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PROGRAMA DE PÓS-GRADUAÇÃO EM BIOMATERIAIS E BIOLOGIA ORAL CONVIDA:

PALESTRA INTERNACIONAL

DATA: 08 DE NOVEMBRO DE 2011.

LOCAL: Departamento de Materiais Dentários da FOUSP

HORÁRIO: 10:00.

A palestra é gratuita.

Não há necessidade de inscrição.

Contato: Prof. Paulo Francisco Cesar ([email protected])

Palestrante: Fernando Jorge Monteiro.

Instituição: Instituto Nacional de Engenharia Biomédica (INEB), Faculdade de

Engenharia da Universidade do Porto, Portugal.

Título: “Revestimentos de sílica bifuncionais, bioativos e microestruturados sobre

materiais dentários”**

**Referência em anexo:

Isotropic micropatterned silica coatings on zirconia induce guided cell growth for

dental implants. Pelaez-Vargas A, Gallego-Perez D, Magallanes-Perdomo M, Fernandes

MH, Hansford DJ, De Aza AH, Pena P, Monteiro FJ. Dent Mater. 2011 Jun;27(6):581-9.

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ARTICLE IN PRESSENTAL-1803; No. of Pages 9

d e n t a l m a t e r i a l s x x x ( 2 0 1 1 ) xxx–xxx

avai lab le at www.sc iencedi rec t .com

journa l homepage: www. int l .e lsev ierhea l th .com/ journa ls /dema

sotropic micropatterned silica coatings on zirconia induceuided cell growth for dental implants

. Pelaez-Vargasa,b,c,∗, D. Gallego-Perezc, M. Magallanes-Perdomod, M.H. Fernandese,

.J. Hansfordc, A.H. De Azad, P. Penad, F.J. Monteiroa,b

INEB – Instituto de Engenharia Biomédica, Divisão de Biomateriais, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto,ortugalDepartamento de Engenharia Metalúrgica e Materiais, FEUP – Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias

/n, 4200-465 Porto, PortugalDepartment of Biomedical Engineering, The Ohio State University, 1080 Carmack Rd., Columbus, OH, USAInstituto de Cerámica y Vidrio, CSIC, C/Kelsen, 5, 28049 Madrid, SpainLaboratório de Farmacologia e Biocompatibilidade Celular, Faculdade de Medicina Dentária, Universidade do Porto, Rua Dr. Manuelereira da Silva, 4200-393 Porto, Portugal

r t i c l e i n f o

rticle history:

eceived 23 July 2010

eceived in revised form

4 February 2011

ccepted 24 February 2011

vailable online xxx

eywords:

irconia

oft lithography

ilica coatings

steoblasts

ol–gel

a b s t r a c t

Titanium implants are the gold standard in dentistry; however, problems such as gingival

tarnishing and peri-implantitis have been reported. For zirconia to become a competitive

alternative dental implant material, surface modification techniques that induce guided

tissue growth must be developed.

Objectives. To develop alternative surface modification techniques to promote guided tissue

regeneration on zirconia materials, for applications in dental implantology.

Methods. A methodology that combined soft lithography and sol–gel chemistry was used

to obtain isotropic micropatterned silica coatings on yttria-stabilized zirconia substrates.

The materials were characterized via chemical, structural, surface morphology approaches.

In vitro biological behavior was evaluated in terms of early adhesion and viability/metabolic

activity of human osteoblast-like cells. Statistical analysis was conducted using one-way

ANOVA/Tukey HSD post hoc test.

Results. Isotropic micropatterned silica coatings on yttria-stabilized zirconia substrates were

obtained using a combined approach based on sol–gel technology and soft lithography.

Micropatterned silica surfaces exhibited a biocompatible behavior, and modulated cell

responses (i.e. inducing early alignment of osteoblast-like cells). After 7 d of culture, the

cells fully covered the top surfaces of pillar microstructured silica films.

Significance. The micropatterned silica films on zirconia showed a biocompatible response,

and were capable of inducing guided osteoblastic cell adhesion, spreading and propaga-

in p
tion. The results here
Please cite this article in press as: Pelaez-Vargas A, et al. Isotropic microdental implants. Dent Mater (2011), doi:10.1016/j.dental.2011.02.014

lithography and sol–gel c

regeneration, thus promo

peri-implantitis.

