Research Article The Influence of Titanium Dioxide on

Research ArticleThe Influence of Titanium Dioxide on Diamond-Like CarbonBiocompatibility for Dental Applications

C C Wachesk1 V J Trava-Airoldi2 N S Da-Silva3 A O Lobo1 and F R Marciano1

1Laboratory of Biomedical Nanotechnology University of Vale do Paraıba Sao Jose dos Campos SP Brazil2Associated Laboratory of Sensors and Materials National Institute for Space Research Sao Jose dos Campos SP Brazil3Laboratory of Cell Biology and Tissue University of Vale do Paraıba Sao Jose dos Campos SP Brazil

Correspondence should be addressed to F R Marciano frmarcianounivapbr

Received 8 May 2016 Revised 15 August 2016 Accepted 23 August 2016

Academic Editor P Davide Cozzoli

Copyright copy 2016 C C Wachesk et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The physical and chemical characteristics of diamond-like carbon (DLC) films make them suitable for implantable medical andodontological interests Despite their good interactions with biological environment incorporated nanoparticles can significantlyenhance DLC properties This manuscript studies the potential of titanium dioxide (TiO

2) incorporated-DLC films in dental

applications In this scene both osteoblasts attachment and spreading on the coatings and their corrosion characteristics inartificial saliva were investigated The films were grown on 304 stainless steel substrates using plasma enhanced chemical vapordeposition Raman scattering spectroscopy characterized the film structure As the concentration of TiO

2increased the films

increased the osteoblast viability (MTT assay) becoming more thermodynamically favorable to cell spreading (119882Ad values becamemore negative) The increasing number of osteoblast nuclei indicates a higher adhesion between the cells and the films Thepotentiodynamic polarization test in artificial saliva shows an increase in corrosion protection when TiO

2are presentThese results

show the potential use of TiO2-DLC films in implantable surfaces

1 Introduction

Implantable surfaces probably have the main contributionfor increasing the implant dentistry Complex reactions attissue-material interface settle the osseointegration and thelong-term success of the implants [1] Therefore a surfacemodification to improve biocompatibility is mostly necessary[2] In this scene diamond-like carbon (DLC) coatings canreveal wear resistance hardness and corrosion resistance to amedical device surface [3ndash6] These coatings consist of denseamorphous carbon or hydrocarbon and their mechanicalproperties fall between those of graphite and diamond [34] Some studies reported modified-DLC films improvedbiocompatibility lubricity stability and cell adhesion [67] However incorporated nanoparticles can change DLCperformance according to the individual properties of eachnanoparticle [8] Yun et al [5] related these characteristics tostructural bonds [9] surface roughness [10] and whether thefilm is hydrophobic or hydrophilic

A thin native oxide layer formed spontaneously ontitanium surface caused by air water or any other electrolyteassigns titanium biocompatibility [11] This layer is respon-sible for bone-bonding characteristics of titanium implants[12] The photocatalytic nature of titanium dioxide (TiO

2)

enables its use as antibacterial agent for decompose organ-isms [13ndash15] These properties are strongly depending on thecrystalline structure morphology and crystallite size [13 15]

In the last recent years previous manuscripts reportedproduction and characterization of TiO

2-DLC films for

biological applications [15ndash17] However besides the cytotox-icity odontological applications also depend on the corrosionresistance [1 18 19] A hostile electrolytic environment as themouth causes gradual degradation of metallic biomaterialsby electrochemical attack [20] This manuscript studies thepotential of TiO

2-DLC films in dental applications In this

scene both osteoblasts attachment and spreading on thecoatings and their corrosion characteristics in artificial salivawere investigated

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2016 Article ID 8194516 7 pageshttpdxdoiorg10115520168194516

2 Journal of Nanomaterials

2 Experimental Procedures

The 304 stainless steel (10mm times 10mm times 1mm) was thesubstrates DLC and TiO

2-DLC films were deposited using

plasma enhanced chemical vapor deposition to a thicknessof around 20 120583m [17] Hexane was the feed gas to produceDLC films Dispersions of TiO

2(Aeroxide from Evonik) in

anatase crystalline form (average particle size of 17 nm) inhexane (01 and 05 gL) substitute hexane to produce TiO

2-

DLC filmsRaman scattering spectroscopy (Renishaw 2000 system

with an Ar+-ion laser (120582 = 514 nm) in backscattering geome-try) analyzed the atomic arrangement of the films

All animal procedures were in agreement with guidelinesof the Research Ethics Committee of the School of Dentistryin Sao Jose dos Campos (0272008-PACEP) Enzymaticdigestion harvested cells from newborn (2ndash4 days) Wistarrat calvaria [21] The cells were plated on samples in 24-wellpolystyrene plates (density of 2 times 104 cellswell) using 120572-MEM (Gibco) supplemented with 10 fetal bovine serum(Gibco) 50mgmL gentamicin (Gibco) 5 120583gL ascorbic acid(Sigma) and 7mM 120573-glycerophosphate (Sigma) During theexperiments cells were incubated (37∘C) in a humidifiedatmosphere (5 CO

2) and the medium was changed every

three daysThe inverted microscope (CK40 Olympus) exam-ined the progression of cultures

