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Novel Fluorapatite-Forsterite Nanocomposite Powder for Oral Bone Defects A. Forghani, M. Mapar, M. Kharaziha,* and M. H. Fathi Biomaterials Research Group, Department of Materials Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran M. Fesharaki Medical Sciences Center, Department of Cell Sciences Research, Isfahan University, 1 Isfahan, 84156-83111, Iran Failing implants lead to osseous defects. Guided tissue regeneration made of bioactive ceramics has been used to promote bone formation in osseous deformation. The aim of this study was to prepare and to characterize the fluorapatite/ forsterite nanocomposite powder for treatment of oral bone defects. In this study, these composite powders with different contents of forsterite nanopowder were prepared via sol-gel process. Characterization of prepared nanocomposite powders and their cytotoxicity evaluation were done and compared with pure forsterite and fluorapatite powders. Results showed that nanocomposite powders with crystallite size of about 2124 nm were fabricated successfully by gel calcination at 600°C. Besides the non-toxicity effects of powders, nanocomposite containing 20 wt% forsterite significantly increased cell viability compared with control groups. According to these results, these nanocomposite powders might be suitable as bioactive material for oral bone defect. Introduction Bone defects around dental implants are often seen in extraction sockets, or around failing implants when implants are placed in areas with inadequate alveolar bone. In these defects, bone regeneration could improve the long-term prognosis implant by means of substitutes or bone grafts. 1 Bone fillers and guided tissue regeneration made of bioactive ceramics such as bioactive glass have been I J A C 0 2 8 2 4 B Dispatch: 15.6.12 Journal: IJAC CE: Deepa R Journal Name Manuscript No. Author Received: No. of pages: 10 PE: Hariprasad *[email protected] © 2012 The American Ceramic Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Int. J. Appl. Ceram. Technol., 1–8 (2012) DOI:10.1111/j.1744-7402.2012.02824.x
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Novel Fluorapatite-Forsterite Nanocomposite Powder for ...Novel Fluorapatite-Forsterite Nanocomposite Powder for Oral Bone Defects A. Forghani, M. Mapar, M. Kharaziha,* and M. H. Fathi

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Page 1: Novel Fluorapatite-Forsterite Nanocomposite Powder for ...Novel Fluorapatite-Forsterite Nanocomposite Powder for Oral Bone Defects A. Forghani, M. Mapar, M. Kharaziha,* and M. H. Fathi

Novel Fluorapatite-Forsterite Nanocomposite Powder forOral Bone Defects

A. Forghani, M. Mapar, M. Kharaziha,* and M. H. Fathi

Biomaterials Research Group, Department of Materials Engineering, Isfahan University of Technology,Isfahan, 84156-83111, Iran

M. Fesharaki

Medical Sciences Center, Department of Cell Sciences Research, Isfahan University,1 Isfahan,84156-83111, Iran

Failing implants lead to osseous defects. Guided tissue regeneration made of bioactive ceramics has been used topromote bone formation in osseous deformation. The aim of this study was to prepare and to characterize the fluorapatite/forsterite nanocomposite powder for treatment of oral bone defects. In this study, these composite powders with differentcontents of forsterite nanopowder were prepared via sol-gel process. Characterization of prepared nanocomposite powders andtheir cytotoxicity evaluation were done and compared with pure forsterite and fluorapatite powders. Results showed thatnanocomposite powders with crystallite size of about 21–24 nm were fabricated successfully by gel calcination at 600°C.Besides the non-toxicity effects of powders, nanocomposite containing 20 wt% forsterite significantly increased cell viabilitycompared with control groups. According to these results, these nanocomposite powders might be suitable as bioactivematerial for oral bone defect.

