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Colloids and Surfaces A: Physicochem. Eng. Aspects 365 (2010) 222–229

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

journa l homepage: www.e lsev ier .com/ locate /co lsur fa

ffect of roughness, wettability and morphology of engineered titaniumurfaces on osteoblast-like cell adhesion

.I. Rosales-Leala, M.A. Rodríguez-Valverdeb,∗, G. Mazzagliaa, P.J. Ramón-Torregrosab,. Díaz-Rodríguezc, O. García-Martínezc, M. Vallecillo-Capillaa, C. Ruizc, M.A. Cabrerizo-Vílchezb

Dental Materials, Faculty of Odontology, University of Granada, Campus de Cartuja, E-18071 Granada, SpainBiocolloid and Fluid Physics Group, Department of Applied Physics, University of Granada, Campus de Fuentenueva, E-18071 Granada, SpainDepartment of Nursing, Physiology Section, School of Health Sciences, University of Granada, Avda. Madrid, s/n E-18071 Granada, Spain

r t i c l e i n f o

rticle history:eceived 26 September 2009eceived in revised form 3 December 2009ccepted 7 December 2009vailable online 16 December 2009

eywords:itanium

a b s t r a c t

Texturization of surfaces is usually advantageous in biomaterial engineering. However, the details of thetextured surfaces can be more determining on cell adhesion and proliferation, rather than their roughnessdegree. Titanium is extensively used as a dental implant material in the human body. In this paper, theeffect of four surface treatments on commercially pure titanium has been evaluated. These treatmentswere polishing (pTi); hydrofluoric acid (HF) etching (eTi); Al2O3 blasting (bTi); Al2O3 blasting + HF etching(beTi). Roughness and fractal dimensions were obtained from atomic force microscopy. Wettability wasmeasured using water sessile drops. Morphology and surface chemical composition were analyzed with

urface treatmentopographyettability

ell culture

scanning electron microscopy and energy dispersive X-ray (EDX). MG-63 cell cultures were performed atdifferent times (180 min, 24 h, 48 h, 72 h). Lowest roughness was found in pTi samples followed by eTi, bTiand beTi samples. Etching generated surfaces with the highest fractal dimension and negative skewness.Young contact angles were similar except for pTi and bTi surfaces. Silicon and aluminum traces werefound in pTi and bTi samples, respectively. Cell adhesion (≤24 h) was greater on bTi and beTi surfaces.After 48 h, cell proliferation, mediated by specific morphologies, was improved in eTi samples followedby beTi surfaces. For the same surface chemistry, cell growth was driven by topography features.

© 2009 Elsevier B.V. All rights reserved.

. Introduction

The success of a dental implant is based on the osseointegra-ion that is defined as the direct contact between the bone tissuend the dental implant surface, without fibrous tissue growing athe interface [1,2]. Surface characteristics of the implant play anmportant role for the evolution of bone tissue of the recipient site,fter implantation [3]. Surface properties control the amount anduality of cells adhered on the implant and consequently, the tissuerowth. Most determining surface properties for cell adhesion areurface topography and surface chemical reactivity. Accordingly,urface engineering of biomaterials is oriented to modify their sur-ace texture and/or surface chemistry.

The topography of a surface substantially affects the macro-copic behaviour of a material [4]. Currently, the influence ofurface topography on biological response is a matter of investiga-ion. At cellular level, biological responses, such as the orientation

∗ Corresponding author. Tel.: +34 958 24 00 25; fax: +34 958 24 32 14.E-mail address: marodri@ugr.es (M.A. Rodríguez-Valverde).

927-7757/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfa.2009.12.017

and migration of cells and the cellular production of organizedcytoskeletal arrangements, are directly influenced by the surfacetopography [5]. There are evidences that a suitable surface rough-ness, at nano- and microscopic scale, can lead to a successfulosseointegration of titanium implants [6]. Osteoblast differen-tiation, proliferation and matrix production [7] as well as theproduction of local growth factors and cytokines are affected bysurface roughness [8].

Biomolecule adsorption onto implant surfaces “in vivo” isindeed a dynamic process driven by the physico-chemical inter-actions between adsorbent surface and macromolecule [9]. Thisprecursor process develops a “conditioning film” which will modu-late the cellular host response. Surface energy, which is intimatelyrelated to wettability [10], is a useful quantity that has oftencorrelated strongly with biological interaction. Hence, implantwettability can become determining for the protein adsorption

and consequently, for the cell adhesion [11–13]. For instance,hydrophobic surfaces (i.e. surfaces with low water wettability)presumably decelerate primary interactions with the aqueousbiosystem. Thus, it is usually reported that biomaterial surfaceswith moderate hydrophilicity, improved cell growth and higher

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J.I. Rosales-Leal et al. / Colloids and Surfaces

iocompatibility [14]. However, cell adhesion can decrease as themplant wettability is further decreased. This points out to thexistence of a range of optimal surface energies [15]. Otherwise,nterfacial reactions “in vivo” change relevant physical and chem-cal surface parameters, such as the surface energy, affecting theong-term stability of implants [16].

Modification of the physico- and physico-chemical surfaceroperties of a biomaterial can improve interaction with cells. Inarticular, several surface treatments have been applied to opti-ize the surface topography of titanium implants in bone-contact

pplications. Most extending treatments are sandblasting and acidtching [17]. The main purpose of these texturization treatments iso achieve greater bone-to-implant contact [18], in order to reduceealing times and accelerate integration into the host tissue. Tex-uring of dental implants improves the mechanical adhesion toone but, at the same time, the asperities and grooves may act asreferable sites for protein adsorption.