The cell viability assay monitors the response and healthof cells quantifying the mitochondrial activity by analyzingformazan crystals formed by reducing the salt 3-[(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide](MTT) (Sigma-Aldrich) After treatment 05mgmL ofMTT was added to the cultures which were incubated for1 h (37∘C) followed by 30min gentle agitation in 200mL ofdimethyl sulfoxide ELISA Spectracount Reader (PackardInstrument) with a 570 nm filter read the plates

Phosphate-buffered saline (PBS) washed the cells Theywere incubated for 10min at room temperature with 300 nM410158406-diamidino-2-phenylindole and dihydrochloride (DAPIMolecular Probes) For fixing 200mL paraformaldehyde(Sigma-Aldrich) at 4 was added to the wells and the plateincubated at room temperature for 10min Fluorescencemicroscope (DMLB Leica) examined the cells and a digitalvideo camera (Leica DFC 300 FX) took the images

One-way ANOVA (Graph Pad Prism 6) analyzed thestatistical differences The populations from stainless steelDLC and TiO

2-DLC films were obtained with normal

distribution and independent to each experiment Statisticaldifferences pointed out 119875 values less than 005

A sessile drop method with a Kruss EasyDrop (DSA 100)measured the contact angle (120579) of the samples Accordingto Owens method [22] the contact angle values with twodifferent liquids (distilled water and diiodomethane) calcu-lated the surface energy Thermodynamically the adhesionand spreading of cells from a liquid suspension onto solidsubstrata can be described by 119882Ad = 120574CS minus 120574CL minus 120574SL [23]where 119882Ad is the interfacial free energy of adhesion 120574CSis the cell-solid interfacial free energy 120574CL the cell-liquidinterfacial free energy and 120574SL is the solid-liquid interfacialfree energy respectively If119882Ad is negative cell spreading is

Relat

ive i

nten

sity

(au

)

TiO2-DLC (05 gL)

TiO2-DLC (01 gL)

DLC

G

band

band

D

750 1000 1250 1500 1750500

Raman shift (cmminus1)

Figure 1 Raman spectra from DLC and TiO2-DLC films The

spectra are vertically shifted for easy of comparison

energetically favorable while if119882Ad is positive cell spreadingis thermodynamically unfavorable

A conventional three-electrode cell performed the elec-trochemical tests In this cell a saturated AgAgCl electrodewas the reference electrode platinum wire was the counterelectrode and the stainless steel DLC and TiO

2-DLC

films were the working electrodes The working electrodeexposed area was 05 cm2 The electrolyte solution was amodified Fusayama artificial saliva [24] which consisted ofNaCl (400mgL) KCl (400mgL) CaCl

2sdot2H2O (795mgL)

NaH2PO4sdotH2O (690mgL) KSCN (300mgL) Na

2Ssdot9H2O

(5mgL) and urea (1000mgL) The electrolyte was adjustedto pH 63 by either lactic acid or sodium hydroxide andmaintained at 37∘C Potentiodynamic tests were carried outby polarization of samples in the anodic direction from minus10to +10 V just after exposition to the electrolyte solution Thepotential sweep rate was 1mVs The electrochemical testswere performed at 37∘C

3 Results and Discussion

Raman scattering spectroscopy evaluated the chemical struc-ture of the DLC films Two broad bands compose the spectrafor visible light one centered in sim1330 cmminus1 (119863 band) andother in sim1530 cmminus1 (119866 band) [25] The 119863 and 119866 band posi-tions were determined by subtracting a linear backgroundand fitting a Gaussian function to the peak of the Ramanspectrum (Figure 1) Table 1 summarizes themain parametersobtained through the spectraThe bond stretching of all pairsof sp2 atoms in both rings and chains causes the 119866 band[26 27]The breathingmodes of sp2 atoms in rings caused the119863 band and appears only in the presence of defects [26 27]The TiO

2-DLC films presented a shift in 119863 and 119866 band

positions towards higher wavenumbersThe full width at halfmaximum (FWHM) of 119866 band measures structural disorderand arises from bond length distortions [27]The presence ofTiO2nanoparticles in DLC films results in the increase of the

intensity ratio of119863 and 119866 area (119860119863119860119866) and a shift of119863 and

Journal of Nanomaterials 3

Table 1 Gaussian fitting results of Raman spectra from DLC and TiO2-DLC films

TiO2

concentration(gL)

119863 band position (cmminus1) G band position (cmminus1) FWHM (119863) FWHM (119866) 119860119863119860119866

FWHM119863FWHM

119866

00 13301 15311 2967 1542 051 19201 13544 15523 2984 1449 081 20605 13344 15314 3330 1622 067 205

Oste

obla

st vi

abili

ty (

)

Control Stainlesssteel

TiO2-DLC(05 gL)

TiO2-DLC(01 gL)

DLC

lowast

0

20

40

60

80

100

120

140

Figure 2 Osteoblast viability assessed by MTT assay in cellscultured for 24 h on stainless steel DLC and TiO

2-DLC surfaces

The percentage was calculated by normalization of optical densityto the control (osteoblast cells) Results are shown as average plusmnstandard error for 119899 = 5 (one-way ANOVA and Tukeyrsquos multiplecomparisons test) Control osteoblasts plated on plastic surfaceslowast119875 lt 005 the interaction is considered significant compared tocontrol (osteoblast cells)