Introduction

Bone defects around dental implants are often seenin extraction sockets, or around failing implants when

implants are placed in areas with inadequate alveolarbone. In these defects, bone regeneration could improvethe long-term prognosis implant by means ofsubstitutes or bone grafts.1

Bone fillers and guided tissue regeneration made ofbioactive ceramics such as bioactive glass have been

I J A C 0 2 8 2 4 B Dispatch: 15.6.12 Journal: IJAC CE: Deepa R

Journal Name Manuscript No. Author Received: No. of pages: 10 PE: Hariprasad

*[email protected]

© 2012 The American Ceramic Society

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Int. J. Appl. Ceram. Technol., 1–8 (2012)DOI:10.1111/j.1744-7402.2012.02824.x

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used to promote bone formation in osseous deforma-tion.2 Literature has showed that the release of ionssuch as Na, Ca, and Si ions from bioactive glass couldcontrol the cell progress leading to the differentiationand proliferation of bone cells, modulation of theexpression of genes that regulate osteogenesis, and thesynthesis of growth factors.3 Hydroxyapatite (HA) hasbeen widely used in medical applications especially asbone filler.4 However, studies showed that substitutingof hydroxylic groups (OH�) by F� ions could improvethe mechanical strength, decrease the dissolution rate ofHA and enhance bone tissue growth.5,6 In addition,results confirmed that presence of F� ion could besuitable for dental application.7,8

Recent study shows that nanoscale forsterite(Mg2SiO4) is a bioactive and biocompatible ceramic.9,10

According to recent studies, forsterite has bettermechanical properties than other bioactive ceramics thatpromote the use of this bioceramic in the oral bonedefects treatment.11 In addition, similar to bioactiveglass, forsterite contains essential ions that release in thebiological environment leading to hydroxi-carbonateapatite layer on the surface of particles which have posi-tive effects on the bone calcification.9 So, forsteritenanopowder is expected to be a suitable candidate asfiller for oral bone defect treatment. According to theabove-mentioned points, nanocomposite powder of flu-orapatite and forsterite could be good candidate for oralbone defects.

Sol-gel technique is a unique method forfabrication of nanoparticles. This method has manyadvantages such as high reactivity of initial materials,lower sintering temperature compared with other pow-der preparation methods, and low cost.12 Therefore,the aim of this work was fabrication and characteriza-tion of fluorapatite-forsterite nanocomposite powder viasol-gel method and studying the effects of forsteritecontents on the structural and morphological propertiesand cytotoxicity of fluorapatite nanopowder.

Experimental Procedures

Preparation of Fluorapatite-Forsterite Powder

To fabricate composite powder, forsterite nano-powder was fabricated, separately. The forsterite nano-powder was fabricated by sol-gel method using organicprecursors; sucrose, and PVA described in a previousreport.9 Briefly, water-based solutions of the magnesium

salts and colloidal silica were prepared. The aqueoussolution of sucrose (sucrose-to-metal ratio = 5:1 mol)was added to the precursor solution. After continuousstirring, PVA solution was added and the pH value wasadjusted to 1 using nitric acid. Then, the solution wasstirred at 80°C and aged for 24 h. After that, it was driedon a hot plate at 100°C and finally, the dried gel was cal-cined up to 800°C in a furnace for 2 h.9

Calcium nitrate tetrahydrate (Ca (NO3)2·4H2O,Merck, Whitehouse Station, NJ) as Ca-precursor,phosphoric pentoxide (P2O5, Merck) as P-precursor,hexafluorophosphoric acid, (HPF6; Sigma-Aldrich,St-Louis, MO) and ethanol (Merck) were used forpreparing fluorapatite powder. A predetermined amountof calcium nitrate was dissolved in ethanol on the stir-rer. Simultaneously, phosphoric pentoxide was dissolvedin ethanol on the stirrer. After that, the first solutionwas added into the second one while stirring. HPF6 as afluorine-containing reagent was added into the mixture.After about 15 min, forsterite powder was directlyadded in the stirred sol. In this way, three solutions withdifferent amounts of forsterite nanopowders (10, 20 and30 wt %) were prepared. Finally, the mixtures werecontinuously stirred for about 24–48 h (depending onthe amount of forsterite) with the stirrer at room tem-perature to form a gel, aged for about 24 h, and driedin an oven at 80°C. As-dried gel was calcined at 600°Cto remove polymeric materials and calcination. To studythe cytotoxicity, pure fluorapatite nanopowder wasfabricated similarly according to the above method.

