Research Article Improved Bonding Strength of ...downloads.hindawi.com/journals/jnm/2016/9143969.pdf · Titanium Dioxide Nanotube Arrays following Alkaline Pretreatment for Orthopedic

Research ArticleImproved Bonding Strength of Hydroxyapatite onTitanium Dioxide Nanotube Arrays following AlkalinePretreatment for Orthopedic Implants

Yardnapar Parcharoen1 Preecha Termsuksawad2 and Sirinrath Sirivisoot3

1Chulabhorn International College of Medicine Thammasat University PathumThani 12121 Thailand2Division of Materials Technology School of Energy Environment and MaterialsKing Mongkutrsquos University of Technology Thonburi Bangkok 10140 Thailand3Biological Engineering Program Faculty of Engineering King Mongkutrsquos University of Technology ThonburiBangkok 10140 Thailand

Correspondence should be addressed to Sirinrath Sirivisoot sirinrathsirkmuttacth

Received 22 April 2016 Accepted 4 July 2016

Academic Editor Lei Liu

Copyright copy 2016 Yardnapar Parcharoen et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Hydroxyapatite (HA) is a bioactive bone substitute used in biomedical applications One approach to use HA for bone implantapplication is to coat it on titanium (Ti) implant However adhesion of HA on Ti is major concern for their long-term usein orthopedic implants To enhance the adhesion strength of HA coating on titanium (Ti) the surface of the Ti was anodizedand alkaline pretreated prior to coating on Ti by electrodeposition Alkaline pretreatment of titanium dioxide nanotubes (ATi)accelerated the formation of HA which mimicked the features and structure of natural bone tissue Nanostructured HA formed onthe ATi and pretreated ATi (P-ATi) unlike on conventional Ti This study is the first to show that the bonding of HA coating to aP-ATi substrate was stronger than those of HA coating to Ti and to ATi The preosteoblast response tests were also conducted Theresults indicated that HA coating improved preosteoblast proliferation after 3 days in standard cell culture

1 Introduction

Recently more than 90 percent of the elderly of worldrsquospopulations suffer from bone-related trauma such as osteo-porosis bone cancers rheumatoid arthritis or accidentswhich require spinal hip and knee replacements [1] Thusa huge success in bone implant development is anticipated in2030 [2] In Thailand the total cost of orthopedic implantsincreased from $49 million in 2002 to $397 million in 2010

Bone is composed of ceramic and biosubstance that drawsattention among material engineers who look for material tosubstitute for bone This substituted material should possessboth high strength and fracture toughness appropriatedfor orthopedic implants [3] Common biomaterials usedfor bone replacements are stainless steels cobalt-chromium(Co-Cr) alloys titanium (Ti) and Ti alloys Among thesemetals the tensile modulus of Ti-based materials is closest

to that of bone Additionally wear and corrosion resistancesof Ti-based materials are higher than those of the othertwo materials because of natural TiO

2formation on the Ti-

based surface Although TiO2is bioinert in physiological

environments Ti-based implants often fail after 10ndash20 yearsof service Failure modes include bone fracture around theimplanted materials (due to lower elastic modulus of bonethan that of the implant) wear or corrosion of the implantinflammation and infection [4] Moreover naturally formedTiO2has low osteoconduction [5 6] which causes implant

loosening leading to failure [7] To provide high bioactivityand to improve bone ingrowth one approach is to use Ti witha coating of nanostructured hydroxyapatite (HA)

HA is a naturally derived ceramic found in bones andits calcium to phosphate atomic ratio (CaP) is 167 [8]Various coating methods of hydroxyapatite (HA) on Ti sur-face have been investigated such as Ti soaking in simulated

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2016 Article ID 9143969 13 pageshttpdxdoiorg10115520169143969

2 Journal of Nanomaterials

body fluid (SBF) (mimicking naturally HA forming) plasmaspraying sol-gel deposition in HA particles solution pulsedlaser deposition hot isostatic pressing and electrochemicaldeposition [9ndash16] HA coating by electrochemical depositionwas shown to possess excellent corrosion protection and goodbiocompatibility under host environment [17] Advantagesof this technique are that it is relatively inexpensive andthickness of the coating can be easily controlled Anotherconcern point is that mechanical bonding strength of the HAcoating should withstand the bone growth stresses inducedduring the bone healing process [18]The adhesion of electro-deposited HA on smooth Ti surface was observed to be veryweek whichmay cause implant loosening [19ndash22]Thereforeto increase the adhesion strength betweenHA and Ti surfaceone suggested method is to use TiO

2nanotube array as a

substrate for HA electrodeposition In addition to improvingbonding strength HA grown on TiO

2nanotube array is more

favorable to osteoblast (or bone-forming cell) growth anddifferentiation when compared with naturally formed TiO

2

layer on conventional Ti It was also found that nanostructuresurface could promote deposition of bone minerals inducedby osteoblasts [23]

TiO2nanotubular can be formed by several methods

such as sol-gel method [24] electrophoretic deposition [25]and anodization [26] These methods could form differenttube alignment on surface The vertically aligned TiO

2

nanotube arrays can be achieved by anodizationmethodwithvarious parameters (electrolytes voltages etc) [27 28] In thepresent study anodization using pulse voltage and neutral-viscous fluoride-containing electrolyte was investigated toachieve a uniform well-oriented TiO

2nanotube arrays (ATi)

The previous study shows that alkaline surface treatment onATi and subsequently annealing of HA-electrodeposited ATiat high temperature (gt300∘C) improve the bonding strengthbetween HA and ATi [29] However the results from anotherstudy suggested that annealed HA grown in simulated bodyfluid (SBF) at high temperature (gt300∘C) was not alwaysnecessary because HA grown in SBF still possesses the CaPratio of 167 closelymimicking natural boneminerals [30 31]

In the present study HA coatings were coated onto alka-line pretreated TiO

2nanotubes The anodization conditions

included two different values of pulse voltage (+20minus4 and+35minus4V) and temperatures (5 and 25∘C) The influence ofalkaline pretreatment on ATi and physical structures (egwall thickness diameter and length) of TiO

2nanotubes on

the growth of bioinspired HA was reported The mechanicalbonding strength of HA on its substrates was studied asan in vitro early assessment of long-term stability in thehuman body The physical and chemical characteristics (egstructure size and a ratio of CaP) of HA were investigatedto elucidate the effect of using this biomaterial to increase thepreosteoblast viability under standard cell culture conditions

2 Experimental Materials and Methods

21 Forming TiO2 Nanotube Arrays Ti plate (Supra AlloysInc USA) of 999 purity was polished using silicon

carbide papers (TOA Thailand) of progressively decreasingroughness of 400 600 800 1000 1500 and 2000 grits Thesamples were then polished with 005 120583m alumina powder(Allied USA) to achieve a mirror quality finish Freshlypolished Ti was then washed with deionized (DI) water andsonicated in acetone (Sigma-Aldrich Thailand) and 95 volethanol (Sigma-Aldrich Thailand) for five minutes eachbefore immediate anodization

Anodizationmethodwas used to produce TiO2nanotube

arrays Ti was used as a positive electrode whereas platinumwas used as a negative electrodeThe electrolytewas amixtureof 90 vol glycerol and 10 vol ammonium fluoride (NH

4F)

(036M) in water Anodization was performed with the pulsevoltage of either +20minus4 or +35minus4V the conditions from aprevious study by Chanmanee et al [32] The anodizationwas carried out at either 5∘C or 25∘C for 90 minutes Thenanodized sample was washed with deionized water before itwas dried with nitrogen gas at room temperature All ATisamples were then annealed at 450∘C for 30minutes to obtainthe anatase phase

22 Electrodeposition of Hydroxyapatite ATi samples werepretreated with 1M NaOH at 50∘C for two minutes Afterthe pretreatment the HA deposition was conducted Theelectrolyte for HA deposition was prepared by dissolv-ing ammonium phosphate (NH

4H2PO4 167mM) (Sigma-

Aldrich Thailand) and calcium nitrate (Ca(NO3)2 25mM)

(Sigma-AldrichThailand) in distilledwater To increase ionicconductivity and to buffer pH of electrolyte at 72 015MNaCl (Ajax Finechem New Zealand) tris(hydroxyl amino-methane) (Ajax Finechem New Zealand) and hydrochloricacid (Ajax Finechem New Zealand) were mixed [29] Thepretreatment ATi (P-ATi) was used as a negative electrodewhereas platinumwas used as a positive electrode Electrode-position of HA was processed at minus25 V and at 80∘C for 10minutes

23 Physical and Chemical Characterizations of AnodizedTitanium and Hydroxyapatite Coating Scanning electronmicroscopy (FE-SEM CamScanMX2600 UK) was used toinvestigate surface morphology of ATi The dimensions ofthe TiO

2nanotubes were measured using image analysis

software (ImageJ version 132 NIH)The crystal structures ofthe surfaces of the NaOH-pretreated ATi and the HA-coatedsamples were investigated byX-ray diffractometer (ShimadzuModel XRD 6000 Japan)

24 The Mechanical Properties of Coated Hydroxyapatite onAnodized Titanium The mechanical bonding strength ofcoated HA on ATi was evaluated by tensile testing followingan ASTM F 1044-05 standard [33] Two HA-coated sampleswere glued together face-to-face using Plasmatex Klebbiadhesive (Plasma-Technik AG Switzerland) and cured at190∘C for two hours Tensile tests were conducted bya computer-controlled universal testing machine (InstronModel 5500R US) at a cross head speed of 025 centimeter

Journal of Nanomaterials 3

perminute (01 inchminute)The degree of bonding strengthwas calculated as shown in

Bonding strength (MPa)

=

Maximum load to failure (N)cross-sectional area (m2)

(1)

25 Preosteoblast Cell Response

251 Cell Culture Preosteoblasts (MC3T3-E passage num-ber = 10 MTECThailand) or bone-forming cells were usedin the present study Cells were cultured in alpha-modifiedminimal essential medium (alpha-MEM Invitrogen Corpo-ration Paisley UK) supplemented with 10 vol fetal calfserum (Dominique Dutcher Brumath France) and 1 volpenicillinstreptomycin (Invitrogen Corporation) at 37∘C inhumidified atmosphere of 5 CO

2in air The cell culture

media were replaced every three days Cells were seeded andcultured on plastic polystyrene (control) Ti ATi P-ATi HA-ATi and HA-P-ATi at the cell density of 5 times 104 cellscm2