119875 lt 0001 the interaction is consid-ered extremely significant compared to stainless steel (noncoatedsubstrate) 119875 lt 005 the interaction is considered significantcompared to DLC (coating without any nanoparticle)

119866 bands towards higher wave numbers These characteristicsimply the increase of the graphite-like bonds in DLC matrix[26ndash28]

In this study mouse osteoblastic cells were the cell culturesystem Calvaria cells have a high proliferation capacitybeing able to expand in vitro which is appropriate to studyinteractions with biomaterials [29]

The MTT assay is a quantitative method for evaluating acell response to external factors It measures the reduction ofa tetrazolium component (MTT) into an insoluble formazanproduct by NAD(P)H-dependent oxidoreductase enzymeslargely in the cytosolic compartment of the cell specificallyin mitochondrial compartment The mitochondrial enzymesuccinate-dehydrogenase within viable cells is able to cleavethe tetrazolium salt into a blue product (formazan)The colorproduced is directly proportional to the number of viable cells[30]

Figure 2 shows the mitochondrial activity for all thestudied samples Stainless steel shows a significant differencewhen compared to control (osteoblast cells) The decreasenumber of viable cells on stainless steel characterizes cell

deathThere was no significant difference amongDLC TiO2-

DLC (01 gL) and the control An increased concentrationof TiO

2nanoparticles (05 gL) in DLC films raised the

mitochondrial activity on these samples which are obviouslynontoxic TiO

2-DLC (05 gL) is significantly different (119875 lt

005) compared toDLC and extremely significant (119875 lt 0001)when compared to stainless steel

DAPI (blue) labeled the cell nuclei during 24 h (Figure 3)All the TiO

2-DLC samples had a high cell attachment The

DAPI test showed a strong compatibility of the TiO2-DLC

films Figure 4 shows the counted osteoblast cells adheredon the samples The number of cells on stainless steel isconsidered different from DLC films (119875 lt 005) and verydifferent from TiO

2-DLC films (119875 lt 001) There was an

increase in cells according to the increased concentration ofTiO2in DLC films TiO

2-DLC films are considered different

from DLC films (119875 lt 005)The study of bone metabolism and biomaterialcell

which is essential for bone tissue engineering generallyuses osteoblasts [29] However not only cell adhesivenessand toxicity have to be investigated to design biomaterialsThe existence of interface phenomena can affect the initialproliferation and cell recruitment at the biomaterial surface[31] The hydrophilicity tries to obtain correlation betweencell response and the biomaterial surface [29]

Table 2 shows the contact angles of the samples formedwith distilled water and diiodomethane As the concentrationof TiO

2nanoparticles in DLC films increased the water

contact angle decreased from 82 to sim53∘ and in the caseof diiodomethane the contact angle had a slight increaseUsually a hydrophobic surface has a contact angle higherthan 70∘ while a hydrophilic surface has a contact anglelower than 70∘ [32] These results indicate that TiO

2-DLC

is hydrophilic This characteristic may be derivate from theamorphous TiO

2surfaces [33]

Table 2 also lists the surface energy components accord-ing to the Owens method [22] Dispersive (120574

119889= 361mNm)

and polar (120574119901= 36mNm) components gave the total

surface energy (120574) of 400mNm for the as-deposited DLCThe interfacial free energy settles the wetting and so thewall shear stress when the liquid touches the surface [22]As the concentration of TiO

2in DLC increased the total

surface energy had a slight increase The increasing in thepolar component (a quantitative indicator of hydrophilicity)increases the total surface energy of TiO

2-DLC Oxide parti-

cles cause a higher polar component on TiO2-DLC surface

[34] The polar component attracts the electric dipole ofwater which minimizes the interfacial energy and the watercontact angle [34] The water contact angle decreased as the


Table 2 Contact angle and surface energy components of DLC and TiO2-DLC films Each mean value corresponds to the average value on

five different areas

Concentration of TiO2

nanoparticles (gL)Contact angle 120579 (∘) Surface energy components (mNm) Work of adhesion (mJm2)

Water Diiodomethane Dispersive Polar Total00 820plusmn 47 385plusmn 10 361plusmn 15 36plusmn 19 400plusmn 33 minus6601 721plusmn 47 427plusmn 15 311plusmn 14 95plusmn 28 406plusmn 42 minus8505 533plusmn 15 441plusmn 54 274plusmn 09 225plusmn 41 499plusmn 50 minus144

30120583m

(a)

30120583m

(b)

30120583m

(c)

30120583m

(d)

Figure 3 Fluorescence images from osteoblast cell nuclei on (a) stainless steel (b) DLC (c) TiO2-DLC (01 gL) and (d) TiO

2-DLC (05 gL)

films labeled with DAPI (blue) at 24 h

polar component in the surface energy increased The polarcomponent attracts electric dipole of water molecule whichreduces the interfacial energy between the surface and thewater and thus the wetting angle of water [35]

A thermodynamic approach offers a powerful tool topredict cell spreading to solids [36 37] For this the surfacefree energy values of the cells (120574

119862) calculated by Schakenraad

et al [23] were used Table 2 lists the average values of workof adhesion (119882Ad) for the studied samples All the calculatedvalues for 119882Ad for cell adhesion on DLC and TiO

2-DLC

films are negative119882Ad point out favorable conditions for celladhesion as provided by Schakenraad et al [23] In additionas the TiO