Characterization of Composite Powder

The phase analysis was carried out using X-raydiffraction (XRD) (CuKa radiation: k = 0.154056 nmat 40 kV and 30 mA). The obtained experimentalpatterns were compared to the standards collected bythe Joint Committee on Powder Diffraction andStandards, which include cards for forsterite andfluorapatite.

The Scherer’s equation (Eq. (1)) was consideredfor calculating the crystallite size of the obtained com-posite. For this goal, three picks of each phase wereselected for measuring in the XRD pattern.

B cosh ¼0:89k

tð1Þ

where t is the apparent crystallite size (nm), k is thewavelength of the X-ray (for Cu tube is about

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0.1542 nm), B is the full width of the selected pick athalf its height, and h is the Bragg angle.

Transmission Electron Microscopy (TEM) tech-nique was utilized to evaluate morphology and particlesize of synthesized forsterite. Scanning Electron Micro-scope (SEM, Philips XL 302 ) equipped with energy dis-persive spectroscopy (EDS) and X-ray map were usedto characterize the morphology and agglomerates sizeof the powders and elemental mapping was used toinvestigate distribution of elements in the structure.

The functional groups of prepared powders wereanalyzed using Fourier Transform Infrared spectroscopy(FT-IR) in the range of 400–4000 cm�1.

Cell culture

Human Osteoblast-like cells were isolated frombone by an enzymatic digestive process: The boneswere washed three times in phosphate-buffered saline(PBS, pH = 7.4) and then minced into fragments.After washing the bone fragments three times withPBS, the chips of calvaria were treated with osteoblast-special cell culture for 90 min at 37°C to release osteo-blast-like cells from the calvaria. The supernatants werecentrifuged at 1000 rpm for 10 min, and then sus-pended in the DMEM F12 medium containing 10%fetal calf serum (FCS) with 1% penicillin/streptomycin,and incubated in a 75 cm2 flask at 37°C under ahumidified atmosphere consisting 5% CO2. Culturemedia were refreshed every 3 days until the cellsreached confluency. The cells were routinely subcul-tured by trypsinization (0.05% [w/v] trypsin and0.02% [w/v] EDTA in PBS). The cells used in thestudy were second passage.

Cytotoxicity Assay

The cells proliferation on different substrates wasdetermined by using the colorimetric MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide)assay. This method was carried out with a dilution offorsterite, fluorapatite, and composite nanopowderextract in contact with human osteoblast-like cells.Prior to test, nanopowders were washed in 75% etha-nol solution, sterilized for 20 min under ultravioletlight, and autoclaved for 30 min at 120°C. For prepa-ration of solution containing the composite extracts,the powders with the ratio of 200 mg/mL (the ratio ofthe powder weight and the PBS) were added to the

PBS. After 24 h-incubation at 37°C, the mixture wascentrifuged and the supernatant was collected.

The cell suspension with the density of 1 9 104

cells/mL was prepared and 180 lL of it was added toeach well of a 96-well plate and incubated at 37°C and5% CO2. After 24 h cell incubation, 50 lL of extractsolution of nanopowders was added to each well of theplate, respectively. The positive control was preparedby the cells in the medium supplemented with 10%FCS without the addition of diluted extracts in eachwell and the negative control was also prepared by50 lL of cell medium supplemented with 10% FCSand 50 lL solution without cells.

After 7 days of incubation, cells were washed withPBS and then media were replaced with a basal med-ium containing MTT solution (400 lL DMEM +

40 lL MTT). After 4 h incubation at 37°C, in 5%CO2, the medium was discarded and formazan was sol-ubilized using DMSO. The plates were incubated for5–10 min, aliquots were pipetted in to a 96-well plateand finally the absorbance of each well was measured at540 nm using a spectrophotometric plate reader (BioRad, Madrid, Spain).

Statistical Analysis

All data were collected with N = 3 and expressedas means ± standard deviation (SD) in each experi-ment. Statistical analysis was done by two-wayANOVA with Duncan test. Differences were consid-ered statistically significant at P < 0.05.

Results and Discussion

Figure 1 shows the TEM micrograph of forsteritenanopowder. The particles of forsterite powder arehomogenous and nearly spherical mainly with a size of18–40 nm. In addition, clear boundary and smoothsurface can be observed.