252 Cell Morphology After three days in culture cells werefixed with 25 vol sodium phosphate buffered glutaralde-hyde (Sigma-AldrichThailand) at pH 7 for 20 minutes Aftertwo washes the cells were postfixed with 1 vol osmiumtetroxide (OsO

4) (Sigma-Aldrich Thailand) in saturated

mercuric chloride (HgCl2) (Sigma-Aldrich Thailand) The

samples were then dehydrated with a series of ethanol washes(20 30 40 50 60 70 80 90 and 100 vol) at roomtemperature All samples were subsequently critical-pointdried (EMSCOPECPD-750 Ashford UK)The samples werecoated with gold (EMSCOPE SC-500 UK) at a thickness of10 nm before being characterized by SEM The cell morphol-ogy on Ti ATi P-ATi HA-coated on ATi (HA-ATi) and HA-coated on P-ATi (HA-P-ATi) was observed using SEM (JEOLJ-SM-5300 Japan) at an accelerating voltage of 20 kV

253 Cell Viability Cells were seeded onto plastic poly-styrene (control) and coated samples at a density of 5 times104 cellssdotcmminus2 in 24-well culture plates Cell viability wastested using a commercial 3-[45-dimethylthiazol-2-yl]-25-diphenyl tetrazolium bromide (MTT) assay (Sigma-AldrichThailand) The 10 vol MTT solution in 1x phosphate buffersaline was mixed with alpha-MEM without phenol red toform a yellowish solution before being added onto the cell-seeded samples at the day 3 of cultures Mitochondrial dehy-drogenases of viable cells cleave the tetrazolium ring yieldingpurple formazan crystals on cell-seeded samples after incu-bation for an hour The absorbance of purple solution wasmeasured at 570 nm wavelength using a spectrophotometer(SynergyMxMultimode Reader US) A concomitant changein the amount of formazan formed correlates to the change inthe number of viable cells on the samples The percentage ofviable cells on the samples was calculated as shown in

Cell viability () =Absorbance of colored solution incubated with samples times 100

Absorbance of colored solution incubated with control (Polystylene plate) (2)

26 Statistical Analysis Two-way analysis of variance(ANOVA) was used in the analysis of wall thicknessdiameter and length of TiO

2nanotubes which were ano-

dized with different voltage pulses and temperaturesANOVA was used to statistically analyze the effects ofmaterials on bonding strength of the HA coatings and cellviabilityThe statistical analysis was performed usingMinitab16 (Minitab Inc USA) software Significant level of 005(119901 lt 005) was used for the test

3 Results and Discussion

31 Characteristics of Anodized Titanium Gong et alreported that a certain anodization potential must be appliedto yield ordered TiO

2nanotube arrays [26]Therefore in this

study to control the size of the nanotubes anodizationpotentials were varied Figure 1 showed top-view SEM imagesof ATi which were anodized under two different tempera-tures and pulse voltages 5∘C +20minus4V (Figure 1(a)) 5∘C+35minus4V (Figure 1(b)) 25∘C +20minus4V (Figure 1(c)) and25∘C +35minus4V (Figure 1(d)) for 90 minutes The insets inFigures 1(a)ndash1(d) showed cross sections of TiO

2nanotube

arrays The SEM images demonstrated that the oxide nan-otubes formed in a uniform shape under the conditions

of +20minus4V at both 5 and 25∘C while the nonuniformnanotubes were found for the sample prepared under theconditions of +35minus4V at both temperatures The formationof the nonhomogeneous nanotubes (at 35V) may be becauseof the high electrical field inducing etching of TiO

2at higher

anodization potential While the electrochemical oxidationrate was increased at the higher anodization potential longertube lengths and formation oxide films were observed (insetof cross sections) At an anodization potential of +20V theformation and etching of TiO

2were slower than those formed

at +35V Thus the applied pulse voltage at +20minus4V inanodization led to the formation of shorter andmore uniformnanotube structures than that formed at +35minus4V

Thewall thickness tube diameter and tube length of TiO2

nanotubes were measured using ImageJ and were plotted asshown in Figure 2 The effects of each anodization parameteron nanotube diameter and wall thickness were summarizedin Table 1 The results suggested that temperature and pulsevoltage in anodization had a remarkable effect on the TiO

2

tubular featuresElectrolytes containing fluorine are known as the most

efficient electrolytes for anodic formation of TiO2nanotube

arrays For electrochemical anodization with pulse potentialof +20minus4V or +35minus4V TiO

2nanotube layers were formed


500nm

500nm

(a)

500nm

500nm

(b)

500nm

500nm

(c)

500nm

500nm

(d)

Figure 1 FE-SEM images of nanotube arrays anodized at 5∘C +20minus4V (a) 5∘C +35minus4V (b) 25∘C +20minus4V (c) and 25∘C +35minus4V (d)for 90 minutes Scale bars are 500 nmThe insets showed cross sections of the anodized TiO

2films

by self-organization of TiO2as a result of the balance between

electrochemical oxidation of Ti and TiO2(reaction (3)) The

induced electrical field caused dissolution of the TiO2by

fluorine ions (reactions (4) and (5)) [34 35] Dissolutionof the TiO

2was partially suppressed by the binding of

NH4

+NH3species on the TiO

2surfaces (reaction (6)) as

described by Chanmanee et al [32]

Ti + 2H2O 997888rarr TiO

2+ 4H+ + 4eminus (3)

Ti4+ + 6Fminus 997888rarr [TiF6]2minus (4)

TiO2+ 6Fminus + 4H+ 997888rarr [TiF

6]2minus

+ 2H2O (5)

Reactions (3) and (5) occur during positive potentialwhereas negative voltage electrostatically induces the bindingof NH

4

+ species with TiO2 as shown in reaction (6) to form

adsorbing TiO2(NH4

+) The role of TiO

2(NH4

+) is to protect

the nanotube walls against chemical etching by fluoride ionseven in short times of negative potential (1 sec) [32 36ndash38]

TiO2+NH

4

+997888rarr TiO

2(NH4

+)ads (6)

In the present study it was found that tube diameter andlength of ATi strongly depended on temperature and appliedpulse voltage (Figures 1 and 2) Figure 2 showed that nanotubediameter and tube length formed at various temperatures

Table 1 The relationship between anodization parameters (i)increasing the system temperature from 5∘C to 25∘C and (ii)increasing positive voltages from +20V to +35V with the formationof TiO

2nanotube arrays in terms of tube width and diameter ldquoTWrdquo

represents tube width ldquoTDrdquo tube diameter and ldquoTLrdquo tube length

Parameter Morphology

(i) 5∘C to 25∘CAt the same voltage

TW not changedTD uarrTL uarr

(ii) +20V to +35VAt the same temperatureA constant voltage of minus4V


were significantly different with significant level of at least005 while the wall thicknesses were comparable The resultsuggested that the rate of TiO

2formation and chemical

dissolution at 25∘C were faster than those at 5∘C while theTiO2formation rate and etching rate of TiO

2induced by the

electric fieldwere similar at both temperatures [28] Howeverat low temperature etching of TiO

2by fluorine ions is largely

suppressed leading to small tube diameter and similar wallthickness The previous study by Wang and Lin found thatat room temperature the TiO

2formation rate was higher

than that in an ice bath [28] while the etching rate of TiO2


Tube wall thicknessTube diameterTube length

lowast

lowast

5∘ C

+20

minus4

V

5∘ C

+35

minus4

V

25∘ C

+20

minus4

V

25∘ C

+35

minus4

V

0

400

800

1200

1600

Dist

ance

(nm

)

Figure 2 Wall thickness diameter and length of anodizedTiO2nanotubes formed from different conditions of anodization

analyzed by ImageJ 119901 value was calculated using two-way ANOVA119899 = 3 lowast119901 lt 005

induced by electric field and fluorine ions remained similarat both temperatures Their results showed a difference ininner diameter but not in the outer diametersTheir workwasconducted in hydrofluoric electrolyte thus the anodizationwas not suppressed by neutral-viscous electrolyte This phe-nomenon may be the reason to obtain different result fromthis present study However Wang and Lin also found thatat low temperature (sim0∘C) even though the potential wasincreased the chemical dissolution of TiO

2by fluorine ions

was still very low in the glycerol solution containing 025wtammonium fluorides Therefore only a small differencein both wall thickness and diameter of the nanotube wasobserved at a constant temperature [28]

32 Hydroxyapatite Electrodeposition

321 Effect of Alkaline Pretreatment From the previouswork the sodium titanate (Na

2Ti5O11

or Na2Ti6O13) was

deposited on the top of TiO2nanotubes and acted as nucle-

ation sites for nanoscale of HA electrodeposition [36] TheSEM images in Figures 3 and 4 showed the HA formation onATi and P-ATi respectively From Figures 3(a)ndash3(d) withoutalkaline pretreatment the HA coatings appeared as an ori-ented rod-like crystal structure on the ATi substrates with theaverage HA crystal sizes of 175 107 155 and 316 nm respec-tively On the P-ATi substrates the HA coatings formed asan unoriented rod-like crystal structure with the average HAcrystal sizes of 171 194 105 and 151 nm respectively as shownin Figures 4(a)ndash4(d) The average size of HA crystal on ATi(107 nm) in Figure 3(b) is smaller than the average HA crystal

Table 2 EDS analysis of HA coating on ATi and conventional Tirepresents the ratio of Ca and P The anodized Ti was performedunder various conditions with a constant voltage of minus4V Datarepresent mean plusmn standard deviation 119901 value was calculated usingANOVA 119899 = 5 lowast119901 lt 005 compared with conventional Ti

Substrate The ratio of Ca and PPretreatment Without pretreatment

ATi 5∘C+20V 156lowast 144lowast




Conventional Ti (control) 089 097

size on P-ATi (194 nm) in Figure 4(b) In this study theresults confirmed that the HA crystal was finer on ATi (5∘C+35minus4V) without NaOH pretreatment

322 Formation of Hydroxyapatite on Different SubstratesFrom Table 2 the EDS analysis of HA-ATi and HA-P-ATirevealed that the calcium to phosphate ratios CaP were inthe range of 143 to 168 when ATi was used as a substrateTheHAonATi and P-ATi had a CaP ratio closelymimicking thatof natural boneTheHA coating on conventional Ti howeverhad a CaP ratio of about 09 This ratio is close to that ofdicalcium phosphate in bone [17] The crystal structures ofgrown calcium phosphateminerals were analyzed by XRD asshown in Figure 5 The XRD spectra and EDS analysisconfirmed that HA (Ca