The evaluation of the topography of biomaterial surfaces ismportant because it usually improves the biological responsesound during osteointegration and the long-term response of theone–implant interface. However, one major difficulty arises in thehoice of the topography parameters, which are actually relevantor the interactions between biomaterial surface and surroundingiological medium [19]. Generally, the description of a surface maye performed at three levels, according to the degree of surface

nformation: height/spatial distribution, topology and morphology.he height/spatial distribution provides a statistical description ofhe surface roughness using amplitude or horizontal lengths andybrid parameters, both in 2D and 3D analysis [20]. The topol-gy description of a surface is understood as the set of intrinsicroperties related to its structure such as connectedness, compact-ess. The topography of most engineering surfaces is topologicallyelf-affine over a range of scales [21]. The fractal dimension (Df)easures, in statistical sense, the structural complexity of a self-

ffine surface, i.e. the random roughness organization [22]. Inther adhesion studies, the fractal dimension correlated better withdhesion than did conventional measurements of surface rough-ess [23,24]. For this reason, fractal analysis might be more helpfulhan conventional roughness descriptions in order to elucidate theomplex mechanisms occurring at the implant surface in contactith the surrounding biological tissues [19]. Otherwise, the mor-hological analysis, usually performed by SEM, provides the detailsnd the figure of the surface topography at higher resolution; evenlthough this raw information without post processing is merelyualitative.

In surface engineering of biomaterials, it is important toetermine when cell spreading is modulated by surface energy,opography or both, at short and long-term times after implan-ation. In addition, rather than conventional approaches, fractalimension might be used to quantify the role of the features of

mplant topography on the cell growth. Accordingly, the aim ofhis work is to evaluate the effect of four texturization treatmentsf titanium surfaces on the adhesion and growth of osteoblast-ike cells, from the topography (roughness, fractal dimension and

orphology) and the water wettability induced by each treatment.

. Materials and methods

.1. Titanium samples preparation

Commercially pure ASTM grade II titanium (cpTi) cylindersManfredi, S. Secondo di Pinerolo, Italy) were suitably cut into smallisks of approximately 12 mm in diameter and 2 mm in thickness.he cpTi disks, previously cleaned with distilled water, were engi-eered as follows:

sicochem. Eng. Aspects 365 (2010) 222–229 223

- Group 1 (control): Polished titanium (pTi). cpTi surfaces weremetallographically polished using silicon carbide (SiC) paperssuccessively from grade 240, 320, 500, 800, 1200, 2000 to 4000grit. Next, an ultra-polishing was achieved using the sequence1–0.3–0.05 �m alumina slurries and gauze. The polished diskswere cleaned in distilled water by immersion in ultrasonic bath(Selecta, Barcelona, Spain).

- Group 2: Etched titanium (eTi). pTi surfaces were etched with asolution of 10% (v/v) hydrofluoric (HF) acid (Panreac, Barcelona,Spain) by complete immersion upon gently agitation for 5 min.

- Group 3: Blasted titanium (bTi). pTi surfaces were blasted by alu-mina (99.78% Al2O3) particles of 110 �m diameter projected atan incidence angle of 60◦ and 0.25 MPa pressure for 3 min.

- Group 4: Blasted + etched titanium (beTi). pTi surfaces wereblasted as in the group 3 but using alumina particles of250–500 �m. Next, these blasted surfaces were etched asdescribed for group 2.

After treatments, pTi, eTi, bTi and beTi samples were degreasedwith a solution of 70% (v/v) acetone (Panreac, Barcelona, Spain)by immersion in ultrasonic bath for 20 min and afterwards, thesamples were ultrasonicated in distilled water for 30 min. eTi andbeTi samples were passivated with 30% (v/v) nitric (HNO3) acid(Panreac, Barcelona, Spain) for 3 min and then again ultrasonicatedin diluted acetone for 20 min and distilled water for 30 min. Alldisks were autoclaved at 121 ◦C and 1 atm for 30 min after prepa-ration.

2.2. Atomic force microscopy

Topographies of the textured titanium surfaces were acquiredby an Atomic Force Microscope Nanoscope IV MultiMode in air(Digital Instruments, Santa Barbara, Ca, USA). The microscope wasoperated in tapping mode on a 2500 �m2 scansize with a Si3N4V-shape cantilever (stiffness, k = 63 ± 8 N/m). The topography datawere sampled in a grid of 256 × 256 points. All images were fittedto a plane and filtered by a Gaussian mask with a cutoff of 2.5 �min order to capture surface features below the average cell size(∼10 �m). Three disks were used per group and three topographieswere acquired per each disk.

2.2.1. RoughnessUsual roughness parameters [20] are described in Table 1.

Amplitude parameters, as the arithmetic average roughness (Ra),the peak roughness (Rp maximum relative height), the valleyroughness (Rv, maximum relative depth) and the absolute height(Rmax = Rp + Rv), usually provide a crude roughness descriptionbecause they just reveal the amplitude of the topography featureswithout information about their spatial distribution. Instead, theroot mean square roughness (Rq), the skewness (Rs) and the kur-tosis (Rk) are statistical moments of the height distribution thatdescribe its width, symmetry and form, respectively. Commonly,the root mean square roughness qualitatively describes the sametrend than the arithmetic average roughness. Otherwise, hybridparameters, as the surface area ratio (Rw), are derivative-basedparameters that quantify topography variations with respect to theplanar coordinates.

The above-mentioned parameters were estimated from thetopography data using the software of the own instrument.Although the results of topographical surface characterization

depend on the length scale employed [25], 50 �m scansizes wereselected in order to cover the cell size range (∼10 �m). More detailsabout the computation of surface area ratio are described elsewhere[26]. The surface area excess with respect to the pTi surface was alsocalculated.

224 J.I. Rosales-Leal et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 365 (2010) 222–229

Table 1Roughness parameters.