Figure 2 illustrates X-ray diffraction patterns of theforsterite nanopowder along with composite powdersand fluorapatite calcined at 600°C. According to stan-dard card of fluorapatite (JCDP#15–0876), fluorapatitediffraction peaks were observed at pure fluorapatite andcomposite samples. At this temperature, the well-crys-talline fluorapatite was fabricated without any impurity.By adding forsterite powder, the new peaks wereobserved. In comparison with the pure forsterite pattern

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and according to the standard card of forsterite(JCDP#34–0189), they belonged to the forsteritephase. By increasing the amount of forsterite nanopow-der, the intensity and number of forsterite peaksincreased. No other peaks were observed, which showsthat there was no other reaction between the fluorapa-tite precursors and forsterite powder. The crystallitesizes of fluorapatite and forsterite powder estimated byScherer’s equation were about 21–24 nm which showstwo powders are nanostructured and no grain growthhappened during calcination.

The SEM micrographs of nanocomposite powdersalong with pure forsterite powders are shown in Fig. 3.Flourapatite powder showed the accumulated fineparticles which were strictly agglomerated and intercon-

nected into the surface of flake-like larger agglomerates.The nano scale nature of the sol-gel derived nanopow-ders causes intense agglomeration of particles with sizeof about 2–5 lm. As shown in Fig. 3, the agglomer-ated particles are composed of very fine particles.However, by increasing the amount of forsterite nano-powder in nanocomposites, the agglomerationdecreased.

To estimate the distribution of forsterite in theflourapatite matrix, powders were mounted and BSEmicrograph of powders were studied. Figure 4 showsthe BSE micrograph and EDS results of nanocompositepowders. Fluorapatite particles appear brighter thanforsterite ones because of their higher molecular weight.The agglomerate sizes of flourapatite phase in the com-posites containing 10, 20, and 30 wt% of forsteritewere about 5–20 lm, 5–15 lm, and 1–6 lm, respec-tively, which shows the positive effects of forsterite onreducing the agglomeration of florapatite.

The EDS results illustrate that the particles ofcomposite are composed of calcium, phosphorus, andalso Mg and Si. Mg and Si ions originate from theforsterite and two other elements are related to fluora-patite particles. No other peaks are identified in thisspectra revealed any impurity in fabricated composites.Furthermore, the molar raitos of Mg and Si ions wereabout 2 in all samples, which corresponds to forsterite.Also, the molar raito of Ca and P is almost likely 1.67which is approxtimately equal to this ratio in fluorapa-tite. The stochimetry Ca/Mg weight ratios for compos-ite samples containing 10, 20, and 30 wt% forsteritewere 10.48, 4.65, 2.71, respectively. These ratios formentioned composites, according to the spectra, wereabout 10.21, 4.67, 2.86, respectively, which confirmedthe correct weight ratios of flourapatite and forsteritecontents in different composites.

Figure 5 shows the results of elemental mapping offluorapatite-20 wt% forsterite nanopowder. It can beseen that the elements have almost covered the entiresurface of the sample, homogenously. The Si and Mgspots partially overlapped, which corresponds to thedispersed forsterite in the structure. These results showhomogenous distribution of both forsterite and fluora-patite powder in the composite.

The FT-IR spectra of the prepared compositepowders with different forsterite contents are shown inFig. 6. In all the spectra, the characteristic peaks of for-sterite and fluorapatite could be noticed. The P–Ostretching υ1 is observed at 960 cm�1 and O–P–O

Fig. 1. 7TEM micrograph of forsterite nanopowder.

Fig. 2. 8X-ray diffraction pattern of the pure forsterite andflourapatite along with forsterite-fluorapatite compositenanopowder calcined at 600°C (, formation of forsterite peaks byincreasing forsterite content in the composite) (FA, Felourapatite;FO, Forsterite).

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(a)

(c) (d)

(b)

Fig. 3. 9SEM micrographs of (a) flourapatite and flourapatite-forsterite composite containing (b) 10, (c) 20 and (d) 30 wt% forsterite.

