assay was
employed to assess cell viability and proliferation. Firstly, the extract of samples was
obtained based on ISO 10993-12 in which 100 mg of powders were added to culture media
and incubate for 7 days at 37°C. To carry out MTT test, MG-63 human osteoblast-like cells
were seeded in 96-well plates at concentration of 1*104 cells/cm2 and incubated at 37°C.
After 7- and 14-days exposure to extract of samples, the cells were incubated with 100 µl of
MTT solution for 4h followed by adding isopropanol. The resulted solutions were transferred
to a 96-well plate and its absorbance was measured by a plate reader at 570 nm and the
results expressed as optical density (OD)[18, 36, 37].
2.9 ALP assay

In order to perform alkaline phosphatase staining, weighed quantities (0.001 g) of the
powders were sterilized by UV rays. In the following, the MG63 cells were seeded and
cultured at a density of 1*103 cells ml-1 on a 6-well plate with a change of culture medium
(RPMI-FBS) every 3-4 days. The plate was incubated in CO2 incubator at 37 ◦ C for two
weeks. Each week, 1 ml of culture medium was added in a sterilized micro-tube and then
kept at 20 ° C for the assay of ALP activity. The ALP activity was determined by using p-
nitrophenyl phosphate as a substrate at 410 nm. Each assay was normalized by concentration
of total protein at the end of experiment. General specifications of synthesized powders are
illustrated in table 1.
3. Results and Discussion