© 2011 Academy

∗ Corresponding author at: INEB – Instituto de Engenharia Biomédica, Uortugal. Tel.: +351 226074900; fax: +351 226094567.

E-mail address: [email protected] (A. Pelaez-Vargas).109-5641/$ – see front matter © 2011 Academy of Dental Materials. Puoi:10.1016/j.dental.2011.02.014

resented suggest that surface-modified ceramic implants via soft

patterned silica coatings on zirconia induce guided cell growth for

hemistry could potentially be used to guide periodontal tissue

ting tight tissue apposition, and avoiding gingival retraction and

of Dental Materials. Published by Elsevier Ltd. All rights reserved.

niversidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto,

blished by Elsevier Ltd. All rights reserved.

dx.doi.org/10.1016/j.dental.2011.02.014


mailto:[email protected]


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1. Introduction

Commercially pure titanium (Ti) is the biomaterial of choicefor dental implants because it is a relatively inexpensivematerial, promotes osseointegration, has well-documentedbiocompatibility, self-passivation, strength, and conforma-bility [1]. However, it has also been reported that Ti couldexhibit problems such as potential allergenic response andpoor esthetics [2,3]. Since the introduction of titanium dentalimplants, many other materials have been explored as poten-tial implant candidates, including zirconia, alumina, tantalumand niobium, but some have never been commercialized, andthe others have achieved very limited commercial success[4–10].

Many patients present large challenges for clinicians,including those requiring implant-supported dental singlecrowns in the anterior segment and patients with highlydemanding esthetic conditions due to the presence of highline upper lip, a thin gingival biotype, or intact natural adjacentteeth [11]. The most common solutions include titanium-based dental implants, metallic or ceramic abutments, andall ceramic crowns (e.g., alumina, zirconia or combination)[12].

Zirconia has been studied due to its potential use indentistry as inlays, onlays, full crowns, bridges, abutments,intra-root posts, scaffolds and implants [4,13–18]. No cyto-toxic or mutagenic effects have been reported, and it hasbeen shown to exhibit osseointegration as well as low inflam-matory infiltrate in gingival tissues [15,19–24]. However, onlypreliminary results have been published, and long term stud-ies are not available. Therefore, further research is requiredto develop an effective and competitive zirconia-based mate-rial candidate as a clinical alternative to titanium implants.Since the initial reports in 1980, dental ceramic implants haveshown superior esthetic behavior, as an adequate appearanceof natural teeth could be obtained; however, their mechanicalproperties have been questionable [6].

For orthopedic and dental applications, a reduction in thecatastrophic failure of all-ceramic restorations was obtainedwith the introduction of yttria-stabilized tetragonal zirconiapolycrystalline (3Y-TZP) [25,26]. However, based on investiga-tions of the use of 3Y-TZP in orthopedics, under physiologicalconditions, yttrium may leach from the ceramic and conse-quently the zirconia may lose its transformational toughening(its crystal structure changes back from tetragonal (t) to mon-oclinic (m) phases, known as “aging”) [27–31]. After more than20 years of research, aging still remains a key issue for zirco-nia implants, with aging of zirconia orthopedic devices (e.g.femoral heads) being by far the most studied example. Agingoccurs by a water-assisted martensitic phase transformation,which propagates at the surface by a nucleation-and-growthmechanism and then invades the bulk structure. Therefore,zirconia could be prone to aging in the presence of humansaliva. Aging results in roughening and microcracking, in themost extreme cases, it leads to catastrophic failure of the com-


ponent [12]. Considering that the use of 3Y-TZP as a dentalmaterial is still increasing [32] and that its low temperaturedegradation is still latent, further extensive clinical researchis necessary to achieve a risk reduction [33].

PRESSx ( 2 0 1 1 ) xxx–xxx

Advanced ceramic processing to obtain 3Y-TZP materials isa demanding process. Tailoring the microstructure by refiningpowder processing can result in zirconia dental structures atthe top end of their strength spectrum [34]. However, since theintroduction of 3Y-TZP as a material for dental implants, onlya limited number of reports concerning surface modificationstrategies have been produced, and the know-how availablefrom decades of surface modifications of titanium implants isnot directly applicable [35].