10(PO4)6(OH)2) coating formed on

ATi and P-ATi but perhaps dicalcium phosphate formed onthe conventional Ti

The formation of HA crystals on Ti and ATi by electrode-position in the solution containing calcium and phosphoruswas investigated in the present study Electrochemical reac-tions of HA formation are shown as follows [39]

2H2O + 2eminus 997888rarr H

2+ 2OHminus (7)

OHminus +H2PO4

minus997888rarr HPO

4

2minus+H2O (8)

HPO4

2minus+OHminus 997888rarr PO

4

3minus+H2O (9)

10Ca2+ + 6PO4

3minus+ 2(OH)minusads

997888rarr Ca10(PO4)6(OH)2

(10)

The reduction of water resulted in the release of H2and

an increase of hydroxide ions as shown in reaction (7) As aresult of reaction (7) [39] the pH between the cathode andelectrolyte interface increases When pH was higher than764 the rates of the HA electrodeposition of reactions (8)and (9) were acceleratedWhen the interfacial pHwas high itprovided an appropriate chemical environment to form HACa10(PO4)6(OH)2 coatings on the cathode (reaction (10))

[40] In addition the electric field draws Ca2+ ions to thecathode surface causing that CaP ratio is closer to that ofnatural bone than the CaP ratio obtained by conservativemethods (precipitation of calcium phosphates from aqueoussolution)


HA crystal thickness (nm)

0

325

65

975

13

Rela

tive f

requ

ency

80ndash1

00

101

ndash120

121

ndash140

141

ndash160

161

ndash180

181

ndash200

201

ndash220

221

ndash240

X = 175nm

(a)


0

325

65

975

13

Relat

ive f

requ

ency

60

ndash80

81ndash1

00

101

ndash120

121

ndash140

141

ndash160

X = 107nm

(b)


0

325

65

975

13

Relat

ive f

requ

ency

100

ndash120

121

ndash140

141

ndash160

161

ndash180

181

ndash200

201

ndash220

X = 155nm

(c)


0

325

65

975

13Re

lativ

e fre

quen

cy

100

ndash101

201

ndash300

301

ndash400

401

ndash500

501

ndash600

X = 316nm

(d)

Figure 3 SEM images of HA coating on different ATi (without 05M NaOH pretreatment) which were anodized under different conditions(a) at 5∘C +20minus4V (b) at 5∘C +35minus4V (c) at 25∘C +20minus4V and (d) at 25∘C +35minus4V for 90 minutes Scale bars are 5 120583m Black arrowsrepresent HA crystal thickness Inset graphs are thickness analysis of HA using ImageJ Data are calculated from three SEM images with119899 = 30

The results from Figures 3 and 4 and Table 2 suggestedthat the chemical composition of HA depends on the surfacestructure of substrates It was known that it is easier forelectrons to travel through TiO

2nanotubes electrode than

through TiO2compact layers [34] Therefore feature of HA

(Ca10(PO4)6(OH)2) coatings on ATi electrode was more

orderly oriented than those on conventional Ti because HAlayers on ATi possess CaP ratios near to that of natural bonedue to Ca2+ ions enrichment on electrode (reaction (10))Since ATi sample can carry more OHminus groups on its surfaceduring NaOH pretreatment it supports a very dense for-mation of apatite nuclei Another study also found that HAdeposition on conventional Ti from the solution containing

Ca2+ and PO4

3minus could obtain four different kinds of calciumphosphates including Ca

10(PO4)6(OH)2 Ca2(PO4)3sdot 119899H2O

Ca8H2(PO4)6sdot5H2O and CaHPO

4sdot2H2O [41]

323 Mechanical Bonding Strength of Hydroxyapatite onAnodized Titanium The bond strength between coated HAand the implant material is very important because the adhe-sive failure of implant after implantation easily takes placewhen the bond strength is low Therefore the present studyaimed to study effect of morphology of substrates used forHA coatings on bond strength between them As shownpreviously that only nanotube length and tube diameter



0

325

65

975

13

Relat

ive f

requ

ency

181

ndash200

201

ndash220

221

ndash240

X = 171nm

141ndash

160

161ndash

180

120ndash

140

(a)

13

975

65

325

0

Relat

ive f

requ

ency


50

ndash100

101

ndash150

151

ndash200

201

ndash250

251

ndash300

301

ndash350

X = 194nm

(b)

13

975

65

325

0

Relat

ive f

requ

ency


60

ndash80

81ndash1

00

101

ndash120

141

ndash160

161

ndash180

X = 105nm

121

ndash140

(c)

13

975

65

325

0

Relat

ive f

requ

ency


100

ndash120

121

ndash140

141

ndash160

161

ndash180

201

ndash220

181

ndash200

221

ndash240

X = 151nm

(d)

Figure 4 SEM images of HA coating on different P-ATi (with 05M NaOH pretreatment) which were anodized under different conditions(a) at 5∘C +20minus4V (b) at 5∘C +35minus4V (c) at 25∘C +20minus4V and (d) at 25∘C +35minus4V for 90 minutes Scale bars are 5 120583m Black arrowsrepresent HA crystal thickness Inset graphs are thickness analysis of HA using ImageJ Data are calculated from three SEM images with119899 = 30

varied with anodization conditions length (119871) to diameter(119863) of TiO

2nanotube was used to test for this effect

Figure 6(a) showed 119871119863 ratio of TiO2nanotube arrays for

ATi and P-ATi anodized at the following conditions 5∘C+20minus4V 119871119863 = 127 5∘C +35minus4V 119871119863 = 292 25∘C+20minus4V 119871119863 = 596 and 25∘C +35minus4V 119871119863 = 703 Theresults suggested that using either higher positive voltage orhigher temperature increased 119871119863 ratios It was found thatbonding strength of the HA coating on P-ATi is higher thanthat of HA-ATi (such that at 5∘C +35minus4V with 119871119863 =292 found bonding strength of the HA coating on P-ATiis 21MPa and via ATi is 12MPa) It is due to the factthat the NaOH pretreatment increases the pH inside the

titania nanopores consequently nucleation of HA crystalswas enhanced during electrodeposition As described by Kimet al during the alkaline treatment the protective oxidelayer on Ti is dissolved into solution because of corrosiveattack by hydroxyl groups [42] Negatively charged hydratesproduced on the ATi substrate surface combine with alkaliions from the aqueous solution to form a hydrogel layer ofsodium titanate After the hydrogel layer was exposed to hightemperature the layer was dehydrated and was densified toform a stable alkali titanate layer The compact layer of alkalititanate facilitates good adhesion between the HA coatingand Ti or ATi substrate The bonding strength progressivelydecreased as 119871119863 increased when 119871119863 was higher than


Rutile TitaniumHydroxyapatite

(Figure 4(d))

(Figure 3(d))

(Figure 4(c))

(Figure 3(c))

(Figure 3(b))

(Figure 4(a))

(Figure 3(a))

(Figure 4(b))

HA-ATi 5∘C 20minus4V

HA-P-ATi 5∘C 20minus4V







HA-Ti (control)

HA-P-Ti (control)

lowast Anatase

lowast

10 20 30 40 50 60 70 80 9002120579 (degrees)

0

200

400

600

800

1000

1200

1400

1600

Inte

nsity

(arb

itrar

y un

it)

Figure 5 X-ray diffraction spectra of the electrodeposited HAcoatings on Ti and ATi at the temperatures of 5∘C and 25∘Cand the applied voltages of +20minus4V and +35minus4V with NaOHpretreatment (P) and without NaOH pretreatment

292 At 119871119863 of 292 the highest bonding strength betweenHA and P-ATi was obtained This phenomenon indicatedthat nanotube geometry is an important factor effecting onbonding strength between coated HA and substrate

According to the ASTM F 1044-05 standard there arethree types of failures that can occur during the tensile testadhesive cohesive and a combination of these two modes(Figures 6(b) and 6(c)) For the HA coated at 5∘C +35minus4V(119871119863 = 292) during the shear bond tests the frac-ture occurred only at the HA coating layers as shown inFigure 6(c)(I) Accordingly it can be stated that cohesionstrength between the HA and TiO

2nanotube was stronger

than that between the HA layers The same fracture surfacewas found for the deposited HA prepared at 25∘C +20minus4V(119871119863 = 596) For the HA coated at 25∘C +35minus4V (119871119863 =703) fracture occurred at the HA and TiO

2interface as

well as at the HA layer as shown in Figure 6(c)(II) FromFigure 6(c)(II) residues of TiO

2nanotubewere found in both

sides of the fracture surfaceThis failure indicated a combina-tion of adhesive and cohesive failures and also occurred forthe HA coated at 5∘C +20minus4V (119871119863 = 127) Using bothbonding strength and fracture surface information the highbonding strength was found when cohesion between HAand TiO

2nanotube is stronger than adhesion between HA

layersThe dependence of bonding strength on119871119863 ratiomaybe described as the following When 119871119863 is 127 the depththat depositedHA could form along nanotube is very shallowAs shown in Figure 2 the nanotube length in this case is onlyabout 120 nm Thus nanotube surface area which acts asnucleation site for HA formation is low leading to less HA-nanotube interface area In addition due to short nanotubelength both the nanotubes and Ti base may be subjectedto the glue Hence the nanotube base was easily destroyedduring testing For the highest 119871119863 ratio of 703 the presence

of nanotube residue indicated that TiO2nanotubes were

damaged From Figure 2 tube length was approximately1120 nm which is almost four times longer than that of thesample whose 119871119863 ratio is 297 In this case the failure ofnanotube could be explained by the Euler equation 120590cr =1205872119864119868(119860119871

2) [43] As shown in the equation critical stress

(120590cr) that the column can bear is inversely proportional to thesecond order of column height 119871 The higher the height ofthe column the lower the critical stress that the column canbear As a result tube strength of nanotube layer is inverselyproportional to nanotube length

For the sample with 119871119863 ratio of 596 the bondingstrength is lower and the tube length is about two timeshigher than that for the sample with 119871119863 ratio of 297 TheEuler equation may be applied for this case neverthelessthe fracture surface of the two samples indicated the similaradhesion mode Further study is needed to analyze the effectof geometry in this range In case of HA on conventional Tisubstrate (Figure 6(c)(III)) fracture during the test occurredat the calcium phosphate-Ti interface which indicated thatonly adhesive failure occurred From these results it can beconcluded that as 119871119863 ratio is approximately 300ndash600 theinterfacial adhesion between HA and TiO