3.1 Powder X-ray diffraction
The X-ray patterns of the HA powder, 5%SrHA, 10%SrHA, FHA, F-5%SrHA and F-
10%SrFHA are shown in Fig. S2. The presence of similar peaks in all samples confirms the
formation of pure apatite powder phase in all samples. The sharp peaks of the patterns
corroborate that the synthesized nano-powders are perfectly crystallized. Although a small
amount of calcium oxide (CaO) is observed in all samples [38-40], there is no trace of
impurities from other calcium phosphates in XRD patterns. It should be noted that this
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amount of CaO does not have a specific effect on biocompatibility and can be readily
disregarded. In the bioceramics, it is important to control the synthesis parameters to increase
the purity and the amount of formed phases. SrHA, FHA and F-SrHA XRD patterns show a
broadening and decrease in peak intensity. This broadening actually suggests the nanoscale
particle size of the synthesis products.
The crystal structure of the HA is hexagonal, which can be used to determine the network
parameters using Bragg's equation for all samples. The diffraction peaks of SrHA are
displaced to lower values of 2θ, resulting in an increase in dspacing and the lattice parameters.
This confirms the strontium substitution [41-43]. On the opposite, X-ray diffraction pattern of
FHA deviated to larger angles which means lattice parameters of FHA are lower than HA.
This is in correspondence with literature [44]. Nevertheless, sharper peaks in FHA in
compare to HA shows that the crystallinity of FHA nano-powders is more than pure HA [34,
45, 46].
Table S2. Compares the lattice parameters of HA, 5%SrHA, 10%SrHA, FHA, 5%SrFHA and
10%SrFHA.
The displacement of the Miller planes by connecting fluorine to the hydroxyapatite crystal
structure, which also occurred in other works, leads to a decrease in a-axis value. The
𝐾𝜆
𝐷ℎ𝑘𝑙 =
𝐹𝑊𝐻𝑀 cos 𝜃
decrease in a-axis value is also due to the smaller ionic radius of F (1.32 Å) in compare to the
OH-ion radius (1.68 Å) which cause the peaks to move to a higher angle. Meanwhile, as the
Sr/Ca molar ratio is increased, the apatite peak moves to a smaller angle due to the larger
ionic radius of strontium (1.13 Å) in compare to calcium (0.19 Å). Simultaneous substitution
of Sr with Ca and F with OH will have simultaneous effect on lattice parameters which
indicate successful substitution of both Sr and F.
The crystal sizes of the different samples obtained from Scherrer equation have been shown
in Table S3.
Eq. 1 Scherrer equation
In this equation, Dhkl is the crystal size calculated for reflection (hkl), λ the X-ray
monochromatic wavelength (related to CuKα beam as high as 1.5406 Å), K is a constant
which changes with the type of crystal. FWHM is the Full Width in Half Maximum of the
diffraction peaks.
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As mentioned in previous works, Increasing the amount of substituted strontium ion in the
apatite structure leads to a smaller particle size [47]. The observation of the mean particle size
for FHA also indicates that the substitution of fluorine into the structure of hydroxyapatite is
also associated with a decrease in particle size; in fact, by introducing the fluorine into the
structure, the growth of the grain is prevented [46].
3.2 Infrared spectroscopy
FTIR spectra the HA powder, 5%SrHA, 10%SrHA, FHA, F-5%SrHA and F-10%SrFHA has
been juxtaposed in the range 400-4000 cm-1 and are shown in Fig. S3, demonstrating
numerous peaks due to the various functional groups exist in samples. In Sr contained
samples, with the increase of strontium, the OH bands decreased by 634 and 3571 cm -1 and
the width of the bands broadened. These changes, indicating the replacement of calcium with
strontium, lead to the reduction of OH-groups and structural irregularities along the c-axis.
PO4 bands will have a shorter displacement to a smaller wavelength numbers and become
broader when they are substituted with strontium. This also increase the irregularity in the
HA structure (around phosphate positions) [48]. The 1037-1117(υ3), 474(υ2), 962(υ1) and
572(υ4) and 603 cm-1 are the molecular vibrations of the phosphate (PO43-), which are visible
in all apatite specimens. In addition, the vibrational modes of the phosphate group correspond
to 1120-1020 m-1. These bands become broader by strontium substitution. These changes in
line of bandwidth, supports the previous argument that irregularity is due to the strontium
substitution. The reduction in wavelength number of P-O band is corresponded to the
substitution of strontium with calcium ions in the apatite structure. The ionic radius of
strontium (1.21 nm) is larger than calcium ionic radius (1.00nm), which leads to the reduction
of P-O bond strength. These changes indicate that Sr2+ ions substituted Ca2+ ions and entered
inside the apatite structure [49]. peaks observed at 1416 and 1465 cm-1 may be due to the
carbonate impurity [49-51]. This carbonate substitution can be due to the reaction absorption
of carbon dioxide from air during the synthesis process. An absorption band of carbonate has
been reported in the region of 1470-1415 cm-1 for SrHA, which its intensity is increased with
increasing strontium. The existence of carbonate in the structure reduces the crystallinity and
increases the solubility [51]. The 1639 cm-1 band is attributed to the absorbed water in
hydroxyapatite, and the peaks of 1950 to 2160 cm-1 are interpreted as a combination of
phosphate bands [52]. Increasing the strontium content reduces the intensity of vibration and
stretching OH-bands. By adding strontium, the OH-band (3571 cm-1) moves toward a higher
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wavelength number and the OH-band (635 cm-1) moves toward a smaller wavelength number
[15, 47].
In fluorinated powders, the small peak between 3561-3556 cm-1 is attributed to the OH-F
bond which indicates the penetration of F ion in the apatite structure. The 1646 cm-1 peak is
water-bending bond. The bending bond of 3556 cm-1 is attributed to hydroxyl groups. The
presence of hydroxyl groups is due to the hydrogen bonding of fluorine ions. This peak
confirms the crystalline structure of fluoro-apatite. The formation of hydrogen bond between
fluorine and hydroxyl (F-OH) indicates the substitution of F-ions instead of OH-ions in the
structure of apatite.
The IR spectrum of all specimens shows the specific apatite phosphate peaks near 473, 571,
605, 641, 961, and 1131-1020 cm-1. In all samples, HPO42- vibrations were observed in the
range of the 881 cm-1 wave length. There are also minor carbonate bands at 1410 and 1460
cm-1 in some samples. The kinetic vibration of OH- in 631 cm-1, indicating hydroxyapatite,
was observed in all specimens. By comparing the spectra of the samples, the vibrational and
kinetic OH bonds of the fluorine samples are displaced very little. The position, number and a
relatively complete absorption of OH bands in the range of 3480-3650 cm-1 can be used to