(a1)

(a2)

(b1)

(b2)

(c1)

(c2)

Fig. 4. 10BSE micrographs and EDS results of flourapatite-forsterite composite powders containing (a)10, (b) 20 and (c) 30 wt%forsterite.

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bending υ2 is observed at 470 cm�1, respectively. Theother important characteristic peak of PO4

�3 tetrahe-dral groups is the P–O stretching υ3 vibration mode,which was visible at 1026 and 1082 cm�1. The bandsat 572 and 601 cm�1 are the υ4 vibration mode of thephosphate group.13 In addition, a doublet appears at1420 and 1462 cm�1 corresponding to the υ3 vibra-tion mode and the band centered at 869 cm�1 for υ2

vibration mode of the carbonated groups. These peaksshowed that the prepared fluorapatite contained some

carbonated groups in PO4�3 sites of apatite lattice (As

a result of decomposition of used alcohol hydrocarbonsas solvent, carbonates imported into the hydroxy apatitestructure and in the wet chemical methods for fabricat-ing fluorapatite such as sol-gel make carbonate hydroxyapatite). As carbonates are constituents of bone tissuestructures, its presence could improve the similarity andbiological response of the repaired fluorapatite to thebone structure.14 According to this spectrum, theabsence of OH� band at 630 cm�1, and the presenceof the band at 738 cm�1 corresponding to the shiftingof OH� liberation mode13 indicate complete transfor-mation of HAp into fluorapatite.

By increasing the amounts of forsterite contents,forsterite peaks appeared or their intensity increased.For example, the intensity of the absorption peaks at873 cm�1 and, 507 cm�1 was related to SiO4 groupsand at 475 cm�1 attributed to MgO6 octahedral itincreased. However, other peaks such as those at961 cm�1 and, 616 cm�1 appeared on increasing theamount of forsterite up to 30 wt%. PO4

�3 tetrahedralgroups appeared in the broad spectrum.

In addition to these characteristic peaks, anotherpeak was observed at 1360 cm�1 and its intensityincreased by increasing the forsterite content. This peakmost likely is due to weak bonding between both theexisting ceramics.

Figure 7 shows the results of the MTT assay ofnanocomposite and pure forsterite and fluorapatitepowder after 3–7 days of cell culturing. The OD valuesare indicators of the relative number of cells. Accordingto other reports, if the OD value has no significant dif-ferences with the positive control, it means that samples

Fig. 5. 11Elemental mapping of fluorapatite-20 wt% forsteritesample.

Fig. 6. 12FTIR spectra of flourapatite-forsterite composite powderscalcined at 600°C.

Fig. 7. 13The effect of powder extracts on cell proliferation. *,The experimental group compared with the positive group,P < 0.05. Ctr+, positive control; Ctr�, negative control.

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have less growth inhibition effects on cells and, on thecontrary, if the value is close to the negative control,growth inhibition effects on cells is expected. It couldbe observed that the cell density increased by increasingincubating time in all samples, which proved that pureforsterite and fluorapatite nanopowders along with theircomposite are non-cytotoxic and their composite couldsupport human osteoblast proliferation. Generally, thedissolution extracts of composite powder show higherosteoblast proliferation than pure ceramic powders. Itcould be seen that the cell proliferation was signifi-cantly higher than the negative control (P < 0.05) afterincubating for 5 days in the nanocomposite containing20 wt% forsterite nanopowder. As can be seen, it hascell viability approximately two times of the positivesamples, after 3 days of incubation. Furthermore, byincreasing forsterite nanopowder concentration, thestimulatory effect decreased. Although the proliferationrate of cells in contact with positive control after 7 daysof cell incubation was higher than that of the othersamples, no significant differences with the positivecontrol (P > 0.05) were observed. The higher rate ofproliferation at 7 day of incubating might be the resultof reduction of ion released in the solution.