Currently, surface modification techniques are based onthe removal of material through low energy processing suchas etching (chemical or electrochemical), or mechanical tech-niques such as grinding, sandblasting or ablation by electronsor lasers [36–41]. These alternatives, however, can lead toyttria leaching, phase transformation and subsequent catas-trophic failure. Alternative additive approaches can be costly,present adhesion problems and introduce heterogeneities[42–46].

This work explored the use of sol–gel processing as aviable approach to modify 3Y-TZP surfaces, based on its ver-satility to yield materials with high chemical homogeneityand fine grained or amorphous microstructure using lowertemperatures, and its ease of implementation for mass pro-duction [7–10,47–51]. In addition, these characteristics alsoallow for the combination with PDMS (polydimethylsiloxane)microstamping via soft lithography; a technique extensivelyused in the last decade to produce ordered (isotropic oranisotropic) or randomized features, to engineer surfaces[52,53] that can modulate cell behavior. Such surfaces havethe potential to induce positive cellular responses in terms ofmorphology, adhesion, migration, proliferation, and differen-tiation [53–68].

The present work describes the preparation of orderedmicropatterned silica thin films on 3Y-TZP, intended toenhance guided tissue regeneration on ceramic dentalimplants. In order to understand how such a combinationwould perform, materials characterization and in vitro biolog-ical behavior in terms of early adhesion and proliferation ofosteoblast-like cells were carried out.

2. Materials and methods

2.1. Substrate processing and characterization

A synthetic 3 mol% Y2O3–ZrO2 (3Y-TZP) powder having anearly uniform submicron particle size was used (TZ-3YS-E,Tosoh, Tokyo, Japan). Chemical analysis of the powder wascarried out by X-ray fluorescence spectroscopy (XRF, Magi X;Phillips, The Netherlands) and flame emission spectroscopyfor alkaline analyses (FES, 2100; Perkin Elmer) to verify thevalues provided by the supplier. The specific surface area ofthe powder was determined by the chromatographic method(Monosorb Surface Area Analyzer MS-13, Quantachrome Cor-poration, UK) using a BET isotherm. Particle size distributionwas measured using a laser scattering size analyzer (Mas-


ter Sizer S; Malvern Instruments, UK). The particle sizemeasurements were done on slurry samples. Dolapix CE-64(Zschimmer & Schwarz GmbH & Co KG, Germany) was usedas dispersant. Dispersion and de-agglomeration of particles



ARTICLE IN PRESSDENTAL-1803; No. of Pages 9

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Fig. 1 – Schematic diagram of the microfabrication. (a) Silicon wafer, (b) photoresist film is deposited, (c) UV light exposuret PDMm

wm

atredidMwtfb5sdtd

dwvHs

2

Sut

hrough the mask, (d) patterned master, (e) PDMS casting, (f)icropatterned thin film on 3-YTZP.

ere further ensured through ultrasonic treatment prior toeasurement.Cylindrical specimens were produced via uniaxial pressing,

nd their sintering behavior was evaluated using a high resolu-ion dilatometer (Setsys 16/18, Setaram, France), at a heatingate of 5 ◦C/min from 20 ◦C to 1550 ◦C. Based on the thermalxpansion/contraction data (dilatometer curve, Fig. 2a), alliscs were sintered at 1480 ◦C for 2 h. Contraction of all spec-

mens was calculated based in the comparison between theiameters of the “green” discs and of the full sintered discs.aximum density was determined using the Archimedesater displacement method, while crystallography and full

ransformation from monoclinic to tetragonal phase wasollowed by XRD analysis (Siemens D5000, Germany). Visi-le Raman spectra (resolution = 4 cm−1, acquisition time = 5 s,14 nm laser) were recorded using a confocal Raman micro-cope (LabRAM HR800 UV, Horiba Jobin-Yvon, Villeneuve’Ascq, France), as a complement to confirm m → t phasesransformation on powders, fully sintered discs, and re-heatediscs (t → m phases transformation) [69].

The sintered samples were polished with a series ofiamond pastes down to 1 �m. Microstructure observationsere performed on polished and thermally etched surfacesia field emission scanning electron microscopy (FE-SEM,itachi-S4700, Tokyo, Japan, fitted with energy X-ray disper-

ive spectroscopy, EDS, Noran System, Japan).