Parameters Description Mathematical definition Typical Units

Rp Maximum relativea height (the highest peak) Zmax �mRv Maximum relative depth (the deepest valley) Zmin �m

Ra Arithmetic average roughness Ra =⟨∣

Z∣⟩

xy

b nm

Rq Root mean square roughness Rq =√⟨

Z2⟩

xynm

Rmax Absolute height Rmax = Rp + Rv �m

Rsk Symmetry of the distribution of heights (skewness) Rsk =⟨

Z3⟩

xy/⟨

Z2⟩3/2

xy–

Rku Form of the distribution of heights (biased kurtosis) Rku =⟨

Z4⟩

xy/⟨

Z2⟩2

xy− 3 –

Rw Surface area ratio (Wenzel factor) Rw ≡ AAz

=⟨√

1 +(

dZdx

)2+(

dZdy

)2

⟩xy

–

a With respect to an ideal average plane⟨

Z⟩

xy= 0.

L

2

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kt(s

N

wBist

2

o(ts

2

pa

b 〈·〉xy = 1LxLy

Lx∫0

dx

y∫0

(·) dy.

.2.2. Fractal analysisAlthough recently there has been much effort devoted to the

evelopment and manufacturing of nanotextured or structuredurfaces [27], the conventional texturization processes provideandomly rough surfaces. Furthermore, these rough surfaces aresually self-affine, i.e. there is an anisotropic disorder becausehe surface remains statistically invariant under dilations of the, y, z coordinates but by different scale factors [21]. Fractalimension provides a scale-independent measure of these stronglyisordered systems, regardless of the observation scale. A frac-al surface typically has irregularities that “fill” the embeddingpace (D = 3). Fractal dimension quantifies the disorder in termsf the space-filling ability of the surface, thus a fractal surfaceust occupy intrinsically more space than a plane surface (i.e. the

opological space, D = 2). In fact, a fractal surface usually becomespace-filling but non-uniformly because it occupies only certainegions of the embedding space. Hence, fractal dimension will beelated to the compactness degree of the space-filling structure21].

In this work, fractal dimensions were computed by the well-nown box-counting method [19,28,29]. In this method, the surfaceopography is covered with boxes of side length d. Once the surfacei.e. the area) is completely covered with N boxes, the followingcaling rule of fractal geometry [21] must be fulfilled:

(d) = ˛d−Df (1)

here ˛ is a geometrical prefactor and Df is the fractal dimension.y continuously changing the magnification scale through chang-

ng the size of the boxes (d), the number of boxes covering theurface (N) is counted. The fractal dimension Df is obtained fromhe slope of the log–log plot of Eq. (1).

.3. Field-emission scanning electron microscopy

The tree titanium disks per group of the Section 2.1 werebserved under a Scanning Electron Microscope Leo Gemini 1530Carl Zeiss, Oberkochen, Germany). In order to improve the elec-rical conductivity of titanium, carbon thin films were produced by

puttering on each sample.

.3.1. MorphologyMorphology of each sample was evaluated from 3072 × 2304

ixel images acquired at ×2000 magnification. Further porositynalysis was accomplished on the eTi and beTi samples.

2.3.2. Surface chemical analysisRoughness is often associated with changes in surface chem-

istry (e.g. plasma deposition). Hence, the texturization treatmentsof unalloyed titanium described in Section 2.1 can produceunexpected modifications in its surface chemical composition.Accordingly, energy dispersive X-ray (EDX) spectra were obtainedat different surface zones of each titanium sample up to a depthof 1 �m. This analytical technique serves to detect the chemicalelements located at the surface [30].

2.4. Wettability

A liquid drop spreads on a solid surface up to cover a particu-lar area, in prejudice of the surrounding liquid vapour, driven bythe solid–liquid intermolecular interactions. This interfacial phe-nomenon is known as wetting and the affinity of a solid surface tobe wetted by a given liquid is referred to as wettability. Wetting,and thus wettability, is strongly influenced by surface roughness[31,32]. Bathomarco et al. found that as the surface area of titaniumimplants increases, the measured contact angle decreases [32]. In1936, Wenzel [33] reported that the roughness of a homogenoussolid surface affects contact angle measurements (referred to asapparent angles) as follows:

cos �app = Rw cos �Y (2)

where Rw symbolizes the surface area ratio, also referred to asWenzel factor, �app is the apparent contact angle associated to thesystem equilibrium state and �Y is the Young contact angle [31].The Wenzel law (2) is valid if the characteristic length of asperitiesis much lower than the size of drop or the distribution of asperitiesis assumed uniform. This law predicts an increase in the experi-mental contact angles with growing roughness for �Y > 90◦ and justthe opposite trend if �Y < 90◦. The thermodynamically meaning-ful angle, i.e. the angle intimately related to surface free energy, isthe Young contact angle. This angle would be the equilibrium con-tact angle of the unattainable smooth surface (i.e. ideal surface).As pointed out by Morra et al. [34], in order to estimate surfacefree energy from experimentally accessible contact angles on roughsurfaces, the concerning Young contact angles must be estimatedthrough Eq. (2). However, the apparent contact angle associated

to the true equilibrium state is hardly measurable due to contactangle hysteresis [16,30,35]. Instead, the employment of advanc-ing contact angles for the solid surface free energy determinationis a common practice [10], if practically no water film is presentbehind the liquid drop. Furthermore, Rupp et al. [6] reported val-

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J.I. Rosales-Leal et al. / Colloids and Surfaces

es of water receding angles equal to zero for acid-etched titaniumurfaces.

The main, commonly used, measure of surface chemistry is theurface free energy. However, the surface energy of solid may note measured directly, at least with relative ease. Instead, therere different approaches for the estimation of surface energy fromontact angle measurements (e.g. critical surface tension, the equa-ion of state, acid–base components of solid surfaces, . . .) [31].evertheless, aside from controversies over the validity of theseethods, this estimation is an unnecessary complication for practi-

al biomaterial applications. Thereby, since the difference betweenolid interfacial energies is indeed the thermodynamically relevantuantity, the Young contact angle becomes more meaningful thanuestionable surface energy values [10].