2concentration increases119882Ad values becamemore

negativeThis result suggests that as the TiO2content in DLC

films increased DLC films became more thermodynamicallyfavorable to cell spreading

Figure 5 shows the correlation between the number ofcells on the samples (Figure 4) and the calculated workof adhesion (Table 2) Besides the fact that there are two

independent results this comparison corroborates that TiO2

content increased the cell adhesion on DLC filmsHowever one of the physical characteristics that settle

the implant corrosion is the thermodynamic force [38 39]It causes corrosion by either oxidation or reduction reaction[38 39] Oral liquids like saliva attacks the metallic implantscausing the dissolution of them [38]Therefore the release ofmetallic ions caused by corrosion also affects the biocompat-ibility of the implant The potentiodynamic polarization testassessed the corrosion susceptibility of the metallic implantin artificial saliva Figure 6 shows the potentiodynamiccurves for stainless steel DLC and TiO

2-DLC films Table 3

summarizes the electrochemical parameters obtained fromthe potentiodynamic polarization curves (Figure 6)

The penetration of the test solution causes the negativeopen circuit potential values [40] TiO

2-DLC (01 gL) films

presented the greatest 119864corr value (minus0146mV) in potentio-dynamic polarization test The corrosion current density(119894corr) reduced from 172 to 0054 nAcm2 and the protection


Table 3 Electrochemical parameters obtained from potentiodynamic polarization curves

Samples 119864corr (mV) 119894corr (nAcm2) Protective efficiency ()

Stainless steel minus0495 117 mdashDLC minus0419 172 985TiO2-DLC (01 gL) minus0146 0151 999

TiO2-DLC (05 gL) minus0220 0054 1000

Num

ber o

f adh

ered

cells

(42

mm2)

Stainlesssteel

TiO2-DLC(05 gL)

TiO2-DLC(01 gL)

DLC

lowast

lowastlowast

lowastlowast

0

20

40

60

80

100

Figure 4 Number of adhered cells labeled with DAPI (blue) at24 h Results are shown as average plusmn standard error for 119899 = 3(one-way ANOVA and Tukeyrsquos multiple comparisons test) lowast119875 lt005 the interaction is considered significant compared to stainlesssteel (substrate without coating) lowastlowast119875 lt 001 the interaction isconsidered very significant compared to stainless steel 119875 lt 005the interaction is considered significant compared to DLC (coatingwithout any nanoparticle)

Num

ber o

f adh

ered

cells

(42

mm2)

TiO2-DLC(05 gL) TiO2-DLC

(01 gL)

DLC

30

40

50

60

70

80

90

minus14 minus12 minus10 minus8 minus6minus16

Work of adhesion (WAd) (mJm2)

Figure 5 Number of osteoblast cells on the samples versus theinterfacial free energy of adhesion of DLC and TiO

2-DLC films

efficiency [41] increased from985 to 1000with the increaseof TiO

2content Lower current density and higher potential

generally points out better corrosion resistance The shift inthe polarization curve towards the region of lower current

minus10

minus05

00

05

10

Pote

ntia

l V v

ersu

s Ag

AgC

l

1E minus 5 1E minus 31E minus 9 1E minus 7

Current density (Acm2)

(a)(b)(c)(d)

Figure 6 Potentiodynamic polarization curves from (a) stainlesssteel uncoated and coatedwith (b)DLC (c) TiO

2-DLC (01 gL) and

(d) TiO2-DLC (05 gL) films

density and higher potential is an indicative of the increasedcorrosion protection with the increased TiO

2concentration

The difference in the filmmicrostructure assigns differentcorrosion behaviors [42] The comparison between electro-chemical parameters (Table 3) and chemical structure(Table 1) shows a tendency to reduce the corrosion currentdensity of the samples as the film disorder increase Robert-son [43] states sp3 bonding of DLC confers on it many of thevaluable properties of diamond itself such as its chemical andelectrochemical inertness Generally the higher the sp3sp2ratio the higher the electrochemical corrosion resistance[44]

4 Conclusion

In this paper the osteoblast attachment and spreading onDLC coatings was studied when TiO

2nanoparticles are

incorporated The presence of TiO2nanoparticles increases

the graphite-like bonds and decreases the DLC disorder AsTiO2increased the films increased the osteoblast viability

(MTT assay) becoming more thermodynamically favorableto cell spreading (119882Ad values became more negative) Thiswas evidenced through the increasing number of osteoblastnuclei suggesting a higher adhesion between the cells andthe films The potentiodynamic polarization test in artificialsaliva shows an increase in corrosion protection with theincreased TiO

2 These results show the potential use of TiO

2-

DLC films in implantable surfaces for dental applications


Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The authors thank Professor Dr Rosilene Fernandes daRocha and Dr Isabel Chaves Silva Carvalho from Schoolof Dentistry Paulista State University Sao Jose dos Cam-pos Brazil for kindly providing the cells from newbornWistar rat calvaria and Sao Paulo Research FoundationFAPESP (201117877-7) (201120345-7) (201215857-1) and(201320054-8) for the financial support

References

[1] M Zafar S Najeeb Z Khurshid et al ldquoPotential of electrospunnanofibers for biomedical and dental applicationsrdquo Materialsvol 9 no 2 article 73 2016