2array is stronger

than cohesion between HA layersIn conclusion the topography and chemical composition

of Ti substrates before the electrodeposition of bioinspiredHA directly influence the crystal structure and phases as wellas mechanical bonding strength of HA coating FollowingISO Standard 13779-42002 bonding strength between coat-ing and substrate of apatite coating implant should be higherthan 15MPa [44] With this result ATi samples anodized at5∘C and +35minus4V (119871119863 = 292) were further studied for cellmorphology and viability with preosteoblasts

324 Responses of Preosteoblast Cells on Different Hydroxyap-atite Coatings ATi and P-ATi anodized at +35minus4V and 5∘Cas shown in Figures 3(b) and 4(b) respectively were chosenfor cell culture testing because the mechanical bondingstrength of HA on these substrates was high as shown in Fig-ure 6 Figure 7 shows cell proliferation on Ti ATi P-ATi HA-ATi andHA-P-ATi Previously the samples were seeded withMC3T3-E1 preosteoblast cells and cultured for three days ina standard cell culture condition The results of the MTTassay indicated that all of the samples had good cytocom-patibility The percentage of viable cells was the highest onHA-ATi The electrodeposited HA whose CaP ratio issimilar to that of natural bone exhibited the oriented rod-like crystal structure when it formed on ATi with an averagecrystal size of 107 nm The results suggested that osteoblastspreferred to grow on HA-ATi compared to HA-P-ATi whoseaverage crystal size was larger 194 nm This result is in anagreement with other works [6 23 45] Webster and Ejioforsuggested that a small HA crystal size improved the biocom-patibility of implants and increases osteoblast adhesion [23]Tsuchiya et al reported that nanostructured materials signif-icantly improved osteoblast growth while they inhibited cellapoptosis [6] Moreover the nanostructure of biomaterialspromoted cell functions such as the synthesis of extracellular


The ratio of nanotube length to diameter of ATi LDPretreatmentWithout pretreatment

292 596 703 Commercialtitanium

127

lowast

lowast

0

55

11

165

22

Bond

ing

stren

gth

(MPa

)

(a)

Titanium substrate

Titanium substrate

GlueCoating

Specimen components

Description of potential failure location

interface

coating interfaceAdhesive

force

Cohesive force

∙ Gluecoating

∙ Glue∙ Coating∙ Substrate

(b)

(I) (II) (III)5mm 5mm 5mm

TiO2 nanotube HA and glue Ti (without TiO2 nanotube arrays)

(c)

Figure 6 (a) Bonding strengths of HA coating on Ti ATi and P-ATi (with 05MNaOH pretreatment) 119901 value was calculated using one-wayANOVA 119899 = 3 lowast119901 lt 005 (b) Nomenclature of specimen components and classification of failure location for the shear testing (c) Lightmicroscopy images of three types of failure for the tensile testing (HA coating on 5∘C +35minus4V ATi HA coating on 25∘C +20minus4V ATi andTi) (I) cohesive (II) combination of cohesive and adhesive and (III) adhesive force

matrix proteins and calcium mineral deposition [45] Thusthe size of the HA crystals formed on ATi and P-ATi directlyaffects osteoblast proliferation in this study

Adhesion of osteoblast cells is a crucial prerequisite toall subsequent cell functions According to Webster et althe use of nanostructured ceramics (such as alumina titaniaand HA) significantly improved osteoblast adhesion [46 47]Importantly the results shown in Figure 7 are in good agree-ment with previous findings that finer HA crystals improvecell proliferation A study by Chou et al demonstrated thatsmall plate-like HA had greater preosteoblast proliferationafter 4 days than large plate-like HA Large plate-like HAhowever induced higher expression of the mature osteogenicmarkers osteocalcin and bone sialoprotein in preosteoblastsafter 21 days of culture compared to small plate-like HA

polystyrene and conventional apatite [48] Further studiesare needed to confirm its effects on osteoblast differentiationand new bone formation juxtaposed to orthopedic implants

Figures 7(b)ndash7(f) show that osteoblasts differed in theirsize spreading and adhesion on the samples Figure 7(a)shows that although Ti is a biocompatible material it is abioinert material which does not adequately increase osteo-blast attachment spreading proliferation or differentiationIn contrast HA as a major inorganic component of hardtissues possesses excellent biocompatibility bioactivity andosteoconductivity [49] Cells exhibited larger spreading on Tithan on all other samples but cell peeling was observed dueto the lower cell adhesion Particularly noteworthy are HA-ATi and HA-P-ATi which induced maximum cell adhesionwith a smaller cell size Cells appeared flattened and spread


Ti ATi P-ATi HA-ATi HA-P-ATi

lowast

Ti ATi P-ATi

HA-ATi HA-P-ATi

(b) (c) (d)

(e) (f)50120583m50120583m

50120583m50120583m 50120583m

(a)

0

200

400

600

800

ce

ll vi

abili

ty

Figure 7 (a) Cell viability of MC3T3-E1 preosteoblasts on Ti ATi P-ATi HA-ATi and HA-P-ATi after three days of cultures Data are meanplusmn a standard error of mean 119901 value was calculated using one-way ANOVA 119899 = 3 lowast119901 lt 005 SEM images of MC3T3-E1 preosteoblasts afterthree days of incubation on (b) conventional Ti (c) ATi (d) P-ATi (e) HA-ATi and (f) HA-P-ATi Arrows show peeling areas of MC3T3-E1cells from the substrates

more widely on Ti ATi and P-ATi samples (Figures 7(b)ndash7(d)) than on the HA-coated samples (Figures 7(e) and 7(f))Osteoblasts with no appendix on their substrates and cellpeeling were observed on Ti ATi and P-ATi (Figures 7(b)ndash7(d)) This is perhaps due to the fact that the cell adhesionstrength on the non-HA-coated samples was lower than thaton HA-ATi and HA-P-ATi HA improves osteoblast adhesionbecause it is a main component of the bone extracellularmatrix The HA coating reduced osteoblast spreading butimproved the elongation of cell projections

The effect of NaOH pretreatment on cell viability wasalso examined Cell morphology of preosteoblasts on the ATiand P-ATi was similar (Figure 7) The ATi (without NaOHpretreatment) however was more favorable for preosteoblast

proliferation than P-ATi possibly due to their differentsurface compositions Sodium titanate was present on the P-ATi surface but not on the ATi surface HA-P-ATi has a lowerratio of CaP (146) and an unoriented rod-like crystal HAcoating (Figure 4(b)) while HA on ATi has a CaP of 167and an oriented rod-like crystal structure The percentage ofviable cells after 3 days of culture was greater onHA-ATi thanon HA-P-ATi but the cell morphology of preosteoblasts onHA-ATi was similar to that on HA-P-ATi A previous studysuggested that osteoblast adhesion and metabolic activitywere very sensitive to surface morphology and roughness[50] Surface energy is also reported to be an influentialsurface characteristic on cellular proliferation [51]Thereforethe higher cell viability on nanoscale HA-ATi could be due to


the positive effects of the surface energy Moreover HA withlow crystallinity may remarkably increase degradation ratesin vitro or in vivo [49] HA is the most stable and least solublein aqueous media of all calcium phosphates [52] It becomesmore acidic and water-soluble as its CaP ratio decreases[53] Thus the dissolution of HA in HA-P-ATi may slowthe growth of preosteoblast cells Approximately 2ndash4mMof Ca2+ is suitable for osteoblast proliferation and survivaland slightly higher concentrations (6ndash8mM) favor osteoblastdifferentiation and mineralization An excessively highconcentration gt10mM can be cytotoxic to cells [54] It ispossible that the high concentration of Ca2+ in HA-P-ATidecreased preosteoblast proliferation in this study On theother hand with a higher CaP ratio (167) of HA-P-ATifree phosphate level in the apatite microenvironment is alsoperhaps lower and thus fewer phosphates participating inosteopontin regulation are transported into the cells [55]Thiscan cause lower cell proliferation on HA-P-ATi than on HA-ATi after 3 days of culture Another possible mechanism isthat high phosphate levels may induce the expression of cal-cium channels and phosphate transporters and activation ofERK12 but notMAPK p38 or JNK signaling pathways [56]This cascade participates in the regulation of a large variety ofprocesses including cell adhesion cell cycle progression cellmigration cell survival differentiation metabolism prolifer-ation and transcription [57] Therefore we conclude that thesize and CaP ratio of the HA crystal directly affect the cellproliferation but not the cell morphology of preosteoblasts

Importantly the nanotube formation of TiO2is the

predominant factor to increase the bonding strength betweenthe HA coating and TiO

2nanotube substrate The nanotube

formation can promote a HA coating in the phase closelymimicking that found in natural bone Therefore the elec-trodeposited HA coating on ATi and P-ATi can illicit morefavorable responses of preosteoblast morphology and prolif-eration than the samples without HA coating

4 Conclusions

TiO2nanotube arrays were anodized for 90min under

various conditions 5∘C +20minus4V 5∘C +35minus4V 25∘C +20minus4V and 25∘C +35minus4VHAwas then electrodeposited ontoATi and P-ATi (with NaOH pretreatment) Increasing thepulse-positive voltage (from +20V to +35V) and tempera-ture (from 5∘C to 25∘C) during anodization increased thenanotube length At 25∘C changing the anodization voltagedid not significantly affect the nanotube wall thickness Thebonding strength of the HA coatings on ATi layers wasstronger than that of the HA coating on conventional TiImportantly the bonding strength of the HA coating washigher on HA-P-ATi than on HA-Ti and HA-ATi The resultssuggested that the HA crystals on ATi had an oriented rod-like crystal structure with an average size of 107 nm anda CaP ratio of 167 The HA crystals on P-ATi had anunoriented rod-like structure with an average size of 194 nmand CaP ratio of 146 The percentage of osteoblast viabilityat day 3 of cell culture was significantly higher on HA-P-ATi(98) than those on Ti (59) and P-ATi (92) but lower

than those on ATi (108) and HA-ATi (117) Higher cellproliferation was observed on the smaller HA crystals ofHA-ATi The CaP ratio (167) of the HA crystals on ATireduced the dissolution of calcium ions and phosphates thusallowing for better cell proliferationThe electrodepositedHAcoating improved preosteoblast adhesion as cell projectionswere elongated In summary P-ATi increases the mechanicalbonding strength of an electrodeposited HA coating butdecreases the CaP ratio of HA with a larger crystal size thusleading to lower preosteoblast proliferation after 3 days ofculture