Since physiological fluids are indeed aqueous saline solutions,ure water is the reference probe liquid used for measuring surfaceettability of biomaterials. Water wettability of titanium surfacesas measured through the contact angle of spreading sessile drops,hich can be considered as the advancing contact angle, as aboveiscussed. MilliQ water drops (2.5 �l of volume) were dispensedsing a micropipette (Eppendorf, Hamburg, Germany) at room tem-erature. Three disks were analyzed per group (see Section 2.1) andhree drops were deposited on each disk. Once the drop was dis-ensed on the substrate, side-view images of drop were acquirednd analyzed by Axisymmetric Drop Shape Analysis. More detailsbout this technique can be found elsewhere [36]. Contact anglesere directly computed by ad-hoc designed software.

.5. Cell culture

Osteoblast-like cell MG-63 [7,17] were cultured in Dulbecco’sodified Eagle medium (Invitrogen, Carlsbad, CA, USA) with

00 IU/ml penicillin (Roger, Barcelona, Spain), 50 �g/ml gentam-cin (Braum Medical, Jaén, Spain), 2.5 �g/ml anfotericin B (Sigma,t. Louis, MO, USA), 1% glutamine (Sigma, St. Louis, MO, USA), 2%EPES (Sigma, St. Louis, MO, USA) and supplemented with 10% fetalovine serum (FBS) (Gibco, Paisley, UK). Cultures were kept at 37 ◦C

Fig. 1. AFM pictures (256 × 256 points) of the textured titanium surfaces: pTi

sicochem. Eng. Aspects 365 (2010) 222–229 225

in a humidified atmosphere of 95% air and 5% CO2. The cells weredetached from the culture flask with a solution of 0.05% trypsinand 0.02% ethylene diamine tetra-acetic acid (EDTA) (Sigma, St.Louis, MO, USA), and were washed and suspended in completeculture medium with 10% FBS. The cells obtained were inoculatedfor tests onto samples at 2 × 105 cell/ml in a 24-well plate (Falcon,Becton Dickinson Labware, NJ, USA) in the ratio of 2 ml/well, inwhich were deposited previously titanium disks. The plates wereincubated to 37 ◦C in CO2 (5%) atmosphere. Assay tests were per-formed at 180 min, 24, 48 and 72 h for each surface. Cell cultureswere repeated four times for each surface and time period. At theend of the culture time, disks were recovered and cleaned withmedium solution. Then, cells were detached from the disks with asolution of 0.05% trypsin and 0.02% ethylene diamine tetra-aceticacid (EDTA) (Sigma, St. Louis, MO, USA), and were washed and sus-pended in complete culture medium with 10% FCS. The numberof adhered cells was determined with a counter cytometer (OrthoDiagnostic System, Raritan, Il, USA). Number of cells was quanti-fied per ml (n·104/ml). One additional sample per treatment andtime was prepared in order to evaluate the cell morphology. Cellculture followed the method above described. After the incuba-tion period, media were removed and specimens were fixed with4% glutaraldehyde in PBS (pH 7.2) for 20 min. After dehydration ingraded alcohols, samples were immersed in hexametildisilazanefor 10 min, air dried and then sputter-coated with gold palladium.Finally, the surface of the specimens was examined by SEM.

3. Results

3.1. Topography

3.1.1. Roughness

In Fig. 1, AFM pictures of titanium disks belonging to the four

groups (see Section 2.1) are displayed keeping the same scaleand scan size. The roughness parameters of the textured titaniumsurfaces are compiled in Table 2. According to the amplitudeparameters (Rp, Rv, Ra and Rmax), the coarsest surfaces were the

(a); eTi (b); bTi (c); beTi (d). The scansize area was fixed to 50 × 50 �m2.

226 J.I. Rosales-Leal et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 365 (2010) 222–229

Table 2Values of roughness parameters of the four engineered titanium surfaces (polished, etched, blasted and blasted-etched surfaces).

Surface Rp (�m) Rv (�m) Ra (nm) Rq (nm) Rmax (�m) Rsk Rku Rw

± 42± 43± 45± 58

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pTi 0.47 ± 0.21 −0.60 ± 0.19 140 ± 17 149eTi 1.15 ± 0.23 −1.60 ± 0.25 209 ± 19 268bTi 3.0 ± 0.9 −1.8 ± 0.7 861 ± 30 1022beTi 5.1 ± 1.6 −3.3 ± 0.9 1370 ± 78 1639

lasted ones with the largest particles (beTi), although they weretched later (see Fig. 1d). In decreasing order of any amplitudearameter, the beTi surfaces were followed by bTi, eTi and pTisee Fig. 1a). Otherwise, from the statistical parameters, the pos-tive biased kurtosis (Rku > 0) and the negative skewness (Rsk < 0)ound on the eTi samples predict a fluctuating morphology (seeig. 1b) with high density of valleys (i.e. microporosity), whereasandblasting seems to produce spiky and coarse morphologiesith negative biased kurtosis and positive skewness (see Fig. 1c

nd d). This is also illustrated in Fig. 2 with the topographyrofiles of each sample. The surface area ratio (see Table 3) justoftened the trend of amplitude parameters since surface area

s a differential parameter which takes into account variationsn planar and perpendicular directions. As expected, the greatestalues of available surface area were found in the beTi samples andhe lowest ones in the pTi samples. However, eTi and bTi surfaces

ig. 2. AFM profiles of the textured titanium surfaces: pTi, eTi, bTi and beTi. Therofiles are intentionally shifted for illustrative purposes only. Sandblasting pro-uced spiky and coarse morphologies, where the coarsest surfaces were the blastednes with the largest particles (beTi) although they were etched later. However, theighly fluctuating morphologies (space-filling ability) of eTi surfaces explain thatTi and bTi surfaces exhibited similar values of surface area.

able 3alues of surface area, fractal dimension and contact angle of the four engineered

itanium surfaces (polished, etched, blasted and blasted-etched surfaces).