[2] E Salgueiredo M Vila M A Silva et al ldquoBiocompatibilityevaluation of DLC-coated Si3N4 substrates for biomedicalapplicationsrdquo Diamond and Related Materials vol 17 no 4-5pp 878ndash881 2008

[3] J Robertson ldquoDiamond-like amorphous carbonrdquoMaterials Sci-ence and Engineering R Reports vol 37 no 4ndash6 pp 129ndash2822002

[4] C Donnet J Fontaine T Le Mogne et al ldquoDiamond-like car-bon-based functionally gradient coatings for space tribologyrdquoSurface amp Coatings Technology vol 120-121 pp 548ndash554 1999

[5] D Y Yun W S Choi Y S Park and B Hong ldquoEffect of H2

and O2plasma etching treatment on the surface of diamond-

like carbon thin filmrdquo Applied Surface Science vol 254 no 23pp 7925ndash7928 2008

[6] A ShirakuraMNakaya Y Koga H Kodama THasebe and TSuzuki ldquoDiamond-like carbon films for PET bottles and medi-cal applicationsrdquo Thin Solid Films vol 494 no 1-2 pp 84ndash912006

[7] R Hauert ldquoA review of modified DLC coatings for biologicalapplicationsrdquo Diamond and Related Materials vol 12 no 3ndash7pp 583ndash589 2003

[8] M Ban and N Hasegawa ldquoDeposition of diamond-like carbonthin films containing photocatalytic titanium dioxide nanopar-ticlesrdquo Diamond and Related Materials vol 25 pp 92ndash97 2012

[9] Q Zhao Y Liu C Wang and S Wang ldquoBacterial adhesionon silicon-doped diamond-like carbon filmsrdquo Diamond andRelated Materials vol 16 no 8 pp 1682ndash1687 2007

[10] W J Ma A J Ruys R S Mason et al ldquoDLC coatings effectsof physical and chemical properties on biological responserdquoBiomaterials vol 28 no 9 pp 1620ndash1628 2007

[11] T Yokota T Terai T Kobayashi T Meguro andM Iwaki ldquoCelladhesion to nitrogen-doped DLCs fabricated by plasma-basedion implantation and deposition method using toluene gasrdquoSurface amp Coatings Technology vol 201 no 19-20 pp 8048ndash8051 2007

[12] E Eisenbarth D Velten K Schenk-Meuser et al ldquoInteractionsbetween cells and titanium surfacesrdquo Biomolecular Engineeringvol 19 no 2ndash6 pp 243ndash249 2002

[13] H Nakano N Hasuike K Kisoda K Nishio T Isshiki and HHarima ldquoSynthesis of TiO

2nanocrystals controlled by means

of the size of magnetic elements and the level of doping with

themrdquo Journal of Physics CondensedMatter vol 21 no 6 ArticleID 064214 2009

[14] P CManess S Smolinski DM Blake ZHuang E JWolfrumand W A Jacoby ldquoBactericidal activity of photocatalytic TiO

2

reaction toward an understanding of its killing mechanismrdquoApplied and Environmental Microbiology vol 65 no 9 pp4094ndash4098 1999

[15] F R Marciano D A Lima-Oliveira N S Da-Silva A V DinizE J Corat and V J Trava-Airoldi ldquoAntibacterial activity ofDLC films containing TiO

2nanoparticlesrdquo Journal of Colloid

and Interface Science vol 340 no 1 pp 87ndash92 2009[16] F RMarciano C CWachesk AO Lobo V J Trava-Airoldi C

Pacheco-Soares and N S Da-Silva ldquoThermodynamic aspectsof fibroblastic spreading on diamond-like carbon films con-taining titanium dioxide nanoparticlesrdquo Theoretical ChemistryAccounts vol 130 no 4 pp 1085ndash1093 2011

[17] C C Wachesk C A F Pires B C Ramos et al ldquoCell via-bility and adhesion on diamond-like carbon films containingtitanium dioxide nanoparticlesrdquo Applied Surface Science vol266 pp 176ndash181 2013

[18] M Saini Y Singh P Arora V Arora and K Jain ldquoImplantbiomaterials a comprehensive reviewrdquoWorld Journal of ClinicalCases vol 3 no 1 pp 52ndash57 2015

[19] Z Khurshid M Zafar S Qasim S Shahab M Naseem and AAbuReqaiba ldquoAdvances in nanotechnology for restorative den-tistryrdquoMaterials vol 8 no 2 pp 717ndash731 2015

[20] A S Litsky ldquoClinical reviews bioabsorbable implants for ortho-paedic fracture fixationrdquo Journal of Applied Biomaterials vol 4no 1 pp 109ndash111 1993

[21] P T de Oliveira and A Nanci ldquoNanotexturing of titanium-based surfaces upregulates expression of bone sialoprotein andosteopontin by cultured osteogenic cellsrdquo Biomaterials vol 25no 3 pp 403ndash413 2004

[22] D K Owens and R C Wendt ldquoEstimation of the surface freeenergy of polymersrdquo Journal of Applied Polymer Science vol 13no 8 pp 1741ndash1747 1969

[23] J M Schakenraad H J Busscher C R H Wildevuur andJ Arends ldquoThermodynamic aspects of cell spreading on solidsubstratardquo Cell Biophysics vol 13 no 1 pp 75ndash91 1988