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

The authors would like to acknowledge financial supportsfrom Commission on Higher Education (the Faculty Devel-opment Scholarships) and a Research Strengthening Projectof the Faculty of Engineering King Mongkutrsquos University ofTechnology Thonburi Bangkok Thailand They also thankAssociate Professor Dr P Kajitvichyanukul Associate Profes-sor Dr K Pasuwat Mr C Manussapol Mr C Jongwanasiriand Ms K Siruksasin for their contributions and usefuladvice in this study Dr W Chanmanee for his advice inanodization and Ms W Chokevivat from National Metaland Materials Technology Center for her help in animalcell experiments They acknowledge Dr Carmen V Gaines(PhD) and Mr Cristian Guajardo for her and his usefuldiscussion

References

[1] S Ramakrishna J Mayer E Wintermantel and K W LeongldquoBiomedical applications of polymer-composite materials areviewrdquo Composites Science and Technology vol 61 no 9 pp1189ndash1224 2001

[2] J B Park and J D Bronzino Biomaterials Principles andApplications CRC Press Boca Raton Fla USA 2003

[3] M J Olszta X Cheng S S Jee et al ldquoBone structure and for-mation a new perspectiverdquo Materials Science and EngineeringR Reports vol 58 no 3ndash5 pp 77ndash116 2007

[4] R S Park and J B LakesBiomaterials An Introduction PlenumPress New York NY USA 1992

[5] P Li and P Ducheyne ldquoQuasi-biological apatite film inducedby titanium in a simulated body fluidrdquo Journal of BiomedicalMaterials Research vol 41 no 3 pp 341ndash348 1998

[6] H Tsuchiya J M Macak L Muller et al ldquoHydroxyapatitegrowth on anodic TiO

2nanotubesrdquo Journal of Biomedical

Materials ResearchmdashPart A vol 77 no 3 pp 534ndash541 2006[7] G Balasundaram and T J Webster ldquoA perspective on

nanophase materials for orthopedic implant applicationsrdquo Jour-nal of Materials Chemistry vol 16 no 38 pp 3737ndash3745 2006

[8] M S Johnsson and G H Nancollas ldquoThe role of brushite andoctacalcium phosphate in apatite formationrdquoCritical Reviews inOral Biology amp Medicine vol 3 pp 61ndash82 1992


[9] T Kokubo T Matsushita and H Takadama ldquoTitania-basedbioactive materialsrdquo Journal of the European Ceramic Societyvol 27 no 2-3 pp 1553ndash1558 2007

[10] A R Boccaccini J Cho T Subhani C Kaya and F Kaya ldquoElec-trophoretic deposition of carbon nanotube-ceramic nanocom-positesrdquo Journal of the European Ceramic Society vol 30 no 5pp 1115ndash1129 2010

[11] J D Haman L C Lucas and D Crawmer ldquoCharacterizationof high velocity oxy-fuel combustion sprayed hydroxyapatiterdquoBiomaterials vol 16 no 3 pp 229ndash237 1995

[12] H Hero HWie R B Jorgensen and I E Ruyter ldquoHydroxyap-atite coatings on Ti produced by hot isostatic pressingrdquo Journalof Biomedical Materials Research vol 28 no 3 pp 343ndash3481994

[13] R Hu C-J Lin and H-Y Shi ldquoA novel ordered nano hydrox-yapatite coating electrochemically deposited on titanium sub-straterdquo Journal of Biomedical Materials Research Part A vol 80no 3 pp 687ndash692 2007

[14] A Montenero G Gnappi F Ferrari et al ldquoSol-gel derivedhydroxyapatite coatings on titanium substraterdquo Journal ofMate-rials Science vol 35 no 11 pp 2791ndash2797 2000

[15] V Nelea C Ristoscu C Chiritescu et al ldquoPulsed laser depo-sition of hydroxyapatite thin films on Ti-5Al-25Fe substrateswith andwithout buffer layersrdquoApplied Surface Science vol 168no 1ndash4 pp 127ndash131 2000

[16] Y C Tsui C Doyle and T W Clyne ldquoPlasma sprayed hydrox-yapatite coatings on titanium substrates part 2 optimisation ofcoating propertiesrdquo Biomaterials vol 19 no 22 pp 2031ndash20431998

[17] X Zhu D W Son J L Ong and K Kim ldquoCharacterization ofhydrothermally treated anodic oxides containing Ca and P ontitaniumrdquo Journal of Materials Science Materials in Medicinevol 14 no 7 pp 629ndash634 2003

[18] R Rodriguez K Kim and J L Ong ldquoIn vitro osteoblastresponse to anodized titanium and anodized titanium followedby hydrothermal treatmentrdquo Journal of Biomedical MaterialsResearch Part A vol 65 no 3 pp 352ndash358 2003

[19] K De Groot R Geesink C P A T Klein and P SerekianldquoPlasma sprayed coatings of hydroxylapatiterdquo Journal ofBiomedicalMaterials Research vol 21 no 12 pp 1375ndash1381 1987

[20] H Kurzweg R B Heimann and T Troczynski ldquoAdhesion ofthermally sprayed hydroxyapatite-bond-coat systemsmeasuredby a novel peel testrdquo Journal of Materials Science Materials inMedicine vol 9 no 1 pp 9ndash16 1998

[21] C-M Lin and S-K Yen ldquoCharacterization and bond strengthof electrolytic HATiO

2double layers for orthopedic applica-

tionsrdquo Journal of Materials Science Materials in Medicine vol15 no 11 pp 1237ndash1246 2004

[22] R McPherson N Gane and T J Bastow ldquoStructural character-ization of plasma-sprayed hydroxylapatite coatingsrdquo Journal ofMaterials Science Materials in Medicine vol 6 no 6 pp 327ndash334 1995

[23] T J Webster and J U Ejiofor ldquoIncreased osteoblast adhesionon nanophase metals Ti Ti6Al4V and CoCrMordquo Biomaterialsvol 25 no 19 pp 4731ndash4739 2004

[24] B B Lakshmi C J Patrissi and C R Martin ldquoSol-gel templatesynthesis of semiconductor oxide micro- and nanostructuresrdquoChemistry of Materials vol 9 no 11 pp 2544ndash2550 1997

[25] Z Miao D Xu J Ouyang G Guo X Zhao and Y Tang ldquoElec-trochemically induced sol-gel preparation of single-crystallineTiO2nanowiresrdquo Nano Letters vol 2 no 7 pp 717ndash720 2002

[26] D Gong C A Grimes O K Varghese et al ldquoTitaniumoxide nanotube arrays prepared by anodic oxidationrdquo Journalof Materials Research vol 16 no 12 pp 3331ndash3334 2001

[27] L-K Tsui and G Zangari ldquoWater content in the anodizationelectrolyte affects the electrochemical and electronic transportproperties of TiO2 nanotubes a study by electrochemicalimpedance spectroscopyrdquo Electrochimica Acta vol 121 pp 203ndash209 2014

[28] J Wang and Z Lin ldquoAnodic formation of ordered TiO2nan-

otube arrays effects of electrolyte temperature and anodizationpotentialrdquo The Journal of Physical Chemistry C vol 113 no 10pp 4026ndash4030 2009

[29] A Kar K S Raja and M Misra ldquoElectrodeposition of hydrox-yapatite onto nanotubular TiO

2for implant applicationsrdquo Sur-

face and Coatings Technology vol 201 no 6 pp 3723ndash37312006

[30] H-M Kim H Kaneko M Kawashita T Kokubo and TNakamura ldquoMechanism of apatite formation on anodically oxi-dized titanium metal in simulated body fluidrdquo Key EngineeringMaterials vol 254ndash256 pp 741ndash744 2004

[31] H-M Kim T HimenoM Kawashita J-H Lee T Kokubo andT Nakamura ldquoSurface potential change in bioactive titaniummetal during the process of apatite formation in simulated bodyfluidrdquo Journal of Biomedical Materials Research Part A vol 67no 4 pp 1305ndash1309 2003

[32] W Chanmanee A Watcharenwong C R ChenthamarakshanP Kajitvichyanukul N R de Tacconi and K RajeshwarldquoTitania nanotubes from pulse anodization of titanium foilsrdquoElectrochemistry Communications vol 9 no 8 pp 2145ndash21492007

[33] ASTM International ldquoStandard test method for shear testingof calcium phosphate coatings and metallic coatingsrdquo ASTMF1044-05 ASTM International West Conshohocken Pa USA2005 httpwwwastmorg

[34] A Ghicov and P Schmuki ldquoSelf-ordering electrochemistry areview on growth and functionality of TiO

2nanotubes and

other self-aligned MO119909structuresrdquo Chemical Communications

no 20 pp 2791ndash2808 2009[35] P Roy S Berger and P Schmuki ldquoTiO

2nanotubes synthesis

and applicationsrdquo Angewandte Chemie-International Editionvol 50 no 13 pp 2904ndash2939 2011

[36] Y Parcharoen P Kajitvichyanukul S Sirivisoot and PTermsuksawad ldquoHydroxyapatite electrodeposition onanodized titanium nanotubes for orthopedic applicationsrdquoApplied Surface Science vol 311 pp 54ndash61 2014

[37] R M Pittman and A T Bell ldquoRaman investigations of NH3

adsorption on TiO2 Nb2O5 and Nb

2O5TiO2rdquo Catalysis Let-

ters vol 24 no 1-2 pp 1ndash13 1994[38] J Ahdjoudj and C Minot ldquoAdsorption of H

2O onmetal oxides

a periodic ab-initio investigationrdquo Surface Science vol 402-404pp 104ndash109 1998

[39] Y-M Liao Z-D Feng and S-W Li ldquoPreparation and char-acterization of hydroxyapatite coatings on human enamel byelectrodepositionrdquo Thin Solid Films vol 516 no 18 pp 6145ndash6150 2008

[40] J M Zhang C J Lin Z D Feng and Z W Tian ldquoMechanisticstudies of electrodeposition for bioceramic coatings of calciumphosphates by an in situ pH-microsensor techniquerdquo Journal ofElectroanalytical Chemistry vol 452 no 2 pp 235ndash240 1998

[41] G H Nancollas and B Tomazic ldquoGrowth of calcium phosphateon hydroxyapatite crystals Effect of supersaturation and ionic


mediumrdquoThe Journal of Physical Chemistry vol 78 no 22 pp2218ndash2225 1974

[42] H-M Kim F Miyaji T Kokubo and T Nakamura ldquoPrepara-tion of bioactive Ti and its alloys via simple chemical surfacetreatmentrdquo Journal of BiomedicalMaterials Research vol 32 no3 pp 409ndash417 1996