estimate the amount of fluoride ions in fluoride samples. The existence of 3570 cm-1 bond
only represents the substitution of less than 5% of hydroxyl ions with fluoride ions, while the
3538 cm-1 bond only represents the substitution of at least 75% OH -ions with F-ions. The
information obtained from our calcined samples is close to the literature [53]. However, the
exact amount of fluorine ions substituted with hydroxyl ions cannot be determined by FTIR
analysis. substituting different ions in the apatite structure reduces the symmetry and causes
the bond to be absorbed in the IR spectrum. The 1956 cm-1 bond is related to the PO43- group,
which is observed in all powder samples. Also, bands of 1992 and 2081 cm-1 of the PO43-
group were also observed for fluoridated samples. The 1400-1500 cm-1 bond is related to the
carbonate group. The carbonate ions present in the 1956-87 bond can be considered to be
CO2-disolution [53]. The two bonds of 3571 and 3556 cm-1, respectively, are the hydrogen
bending bond of OH- ions (OH—OH and OH—F). The OH- bond, which appeared only in
3556 cm-1, indicates the interaction of OH—F with OH-ions present in the structure [54, 55].
As fluorine increases, OH- bonds disappear completely, which suggests that a significant
amount of fluoride is substituted by hydroxyl groups.
As a result, FHA powder has the same bond structure as the HA powder, but bending bond of
OH-F has also been observed at 3561 cm-1, and the vibrating bond of OH- in the 630 cm-1 is
also very weak. In the spectrum of fluoridated samples, the intensity of the two bonds is
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reduced to 3570 and 634 cm-1 OH- groups, which indicates the substitution of fluorine with
hydroxyl. This effect is due to an additional OH-F bending bond that appears at a lower
frequency and with the substitution of the F- with OH- ions. It is worth noting that these two
vibrations are OH- immersed in unlimited chains of OH-OH, but this chain is terminated by
F- when F- enters the apatite structure. These observations for the FHA powder indicate the
formation of a hydrogen bond between F and OH (OH-F), which shows partial substitution of
F ions with OH- ions in the apatite structure, and, as already mentioned, the exact amount of
substituted fluorine ions, instead of ions hydroxyl is not detectable from the FTIR analysis
[45, 56, 57].
3.3 Inductively coupled plasma spectroscopy
𝐹 𝑤𝑒𝑖𝑔ℎ𝑡
F wt% = × 100
𝐹 − 𝑆𝑟𝐻𝐴 𝑤𝑒𝑖𝑔ℎ𝑡
The chemical composition of the hydroxyapatite powders and substituted samples were
reported by ICP (Inductively Coupled Plasma spectroscopy) on a AGILENT ICP-7900
spectrometer [4, 30, 58].
3.4 F-selective electrode
A F-selective electrode analysis was used to measure the amount of F- ions in a crystalline
structure of powders. According to the results of the X-ray diffraction pattern and FTIR
spectroscopy, it was not expected that the all F- ions substitute OH- ion. The values of
fluorine measured in the fluorinated samples are summarized in Table S4., which confirms
the result obtained from the XRD and FTIR tests. The general formula of F-SrHA is
(Ca,Sr)10(PO4)6(OH)2-2xF2x. To calculate x in this formula, we used the following equation.
Eq. 2. Weight percentage of F- ions
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3.5 Scanning Electron Microscope
A comparison between the morphology and grain size of the HA, SrHA, FHA and F-SrHA
was performed by SEM micrograms (Fig. S4.). The SEM analysis of the HA powder shows
that the hydroxyapatite particles synthesized by precipitation method are spherical and these
nanoparticles tend to be agglomerated. Many agglomerates of spherical particles physically
connected to each other are readily visible on the nanometer scale, with approximate
proportions of these particles for HA, 5%SrHA and 10%SrHA, respectively, between 65-40
nm, 60-25 nm, and 45-20 nm. The addition of strontium to the structure of hydroxyapatite did
not change the morphology of HA powders. Despite this, measuring the dimensions of
spherical nanoparticles showed that by increasing the percentage of strontium, the size of the
particles of strontium hydroxyapatite becomes smaller. Also, with increasing strontium
content, it is observed that, in addition to smaller particles, their distribution is become more
homogeneous. Substituting larger Sr2+ ions with Ca2+ ions results in a more compact atomic
structure, which can delay crystal growth. When the crystals have less freedom to grow, the
particle size becomes smaller [56]. In the analysis of the XRD results, it was found that
increasing the percentage of strontium is associated with an increase in peak width and
particle size reduction. Also, the FTIR spectroscopy of SrHA specimens was accompanied by
wider and lesser OH-band banding depths. Shrinking the size of the SrHA particles by
increasing the strontium content of the sample in the SEMs suggests that the results
correspond with XRD and FTIR.
SEM images of fluorinated samples of apatites indicate that fluorine enters no change in the
structure of the morphology of the powders, but the particle size is smaller than that of
fluorine-free specimens. In fact, fluorine is a stimulant to prevent the growth of apatite
crystals [55]. As previously mentioned, the fluorine substitution in the structure of apatite
microcrystalline has been increased and this strain can lead to a change in unit cell
parameters and a decrease in particle size. The broadening of the FTIR peaks and the grain
size calculated by the Sherer formula also showed a decrease in the particle size by adding
fluorine.
Fig S6. Shows cumulative and normal particle size distribution curves for different samples
that were obtained by ImageJ software. It can be seen that by adding strontium in HA and
FHA samples the curves shift leftward which shows the reduced particle size. In addition, in
samples which include fluoride by adding strontium the border particle size distribution
obtained. These results approve the data which was obtained by XRD test.
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3.6 MTT assay
In order to evaluate cell viability and proliferation MTT assay was carried out at intervals for
up to 14 days. Fig S7. shows that the presence of strontium and fluorine in the structure of
HA increased cell proliferation as compared to the control sample. In SrHA groups,
increasing the percentage of Sr has had positive effects on cell proliferation after 7 days of
culture. Also, in FHA and F-5%SrHA samples, the percentage of cell proliferation was higher
than that of HA and 5%SrHA, respectively. However, results indicated that MG63 cells
viability in F-10% SrHA sample is lower than other samples. From days 7 to 14, a down-
regulation is observed for all the samples which is significant for 10%SrHA. The decrease in
cell proliferation can be explained by the fact that rate of cell division decreases after 7 days.
By examining the results of MTT after 7 and 14 days, it can be suggested that in general,