[24] J Geis-Gerstorfer and H Weber ldquoEffect of potassium thio-cyanate on corrosion behavior of non-precious metal dentalalloysrdquo Deutsche zahnarztliche Zeitschrift vol 40 no 2 pp 87ndash91 1985

[25] F Tuinstra and J L Koenig ldquoRaman spectrum of graphiterdquoJournal of Chemical Physics vol 53 no 3 pp 1126ndash1130 1970

[26] C Casiraghi F Piazza A C Ferrari D Grambole and JRobertson ldquoBonding in hydrogenated diamond-like carbon byRaman spectroscopyrdquo Diamond and Related Materials vol 14no 3ndash7 pp 1098ndash1102 2005

[27] L Ji H X Li F Zhao J M Chen andH D Zhou ldquoMicrostruc-ture and mechanical properties of MoDLC nanocompositefilmsrdquo Diamond and Related Materials vol 17 no 11 pp 1949ndash1954 2008

[28] A Madhan Kumar and N Rajendran ldquoElectrochemical aspectsand in vitro biocompatibility of polypyrroleTiO

2ceramic

nanocomposite coatings on 316L SS for orthopedic implantsrdquoCeramics International vol 39 no 5 pp 5639ndash5650 2013

[29] C Wirth B Grosgogeat C Lagneau N Jaffrezic-Renaultand L Ponsonnet ldquoBiomaterial surface properties modulate invitro rat calvaria osteoblasts response roughness and or chem-istryrdquo Materials Science amp Engineering CmdashBiomimetic andSupramolecular Systems vol 28 no 5-6 pp 990ndash1001 2008


[30] H Wan R Williams P Doherty and D F Williams ldquoA studyof the reproducibility of the MTT testrdquo Journal of Materials Sci-ence Materials in Medicine vol 5 no 3 pp 154ndash159 1994

[31] J Pino-Mınguez A Jorge-Mora R Couceiro-Otero and CGarcıa-Santiago ldquoStudy of the viability and adhesion of osteo-blast cells to bone cements mixed with hydroxyapatite at dif-ferent concentrations to use in vertebral augmentation tech-niquesrdquo Revista Espanola de Cirugıa Ortopedica y Trauma-tologıa (English Edition) vol 59 no 2 pp 122ndash128 2015

[32] HW Choi R H Dauskardt S-C Lee K-R Lee and K H OhldquoCharacteristic of silver doped DLC films on surface propertiesand protein adsorptionrdquo Diamond and Related Materials vol17 no 3 pp 252ndash257 2008

[33] A Sobczyk-Guzenda M Gazicki-Lipman H Szymanowski etal ldquoCharacterization of thin TiO

2films prepared by plasma

enhanced chemical vapour deposition for optical and photocat-alytic applicationsrdquo Thin Solid Films vol 517 no 18 pp 5409ndash5414 2009

[34] R K Roy H W Choi J W Yi et al ldquoHemocompatibility ofsurface-modified silicon-incorporated diamond-like carbonfilmsrdquo Acta Biomaterialia vol 5 no 1 pp 249ndash256 2009

[35] R K Roy H-W Choi S-J Park and K-R Lee ldquoSurface energyof the plasma treated Si incorporated diamond-like carbonfilmsrdquo Diamond and Related Materials vol 16 no 9 pp 1732ndash1738 2007

[36] E Flahaut M C Durrieu M Remy-Zolghadri R Bareille andC Baquey ldquoInvestigation of the cytotoxicity of CCVD carbonnanotubes towards human umbilical vein endothelial cellsrdquoCarbon vol 44 no 6 pp 1093ndash1099 2006

[37] S M Hussain K L Hess J M Gearhart K T Geiss and J JSchlager ldquoIn vitro toxicity of nanoparticles in BRL 3A rat livercellsrdquo Toxicology in Vitro vol 19 no 7 pp 975ndash983 2005

[38] J J Jacobs J L Gilbert and R M Urban ldquoCorrosion of metalorthopaedic implantsrdquo The Journal of Bone amp Joint SurgerymdashAmerican Volume vol 80 no 2 pp 268ndash282 1998

[39] J J Jacobs R M Latanision R M Rose and S J Veeck ldquoTheeffect of porous coating processing on the corrosion behavior ofcast Co-Cr-Mo surgical implant alloysrdquo Journal of OrthopaedicResearch vol 8 no 6 pp 874ndash882 1990

[40] C L Liu M Xu W Zhang S H Pu and P K Chu ldquoEffects oftungsten pre-implanted layer on corrosion and electrochemicalcharacteristics of amorphous carbon films on stainless steelrdquoDiamond and Related Materials vol 17 no 7ndash10 pp 1738ndash17422008

[41] H-G Kim S-H Ahn J-G Kim S J Park and K-R LeeldquoElectrochemical behavior of diamond-like carbon films forbiomedical applicationsrdquo Thin Solid Films vol 475 no 1-2 pp291ndash297 2005

[42] Z Wang C Wang Q Wang and J Zhang ldquoElectrochemicalcorrosion behaviors of a-CH and a-CN

119883H filmsrdquo Applied

Surface Science vol 254 no 10 pp 3021ndash3025 2008[43] J Robertson ldquoDiamond-like amorphous carbonrdquoMaterials Sci-

ence and Engineering R Reports vol 37 no 4ndash6 pp 129ndash2812002

[44] A Dorner-Reisel C Schurer G Irmer and E Muller ldquoElec-trochemical corrosion behaviour of uncoated and DLC coatedmedical grade Co28Cr6Mordquo Surface and Coatings Technologyvol 177-178 pp 830ndash837 2004

