(FA, Ca10(PO4)6F2) has potential
applications in dentistry and orthopedics. FA nanopowder was synthesized by
modified wet-chemical precipitation method. Sodium fluoride was added to the
hydroxyapatite preparation for the synthesis of fluorapatite. The synthesized
samples were characterized by using FTIR, XRD, SEM, EDX, TEM, and in vitro antimicrobial activity. From
the size and morphology analysis it is confirmed that the synthesized sample is
FA nanopowder and from the in vitro activity
it is confirmed that the FA nanopowder can be used for biomedical applications.
Fluorapatite; wet chemical method; XRD; TEM; in vitro antimicrobial activity
Hydroxyapatite (HAp, Ca10(PO4)6OH2)
belongs to the calcium phosphate (CaP) family and is the main mineral component
of human bone and teeth 1. The application of calcium phosphates, especially
HAp, for bone regeneration is known for a hundred years, but only last fifteen
years CaP have been studied extensively as promoters of tooth enamel and
dentine remineralisation. Since 96-97% of tooth enamel consists of mineral
phase, mainly HAp 2, 3, and fluorine is known for its anti-caries effect 4,
several scientific papers 5-7 are devoted to synthesis and characterization
of F-doped hydroxyapatite (FHAp, Ca10(PO4)6OH2-xF2x).
FHAp is of high interest due to its bioactivity, mechanical properties and
chemical similarity to teeth 4. The structural and chemical stability of FHAp
against chemical and physical factors are the main properties promising for
remineralisation of tooth enamel. As reported in 8, too high intake of
fluorine can lead to bone and teeth diseases like osteomalacia and dental
fluorosis. Therefore it is necessary to control F- content in the
HAp structure and also release afterwards.
Once the OH
groups were partially substituted by the F- ions, the existing
hydrogen ions of the OH groups were bound to the nearby F ions because of the
higher affinity of the F ions with respect to the oxygen ions, producing a quite
well-ordered apatite structure, which caused an increase of the thermal and
chemical stabilities of the HA matrix. Therefore, when a certain amount of F
ions substituted the OH groups in the HA matrix, a certain level of chemical
and thermal stability of the FHA ceramics was achieved. Theoretically, a F ion
concentration of 50% in the FHA should be enough to remove the disorder of the
crystal structure of HA and hence stabilize the structure F due to the
alternating arrangement of the F ions between each pair of OH groups.9 In this research, synthesis of
fluorapatite nanopowder via modified wet-chemical method by using sodium
fluoride and the resulting FA powder was characterized by instrumentation
II. Experimental procedure
Synthesis of FA nanopowder
Modified wet chemical method was used to prepare the pure FA sample.
The precursors for synthesis were
calcium hydroxide(Ca(OH)2)(Merck,India), ammonium dihydrogen
phosphate (NH4)H2PO4 (Merck, India)(ADP)
and sodium fluoride (NaF) (Sigma Aldrich, India). 1.0M calcium hydroxide
and 0.67M ammonium dihydrogen phosphate were dissolved in 100ml of double
distilled water for 1hour in magnetic stir. The dissolved solution of ADP was
added drop wise to the (Ca(OH)2) solution for 35mins and
continuously stirred for 18hours. The pH of the solution was maintained in 11.
The obtained slurry was irradiated to microwave radiation for 30mins and the
dried powder were grinded well by using mortar and pestle.
The size, morphology
and in vitro activity for the synthesized FA nanopowder
were characterized by using Fourier transmission infrared spectroscopy (FTIR),
X-Ray diffraction(XRD), Scanning electron microscope (SEM), Energy-dispersive X-Ray
spectroscopy (EDX), Transmission electron microscope (TEM) and in vitro antibacterial activity.
III. Results and disscussion
Figure 1.shows the FT-IR spectrum of FA nanopowder. The
characteristic bands exhibited in the range 400cm-1 to 4000cm-1
are assigned here: a) the bands at 1095.47 cm-1 and 1044.69 cm-1
arise from ?3 , the bands at
604 cm-1 and 566 cm-1
arises from ?4 , the band at 473.4 cm-1 arises
from ?2 (b) the bands at 1420.24 cm-1 and
1456.69 cm-1 arises from (c) the
band at 3438.56 cm-1 arises due to OH….F bond.
The XRD analysis was performed using the X-Ray diffractrometer.
Figure 2.shows the XRD spectrum of the synthesized FA nanopowder. The pattern matches the FA (JCPDS PDF#15-0876)
data indicating that the synthesized were FA. The average crystalline size of
the synthesized powder was calculated by using debye scherrer equation. It is
observed that the value of average crystallite size calculated from the
reflection of the planes: (002),(211), (300) and (202) are 43.31nm,35.12nm,
22.02nm and 29.44nm.
1. FTIR spectrum of FA nanopowder
2. XRD spectrum of FA nanopowder
SEM and EDX analysis
The SEM analysis was observed using VEGAS
TESCAN SEM instrument. The morphology of the synthesized FA nanopowder was
shown in figure 3. The elemental composition of the FA nanopowder is observed
in the figure 4. The chemical composition of the synthesized FA nanopowder are
Ca, P, Na, F and O.