Surface Surface area Fractal dimension Contactangle (◦)

Real areaa

(�m2)Areaexcessb (%)

pTi 2540 ± 100 0 2.33 ± 0.09� 62 ± 4eTi 2760 ± 110 9 ± 2 2.56 ± 0.11 53 ± 3bTi 2890 ± 110 14 ± 1 2.26 ± 0.13� 46 ± 2beTi 3340 ± 120 32 ± 2 2.24 ± 0.12� 40 ± 2

ean values with symbol (�) were statistically similar (p > 0.05). (One-way ANOVAnd Tukey’s test).

a Apparent area: 2500 �m2.b With respect to ultrapolished surface.

2.3 ± 0.1 −0.07 ± 0.23 1.4 ± 0.7 1.02 ± 0.043.1 ± 0.1 −0.36 ± 0.12 0.71 ± 0.19 1.10 ± 0.115.7 ± 0.3 0.40 ± 0.19 −0.75 ± 0.25 1.15 ± 0.168.2 ± 0.5 0.38 ± 0.11 −0.31 ± 0.21 1.33 ± 0.14

exhibited similar values of surface area. The highly fluctuatingmorphologies induced by etching (Fig. 2) compensated the coarsemorphologies blasted with the smallest particles (bTi).

3.1.2. Fractal dimensionThe values of fractal dimension computed by box-counting

method are presented in Table 3. All samples were recognizedas fractal surfaces because Eq. (1) was fulfilled up to a limit boxsize, where fractal behaviour disappeared. No significant differ-ences between pTi, bTi and beTi groups were found, though this didnot mean similar morphologies. However, eTi surfaces obtained thehighest fractal dimension due to their highly fluctuant morphology(space-filling ability). In spite of common belief, a fractal surfacemay be less rough and have more space-filling irregularities, i.e.it occupies only certain regions of the embedding space but morecompactly [21].

3.1.3. MorphologyFig. 3 compiles the SEM micrographs of the four groups. These

images qualitatively confirmed the results obtained from AFMtopographies. The reference surface (pTi) showed a smooth mor-phology without characteristic features (see Fig. 3a). Instead, acidetching produced angular stepped morphologies with microporesfrom 1.5 to 2.5 �m (see Fig. 3b). This random structure manifestedcertain hierarchy with double scale roughness, i.e. a nano-roughtexture. This indeed revealed the grain-structured titanium. Oth-erwise, blasting generated very irregular and coarse morphologies(see Fig. 3c) with wide cavities (10–50 �m in diameter), as the sizeof shot alumina particles. Even it was observed the presence ofalumina particles incrusted in the titanium surface, as reported[37]. These surface alterations might significantly hinder the celladhesion. Finally, the beTi surfaces exhibited a coarse morphologythough less chaotic than the bTi samples and with micropores likethe eTi samples (see Fig. 3d).

3.2. Surface chemical composition

The EDX spectra of all samples (not shown) pointed out thatsurface chemistry of the textured surfaces was mostly based ontitanium dioxide with a constant percentage of metallic titaniumbehind the protective oxide film (see the ratios of surface chemi-

cal species in Table 4). Silicon traces appeared on the pTi samplesdue to polishing with SiC papers; just as well the bTi samples con-tained aluminium traces due to the alumina particles trapped intothem (as pointed out in Section 3.1.3). Indeed, the carbon from SiCwas overshadowed in the spectra by the residual carbon originating

Table 4Values of Young contact angle and surface chemical species ratio of the four engi-neered titanium surfaces (polished, etched, blasted and blasted-etched surfaces).

Surface Young contact angle (◦) Ti/TiO2 Al/TiO2 Si/TiO2

pTi 63 ± 6© 0.14 0.16 0.34eTi 57 ± 8 0.13 0.00 0.00bTi 50 ± 10© 0.14 0.28 0.00beTi 55 ± 7 0.13 0.03 0.00

Mean values with symbol (©) were statistically dissimilar (p < 0.05). (One-wayANOVA and Tukey’s test).

J.I. Rosales-Leal et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 365 (2010) 222–229 227

: pTi (

fbmori

3

notahspbiioaioantta

fTfirsp

ently the main events of cell adhesion. After 180 min and 24 h, cellcontact and attachment were more marked on bTi and beTi sur-faces than pTi and eTi surfaces. However, after 48 and 72 h, cellspreading and proliferation were enhanced on eTi surfaces, fol-

Fig. 3. SEM microimages of the textured titanium surfaces

rom the reflecting film for SEM experiments. Unlike bTi samples,eTi surfaces showed smaller aluminium amounts because the alu-ina particles were mostly etched. Likewise, no SiC trace was found

n eTi samples due to etching. Hence, whereas acid etching justecovered the original surface chemical composition of cpTi, blast-ng and polishing produced undesired surface doping.

.3. Wettability

The apparent contact angles of water drops on the textured tita-ium surfaces are compiled in Table 3. These values, measurednce drop spreading was finished (1–2 s), corresponded to sys-em metastates very close to advancing mode because the recedingngles reported in literature are practically zero [6]. As expected forydrophilic surfaces (see Eq. (2)), greater surface area ratio corre-ponds to higher wettability (lower apparent contact angle). Hence,Ti surfaces showed the lowest wettability followed by eTi, bTi andeTi surfaces. It is worth to highlight that this result can be mislead-

ng because the effect of roughness overshadows the influence ofnterfacial energetics. The influence of roughness on contact anglef homogeneous surfaces is a twofold issue. Phenomenologically,dvancing contact angles often increase linearly with the concern-ng surface texture parameter [38], and just the opposite trend isbserved for the receding angles. However, in addition to this effect,drop (meniscus) in contact with a rough and chemically homoge-eous surface undergoes the second-order effect [32] predicted byhe Wenzel law (2). Although both effects can appear at once, lat-er effect typically dominates from intermediate values of contactngle hysteresis.