[43] B I Yakobson M P Campbell C J Brabec and J BernholcldquoHigh strain rate fracture and C-chain unraveling in carbonnanotubesrdquo Computational Materials Science vol 8 no 4 pp341ndash348 1997

[44] ISO ldquoImplants for surgery (Hydroxyapatite) Part 4 determina-tion of coating adhesion strengthrdquo Tech Rep ISOCD 13779-4 The International Organization for Standardisation GenevaSwitzerland 2002 httpwwwisoorg

[45] K C Popat L Leoni C A Grimes and T A Desai ldquoInfluenceof engineered titania nanotubular surfaces on bone cellsrdquoBiomaterials vol 28 no 21 pp 3188ndash3197 2007

[46] T J Webster C Ergun R H Doremus R W Siegel andR Bizios ldquoEnhanced functions of osteoblasts on nanophaseceramicsrdquo Biomaterials vol 21 no 17 pp 1803ndash1810 2000

[47] T J Webster L S Schadler R W Siegel and R BiziosldquoMechanisms of enhanced osteoblast adhesion on nanophasealumina involve vitronectinrdquo Tissue Engineering vol 7 no 3pp 291ndash301 2001

[48] Y-F Chou W Huang J C Y Dunn T A Miller and B MWu ldquoThe effect of biomimetic apatite structure on osteoblastviability proliferation and gene expressionrdquo Biomaterials vol26 no 3 pp 285ndash295 2005

[49] W Chen B Tian Y Lei Q-F Ke Z-A Zhu and Y-PGuo ldquoHydroxyapatite coatings with oriented nanoplate andnanorod arrays fabrication morphology cytocompatibilityand osteogenic differentiationrdquoMaterials Science and Engineer-ing C vol 67 pp 395ndash408 2016

[50] N Ribeiro S R Sousa and F J Monteiro ldquoInfluence ofcrystallite size of nanophased hydroxyapatite on fibronectin andosteonectin adsorption and on MC3T3-E1 osteoblast adhesionand morphologyrdquo Journal of Colloid and Interface Science vol351 no 2 pp 398ndash406 2010

[51] B Feng J Weng B C Yang S X Qu and X D Zhang ldquoChar-acterization of surface oxide films on titanium and adhesion ofosteoblastrdquo Biomaterials vol 24 no 25 pp 4663ndash4670 2003

[52] J C Elliott Structure and Chemistry of the Apatites andOther Calcium Orthophosphates vol 18 of Studies in InorganicChemistry Elsevier Amsterdam The Netherlands 2003

[53] V Nelea I N Mihailescu and M Jelınek ldquoBiomaterials newissues and breakthroughs for biomedical applicationsrdquo inPulsedLaser Deposition of Thin Films Applications-Led Growth ofFunctional Materials R Eason Ed chapter 18 John Wiley ampSons Hoboken NJ USA 2006

[54] S Maeno Y Niki H Matsumoto et al ldquoThe effect of calciumion concentration on osteoblast viability proliferation anddifferentiation in monolayer and 3D culturerdquo Biomaterials vol26 no 23 pp 4847ndash4855 2005

[55] G R Beck Jr E C Sullivan E Moran and B ZerlerldquoRelationship between alkaline phosphatase levels osteopontinexpression and mineralization in differentiating MC3T3-E1osteoblastsrdquo Journal of Cellular Biochemistry vol 68 no 2 pp269ndash280 1998

[56] G R Beck Jr ldquoInorganic phosphate as a signaling molecule inosteoblast differentiationrdquo Journal of Cellular Biochemistry vol90 no 2 pp 234ndash243 2003

[57] R Roskoski Jr ldquoERK12 MAP kinases structure function andregulationrdquo Pharmacological Research vol 66 no 2 pp 105ndash143 2012

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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


body fluid (SBF) (mimicking naturally HA forming) plasmaspraying sol-gel deposition in HA particles solution pulsedlaser deposition hot isostatic pressing and electrochemicaldeposition [9ndash16] HA coating by electrochemical depositionwas shown to possess excellent corrosion protection and goodbiocompatibility under host environment [17] Advantagesof this technique are that it is relatively inexpensive andthickness of the coating can be easily controlled Anotherconcern point is that mechanical bonding strength of the HAcoating should withstand the bone growth stresses inducedduring the bone healing process [18]The adhesion of electro-deposited HA on smooth Ti surface was observed to be veryweek whichmay cause implant loosening [19ndash22]Thereforeto increase the adhesion strength betweenHA and Ti surfaceone suggested method is to use TiO

2nanotube array as a

substrate for HA electrodeposition In addition to improvingbonding strength HA grown on TiO

2nanotube array is more

favorable to osteoblast (or bone-forming cell) growth anddifferentiation when compared with naturally formed TiO

2

layer on conventional Ti It was also found that nanostructuresurface could promote deposition of bone minerals inducedby osteoblasts [23]

TiO2nanotubular can be formed by several methods

such as sol-gel method [24] electrophoretic deposition [25]and anodization [26] These methods could form differenttube alignment on surface The vertically aligned TiO

2

nanotube arrays can be achieved by anodizationmethodwithvarious parameters (electrolytes voltages etc) [27 28] In thepresent study anodization using pulse voltage and neutral-viscous fluoride-containing electrolyte was investigated toachieve a uniform well-oriented TiO

2nanotube arrays (ATi)

The previous study shows that alkaline surface treatment onATi and subsequently annealing of HA-electrodeposited ATiat high temperature (gt300∘C) improve the bonding strengthbetween HA and ATi [29] However the results from anotherstudy suggested that annealed HA grown in simulated bodyfluid (SBF) at high temperature (gt300∘C) was not alwaysnecessary because HA grown in SBF still possesses the CaPratio of 167 closelymimicking natural boneminerals [30 31]

In the present study HA coatings were coated onto alka-line pretreated TiO

2nanotubes The anodization conditions

included two different values of pulse voltage (+20minus4 and+35minus4V) and temperatures (5 and 25∘C) The influence ofalkaline pretreatment on ATi and physical structures (egwall thickness diameter and length) of TiO

2nanotubes on

the growth of bioinspired HA was reported The mechanicalbonding strength of HA on its substrates was studied asan in vitro early assessment of long-term stability in thehuman body The physical and chemical characteristics (egstructure size and a ratio of CaP) of HA were investigatedto elucidate the effect of using this biomaterial to increase thepreosteoblast viability under standard cell culture conditions

2 Experimental Materials and Methods

21 Forming TiO2 Nanotube Arrays Ti plate (Supra AlloysInc USA) of 999 purity was polished using silicon

carbide papers (TOA Thailand) of progressively decreasingroughness of 400 600 800 1000 1500 and 2000 grits Thesamples were then polished with 005 120583m alumina powder(Allied USA) to achieve a mirror quality finish Freshlypolished Ti was then washed with deionized (DI) water andsonicated in acetone (Sigma-Aldrich Thailand) and 95 volethanol (Sigma-Aldrich Thailand) for five minutes eachbefore immediate anodization

Anodizationmethodwas used to produce TiO2nanotube

arrays Ti was used as a positive electrode whereas platinumwas used as a negative electrodeThe electrolytewas amixtureof 90 vol glycerol and 10 vol ammonium fluoride (NH

4F)

(036M) in water Anodization was performed with the pulsevoltage of either +20minus4 or +35minus4V the conditions from aprevious study by Chanmanee et al [32] The anodizationwas carried out at either 5∘C or 25∘C for 90 minutes Thenanodized sample was washed with deionized water before itwas dried with nitrogen gas at room temperature All ATisamples were then annealed at 450∘C for 30minutes to obtainthe anatase phase

22 Electrodeposition of Hydroxyapatite ATi samples werepretreated with 1M NaOH at 50∘C for two minutes Afterthe pretreatment the HA deposition was conducted Theelectrolyte for HA deposition was prepared by dissolv-ing ammonium phosphate (NH

4H2PO4 167mM) (Sigma-

Aldrich Thailand) and calcium nitrate (Ca(NO3)2 25mM)

(Sigma-AldrichThailand) in distilledwater To increase ionicconductivity and to buffer pH of electrolyte at 72 015MNaCl (Ajax Finechem New Zealand) tris(hydroxyl amino-methane) (Ajax Finechem New Zealand) and hydrochloricacid (Ajax Finechem New Zealand) were mixed [29] Thepretreatment ATi (P-ATi) was used as a negative electrodewhereas platinumwas used as a positive electrode Electrode-position of HA was processed at minus25 V and at 80∘C for 10minutes

23 Physical and Chemical Characterizations of AnodizedTitanium and Hydroxyapatite Coating Scanning electronmicroscopy (FE-SEM CamScanMX2600 UK) was used toinvestigate surface morphology of ATi The dimensions ofthe TiO

2nanotubes were measured using image analysis

software (ImageJ version 132 NIH)The crystal structures ofthe surfaces of the NaOH-pretreated ATi and the HA-coatedsamples were investigated byX-ray diffractometer (ShimadzuModel XRD 6000 Japan)

24 The Mechanical Properties of Coated Hydroxyapatite onAnodized Titanium The mechanical bonding strength ofcoated HA on ATi was evaluated by tensile testing followingan ASTM F 1044-05 standard [33] Two HA-coated sampleswere glued together face-to-face using Plasmatex Klebbiadhesive (Plasma-Technik AG Switzerland) and cured at190∘C for two hours Tensile tests were conducted bya computer-controlled universal testing machine (InstronModel 5500R US) at a cross head speed of 025 centimeter




=


(1)



















2nanotube



2at higher






2






500nm

500nm

(a)

500nm

500nm

(b)

500nm

500nm

(c)

500nm

500nm

(d)


2films






NH4





2+ 4H+ + 4eminus (3)



6]2minus

+ 2H2O (5)


4


adsorbing TiO2(NH4

+) The role of TiO

2(NH4

+) is to protect


TiO2+NH

4

+997888rarr TiO

2(NH4

+)ads (6)













2induced by the








lowast

lowast

5∘ C

+20

minus4

V

5∘ C

+35

minus4

V

25∘ C

+20

minus4

V

25∘ C

+35

minus4

V

0

400

800

1200

1600

Dist

ance

(nm

)




2by fluorine ions




2Ti5O11

or Na2Ti6O13) was
















2+ 2OHminus (7)

OHminus +H2PO4

minus997888rarr HPO

4

2minus+H2O (8)

