Abstract— Fluorapatite(FA, Ca10(PO4)6F2) has potentialapplications in dentistry and orthopedics. FA nanopowder was synthesized bymodified wet-chemical precipitation method. Sodium fluoride was added to thehydroxyapatite preparation for the synthesis of fluorapatite. The synthesizedsamples were characterized by using FTIR, XRD, SEM, EDX, TEM, and in vitro antimicrobial activity. Fromthe size and morphology analysis it is confirmed that the synthesized sample isFA nanopowder and from the in vitro activityit is confirmed that the FA nanopowder can be used for biomedical applications.Keywords—Fluorapatite; wet chemical method; XRD; TEM; in vitro antimicrobial activity I.
Introduction Hydroxyapatite (HAp, Ca10(PO4)6OH2)belongs to the calcium phosphate (CaP) family and is the main mineral componentof human bone and teeth 1. The application of calcium phosphates, especiallyHAp, for bone regeneration is known for a hundred years, but only last fifteenyears CaP have been studied extensively as promoters of tooth enamel anddentine remineralisation. Since 96-97% of tooth enamel consists of mineralphase, mainly HAp 2, 3, and fluorine is known for its anti-caries effect 4,several scientific papers 5-7 are devoted to synthesis and characterizationof F-doped hydroxyapatite (FHAp, Ca10(PO4)6OH2-xF2x).FHAp is of high interest due to its bioactivity, mechanical properties andchemical similarity to teeth 4.
The structural and chemical stability of FHApagainst chemical and physical factors are the main properties promising forremineralisation of tooth enamel. As reported in 8, too high intake offluorine can lead to bone and teeth diseases like osteomalacia and dentalfluorosis. Therefore it is necessary to control F- content in theHAp structure and also release afterwards.Once the OHgroups were partially substituted by the F- ions, the existinghydrogen ions of the OH groups were bound to the nearby F ions because of thehigher affinity of the F ions with respect to the oxygen ions, producing a quitewell-ordered apatite structure, which caused an increase of the thermal andchemical stabilities of the HA matrix. Therefore, when a certain amount of Fions substituted the OH groups in the HA matrix, a certain level of chemicaland thermal stability of the FHA ceramics was achieved. Theoretically, a F ionconcentration of 50% in the FHA should be enough to remove the disorder of thecrystal structure of HA and hence stabilize the structure F due to thealternating arrangement of the F ions between each pair of OH groups.9 In this research, synthesis offluorapatite nanopowder via modified wet-chemical method by using sodiumfluoride and the resulting FA powder was characterized by instrumentationtechniques II.
Experimental procedureA. Synthesis of FA nanopowderModified wet chemical method was used to prepare the pure FA sample.The precursors for synthesis werecalcium hydroxide(Ca(OH)2)(Merck,India), ammonium dihydrogenphosphate (NH4)H2PO4 (Merck, India)(ADP)and sodium fluoride (NaF) (Sigma Aldrich, India). 1.0M calcium hydroxideand 0.67M ammonium dihydrogen phosphate were dissolved in 100ml of doubledistilled water for 1hour in magnetic stir.
The dissolved solution of ADP wasadded drop wise to the (Ca(OH)2) solution for 35mins andcontinuously stirred for 18hours. The pH of the solution was maintained in 11.The obtained slurry was irradiated to microwave radiation for 30mins and thedried powder were grinded well by using mortar and pestle. B. CharacterisationThe size, morphologyand in vitro activity for the synthesized FA nanopowderwere characterized by using Fourier transmission infrared spectroscopy (FTIR),X-Ray diffraction(XRD), Scanning electron microscope (SEM), Energy-dispersive X-Rayspectroscopy (EDX), Transmission electron microscope (TEM) and in vitro antibacterial activity. III. Results and disscussionA. FTIR analysisFigure 1.
shows the FT-IR spectrum of FA nanopowder. Thecharacteristic bands exhibited in the range 400cm-1 to 4000cm-1are assigned here: a) the bands at 1095.47 cm-1 and 1044.69 cm-1arise from ?3 , the bands at604 cm-1 and 566 cm-1arises from ?4 , the band at 473.4 cm-1 arisesfrom ?2 (b) the bands at 1420.24 cm-1 and1456.69 cm-1 arises from (c) theband at 3438.56 cm-1 arises due to OH….
F bond.B. XRD analysisThe 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 ofthe synthesized powder was calculated by using debye scherrer equation. It isobserved that the value of average crystallite size calculated from thereflection of the planes: (002),(211), (300) and (202) are 43.31nm,35.12nm,22.02nm and 29.
44nm.Fig1. FTIR spectrum of FA nanopowderFig2. XRD spectrum of FA nanopowderC. SEM and EDX analysisThe SEM analysis was observed using VEGASTESCAN SEM instrument. The morphology of the synthesized FA nanopowder wasshown in figure 3. The elemental composition of the FA nanopowder is observedin the figure 4. The chemical composition of the synthesized FA nanopowder areCa, P, Na, F and O.