.2. Thin film production


ilica sol was produced via acid catalysis in a single stepsing tetraethylorthosilicate (TEOS, Aldrich, USA) and methyl-riethoxysilane (MTES, Aldrich, USA) as precursors, following

S negative mold, (g) silica casting and drying, and (h) silica

a previously reported method [50]. The solution was aged for24 h and applied via spin coating (3000 rpm for 45 s) on 3Y-TZPbare (3Y-TZP) substrates to produce flat silica coatings (fSiO2).

Standard UV photolithography was used initially to cre-ate a silicon master with the pattern of interest. First, achromium photomask was designed using L-Edit software(Tanner EDA, USA) and produced at a mail order mask-makingfoundry (Martin Photomask Service, USA). A negative tone SU-8 2005 (Microchem Corp, USA) photoresist was spin-coatedon a silicon wafer at 3000 rpm to produce a ∼5 �m thick film(Fig. 1a and b). The photoresist was exposed to ultravioletlight through the photomask, and post-processed followingpreviously established parameters (Fig. 1c and d). Finally, themaster was used to create PDMS (Silastic T2, Dow Corning,USA) negative molds (Fig. 1e and f) with an ordered texturecomposed of wells with 5 �m diameter, 5 �m deep, and 5 �medge-edge spacing.

3Y-TZP substrates were cleaned and dried. Micropatternedsilica coatings (mpSiO2) were prepared by pouring (40 ul) thesol on the PDMS mold and then pressing against the sub-strate (Fig. 1g). After drying for 2 h, the mold was removed(Fig. 1h). This technique is a modification of the pioneeringwork by Marzolin et al. [70]. Afterwards, the samples were eval-uated under light microscopy, and then sintered at 500 ◦C for60 min [71] to produce micropatterned silica coating on 3Y-TZPsubstrates. The samples were characterized post-sintering viaSEM.


2.3. Cell culture

MG63 cells (ATCC, USA) were cultured in �-minimal essen-tial medium (�-MEM, Sigma, USA) containing 10% fetal bovine




4 d e n t a l m a t e r i a l s x x x ( 2 0 1 1 ) xxx–xxx

Fig. 2 – Thermal contraction, X-ray diffraction and Raman microanalysis. (a) Thermal contraction using a 5 ◦C/min heatingrate: absolute (dotted line) and differential contraction (continuous line). (b) XRD diffraction and (c) Raman spectra of rawcommercial powders, and fully sintered or re-heated 3Y-TZPdiscs. Labels on the peaks correspond to defined wavelengths

, i = 5
(a = 146, b = 176, c = 188, d = 218, e = 260, f = 330, g = 377, h = 471
serum (ATCC, USA), penicillin–streptomycin (100 IU/ml and10 mg/ml, respectively) (Sigma), gentamicin–amphotericin B(10 �g/ml and 0.25 �g/ml, Invitrogen, USA), ascorbic acid(50 �g/ml, Sigma), and l-glutamine (2 mM, Sigma). The cellswere seeded at a density of 2 × 104 cells/cm2 on bare and silicacoated (micropatterned and flat) zirconia and control groupsamples (tissue culture-treated polystyrene, TCP). All cultureswere incubated for 3 different time points (1, 4 and 7 d). Then,the cells were washed twice with 37 ◦C PBS, fixed in 10% (v/v)neutral buffered formalin (Ted Pella, USA) for 15 min, and pre-pared for SEM and epifluorescence microscopy.

2.4. Morphology and proliferation

For morphology evaluation via MIF (Eclipse TE2000-5, Nikon,Japan), the cells were washed and permeabilized with 0.1%(v/v) Triton X-100 for 30 min. F-actin filaments were stainedusing alexafluor phalloidin (Invitrogen, USA) for 30 min. Nucleiwere stained with a buffer of propidium iodide and RNase (BDPharmigen, USA) at 4 ◦C for 15 min and washed with PBS.

For morphology evaluation via SEM, the cells were dehy-drated in graded ethanol and hexamethyldisilazane (HMDS,Ted Pella, USA) solutions (50–100%), respectively [72]. The sam-ples were then gold-coated and visualized by SEM.