irrespective of the any time period of cell culture, strontium-containing samples are not only
non-toxic to cells, but also enhanced cell viability and growth. Fluorine incorporation in
SrHA samples (89.8% mol) also improved biocompatibility , division and proliferation of
MG63 cells.
3.7 ALP activity
The ALP assay was carried out in order to evaluate the functional activity of proliferated
cells. Figure S8 shows the ALP activity of MG63 cells on different apatite samples at 7 and
14 days of culture. It was observed that the ALP activity of HA and 5%Sr-HA is less than
control sample after 7 days of culture. The ALP level for 5%Sr-HA sample was slightly
lower than that of HA at 7th day of culture which increased by increasing both culture
period(up 14 days) and amount of Sr (up to 10%) in the structure. It should be noted that the
effect of culture time is more significant than increasing amount of Sr in the samples.
In the case of fluoridated apatite and its different composites, there was no significant
difference in ALP activity between FHA and HA sample in the first 7 days in culture.
However, after 14 days, a higher ALP level was observed in in FHA sample than that of HA.
It was observed that that an 80% increase in ALP activity occurred after 7 days of culture in
the F-5% SrHA sample compared to the pure 5% SrHA sample. After 14 days of culture
period, this increase in ALP activity was reached to a maximum value of 100 percent. MG63
cells on F-10%SrHA had lower ALP activity at 7 and 14 days of culture than those on 10%
SrHA. It can be argued that the presence of fluorine in 10% SrHA leads to apatite
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crystallization and a reduction in calcium release, while in the 5% SrHA structure, it has a
very small effect on calcium ion release and consequently ALP activity.
In general, we can conclude that Strontium incorporated into HA has positive and stimulating
effects on the cell growth and differentiation. Fluorine also has a great influence on
stimulating the differentiation of cells and their growth when it incorporates into the
hydroxyapatite and strontium hydroxyapatite (up to 5%).
4 Conclusion
The synthesis of nano-size strontium hydroxyapatite powders was carried out using the
precipitation method. Addition of fluorine to the apatite structure by pH-cycling also
produced nanosize powder. The XRD pattern of samples shows that hydroxyapatite has been
formed in all of them, and no trace of impurities of other calcium phosphates has been
observed with this technique. Of course, there is a small amount of CaO in synthetic powders,
with no effect on biocompatibility. Broadening and decrease in peak intensity is observed
with the introduction of strontium into the structure of apatite. While the fluorine makes HA
peaks sharper. In fact, the increase in strontium is accompanied by a structural disorder and
the addition of fluorine make the structure more arranged. As the ratio of strontium molar to
calcium increases, the apatite peaks move toward smaller angles due to the larger ions of
strontium compared to calcium. While the fluoride enters the apatite structure, due to the
small amount of ionic fluorine radius relative to hydroxyl, it moves apatite peaks to larger
angles. The substitution of strontium with calcium in the apatite structure leads to an increase
in the a-axis and c-axis parameters, but the substitution of the hydroxylapatite group with
fluorine results in a decrease in the parameter a and a non-change (or increase) of the
parameter c. The results of the IR spectrum show that apatite bonds do not change in
strontium-containing specimens, but they become wider by increasing strontium. In addition
to these bonds, the band OH-- F has been formed in 3556 cm-1, representing the direct effect
of OH-- F on total OH- ions present in the specimens. The SEM images show the nanoscale
size of the synthesized samples, which also corresponds to the results of the XRD. This
decrease is especially noticeable in the case of an increase in the percentage of substituted
strontium with calcium. ICP and F-selective also proved the successful substitution. For
5%SrHA & 10%SrHA the amount of Sr substitution was 4.29 & 8.36 wt%, However in F-
5%SrHA & F-10%SrHA this amount was 4.26 & 8.34 wt% respectively. The amount of F
substitution in FHA, F-5%SrHA & F-10%SrHA was 2.56, 2.55 & 2.56 wt% respectively.
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The morphology of all the synthetic samples were spherical. The biological evaluation shows
that the addition of fluorine and strontium in the long intervals produces a little toxicity,
while the hydroxyapatite sample has the lowest toxicity after 14 days and the F-10% SrHA
sample has the highest toxicity among all the synthetic powder compounds. The increase in
strontium in the structure of hydroxyapatite has positive and stimulating effects on the growth
and differentiation of cells, which indicates that strontium will be effective in long-term and
longer intervals. Fluorine also has a great influence on stimulating the differentiation of cells
and their growth by replacing them with the structure of hydroxyapatite and SrHA (up to
5%). As the activity of alkaline phosphatase (ALP) is associated with the process of cloning
in hard tissues (bones and teeth), F-5% SrHA powder is the best synthetic sample for dental
use (a study of the process of remineralization and decay).
Conflict of Interest
We wish to draw the attention of the Editor to the following facts which may be considered as
potential conflicts of interest and to significant financial contributions to this work.
We wish to confirm that there are no known conflicts of interest associated with this
publication and there has been no significant financial support for this work that could have
influenced its outcome.
We further confirm that any aspect of the work covered in this manuscript that has involved
either experimental animals or human patients has been conducted with the ethical approval
of all relevant bodies and that such approvals are acknowledged within the manuscript.
AUTHORSHIP STATEMENT
All persons who meet authorship criteria are listed as authors, and all authors certify that they
have
participated sufficiently in the work to take public responsibility for the content, including
participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore,
each author certifies that this material or similar material has not been and will not be submitted
to or published in any other publication before its appearance in the Microchemical Journal.
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Acknowledgement
Authors would like to thank Mr. Asad Shahin Moghadam for his efforts in graphical
development of this work.
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Fig. 1: Flowchart of synthesis processes.
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Table 1: General Specifications of Synthesized Powders.
Sample Abbreviation chemical formula