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


2 Experimental Procedures

The 304 stainless steel (10mm times 10mm times 1mm) was thesubstrates DLC and TiO

2-DLC films were deposited using

plasma enhanced chemical vapor deposition to a thicknessof around 20 120583m [17] Hexane was the feed gas to produceDLC films Dispersions of TiO

2(Aeroxide from Evonik) in

anatase crystalline form (average particle size of 17 nm) inhexane (01 and 05 gL) substitute hexane to produce TiO

2-

DLC filmsRaman scattering spectroscopy (Renishaw 2000 system

with an Ar+-ion laser (120582 = 514 nm) in backscattering geome-try) analyzed the atomic arrangement of the films

All animal procedures were in agreement with guidelinesof the Research Ethics Committee of the School of Dentistryin Sao Jose dos Campos (0272008-PACEP) Enzymaticdigestion harvested cells from newborn (2ndash4 days) Wistarrat calvaria [21] The cells were plated on samples in 24-wellpolystyrene plates (density of 2 times 104 cellswell) using 120572-MEM (Gibco) supplemented with 10 fetal bovine serum(Gibco) 50mgmL gentamicin (Gibco) 5 120583gL ascorbic acid(Sigma) and 7mM 120573-glycerophosphate (Sigma) During theexperiments cells were incubated (37∘C) in a humidifiedatmosphere (5 CO

2) and the medium was changed every

three daysThe inverted microscope (CK40 Olympus) exam-ined the progression of cultures

The cell viability assay monitors the response and healthof cells quantifying the mitochondrial activity by analyzingformazan crystals formed by reducing the salt 3-[(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide](MTT) (Sigma-Aldrich) After treatment 05mgmL ofMTT was added to the cultures which were incubated for1 h (37∘C) followed by 30min gentle agitation in 200mL ofdimethyl sulfoxide ELISA Spectracount Reader (PackardInstrument) with a 570 nm filter read the plates

Phosphate-buffered saline (PBS) washed the cells Theywere incubated for 10min at room temperature with 300 nM410158406-diamidino-2-phenylindole and dihydrochloride (DAPIMolecular Probes) For fixing 200mL paraformaldehyde(Sigma-Aldrich) at 4 was added to the wells and the plateincubated at room temperature for 10min Fluorescencemicroscope (DMLB Leica) examined the cells and a digitalvideo camera (Leica DFC 300 FX) took the images

One-way ANOVA (Graph Pad Prism 6) analyzed thestatistical differences The populations from stainless steelDLC and TiO

2-DLC films were obtained with normal

distribution and independent to each experiment Statisticaldifferences pointed out 119875 values less than 005

A sessile drop method with a Kruss EasyDrop (DSA 100)measured the contact angle (120579) of the samples Accordingto Owens method [22] the contact angle values with twodifferent liquids (distilled water and diiodomethane) calcu-lated the surface energy Thermodynamically the adhesionand spreading of cells from a liquid suspension onto solidsubstrata can be described by 119882Ad = 120574CS minus 120574CL minus 120574SL [23]where 119882Ad is the interfacial free energy of adhesion 120574CSis the cell-solid interfacial free energy 120574CL the cell-liquidinterfacial free energy and 120574SL is the solid-liquid interfacialfree energy respectively If119882Ad is negative cell spreading is

Relat

ive i

nten

sity

(au

)

TiO2-DLC (05 gL)

TiO2-DLC (01 gL)

DLC

G

band

band

D

750 1000 1250 1500 1750500

Raman shift (cmminus1)

Figure 1 Raman spectra from DLC and TiO2-DLC films The

spectra are vertically shifted for easy of comparison

energetically favorable while if119882Ad is positive cell spreadingis thermodynamically unfavorable

A conventional three-electrode cell performed the elec-trochemical tests In this cell a saturated AgAgCl electrodewas the reference electrode platinum wire was the counterelectrode and the stainless steel DLC and TiO

2-DLC

films were the working electrodes The working electrodeexposed area was 05 cm2 The electrolyte solution was amodified Fusayama artificial saliva [24] which consisted ofNaCl (400mgL) KCl (400mgL) CaCl

2sdot2H2O (795mgL)

NaH2PO4sdotH2O (690mgL) KSCN (300mgL) Na

2Ssdot9H2O

(5mgL) and urea (1000mgL) The electrolyte was adjustedto pH 63 by either lactic acid or sodium hydroxide andmaintained at 37∘C Potentiodynamic tests were carried outby polarization of samples in the anodic direction from minus10to +10 V just after exposition to the electrolyte solution Thepotential sweep rate was 1mVs The electrochemical testswere performed at 37∘C

3 Results and Discussion

Raman scattering spectroscopy evaluated the chemical struc-ture of the DLC films Two broad bands compose the spectrafor visible light one centered in sim1330 cmminus1 (119863 band) andother in sim1530 cmminus1 (119866 band) [25] The 119863 and 119866 band posi-tions were determined by subtracting a linear backgroundand fitting a Gaussian function to the peak of the Ramanspectrum (Figure 1) Table 1 summarizes themain parametersobtained through the spectraThe bond stretching of all pairsof sp2 atoms in both rings and chains causes the 119866 band[26 27]The breathingmodes of sp2 atoms in rings caused the119863 band and appears only in the presence of defects [26 27]The TiO