2.5. Viability/proliferation

Proliferation was evaluated using the alamar blue assay, a sim-ple, non-reactive assay based on the oxidation–reduction ofresuzarim, a non-fluorescent blue component that is reducedby living cells to a fluorescent pink component. Briefly, freshmedium with 10% alamar blue (v/v) (Sigma) was added, andincubated for 3 h. 100 �l of the medium was then transferredto a 96-well plate and a microplate reader (Infinite F-500,Tecan Trading AG, Switzerland) was used to quantify the flu-orescence (at 535 nm excitation wavelength and 590 emissionwavelength).

All cell culture experiments were carried out in duplicated


(n = 3) and results were expressed in relative to TCP Statisti-cal analysis of the viability/metabolic activity was performedusing one-way ANOVA/Tukey HSD post hoc test. Levels ofp < 0.05 were considered to be statistically significant.

40, j = 557, k = 614, and l = 640 cm−1).

3. Results

The 3Y-TZP powder presents a specific surface of 6.2 m2/g,with a mean grain size of 0.38 �m (d10 = 0.19 �m, d50 = 0.38 �mand d90 = 1.17 �m). The crystallite size reported by the supplierwas 36 nm. The result of the chemical analysis was (wt%(SD)oxide): 5.31(0.02) Y2O3; 0.244(0.003) Al2O3; 0.005(0.002) SiO2;<0.002 Fe2O3; and 0.006(0.002) N2O. These values agree withthe ones reported by the supplier. Small traces of HfO2 werealso detected.

Fig. 2a shows the sintering curve of “green” compacted 3Y-TZP powders. The graph shows that the contraction startedat around 1050 ◦C (Fig. 2a, single arrow), and it continued pro-gressively up to 1500 ◦C (Fig. 2a, triple arrow), with the highestcontraction reached at 1300 ◦C (Fig. 2a, double arrow). XRDanalysis carried out on raw 3Y-TZP powders (Fig. 2b) show twosmall peaks corresponding to the monoclinic phase (Fig. 2b,arrows) and all the peaks of the major tetragonal phase. Opti-mum sintering temperature, to ensure high densification andfull transformation of zirconia from monoclinic to tetrago-nal phase was established at 1480 ◦C (Fig. 2a, triple arrow), asconfirmed by density data and XRD (Fig. 2b). Raman spectra(Fig. 2c) exhibited characteristic peaks at 146, 176, 188, 218, 260,330, 377, 471 540, 557, 614 and 640 cm−1 for raw powders, andpeaks at 145, 260, 318, 400, 610 and 640 cm−1 for fully sinteredand re-heated discs, respectively (Fig. 2c).

Geometric density of 3Y-TZP green-stage discs was2.85 ± 0.03 g/cm3, and the apparent density of sintered discswas 6.04 ± 0.1 g/cm3, which is close to the X-ray theoreticaldensity value of Y-TZP (6.07 g/cm3) [30]. A maximum contrac-tion of 25% was seen during the thermal contraction analysis.This value is 3.5% higher than the diametral contractionobserved on the discs (mean = 21.5% and SD = 0.67%).

SEM characterization (Fig. 3c and d) shows that thetetragonal zirconia ceramic presents a dense and finegrained microstructure with a mean submicron grain size of0.3 ± 0.2 �m. No impurity concentrations (data not shown), atthe level of resolution employed (FE-SEM fitted with energyX-ray dispersive spectroscopy, EDS, Noran System, Japan), or


singular large defects were found.Fig. 3b shows an SEM micrograph of a PDMS mold, with

5 �m diameter wells separated by 5 �m (edge–edge). Fig. 3cshows a defect-free micropatterned coating. Polishing marks




d e n t a l m a t e r i a l s x x x ( 2 0 1 1 ) xxx–xxx 5

Fig. 3 – SEM images showing the 1 �m polished (a) surface after thermal etching (1330 ◦C for 30 min), the samples show adense and d = 0.3 (0.2 �m) fine grained microstructure of 3Y-TZP. (b) PDMS mold and (c) micropatterned silica coating with5

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�m diameter pillars on a 3Y-TZP substrate.

n the 3Y-TZP surface can be observed through the silica coat-ng.