HPO4


4

3minus+H2O (9)

10Ca2+ + 6PO4


997888rarr Ca10(PO4)6(OH)2

(10)






0

325

65

975

13

Rela

tive f

requ

ency

80ndash1

00

101

ndash120

121

ndash140

141

ndash160

161

ndash180

181

ndash200

201

ndash220

221

ndash240

X = 175nm

(a)


0

325

65

975

13

Relat

ive f

requ

ency

60

ndash80

81ndash1

00

101

ndash120

121

ndash140

141

ndash160

X = 107nm

(b)


0

325

65

975

13

Relat

ive f

requ

ency

100

ndash120

121

ndash140

141

ndash160

161

ndash180

181

ndash200

201

ndash220

X = 155nm

(c)


0

325

65

975

13Re

lativ

e fre

quen

cy

100

ndash101

201

ndash300

301

ndash400

401

ndash500

501

ndash600

X = 316nm

(d)







Ca2+ and PO4


10(PO4)6(OH)2 Ca2(PO4)3sdot 119899H2O


4sdot2H2O [41]




0

325

65

975

13

Relat

ive f

requ

ency

181

ndash200

201

ndash220

221

ndash240

X = 171nm

141ndash

160

161ndash

180

120ndash

140

(a)

13

975

65

325

0

Relat

ive f

requ

ency


50

ndash100

101

ndash150

151

ndash200

201

ndash250

251

ndash300

301

ndash350

X = 194nm

(b)

13

975

65

325

0

Relat

ive f

requ

ency


60

ndash80

81ndash1

00

101

ndash120

141

ndash160

161

ndash180

X = 105nm

121

ndash140

(c)

13

975

65

325

0

Relat

ive f

requ

ency


100

ndash120

121

ndash140

141

ndash160

161

ndash180

201

ndash220

181

ndash200

221

ndash240

X = 151nm

(d)









(Figure 4(d))

(Figure 3(d))

(Figure 4(c))

(Figure 3(c))

(Figure 3(b))

(Figure 4(a))

(Figure 3(a))

(Figure 4(b))









HA-Ti (control)

HA-P-Ti (control)

lowast Anatase

lowast

10 20 30 40 50 60 70 80 9002120579 (degrees)

0

200

400

600

800

1000

1200

1400

1600

Inte

nsity

(arb

itrar

y un

it)






2interface as











2array is stronger







127

lowast

lowast

0

55

11

165

22

Bond

ing

stren

gth

(MPa

)

(a)

Titanium substrate

Titanium substrate

GlueCoating

Specimen components


interface


force

Cohesive force

∙ Gluecoating


(b)



(c)








lowast

Ti ATi P-ATi

HA-ATi HA-P-ATi

(b) (c) (d)

(e) (f)50120583m50120583m

50120583m50120583m 50120583m

(a)

0

200

400

600

800

ce

ll vi

abili

ty











4 Conclusions




Competing Interests


Acknowledgments


References











































2nanotubes and










2O onmetal oxides






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






=


(1)



















2nanotube



2at higher






2






500nm

500nm

(a)

500nm

500nm

(b)

500nm

500nm

(c)

500nm

500nm

(d)


2films






NH4





2+ 4H+ + 4eminus (3)



6]2minus

+ 2H2O (5)


4


adsorbing TiO2(NH4

+) The role of TiO

2(NH4

+) is to protect


TiO2+NH

4

+997888rarr TiO

2(NH4

+)ads (6)













2induced by the








lowast

lowast

5∘ C

+20

minus4

V

5∘ C

+35

minus4

V

25∘ C

+20

minus4

V

25∘ C

+35

minus4

V

0

400

800

1200

1600

Dist

ance

(nm

)




2by fluorine ions




2Ti5O11

or Na2Ti6O13) was
















2+ 2OHminus (7)

OHminus +H2PO4

minus997888rarr HPO

4

2minus+H2O (8)

HPO4


4

3minus+H2O (9)

10Ca2+ + 6PO4


997888rarr Ca10(PO4)6(OH)2

(10)






0

325

65

975

13

Rela

tive f

requ

ency

80ndash1

00

101

ndash120

121

ndash140

141

ndash160

161

ndash180

181

ndash200

201

ndash220

221

ndash240

X = 175nm

(a)


0

325

65

975

13

Relat

ive f

requ

ency

60

ndash80

81ndash1

00

101

ndash120

121

ndash140

141

ndash160

X = 107nm

(b)


0

325

65

975

13

Relat

ive f

requ

ency

100

ndash120

121

ndash140

141

ndash160

161

ndash180

181

ndash200

201

ndash220

X = 155nm

(c)


0

325

65

975

13Re

lativ

e fre

quen

cy

100

ndash101

201

ndash300

301

ndash400

401

ndash500

501

ndash600

X = 316nm

(d)







Ca2+ and PO4


10(PO4)6(OH)2 Ca2(PO4)3sdot 119899H2O


4sdot2H2O [41]




0

325

65

975

13

Relat

ive f

requ

ency

181

ndash200

201

ndash220

221

ndash240

X = 171nm

141ndash

160

161ndash

180

120ndash

140

(a)

13

975

65

325

0

Relat

ive f

requ

ency


50

ndash100

101

ndash150

151

ndash200

201

ndash250

251

ndash300

301

ndash350

X = 194nm

(b)

13

975

65

325

0

Relat

ive f

requ

ency


60

ndash80

81ndash1

00

101

ndash120

141

ndash160

161

ndash180

X = 105nm

121

ndash140

(c)

13

975

65

325

0

Relat

ive f

requ

ency


100

ndash120

121

ndash140

141

ndash160

161

ndash180

201

ndash220

181

ndash200

221

ndash240

X = 151nm

(d)









(Figure 4(d))

(Figure 3(d))

(Figure 4(c))

(Figure 3(c))

(Figure 3(b))

(Figure 4(a))

(Figure 3(a))

(Figure 4(b))









HA-Ti (control)

HA-P-Ti (control)

lowast Anatase

lowast

10 20 30 40 50 60 70 80 9002120579 (degrees)

0

200

400

600

800

1000

1200

1400

1600

Inte

nsity

(arb

itrar

y un

it)






2interface as











2array is stronger







127

lowast

lowast

0

55

11

165

22

Bond

ing

stren

gth

(MPa

)

(a)

Titanium substrate

Titanium substrate

GlueCoating

Specimen components


interface


force

Cohesive force

∙ Gluecoating


(b)



(c)








lowast

Ti ATi P-ATi

HA-ATi HA-P-ATi

(b) (c) (d)

(e) (f)50120583m50120583m

50120583m50120583m 50120583m

(a)

0

200

400

600

800

ce

ll vi

abili

ty











4 Conclusions




Competing Interests


Acknowledgments


References











































2nanotubes and










2O onmetal oxides






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




500nm

500nm

(a)

500nm

500nm

(b)

500nm

500nm

(c)

500nm

500nm

(d)


2films






NH4





2+ 4H+ + 4eminus (3)



6]2minus

+ 2H2O (5)


4


adsorbing TiO2(NH4

+) The role of TiO

2(NH4

+) is to protect


TiO2+NH

4

+997888rarr TiO

2(NH4

+)ads (6)













2induced by the








lowast

lowast

5∘ C

+20

minus4

V

5∘ C

+35

minus4

V

25∘ C

+20

minus4

V

25∘ C

+35

minus4

V

0

400

800

1200

1600

Dist

ance

(nm

)




2by fluorine ions




2Ti5O11

or Na2Ti6O13) was
















2+ 2OHminus (7)

OHminus +H2PO4

minus997888rarr HPO

4

2minus+H2O (8)

HPO4


4

3minus+H2O (9)

10Ca2+ + 6PO4


997888rarr Ca10(PO4)6(OH)2

(10)






0

325

65

975

13

Rela

tive f

requ

ency

80ndash1

00

101

ndash120

121

ndash140

141

ndash160

161

ndash180

181

ndash200

201

ndash220

221

ndash240

X = 175nm

(a)


0

325

65

975

13

Relat

ive f

requ

ency

60

ndash80

81ndash1

00

101

ndash120

121

ndash140

141

ndash160

X = 107nm

(b)


0

325

65

975

13

Relat

ive f

requ

ency

100

ndash120

121

ndash140

141

ndash160

161

ndash180

181

ndash200

201

ndash220

X = 155nm

(c)


0

325

65

975

13Re

lativ

e fre

quen

cy

100

ndash101

201

ndash300

301

ndash400

401

ndash500

501

ndash600

X = 316nm

(d)







Ca2+ and PO4


10(PO4)6(OH)2 Ca2(PO4)3sdot 119899H2O


4sdot2H2O [41]




0

325

65

975

13

Relat

ive f

requ

ency

181

ndash200

201

ndash220

221

ndash240

X = 171nm

141ndash

160

161ndash

180

120ndash

140

(a)

13

975

65

325

0

Relat

ive f

requ

ency


50

ndash100

101

ndash150

151

ndash200

201

ndash250

251

ndash300

301

ndash350

X = 194nm

(b)

13

975

65

325

0

Relat

ive f

requ

ency


60

ndash80

81ndash1

00

101

ndash120

141

ndash160

161

ndash180

X = 105nm

121

ndash140

(c)

13

975

65

325

0

Relat

ive f

requ

ency


100

ndash120

121

ndash140

141

ndash160

161

ndash180

201

ndash220

181

ndash200

221

ndash240

X = 151nm

(d)









(Figure 4(d))

(Figure 3(d))

(Figure 4(c))

(Figure 3(c))

(Figure 3(b))

(Figure 4(a))

(Figure 3(a))

(Figure 4(b))









HA-Ti (control)

HA-P-Ti (control)

lowast Anatase

lowast

10 20 30 40 50 60 70 80 9002120579 (degrees)

0

200

400

600

800

1000

1200

1400

1600

Inte

nsity

(arb

itrar

y un

it)






2interface as











2array is stronger







127

lowast

lowast

0

55

11

165

22

Bond

ing

stren

gth

(MPa

)

(a)

Titanium substrate

Titanium substrate

GlueCoating

Specimen components


interface


force

Cohesive force

∙ Gluecoating


(b)



(c)








lowast

Ti ATi P-ATi

HA-ATi HA-P-ATi

(b) (c) (d)

(e) (f)50120583m50120583m

50120583m50120583m 50120583m

(a)