Recent studies have studied the positive stimulatoryeffects of Ca-, Si-, and Mg-containing ionic productsfrom the dissolution of MgO·SiO2·CaO ceramicsystems at a certain concentration range on the osteo-blast�like cells.15 Results showed that, the released Siions could promote mineralized nodule formation ofhuman primary osteoblasts.16,17 In addition, itcould stimulate gene expression and osteoblast prolifer-ation.18–21 Mg ion is the other ion released during theimmersion of forsterite nanopowder in solutions.Results showed that, Mg ion closely associated withmineralization of calcined tissues and indirectly influ-enced mineral metabolism.22,23 In spite of the impor-tant role of Mg ion dissolution in the bioactivity ofinorganic biomaterials, at high concentration ofextracts, the inhibitory effect on cell proliferationmight be observed.15 These results are reported previ-ously in the cytotoxicity evaluation report of forsteritenanopowder.11

Besides forsterite, presence of fluorapatite in thecomposite powder let to the release of Ca and Pions in the solution. Reports showed that the releasedCa ions could induce osteoblast proliferation andchemotaxis through binding to a G-protein coupledwith extracellular calcium sensing receptor.21 Accord-

ing to the above results, the release of inorganic ions,such as Ca, Mg, and Si ions together could stimulatethe cell proliferation which might be one of the eval-uation criteria for bioactivity of the biomaterials.However, to identify the mechanisms of the specificstimulatory effects of different ions and ion combina-tions, further investigations are required. So, Si ionsfrom composite powder containing 20 wt% forsteritenanopowder significantly stimulated osteoblastproliferation.

Conclusion

In this study fluorapatite- forsterite nanocompositepowder was fabricated and characterized for oral bonedefects. Nanopowder forsterite (18–40 nm) fabricatedusing sol-gel method was used for this purpose.Composite nanopowder with 10, 20, and 30 wt% forste-rite nanopowder was prepared using sol-gel method.Results showed that calcined gel at 600°C producedpowders with forsterite and fluorapatite without impu-rity. The crystallite size of fluorapatite and forsteritereported about 21–24 nm. Presence of forsteritedecreased powder agglomeration. In addition, thecomposite powders and the pure ceramic powders arenon-cytotoxic and nanocomposite powder containing20 wt% powder showed the best biocompatibility. Inconclusion, fluorapatite-forsterite nanocomposite powderpossessed good biocompatibility and might be suitablefor oral hard tissue repair.

Acknowledgment

The authors are grateful to Isfahan University ofTechnology for supporting the present research.

References

1. M. S. Block and J. N. Kent, “Prospective Review of Integral Implants,”Dent. Clin. North Am., 36 [1] 27–37 (1992).

2. M. H. Fathi, V. Mortazavi, and A. Doostmohammadi, “Bioactive GlassNanopowder for the Treatment of Oral Bone Defects,” J. Dent., 4 [2] 119–122 (2007).

3. R. Stvrtecky, A. Gorustovich, C. Perio, and M. B. Gugliel-motti, “A His-tologic Study of Bone Response to Bioactive Glass Particles used BeforeImplant Placement: A Clinical Report,” J. Prosthet. Dent., 90 [5] 424–428(2003).

4. L. L. Hench and J. Wilson, An Introduction to Bioceramics, Advanced Cera-mic Society, Vol. 1, World Scientific Publishing Co, Singapore, 1998.

5. M. Kheradmandfard and M. H. Fathi, “Preparation and Characterizationof Mg-Doped Fluorapatite Nanopowders by sol–Gel Method,” J. AlloysCompd., 504 [1] 141–145 (2010).

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6. M. H. Fathi and E. Mohammadi Zahrani, “Fabrication and Characteriza-tion of Fluoridated Hydroxyapatit Nanopowders via Mechanical Alloying,”J. Alloys Compd., 475 [1–2] 408–414 (2009).

7. H.-W. Kim, H.-E. Kim, and C. J. Knowles, “Fluor-Hydroxyapatite sol–Gel Coating on Titanium Substrate for Hard Tissue Implants,” Biomateri-als., 25 [17] 3351–3358 (2004).

8. M. H. Fathi and M. Kharaziha, “The Effect of Fluorine ion on Fabrica-tion of Nanostructure Forsterite During Mechanochemical Synthesis,”J. Alloys Compd., 472 [1] 540–545 (2008).