Table 4 collects the values of Young contact angle computedrom Eq. (2) and the concerning values of surface area ratio (see

able 3) for each surface treatment. The use of Eq. (2) was justi-ed by the larger drop volume (2.5 �l) as regards the maximumoughness amplitude (see Table 2). The Young contact angles weretatistically similar, except for pTi and bTi surfaces. The bTi sam-les exhibited the lowest value of Young contact angle due to the

a); eTi (b); bTi (c); beTi (d) (original magnification ×2000).

hydrophilic alumina content whereas the pTi samples were anoma-lously less hydrophilic due to the carbon excess originating fromthe SiC traces.

3.4. Cell culture

Fig. 4 shows cell culture results. Fig. 5a and b show representa-tive cell morphologies. As expected for biocompatible surfaces, thecell adhesion rate increased on all treated titanium surfaces, thoughspecific morphologies developed by each texturing affected differ-

Fig. 4. Cell adhesion and proliferation on the engineered titanium surfaces as afunction of time. Cell cultures were repeated four times for each treatment andtime period.

228 J.I. Rosales-Leal et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 365 (2010) 222–229

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Fig. 5. Scanning electron micrographs of osteablast-like cells adhered to an

owed by beTi, bTi and pTi surfaces as decreasing surface area.G63 osteoblastic-like cells showed high adhesion on eTi surfaces,

wing to an enhanced filopodial anchorage at the nanoasperitiesnd micropores. Otherwise, on the bTi samples, the cells weredherent but not confluent.

. Discussion

In culturing cells on biomaterial surfaces, surface free energy isn important parameter that guides the first events occurring at theiomaterial/biological interface, such as interaction of water androteins with biomaterial, and these events guide further response8,38]. Nevertheless, surface energy estimation from contact angle

easurements is a hard task because biomaterial surfaces arelways rough and/or heterogeneous. If surface roughness is therimary cause of hysteresis, i.e. Ra ≥ 0.1 �m [31], the advancingontact angle is influenced more by the microscopic relief thany interfacial energetics. However, surface energy estimation justequires thermodynamically significant angles. Hence, for roughitanium-based surfaces, the Young contact angles computed byq. (2) from advancing contact angles can serve to detect relativelyross changes in surface energy.

The good cell response at long times on eTi samples, as regardshe rest of textured groups, was explained by their particular mor-hology (the greatest fractal dimension and negative skewness)nd surface chemical homogeneity (no traces). Chemically etchedurfaces have long been recognized as fractal, due to the stochas-ic character of wet etching. Particularly, the morphologies of eTiamples were angular and stepped due to the crystalline grainetachment from the amorphous structure of titanium. Moreover,t dimensions smaller than the grain structure of titanium, a sec-ndary texture was visualized. Hence, eTi samples, even beingess rough than bTi and beTi groups, exhibited microporous andano-rough texture. It is worth to mention that surface rough-ess in the range from 10 nm to 10 �m may influence the interfaceiology, since it is of the same order in size as cells and largeiomolecules. Microporous surfaces enhance osteointegration ofitanium implants, indicated by mechanical pull-out tests and his-omorphometric analysis [39] or osteoblast spreading tests [40].

As far as biomaterial interactions with proteins are concerned,urface features on the nanometer scale are important. Changes inurface chemistry or roughness, of the same dimension as a proteinill affect its adsorption characteristics. Instead, if the rough sur-

ace is treated as chemically homogeneous and the size of asperities

s larger than the protein dimension, then roughness will sim-ly add available surface area for adsorbing protein. This explainshat beTi and bTi samples were more bioadhesive at early stagesf cell attachment [6,12,41]. At late stages, the presence of alu-ina particles and the coarsely blasted morphology, self-affine but

tched titanium surface (a) and to a polished titanium surface (b) after 72 h.

uncomfortable for cell spreading, impaired the cell proliferation asexpected [22]. Furthermore, sandblasting can also affect the cor-rosion resistance of titanium due to the residual surface stressesinduced [37].

Fractal dimension arises as a more appropriate index of surfacedisorder [22,42] rather than the amplitude roughness parame-ters. However, a single exponent is not enough for the completedescription of fractal surfaces. In this work, the partial informa-tion provided by fractal dimension was refined by the values ofkurtosis and skewness, and confirmed by SEM pictures. Alterna-tively, lacunarity becomes a counterpart to the fractal dimensionbecause it just describes the texture of a fractal surface [21]. Fractalsurfaces having same fractal dimensions can look widely differentbecause of having different lacunarity. This parameter is related tothe prefactor ˛ of Eq. (1), known as topothesy.

HF acid etching is recommended for titanium-based dentalimplants rather than sandblasting based in the results of this study.As suggested in the literature, the HF treatment creates a micro-and nano-level topography that enhances adherent cells [43,44].Moreover, since surface characterization of biomaterials is crucialto understand the concerning biological events, the use of comple-mentary topography analysis, like fractal analysis, in addition tostatistical roughness description are highly recommended. Finally,theories and experimental methods related to wetting phenomenashould be carefully employed in biomaterial engineering.

5. Conclusions

The conclusions of this work are summarized as follows:

1. The traces of silicon carbide and alumina found in the polishedand blasted surfaces modified their surface energy, accordingly.

2. Cell adhesion and proliferation on the textured titanium surfaceswith similar surface chemistry (acid-etched and blasted-etchedsurfaces) were driven by topography features.

3. Cell adhesion (≤24 h) depended on the available surface area(more roughness, more cell adhesion).

4. Cell proliferation (≥48 h) was mediated by specific morpholo-gies: highly fluctuating surfaces (greater fractal dimension) andpunching surfaces (negative skewness). Unlike, for surfaces withsimilar fractal dimension and positive skewness, the cell growthbasically depended on their surface area.