As the applied thin silica coatings needed to be sinteredt 500 ◦C, XRD analysis (Fig. 2b) was conducted on a fully sin-ered disc, with an additional 500 ◦C heat-treatment, to verifyf there were no changes to the crystalline structure of theulk material. This was confirmed by the similarity betweenaman spectra of the fully sintered and re-heated specimens

Fig. 2c).Early stages of MG63 cell adhesion and propagation (1 d and

d) on flat and micropatterned silica surfaces, and on bare 3Y-ZP surfaces, are shown in Fig. 4. Lamellopodia extensions cane observed on the micropatterned surface directed towardshe micropillars. This effect is further enhanced at 4 d of cul-ure, when compared to what could be observed for flat silicar bare 3Y-TZP surfaces. However, there was more area sur-ace coverage by the cells on both flat surfaces than on the

icropatterned ones.Further characterization of the cells on the micropatterned

urfaces via SEM (Fig. 5a) revealed that during initial stages


t = 1 d), some cells appear to exhibit a rounded morphologypon initial contact with the surface (Fig. 5b and c, singlerrow), and an approximate aspect ratio of 2:1 between the cellnd micropillars, which decreases to 1:1 for the cells already

ig. 4 – Early stages of adhesion and spreading of osteoblast-likeY-TZP (e,f) after 1 d (a,c,e) and 4 d (b,d,f) of culture. Scale bar = 50

showing signs of initial spreading (double arrow) and sub-sequent alignment on the top of the pillars (triple arrow).Fig. 5d shows low and high magnification images at 7-d cul-tures, where a dense cell monolayer growing on the top of themicropillars can be observed.

The alamar blue assay was selected to test the viabil-ity/metabolic activity of osteoblast-like MG63 cells on differentsurfaces. A one-way ANOVA test was used to evaluate dif-ferences in the percentage of viability/metabolic activity ofMG63 cells on three different surfaces (using TCP as a con-trol surface). This analysis showed a statistically significantdifference between the surfaces (F(6, 26) = 8.84, p < .001) at alltested time points. Tukey post-hoc comparisons indicated thatcells on micropatterned silica coatings (mpSiO2: M = 95.60%,SD = 3.22%, p = .003) had a statistically significant highermetabolic activity than those on flat silica coatings (fSiO2:M = 89.27%, SD = 1.91%) and bare Y-TZP (3Y-TZP: M = 92.50%,SD = 2.67%) surfaces at 1 d. No statistically significant differ-ences were found at 4 d (mpSiO2: M = 79.03%, SD = 1.77%; fSiO2:M = 73.82%, SD = 5.43% and 3Y-TZP: M = 79.96%, SD = 6.27%).


Finally, at 7 d cells on micropatterned silica coatings showeda significantly lower activity (M = 61.90%, SD = 7.19%, p = .001)than cells on flat silica (M = 84.62%, SD = 1.25%) and on bareY-TZP (M = 81.85%, SD = 5.97%) surfaces.

MG63 on micropatterned (a,b), flat silica coatings (c,d), bare�m.




6 d e n t a l m a t e r i a l s x x x ( 2 0 1 1 ) xxx–xxx

Fig. 5 – SEM images of adhesion and proliferation of osteoblast-like MG63 cells on micropatterned silica coatings. Cells withtage
round shape (adhesion stage), elongated shape (spreading s
was observed at 7 d (d,e).

4. Discussion

This study was proposed with the aim to microfabricate pillarpatterned silica thin films on zirconia, using a strategy thatcombines soft lithography and sol–gel technologies. Based onseveral recent reviews on dental implants [7–10,19], it is evi-dent that titanium is the gold standard in clinical applicationsand many efforts have been proposed to improve its biologicalbehavior through surface modification. It is also evident thatthere is a growing interest in zirconia implants but ceramicsimplants are in the initial stages of development and furtherresearch is required to produce an effective and competitivecandidate to become a clinical alternative to titanium.