9. M. Kharaziha and M. H. Fathi, “Synthesis and Characterization ofBioactive Forsterite Nanopowder,” Ceram. Int., 35 [6] 2449–2454(2008).

10. M. H. Fathi and M. Kharaziha, “Two-Step Sintering of Dense, Nanostruc-tural Forsterite,” Mater. Lett., 63 [17] 1455–1458 (2009).

11. M. Kharaziha and M. H. Fathi, “Improvement of Mechanical Propertiesand Biocompatibility of Forsterite Bioceramic Addressed to Bone TissueEngineering Materials,” J. Mech. Behav. Biomed. Mater., 3 [7] 530–537(2010).

12. G. Bezzi, G. Celotti, E. Landi, T. M. G. La Torretta, I. Sopyan, andA. Tampieri, “Novel sol–Gel Technique for Hydroxyapatite Preparation,”Mater. Chem. Phys., 78 816–824 (2003).

13. I. Nikcevic, V. Jokanovic, M. Mitric, Z. Nedic, D. Makovec, and D.Uskokovic, “Mechanochemical Synthesis of Nanostructured Fluorapatite/Fluorhydroxyapatite and Carbonated Fluorapatite/Fluorhydroxyapatite,”J. Solid State Chem., 177 [7] 2565–2574 (2004).

14. E. Mohammadizahrani, M. H. Fathi, and A. M. Alfantazi, “Sol-GelDerived Nanocrystalline Fluoridated Hydroxyapatite Powders and Nano-structured Coatings for Tissue Engineering Applications,” Metal. andMater. Trans. A. Phys. Metal. and Mater. Sci., 42A [11] 3291–3309(2011).3

15. C. Wu, J. Chang, J. Wang, S. Ni, and W. Zhai, “Preparation and Charac-teristics of a Calcium Magnesium Silicate (Bredigite) Bioactive Ceramic,”Biomaterials, 26 [16] 2925–2931 (2005).

16. J. E. Gough, I. Notingher, and L. L. Hench, “Osteoblast Attachment andMineralized Nodule Formation on Rough and Smooth 45S5 BioactiveGlass Monoliths,” J. Biomed. Mater. Res., 68A [4] 640–??? (2004). 4

17. J. E. Gough, D. C. Clupper, and L. L. Hench, “Osteoblast Responses toTape Cast and Sintered Bioactive Glass Ceramics,” J. Biomed. Mater. Res.,69A [4] 621–??? (2004). 5

18. I. D. Xynos, A. J. Edgar, L. D. Buttery, L. L. Hench, and J. M. Polak,“Ionic Products of Bioactive Glass Dissolution Increase Proliferation ofHuman Osteoblasts and Induce Insulin-Like Growth Factor II MRNAExpression and Protein Synthesis,” Biochem. Biophys. Res. Commun., 276[2] 461–465 (2000).

19. L. L. Hench and J. K. West, “Biological Applications of Bioactive Glasses,”Life Chem. Rep., 13 [1] 187–241 (1996).

20. P. V. Phan, et al. “The Effect of Silica-Containing Calcium-PhosphateParticles on Human Osteoblasts In Vitro,” J. Biomed. Mater. Res., 67A [1]1001–1008 (2003).

21. I. D. Xynos, A. J. Edgar, L. D. Buttery, L. L. Hench, and J. M. Polak,“Gene-Expression Profiling of Human Osteoblasts Following Treatmentwith the Ionic Products of Bioglass 45S5 Dissolution,” J. Biomed. Mater.Res., 55 151–157 (2001).

22. R. Z. LeGeros, Calcium Phosphates in Oral Biology and Medicine, ????,Basel, Switzerland, 1991. 6

23. J. Althoff, P. Quint, E. R. Krefting, and H. J. Hohling, “MorphologicalStudies on the Epiphyseal Growth Plate Combined with Biochemiacal andX-ray Microprobe Analyasis,” Histochemistry, 74 [1] 541–552(1982).

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USING e-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION

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rkevwtgf"qrrqukvg0"YgÓxg"rkemgf"qwv"uqog"qh"vjgug"vqqnu"dgnqy<

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