5. Acid etching yielded angular stepped morphologies due to thecrystalline grain detachment, with horizontal length scale close

to the cell size (∼10 �m).

6. Blasting treatment generated pit-like surface features with highaspect ratios and chaotic morphologies. These physical surfacemodifications, aside from the small alumina doping, hinderedthe cell growth.

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J.I. Rosales-Leal et al. / Colloids and Surfaces

cknowledgements

This work was supported by the “Ministerio Espanol de Edu-ación y Ciencia” (project MAT2007-66117 and contract “Ramón yajal” RYC-2005-000983), Junta de Andalucía (projects P07-FQM-2517 and P08-FQM-4325) and the European Social Fund (ESF). Theuthors are grateful to Yudi Gómez-Villaescusa for the laboratoryssistance.

eferences

[1] P.I. Branemark, B.O. Hansonn, R. Adell, U. Breine, J. Lindstrom, O. Hallen, A.Ohman, Osseointegrated titanium implants in the treatment of the edentulousjaw, Scand. J. Plast. Reconstr. Surg. 11 (1977) 171–175.

[2] T. Albrektsson, P.I. Branemark, H.A. Hanson, J. Lindstrom, Osseointegratedtitanium implants. Requirements for ensuring a long-lasting, direct boneanchorage in man, Acta Orthop. Scand. 52 (1981) 155–170.

[3] D. Botticelli, T. Berglundh, D. Buser, J. Lindhe, The jumping distance revisited.An experimental study in the dog, Clin. Oral Implant. Res. 14 (2003) 35–42.

[4] H. Assender, V. Bliznyuk, K. Porfyrakis, How surface topography relates to mate-rials’ properties, Science 297 (2002) 973–976.

[5] R.G. Flemming, C.J. Murphy, G.A. Abrams, S.L. Goodman, P.F. Nealy, Effects ofsynthetic micro- and nano-structured surfaces on cell behaviour, Biomaterials20 (1999) 573–588.

[6] F. Rupp, L. Scheideler, D. Rehbein, D. Axmann, J. Geis-Gerstorfer, Roughnessinduced dynamic changes of wettability of acid etched titanium implant mod-ifications, Biomaterials 25 (2004) 1429–1438.

[7] J.Y. Martin, Z. Schwartz, T.W. Hummert, D.M. Schraub, J. Simpson, J. Lankford,D.D. Dan, D.L. Cochran, B.D. Boyan, Effect of titanium surface roughness onproliferation, differentiation, and protein synthesis of human osteoblast-likecells (MG63), J. Biomed. Mater. Res. 29 (1995) 389–401.

[8] K. Kieswetter, Z. Schwartz, T.W. Hummert, D.L. Cochran, J. Simpson, D.D. Dean,B.D. Boyan, Surface roughness modulates the local production of growth factorsand cytokines by osteoblast-like MG-63 cells, J. Biomed. Mater. Res. A 32 (1996)55–63.

[9] D.A. Puleo, A. Nance, Understanding and controlling the bone-implant interface,Biomaterials 20 (1999) 2311–2321.

10] A.W. Adamson, A.P. Gast, Physical Chemistry of Surfaces, 6th ed., New York,Wiley, 1997.

11] X. Zhu, J. Chen, L. Scheideler, R. Reichl, J. Geis-Gerstorfer, Effects of topographyand composition of titanium surface oxides on osteoblast responses, Biomate-rials 25 (2004) 4087–4103.

12] L. Hao, J. Lawrence, K.S. Chian, Osteoblast cell adhesion on a laser modifiedzirconia based bioceramic, J. Mater. Sci. Mater. Med. 16 (2005) 719–726.

13] M.C. Advincula, F.G. Rahemtull, R.C. Advincula, E.T. Ada, J.E. Lemons, S.L. Bellis,Osteoblast adhesion and matrix mineralization on sol–gel derived titaniumoxide, Biomaterials 27 (2006) 2201–2212.

14] M.I. Janssen, M.B. Van Leeuwen, T.J. van Coten, J. de Vries, L. Dijkhuizen, H.A.Wösten, Promotion of fibroblast activity by coating with hydrophobins in thebeta-sheet end state, Biomaterials 25 (2004) 2731–2739.

15] R.E. Baier, Surface behaviour of biomaterials: the theta surface for biocompat-ibility, J. Mater. Sci. Mater. Med. 17 (2006) 1057–1062.

16] F. Rupp, L. Scheideler, J. Geis-Gerstorfer, Effect of heterogenic surfaces on con-tact angle hysteresis: dynamic contact angle analysis in material sciences,Chem. Eng. Technol. 25 (2002) 877–882.

17] M. Bächle, R.J. Kohal, A systematic review of the influence of different titaniumsurfaces on proliferation, differentiation and protein synthesis of osteoblast-like MG63 cells, Clin. Oral Implant. Res. 15 (2004) 683–692.

18] A. Bagno, C. Di Bello, Surface treatments and roughness properties of Ti-basedbiomaterials, J. Mater. Sci. Mat. Med. 15 (2004) 935–949.

19] S.W. Ha, A. Gisep, J. Mayer, E. Wintermantel, Topographical char-acterization and microstructural interface analysis of vacuum-plasma-sprayed titanium and hydroxyapatite coatings on carbon fibre-reinforced

poly(etheretherketone), J. Mater. Sci. Mat. Med. 8 (1997) 891–896.

20] E.S. Gadelmawla, M.M. Koura, T.M.A. Maksoud, I.M. Elewa, H.H. Soliman, Rough-ness parameters, J. Mater. Process. Technol. 123 (2002) 133–145.