SEM and EDX spectrum of FA nanopowder
The size and
morphology of the fine powder may be determined with TEM. The bright field
transmission electrn microscopic images of FA is shown in figure 4. The FA
nanoparticles are rod like morphology and does not posses regularity in shape
due to the porous surface. The particles show a high tendency towards
4. TEM images of synthesized FA nanopowder
E. In vitro anti microbial activity
The antimicrobial activity
was performed by disc diffusion method.
Antibiogram was done by disc diffusion method (NCCLS, 1993; Awoyinka et al., 2007) using sample. Petri plates
were prepared by pouring 30 ml of NA medium for bacteria/fungi. The test
organism was inoculated on solidified agar plate with the help of micropipette
and spread and allowed to dry for 10 mints. The surfaces of media were
inoculated with bacteria from a broth culture. A sterile cotton swab is dipped
into a standardized bacterial test suspension and used to evenly inoculate the
entire surface of the Nutrient agar plate. Briefly, inoculums containing Staphylococcus aureus and Escherichia
coli specie of bacteria were
spread on Nutrient agar plates. Using sterile forceps, the sterile filter
papers (6 mm diameter) containing the crude extracts (50?l) were laid down on
the surface of inoculated agar plate. The plates were incubated at 37?C for 24
h for the bacteria and at room temperature (30±1) for 24-48 hr. for yeasts
strains. Each sample was tested in triplicate.
zone of inhibition
The antimicrobial potential of test
compounds was determined on the basis of mean diameter of zone of inhibition
around the disc in millimeters. The zones of inhibition of the tested
microorganisms by the samples were measured using a millimeter scale.
Escherichia coli Staphylococcus aureus
Escherichia coli (mm)
Staphylococcus aureus (mm)
Table 1. Antimicrobial activity of FA
were expressed as Mean ± SD
standard : Chloramphenicol
standard : Fluconazole
Water + Alcohol
Fluorapatite nanopowder was synthesized
successfully by using modified wet chemical method by using sodium fluoride in
hydroxyapatite suspension. The synthesized FA powder were characterized by
FTIR, XRD, SEM, EDX, TEM and in vitro
antimicrobial activity. The FTIR analysis reveals that the characteristics band
corresponding to the FA appears in the synthesized sample. The disappearance of
the hydroxyl vibration band in the FTIR spectrum confirms the fluorine
substitution in the HA. The XRD analysis reveals that the particles of the FA nanopowder are of nano size
and homogeneous in composition. The EDX spectra confirm the presence of
chemical composition of the FA sample. The morphological evaluation shows that the
morphology is rod like and agglomerated. The in vitro antimicrobial activity was done by disc method by using
gram positive and gram negative bacteria like E.coli and S.aureus. From the in vitro studies it is confirmed that
the synthesized FA nanopowder has antimicrobial property and the sample can be
used for biomedical applications.
The author would like to extend their
gratitude to Alagappa university, Karaikudi and Gandhigram university, Tindigul
for the characterization techniques provided which have been helpful for
the research work to make it a success.
Epple, E. Baeuerlein, Handbook of
Biomineralization, Medical and Clinical Aspects, WileVCH Verlag GmbH &
Eslami, M. Solati-Hashjin, M. Tahriri, The
comparison of powder characteristics and physicochemical, mechanical and
biological properties between nanostructure ceramics of hydroxyapatite and
fluoridated hydroxyapatite, Mater. Sci. Eng. C. 29 (2009) 1387–1398.
Chen, K. Liang, J. Li, D. Wu, X. Zhou, J. Li, Regeneration of biomimetic hydroxyapatite on etched human enamel by
anionic PAMAM template in vitro, Arch. Oral Biol. 58 (2013) 975–80.
D. R. Clark, a Czajka-Jakubowska, C. Rick, J. Liu, S. Chang,
B. H. Clarkson, In vitro anti-caries
effect of fluoridated hydroxyapatite-coated preformed metal crowns, Eur.
Arch. Paediatr. Dent. 14 (20013) 253–8.
H. Eslami, F. Moztarzadeh, K. Khoshroo, M. Ashuri, M. Tahriri,
Synthesis and characterization of Ca5(PO4)3(OH)1-xFx
(0?x?1) nanopowders via pH-cycling method as bioceramics, Proc. 4th Int. Conf.
Nanostructures. (2012) 794–796.
M. H. Fathi, E. M. Zahrani, Fabrication and characterization of fluoridated hydroxyapatite nanopowders
via mechanical alloying, J. Alloys Compd. 475 (2009) 408–414.
M. T. Hossein Eslami, Mehran Solati-Hashjin, Synthesis and characterization of
nanocrystalline fluorinated hydroxyapatite powder by a modified wet-chemical
process, J. Ceram. Process. Res. 9 (2008) 224–229.
J. Abanto Alvarez, K. M. P. C. Rezende, S. M. S. Marocho, F.
B. T. Alves, P. Celiberti, A. L. Ciamponi,
Dental fluorosis: exposure, prevention and management, Med. Oral Patol. Oral
Cir. Bucal, 14 (2009) E103–7.
Y. Chen and X. Miao, Biomaterials 26 (2005) 1205-1210.