2-DLC films presented a shift in 119863 and 119866 band

positions towards higher wavenumbersThe full width at halfmaximum (FWHM) of 119866 band measures structural disorderand arises from bond length distortions [27]The presence ofTiO2nanoparticles in DLC films results in the increase of the

intensity ratio of119863 and 119866 area (119860119863119860119866) and a shift of119863 and



TiO2

concentration(gL)


FWHM119863FWHM

119866

00 13301 15311 2967 1542 051 19201 13544 15523 2984 1449 081 20605 13344 15314 3330 1622 067 205

Oste

obla

st vi

abili

ty (

)


TiO2-DLC(05 gL)

TiO2-DLC(01 gL)

DLC

lowast

0

20

40

60

80

100

120

140


2-DLC surfaces

























2-DLC


2surfaces [33]


119889= 361mNm)





2-DLC Oxide parti-









30120583m

(a)

30120583m

(b)

30120583m

(c)

30120583m

(d)


2-DLC (05 gL)






2-DLC









2-DLC films Table 3



2-DLC (01 gL) films






TiO2-DLC (05 gL) minus0220 0054 1000

Num

ber o

f adh

ered

cells

(42

mm2)

Stainlesssteel

TiO2-DLC(05 gL)

TiO2-DLC(01 gL)

DLC

lowast

lowastlowast

lowastlowast

0

20

40

60

80

100


Num

ber o

f adh

ered

cells

(42

mm2)


(01 gL)

DLC

30

40

50

60

70

80

90




2-DLC films




minus10

minus05

00

05

10

Pote

ntia

l V v

ersu

s Ag

AgC

l



(a)(b)(c)(d)


2-DLC (01 gL) and



2concentration


4 Conclusion


2nanoparticles are





2-



Competing Interests


Acknowledgments


References




















2


















2ceramic






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





TiO2

concentration(gL)


FWHM119863FWHM

119866

00 13301 15311 2967 1542 051 19201 13544 15523 2984 1449 081 20605 13344 15314 3330 1622 067 205

Oste

obla

st vi

abili

ty (

)


TiO2-DLC(05 gL)

TiO2-DLC(01 gL)

DLC

lowast

0

20

40

60

80

100

120

140


2-DLC surfaces

























2-DLC


2surfaces [33]


119889= 361mNm)





2-DLC Oxide parti-









30120583m

(a)

30120583m

(b)

30120583m

(c)

30120583m

(d)


2-DLC (05 gL)






2-DLC









2-DLC films Table 3



2-DLC (01 gL) films






TiO2-DLC (05 gL) minus0220 0054 1000

Num

ber o

f adh

ered

cells

(42

mm2)

Stainlesssteel

TiO2-DLC(05 gL)

TiO2-DLC(01 gL)

DLC

lowast

lowastlowast

lowastlowast

0

20

40

60

80

100


Num

ber o

f adh

ered

cells

(42

mm2)


(01 gL)

DLC

30

40

50

60

70

80

90




2-DLC films




minus10

minus05

00

05

10

Pote

ntia

l V v

ersu

s Ag

AgC

l



(a)(b)(c)(d)


2-DLC (01 gL) and



2concentration


4 Conclusion


2nanoparticles are





2-



Competing Interests


Acknowledgments


References




















2


















2ceramic






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials









30120583m

(a)

30120583m

(b)

30120583m

(c)

30120583m

(d)


2-DLC (05 gL)






2-DLC









2-DLC films Table 3



2-DLC (01 gL) films






TiO2-DLC (05 gL) minus0220 0054 1000

Num

ber o

f adh

ered

cells

(42

mm2)

Stainlesssteel

TiO2-DLC(05 gL)

TiO2-DLC(01 gL)

DLC

lowast

lowastlowast

lowastlowast

0

20

40

60

80

100


Num

ber o

f adh

ered

cells

(42

mm2)


(01 gL)

DLC

30

40

50

60

70

80

90




2-DLC films




minus10

minus05

00

05

10

Pote

ntia

l V v

ersu

s Ag

AgC

l



(a)(b)(c)(d)


2-DLC (01 gL) and



2concentration


4 Conclusion


2nanoparticles are





2-



Competing Interests


Acknowledgments


References




















2


















2ceramic






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







TiO2-DLC (05 gL) minus0220 0054 1000

Num

ber o

f adh

ered

cells

(42

mm2)

Stainlesssteel

TiO2-DLC(05 gL)

TiO2-DLC(01 gL)

DLC

lowast

lowastlowast

lowastlowast

0

20

40

60

80

100


Num

ber o

f adh

ered

cells

(42

mm2)


(01 gL)

DLC

30

40

50

60

70

80

90




2-DLC films




minus10

minus05

00

05

10

Pote

ntia

l V v

ersu

s Ag

AgC

l



(a)(b)(c)(d)


2-DLC (01 gL) and



2concentration


4 Conclusion


2nanoparticles are





2-



Competing Interests


Acknowledgments


References




















2


















2ceramic






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Competing Interests


Acknowledgments


References




















2


















2ceramic






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



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