21] B.B. Mandelbrot, The Fractal Geometry of Nature, Freeman, New York, 1983.22] K. Anselme, M. Bigerelle, B. Noel, E. Dfresne, D. Judas, A. Iost, P. Hardouin,

Qualitative and quantitative study of human osteoblast adhesion on materi-

[

sicochem. Eng. Aspects 365 (2010) 222–229 229

als with various surface roughnesses, J. Biomed. Mater. Res. A 49A (2000) 155–166.

23] S. Amada, A. Satoh, Fractal analysis of surfaces roughened by grit blasting, J.Adhes. Sci. Technol. 14 (2000) 2741.

24] G. Palasantzas, J.T.M. De Hosson, Influence of surface roughness on the adhesionof elastic films, Phys. Rev. E 67 (2003) 021604.

25] P.F. Chauvy, C. Madore, D. Landolt, Variable length scale analysis of surfacetopography: characterization of titanium surfaces for biomedical applications,Surf. Coat. Technol. 110 (1998) 48–56.

26] P.J. Ramon-Torregrosa, M.A. Rodríguez-Valverde, A. Amirfazli, M.A. Cabrerizo-Vílchez, Factors affecting the measurement of roughness factor of surfaces andits implications for wetting studies, Colloids Surf. A: Physicochem. Eng. Aspects323 (2008) 83–94.

27] T.J. Webster, E.S. Ahn, Nanostructured biomaterials for tissue engineering bone,Adv. Biochem. Eng. Biotechnol. 103 (2006) 275–308.

28] C. Douketis, Z. Wang, T.L. Haslett, M. Moskovits, Fractal character of cold-deposited silver films determined by low-temperature scanning tunnellingmicroscopy, Phys. Rev. B 51 (1995) 11022–11031.

29] W. Zahn, A. Zösch, The dependance of fractal dimension on measuring con-ditions of scanning probe microscopy, Fresenius J. Anal. Chem. 365 (1999)168–172.

30] M. Morra, C. Cassinelli, Evaluation of surface contamination of titanium dentalimplants by LV-SEM: comparison with XPS measurements, Surf. Interface Anal.25 (1997) 983–988.

31] M. Morra, E. Occhiello, F. Garbassi, Knowledge about polymer surfaces fromcontact angle measurements, Adv. Colloid Interface Sci. 32 (1990) 79–116.

32] R.V. Bathomarco, G. Solorzano, C.N. Elias, R. Prioli, Atomic force microscopyanalysis of different surface treatments of Ti dental implant surfaces, Appl.Surf. Sci. 233 (2004) 29–34.

33] R.N. Wenzel, Resistance of solid surfaces to wetting by water, Ind. Eng. Chem.28 (1936) 988–994.

34] M. Morra, C. della Volpe, S. Siboni, Comment to the paper: enhancing surfacefree energy and hydrophilicity through chemical modification of microstruc-tured titanium implant surfaces, by F. Rupp, L. Scheideler, N. Olshanska, M.de Wild, M. Wieland, J. Geis-Gerstorfer, J. Biomed. Mater. Res. A 79A (2006)752–754.

35] M.A. Rodríguez-Valverde, F.J. Ruiz-Cabello, M.A. Cabrerizo-Vilchez, Wetting onaxially-patterned heterogeneous surfaces, Adv. Colloid Interface Sci. 138 (2008)84–100.

36] H.A. Wege, J.A. Holgado-Terriza, J.I. Rosales-Leal, R. Osorio, M. Toledano, M.A.Cabrerizo-Vílchez, Contact angle hysteresis on dentin surfaces measured withADSA on drops and bubbles, Colloids Surf. A: Physicochem. Eng. Aspects 206(2002) 469–483.

37] C. Aparicio, F.J. Gil, C. Fonseca, M. Barbosa, J.A. Planell, Corrosion behaviour ofcommercially pure titanium shot blasted with different materials and sizes ofshot particles for dental implant applications, Biomaterials 24 (2003) 263–273.

38] F. Rupp, L. Scheideler, N. Olshanska, M. de Wild, M. Wieland, J. Geis-Gerstorfer,Enhancing surface free energy and hydrophilicity through chemical modifica-tion of microstuctured titanium implant surfaces, J. Biomed. Mater. Res. A 76A(2006) 323–334.

39] G. Giavaresi, M. Fini, A. Cigada, R. Chiesa, G. Rondelli, L. Rimondini, P. Torri-celli, N.N. Aldini, R. Giardino, Mechanical and histomorphometric evaluations oftitanium implants with different surface treatments inserted in sheep corticalbone, Biomaterials 24 (2003) 1538–1594.

40] R.L. Sammons, N. Lumbikanonda, M. Gross, P. Cantzler, Comparison ofosteoblast spreading on microstructured dental implant surfaces and cellbehaviour in an explant model of osseointegration: a scanning electron micro-scopic study, Clin. Oral Implant. Res. 16 (2005) 657–666.

41] C.N. Elias, Y. Oshida, J.H. Lima, C.A. Muller, Relationship between surface prop-erties (roughness, wettability and morphology) of titanium and dental implantremoval torque, J. Mech. Behav. Biomed. Mater. 1 (3) (2008) 234–242.

42] D. Chappard, I. Degasne, G. Huré, E. Legrand, M. Audran, M.F. Bas, Image anal-ysis measurements of roughness by texture and fractal analysis correlate withcontact profilometry, Biomaterials 24 (2003) 1399–1407.

43] J. Guo, R.J. Padilla, W. Ambrose, I.J. De Kok, L.F. Cooper, The effect of hydrofluoric

gene expression in vitro and in vivo, Biomaterials 28 (2007) 5418–5425.44] S.F. Lamolle, M. Monjo, M. Rubert, H.J. Haugen, S.P. Lyngstadaas, J.E. Ellingsen,

The effect of hydrofluoric acid treatment of titanium surface on nanostruc-tural and chemical changes and the growth of MC3T3-E1 cells, Biomaterials 30(2009) 736–742.