0

200

400

600

800

ce

ll vi

abili

ty











4 Conclusions




Competing Interests


Acknowledgments


References











































2nanotubes and










2O onmetal oxides






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





lowast

lowast

5∘ C

+20

minus4

V

5∘ C

+35

minus4

V

25∘ C

+20

minus4

V

25∘ C

+35

minus4

V

0

400

800

1200

1600

Dist

ance

(nm

)




2by fluorine ions




2Ti5O11

or Na2Ti6O13) was
















2+ 2OHminus (7)

OHminus +H2PO4

minus997888rarr HPO

4

2minus+H2O (8)

HPO4


4

3minus+H2O (9)

10Ca2+ + 6PO4


997888rarr Ca10(PO4)6(OH)2

(10)






0

325

65

975

13

Rela

tive f

requ

ency

80ndash1

00

101

ndash120

121

ndash140

141

ndash160

161

ndash180

181

ndash200

201

ndash220

221

ndash240

X = 175nm

(a)


0

325

65

975

13

Relat

ive f

requ

ency

60

ndash80

81ndash1

00

101

ndash120

121

ndash140

141

ndash160

X = 107nm

(b)


0

325

65

975

13

Relat

ive f

requ

ency

100

ndash120

121

ndash140

141

ndash160

161

ndash180

181

ndash200

201

ndash220

X = 155nm

(c)


0

325

65

975

13Re

lativ

e fre

quen

cy

100

ndash101

201

ndash300

301

ndash400

401

ndash500

501

ndash600

X = 316nm

(d)







Ca2+ and PO4


10(PO4)6(OH)2 Ca2(PO4)3sdot 119899H2O


4sdot2H2O [41]




0

325

65

975

13

Relat

ive f

requ

ency

181

ndash200

201

ndash220

221

ndash240

X = 171nm

141ndash

160

161ndash

180

120ndash

140

(a)

13

975

65

325

0

Relat

ive f

requ

ency


50

ndash100

101

ndash150

151

ndash200

201

ndash250

251

ndash300

301

ndash350

X = 194nm

(b)

13

975

65

325

0

Relat

ive f

requ

ency


60

ndash80

81ndash1

00

101

ndash120

141

ndash160

161

ndash180

X = 105nm

121

ndash140

(c)

13

975

65

325

0

Relat

ive f

requ

ency


100

ndash120

121

ndash140

141

ndash160

161

ndash180

201

ndash220

181

ndash200

221

ndash240

X = 151nm

(d)









(Figure 4(d))

(Figure 3(d))

(Figure 4(c))

(Figure 3(c))

(Figure 3(b))

(Figure 4(a))

(Figure 3(a))

(Figure 4(b))









HA-Ti (control)

HA-P-Ti (control)

lowast Anatase

lowast

10 20 30 40 50 60 70 80 9002120579 (degrees)

0

200

400

600

800

1000

1200

1400

1600

Inte

nsity

(arb

itrar

y un

it)






2interface as











2array is stronger







127

lowast

lowast

0

55

11

165

22

Bond

ing

stren

gth

(MPa

)

(a)

Titanium substrate

Titanium substrate

GlueCoating

Specimen components


interface


force

Cohesive force

∙ Gluecoating


(b)



(c)








lowast

Ti ATi P-ATi

HA-ATi HA-P-ATi

(b) (c) (d)

(e) (f)50120583m50120583m

50120583m50120583m 50120583m

(a)

0

200

400

600

800

ce

ll vi

abili

ty











4 Conclusions




Competing Interests


Acknowledgments


References











































2nanotubes and










2O onmetal oxides






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





0

325

65

975

13

Rela

tive f

requ

ency

80ndash1

00

101

ndash120

121

ndash140

141

ndash160

161

ndash180

181

ndash200

201

ndash220

221

ndash240

X = 175nm

(a)


0

325

65

975

13

Relat

ive f

requ

ency

60

ndash80

81ndash1

00

101

ndash120

121

ndash140

141

ndash160

X = 107nm

(b)


0

325

65

975

13

Relat

ive f

requ

ency

100

ndash120

121

ndash140

141

ndash160

161

ndash180

181

ndash200

201

ndash220

X = 155nm

(c)


0

325

65

975

13Re

lativ

e fre

quen

cy

100

ndash101

201

ndash300

301

ndash400

401

ndash500

501

ndash600

X = 316nm

(d)







Ca2+ and PO4


10(PO4)6(OH)2 Ca2(PO4)3sdot 119899H2O


4sdot2H2O [41]




0

325

65

975

13

Relat

ive f

requ

ency

181

ndash200

201

ndash220

221

ndash240

X = 171nm

141ndash

160

161ndash

180

120ndash

140

(a)

13

975

65

325

0

Relat

ive f

requ

ency


50

ndash100

101

ndash150

151

ndash200

201

ndash250

251

ndash300

301

ndash350

X = 194nm

(b)

13

975

65

325

0

Relat

ive f

requ

ency


60

ndash80

81ndash1

00

101

ndash120

141

ndash160

161

ndash180

X = 105nm

121

ndash140

(c)

13

975

65

325

0

Relat

ive f

requ

ency


100

ndash120

121

ndash140

141

ndash160

161

ndash180

201

ndash220

181

ndash200

221

ndash240

X = 151nm

(d)









(Figure 4(d))

(Figure 3(d))

(Figure 4(c))

(Figure 3(c))

(Figure 3(b))

(Figure 4(a))

(Figure 3(a))

(Figure 4(b))









HA-Ti (control)

HA-P-Ti (control)

lowast Anatase

lowast

10 20 30 40 50 60 70 80 9002120579 (degrees)

0

200

400

600

800

1000

1200

1400

1600

Inte

nsity

(arb

itrar

y un

it)






2interface as











2array is stronger







127

lowast

lowast

0

55

11

165

22

Bond

ing

stren

gth

(MPa

)

(a)

Titanium substrate

Titanium substrate

GlueCoating

Specimen components


interface


force

Cohesive force

∙ Gluecoating


(b)



(c)








lowast

Ti ATi P-ATi

HA-ATi HA-P-ATi

(b) (c) (d)

(e) (f)50120583m50120583m

50120583m50120583m 50120583m

(a)

0

200

400

600

800

ce

ll vi

abili

ty











4 Conclusions




Competing Interests


Acknowledgments


References











































2nanotubes and










2O onmetal oxides






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





0

325

65

975

13

Relat

ive f

requ

ency

181

ndash200

201

ndash220

221

ndash240

X = 171nm

141ndash

160

161ndash

180

120ndash

140

(a)

13

975

65

325

0

Relat

ive f

requ

ency


50

ndash100

101

ndash150

151

ndash200

201

ndash250

251

ndash300

301

ndash350

X = 194nm

(b)

13

975

65

325

0

Relat

ive f

requ

ency


60

ndash80

81ndash1

00

101

ndash120

141

ndash160

161

ndash180

X = 105nm

121

ndash140

(c)

13

975

65

325

0

Relat

ive f

requ

ency


100

ndash120

121

ndash140

141

ndash160

161

ndash180

201

ndash220

181

ndash200

221

ndash240

X = 151nm

(d)









(Figure 4(d))

(Figure 3(d))

(Figure 4(c))

(Figure 3(c))

(Figure 3(b))

(Figure 4(a))

(Figure 3(a))

(Figure 4(b))









HA-Ti (control)

HA-P-Ti (control)

lowast Anatase

lowast

10 20 30 40 50 60 70 80 9002120579 (degrees)

0

200

400

600

800

1000

1200

1400

1600

Inte

nsity

(arb

itrar

y un

it)






2interface as











2array is stronger







127

lowast

lowast

0

55

11

165

22

Bond

ing

stren

gth

(MPa

)

(a)

Titanium substrate

Titanium substrate

GlueCoating

Specimen components


interface


force

Cohesive force

∙ Gluecoating


(b)



(c)








lowast

Ti ATi P-ATi

HA-ATi HA-P-ATi

(b) (c) (d)

(e) (f)50120583m50120583m

50120583m50120583m 50120583m

(a)

0

200

400

600

800

ce

ll vi

abili

ty











4 Conclusions




Competing Interests


Acknowledgments


References











































2nanotubes and










2O onmetal oxides






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





(Figure 4(d))

(Figure 3(d))

(Figure 4(c))

(Figure 3(c))

(Figure 3(b))

(Figure 4(a))

(Figure 3(a))

(Figure 4(b))









HA-Ti (control)

HA-P-Ti (control)

lowast Anatase

lowast

10 20 30 40 50 60 70 80 9002120579 (degrees)

0

200

400

600

800

1000

1200

1400

1600

Inte

nsity

(arb

itrar

y un

it)






2interface as











2array is stronger







127

lowast

lowast

0

55

11

165

22

Bond

ing

stren

gth

(MPa

)

(a)

Titanium substrate

Titanium substrate

GlueCoating

Specimen components


interface


force

Cohesive force

∙ Gluecoating


(b)



(c)








lowast

Ti ATi P-ATi

HA-ATi HA-P-ATi

(b) (c) (d)

(e) (f)50120583m50120583m

50120583m50120583m 50120583m

(a)

0

200

400

600

800

ce

ll vi

abili

ty











4 Conclusions




Competing Interests


Acknowledgments


References











































2nanotubes and










2O onmetal oxides






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






127

lowast

lowast

0

55

11

165

22

Bond

ing

stren

gth

(MPa

)

(a)

Titanium substrate

Titanium substrate

GlueCoating

Specimen components


interface


force

Cohesive force

∙ Gluecoating


(b)



(c)








lowast

Ti ATi P-ATi

HA-ATi HA-P-ATi

(b) (c) (d)

(e) (f)50120583m50120583m

50120583m50120583m 50120583m

(a)

0

200

400

600

800

ce

ll vi

abili

ty











4 Conclusions




Competing Interests


Acknowledgments


References











































2nanotubes and










2O onmetal oxides






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





lowast

Ti ATi P-ATi

HA-ATi HA-P-ATi

(b) (c) (d)

(e) (f)50120583m50120583m

50120583m50120583m 50120583m

(a)

0

200

400

600

800

ce

ll vi

abili

ty











4 Conclusions




Competing Interests


Acknowledgments


References











































2nanotubes and










2O onmetal oxides






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials









4 Conclusions




Competing Interests


Acknowledgments


References











































2nanotubes and










2O onmetal oxides






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



































2nanotubes and










2O onmetal oxides






























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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Research Article Improved Bonding Strength of ...downloads.hindawi.com/journals/jnm/2016/9143969.pdf · Titanium Dioxide Nanotube Arrays following Alkaline Pretreatment for Orthopedic