Hydroxyapatite HA Ca10(PO4)6(OH)2
5% Strontium hydroxyapatite 5SrHA Ca9/5F0/5(PO4)6(OH)2
10% Strontium hydroxyapatite 10SrHA Ca9F (PO4)6(OH)2
Fluoro-hydroxyapatite F-HA Ca10(PO4)6(OH)0/64F1/3
5% Strontium fluoro-hydroxyapatite F-5SrHA Ca9/5F0/5(PO4)6(OH)0/62F1/38
10% Strontium fluoro-hydroxyapatite F-10SrHA Ca9F (PO4)6(OH)0/58F1/42
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Synthesis and Characterization of Strontium Fluor-Hydroxyapatite Nanoparticles For Dental Applications

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S2.0 S0925838816320382 main

Synthesis and characterization of Strontium Fluor- Hydroxyapatite

Alireza Rajabnejadkeleshteri , Armin Kamyar , Mehrdad Khakbiz ,

To appear in: Microchemical Journal

Received date: 24 August 2019

© 2019 Published by Elsevier B.V.

Alireza Rajabnejadkeleshteria, Armin Kamyara, Mehrdad Khakbiza*,

2. Materials and methods

Hydroxyapatite powder (Ca10(PO4)6(OH)2) was synthesized by precipitation technique using

2.2 Synthesis of SrHA

Strontium hydroxyapatites (Ca10-xSrx(PO4)6(OH)2) was synthesized by precipitation technique

2.3 Synthesis of FHA

Fluoride hydroxyapatite (Ca10(PO4)6(OH)2-2xF2x) was synthesized by pH cycling technique. 5

2.4 Synthesis of F-SrHA

Strontium Fluoro-hydroxyapatite (Ca10-xSrx(PO4)6(OH)2-2yF2y) was synthesized by pH cycling

2.5 Characterization techniques (XRD, FTIR and SEM)

2.6 Chemical analysis of different samples

2.7 F-selective electrode

2.9 ALP assay

3. Results and Discussion

3.2 Infrared spectroscopy

3.3 Inductively coupled plasma spectroscopy

3.4 F-selective electrode

3.7 ALP activity

Fig. 1: Flowchart of synthesis processes.

Table 1: General Specifications of Synthesized Powders.

Sample Abbreviation chemical formula

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