The Y2O3 stabilized tetragonal ZrO2 polycrystalline ceramic(1480 ◦C by 2 h), used in the present work, consists ofhomogeneous and fine equiaxed grains of tetragonal ZrO2

(D50 = 0.3 ± 0.2 �m) sintered to ∼99% of the theoretical density.The small amounts of stabilizing Y2O3 (3 mol%) maintain themetastable tetragonal phase following sintering. This addi-tive is uniformly distributed, forming a ZrO2 solid solutionas confirmed by FE-SEM/EDS. The homogeneous monopha-sic microstructure, as well as the lack of segregation ofnonequilibrium phases, confirmed chemical and microstruc-tural equilibrium. The low intensity peaks in the Ramanspectra of raw powders might be interpreted as monoclinicphase (176, 187 cm−1), while fully sintered and re-heated discs(Fig. 2b and c) exhibit the well defined high intensity peaks(146, 260, 325, 473, 614 and 641 cm−1) corresponding to tetrag-onal phase [27–29,69]. According to the literature, this materialcould present high bending strength (�f = 1000–1200 MPa) andtoughness (KIC = 6–9 MPa m1/2), depending on the microstruc-ture and critical flaw size. The reported values for hardness(Hv) and Young modulus are around 13 and 210 GPa, respec-tively [12,32].

After re-heating for one hour at 500 ◦C, which is close to theeutectoid temperature (located at 4.5 mol% Y2O3 and 490 ◦C)for the ZrO2–Y2O3 binary system [31], the eutectoid reac-tion (tetragonal zirconia solid solution = monoclinic + cubiczirconia solid solutions) was not detected. Therefore, de-stabilization of the tetragonal zirconia solid solution, in thebulk of the structure, did not occur. The retention of thetetragonal zirconia is explained by the slowness of the solid


state reactions at such low temperatures. Visible Raman spec-troscopy, a technique used to characterize both bulk andsurface properties, confirmed that there were no differencesin the crystalline structure between sintered and re-heated

) at 1 d (a–c). Monolayer of cells on the micropillar patterns

(500 ◦C for 1 h) specimens. For further studies, UV Raman spec-troscopy could be explored, as it is more sensitive for surfaceanalysis [27–29].

Surface modification on zirconia presents a technologicalchallenge with few alternatives reported until now. In gen-eral, subtractive processes produce changes in the surfaceroughness. This type of surface modification is well studiedon titanium, where an increased roughness has been reportedto improve in vitro osteoblast adhesion/proliferation [66], how-ever, it also facilitates in vitro bacterial adhesion [67,68]. Froman in vivo perspective, strong adhesion of peri-implant soft tis-sues helps to prevent bacterial colonization and subsequentchronic inflammation, which increases the risk of developingperi-implantitis.

Zirconia surface modifications using additive techniquesmay increase bioactivity. Kim et al. [35] reported the pro-duction of homogeneous fluoro-hydroxyapatite coatings with∼1 �m thickness on zirconia using a sol–gel approach. TheirSEM images apparently showed a uniform interface withoutthe presence of defects, intuitively indicating the occurrenceof a chemical and mechanical compatibility between coatingand substrate. In the present work, homogeneous crack-freeflat silica thin films were successfully obtained using spincoating. These films were optimized in a previous study usingfractional factorial statistical experimental design, where athickness dependence on the rotational speed, accelerationand aging of the sol was found (data not published).

Sollazo et al. reported an interesting technique to intro-duce high roughness coatings on titanium or zirconia. In theirwork, zirconium dioxide was produced by colloidal precipita-tion, and deposited on the substrates by dip coating, followedby heat treatment at 700 ◦C for 60 min. The biological behav-ior was evaluated both in vitro and in vivo. The animal studyshowed that the coating promoted increased bone integra-tion [43]. The in vitro studies with MG63 cells and microarraytechnology showed promising results as well. However, eventhough their coatings showed appropriate thickness (∼1 �m)for dental implants, it is also evident, based on their SEMmicrographs, that the coatings presented adhesion problemsand discontinuities [44]. In the present work, microfabricatedand flat silica thin films exhibited stable interfacial adhesionto the 3Y-TZP substrate.