. Fig 3SEM and EDX spectrum of FA nanopowderD. TEM analysisThe size andmorphology of the fine powder may be determined with TEM. The bright fieldtransmission electrn microscopic images of FA is shown in figure 4. The FAnanoparticles are rod like morphology and does not posses regularity in shapedue to the porous surface. The particles show a high tendency towardsagglomeration.Fig4. TEM images of synthesized FA nanopowder E.
In vitro anti microbial activity The antimicrobial activitywas performed by disc diffusion method..Antimicrobial assay Antibiogram was done by disc diffusion method (NCCLS, 1993; Awoyinka et al., 2007) using sample.
Petri plateswere prepared by pouring 30 ml of NA medium for bacteria/fungi. The testorganism was inoculated on solidified agar plate with the help of micropipetteand spread and allowed to dry for 10 mints. The surfaces of media wereinoculated with bacteria from a broth culture. A sterile cotton swab is dippedinto a standardized bacterial test suspension and used to evenly inoculate theentire surface of the Nutrient agar plate. Briefly, inoculums containing Staphylococcus aureus and Escherichiacoli specie of bacteria werespread on Nutrient agar plates. Using sterile forceps, the sterile filterpapers (6 mm diameter) containing the crude extracts (50?l) were laid down onthe surface of inoculated agar plate. The plates were incubated at 37?C for 24h for the bacteria and at room temperature (30±1) for 24-48 hr. for yeastsstrains.
Each sample was tested in triplicate. Measurement ofzone of inhibition The antimicrobial potential of testcompounds was determined on the basis of mean diameter of zone of inhibitionaround the disc in millimeters. The zones of inhibition of the testedmicroorganisms by the samples were measured using a millimeter scale. Antimicrobialactivity Escherichia coli Staphylococcus aureus Microorganisms (50?l) (100µl) (150µl) Standard (30µl) Control (solvent) (30µl) Bacteria Escherichia coli (mm) 0.90±0.06 3.10±0.21 5.
90±0.41 8.70±0.60 0.20±0.01 Staphylococcus aureus (mm) 0.50±0.
03 2.70±0.18 5.30±0.37 8.
50±0.59 0.10±0.01 Table 1. Antimicrobial activity of FAnanopowderValueswere expressed as Mean ± SDBacterialstandard : ChloramphenicolFungalstandard : Fluconazole Control : Water + Alcohol IV. Conclusion Fluorapatite nanopowder was synthesizedsuccessfully by using modified wet chemical method by using sodium fluoride inhydroxyapatite suspension.
The synthesized FA powder were characterized byFTIR, XRD, SEM, EDX, TEM and in vitroantimicrobial activity. The FTIR analysis reveals that the characteristics bandcorresponding to the FA appears in the synthesized sample. The disappearance ofthe hydroxyl vibration band in the FTIR spectrum confirms the fluorinesubstitution in the HA. The XRD analysis reveals that the particles of the FA nanopowder are of nano sizeand homogeneous in composition. The EDX spectra confirm the presence ofchemical composition of the FA sample.
The morphological evaluation shows that themorphology is rod like and agglomerated. The in vitro antimicrobial activity was done by disc method by usinggram positive and gram negative bacteria like E.coli and S.
aureus. From the in vitro studies it is confirmed thatthe synthesized FA nanopowder has antimicrobial property and the sample can beused for biomedical applications. Acknowledgement The author would like to extend theirgratitude to Alagappa university, Karaikudi and Gandhigram university, Tindigulfor the characterization techniques provided which have been helpful forthe research work to make it a success. References 1 M.Epple, E. Baeuerlein, Handbook ofBiomineralization, Medical and Clinical Aspects, WileVCH Verlag GmbH &Co, 2007. 2 H.Eslami, M.
Solati-Hashjin, M. Tahriri, Thecomparison of powder characteristics and physicochemical, mechanical andbiological properties between nanostructure ceramics of hydroxyapatite andfluoridated hydroxyapatite, Mater. Sci. Eng.
C. 29 (2009) 1387–1398. 3 L.Chen, K. Liang, J. Li, D.
Wu, X. Zhou, J. Li, Regeneration of biomimetic hydroxyapatite on etched human enamel byanionic PAMAM template in vitro, Arch.
Oral Biol. 58 (2013) 975–80. 4 D. R.
Clark, a Czajka-Jakubowska, C. Rick, J. Liu, S. Chang,B.
H. Clarkson, In vitro anti-carieseffect of fluoridated hydroxyapatite-coated preformed metal crowns, Eur.Arch. Paediatr.
Dent. 14 (20013) 253–8. 5 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. 6 M. H. Fathi, E. M. Zahrani, Fabrication and characterization of fluoridated hydroxyapatite nanopowdersvia mechanical alloying, J. Alloys Compd.
475 (2009) 408–414. 7 M. T. Hossein Eslami, Mehran Solati-Hashjin, Synthesis and characterization ofnanocrystalline fluorinated hydroxyapatite powder by a modified wet-chemicalprocess, J. Ceram.
Process. Res. 9 (2008) 224–229. 8 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. OralCir. Bucal, 14 (2009) E103–7. 9 Y.
Chen and X. Miao, Biomaterials 26 (2005) 1205-1210.