Calcium phosphate glass-hydroxyapatite (HA) composites


have also been studied as coatings on zirconia. Differentmixture ratios were evaluated in terms of heat-treatmenttemperature, morphology, adhesion strength, solubility andcell responses. Their SEM images showed a highly dense and



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omogeneous continuous coating on zirconia substrates [45].roliferation and ALP activity of osteoblast-like cells on theseurfaces were improved in comparison to bare zirconia. Theell proliferation results are in agreement with our observa-ions after 7 d of culture. This trend was also observed in atudy about silica-containing HA nanospheres, where in addi-ion, an optimum amount of Si+ was found to increase theioactivity without accelerating the coating dissolution [46].

A host of biomedical applications using sol–gel technologyave been published [35,47–51]. In the present work, sol–geloatings were sintered at 500 ◦C for 1 h, these conditions wereelected in order to ensure effective removal of the ethylnd methyl groups from the alkoxides, producing a changerom Si–O–C2H5 or Si–CH3 to Si–O bonds, and formation of aomplete SiO2 lattice, as it was described by Santucci et al.71].

The introduction of microfabrication techniques intoiomedical applications has allowed for the study of theffects of surface topography on cellular behavior, using poly-ers, silicon or metal thin films; materials that presentechanical disadvantages for structural applications in den-

istry or orthopedics [53]. With current technology, theicrofabrication of ceramics is associated with hard lithog-

aphy techniques that in many cases are expensive, timeonsuming and have low yield. The combination of silicaol–gel chemistry and soft lithography was initially reportedo produce optical waveguides and glass surfaces [70], but tohe best of the authors knowledge, there is a limited numberf reports about the use of this approach for dental ceramics

62].Considering the effects of the topography on cellular

ehavior, it is accepted that micro-engineered surfaces pro-uce unique and reproducible cellular responses [63,64];owever, such behaviors are not universal for all types of cellsnd they are apparently associated with specific aspects suchs origin, size and function of the cells specifically studied inach case. In this work, a strategy to produce micropillared sil-ca thin films on 3Y-TZP was presented. MG63 osteoblast-likeells showed a preference to adhere and grow on the top ofhe micropillars at all time points. No apparent interactionsere seen between the cellular processes and the sidewallsf the pillars, or the underlying flat surface. This preferenceas consistent with observations made by Turner et al. [54]here astrocyte-like cells were cultured on single-crystal sili-

on micropillars.In the current work, the viability/metabolic activity of MG63

steoblast-like cells that was evaluated using an alamar bluessay was higher on the micropillars and flat silica coatingshat on the 3Y-TZP surfaces. These results could be explaineds being associated with the surface chemistry [46], becausef the high silicon content. No significant differences wereeen between the patterned and flat coatings. Other studiesave reported higher proliferation of human bone marrowells on PDMS micropillars [53] than on flat surfaces. Theifferences between both findings could be due to differ-nces in the stiffness of the substrate, material chemistry,


nd micropattern geometry. Studies on the effect of silicaicropatterned geometry are currently being conducted by

ur research team, and will be addressed in future publica-ions.

PRESS( 2 0 1 1 ) xxx–xxx 7

5. Conclusions

The current study described a technique that combinedsol–gel and soft lithography to modify the surfaces of 3Y-TZP with micropatterned silica for potential use in dentalimplants. This technique allowed the fabrication of micropil-lared silica surfaces with 5 �m diameter and 5 �m wide gapsbetween pillars, at low temperatures (500 ◦C). In vitro stud-ies showed that cells were able to adhere, elongate, migrateand proliferate on the coatings, preferably covering the topsurfaces of the pillars, and mostly avoiding contact with thebottom surface or the pillar side walls. This behavior indicateda cytocompatible pattern, alluding to their potential applica-tions as coatings for use in dental implantology.

Acknowledgements

This work was supported partially by the Por-tuguese Science and Technology Foundation(Scholarship FCT/SFRH/BD/36220/2007 and Grant No.FCT/PTDC/CTM/100120/2008 “Bonamidi”), CRUP – Accõesintegradas Luso-Espanholas: E46/09, Acciones integradas Hispano-Portuguesas, MICINN: HP2008-0075. NSF Nanoscale Science andEngineering Center fellowship (NF, Grant No. EEC-0425626)and U.S. Air Force Office of Scientific Research MURI (GrantNo. F49620-03-1-0421). The support provided by Dr. C. Ribeiro(Raman microanalysis) is gratefully acknowledged.

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