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Biomaterials 26 (2005) 12191229

Surface properties and cell response of low metal ion release Ti-6Al-7Nb alloy after multi-step chemical and thermal treatments
" a, Giovanni Mainac, Silvia Sprianoa,*, Michela Bosettib, Marco Bronzonia, Enrica Verne Valerio Bergoc, Mario Cannasb
a Material Science and Chemical Engineering Department, University of Turin, Turin, Italy Department of Medical Sciences, Human Anatomy, University of Eastern Piedmont, Novara, Italy c Traumatology Orthopaedics and Occupational Medicine Department, University of Turin, Turin Italy b

Received 18 February 2004; accepted 8 April 2004 Available online 4 June 2004

Abstract Ti-6Al-7Nb samples treated by innovative multi-step chemical and thermal processes were characterized in order to evaluate their surface properties and cell interaction. The main object was to asses if the treatments were effective in order to obtain a surface presenting at the same time bone-like apatite induction ability, low metal ion release, good cell response and high protein binding. The morphology, crystallographic structure, porosity and wettability of the treated materials were investigated, as well as their interaction with simulated body uid during soaking for different times. Cytotoxicity, protein adsorption tests and in vitro broblast and osteoblast-like cell cultures were also performed. r 2004 Elsevier Ltd. All rights reserved.
Keywords: Titanium alloys; Surface modication; Osteointegration; Ion release; Protein adsorption; Cell culture

1. Introduction Titanium and its alloys are widely used in dentistry and orthopedics because of their outstanding biocompatibility and mechanical properties. Their interaction with cells depends most on surface properties (crystallographic structure, morphology and composition of surface layers, wettability) and on metal ion release. They spontaneously form in air an oxide layer containing dense and stable TiO2, with a thickness of a few nanometers [1]. When implanted, they form a morphological connection with living tissues (bioinert behavior), and also show some osteointegration ability with direct boneimplant contact. Problems arise when a low-quality bone is present or when faster healing is required [1]. Macroscopical modications in morphology are often used to obtain a better connection: surface is made porous by plasma-spray coating or simply rougher by blasting, or covered with titanium beads [2,3]. Plasma spray is an expensive technique and many authors have
*Corresponding author. Fax: +39-011-564-4699. E-mail address: silvia.spriano@polito.it (S. Spriano). 0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.04.026

reported that plasma-sprayed hydroxyapatite (HA) coatings enhanced implant performance at an early stage after implantation, but caused poorer long-term performances because of low adhesion of the coating to the metal and low crystallinity of the HA [2,3]. Furthermore, it is difcult to obtain uniform and thin coating on implants with a complex geometry. On the other hand, some simple chemical and thermal treatments have been developed and tested in order to modify composition and morphology of surface layers, leading to a moderate bioactive behavior [48]. These surfaces are characterized by a sub-micrometric or nanometric texture. In the present study, new multi-step chemical and thermal treatments were tested and compared with single-step treatments applied on a Ti-6Al-7Nb alloy used for prosthetic implants. Morphology, crystallographic structure, porosity and wettability of the treated surface were investigated, together with some compositional analyses. Ion release tests in simulated body uid (SBF), cytotoxicity and protein adsorption tests were performed, followed by a wide in vitro characterization based on broblast and osteoblast-like cell cultures.

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2. Materials and methods 2.1. Materials A Ti-6Al-7Nb titanium alloy for prosthetic implants supplied by Centerpulse Orthopedics and commercialized as Protasul100 (composition given in Table 1) was employed, and all experiments were carried out on diskshaped samples (18 mm diameter, 2 mm thickness). Their surface was grit blasted with corundum, giving a roughness of Ra 4 6 mm. This will be indicated by letter B (blasted) in every acronym for samples. Two kinds of chemical treatment were used: a passivation in nitric acid, 30 min in 35 wt% HNO3 aqueous solution at room temperature (this treatment will be indicated by letter P, standing for passivation) and/or an alkali treatment, 24 h in 5 m NaOH aqueous solution at 60 C (this treatment will be indicated by S, standing for [caustic] soda). Two kinds of thermal treatments were performed: a heat treatment in air, 1 h at 600 C in a standard electric furnace, with a heating rate of 300 C/h as well as the initial cooling rate (this treatment will be indicated by A), or a heat treatment in vacuum, 1 h at 600 C under rotative pump dynamic vacuum conditions (5 101 bar), same heating/cooling rates as above (this treatment will be indicated by V). After P and S treatments, the samples were washed with distilled water and dried at room temperature. After A and V treatments, they were washed with acetone and dried at room temperature. The innovative multi-step treatments consist of passivation followed by alkali treatment, and a subsequent heat treatment. Therefore, the acronyms will be BPSA or BPSV depending on the atmosphere used for the heat treatment. If the samples underwent only an alkali treatment and a heat treatment in air, they will be named bovine serum albumin (BSA) and so on. 2.2. Surface characterization Surface morphology and composition were assessed by scanning electron microscopy (SEM Philips 525 M) and energy dispersion spectrometry (EDS PhilipsEDAX 9100). Surface crystalline structure was investigated by Xray diffraction analysis (XPert Philips diffractometer), using a parallel-beam conguration (PB-XRD) and Cu-

Ka incident radiation. The incident angle was set at 1.2 . Surface porosity of the modied samples was evaluated by mercury intrusion porosimetry (FISONS Porosimeter 2000), using a cylindrical geometry as a model for pore shape. 2.3. Wettability tests Contact angle measurements were carried out in order to evaluate the wettability of the surface-modied alloy as resulting from every treatment. An equal volume of distilled water was placed on every sample by means of a micropipette, forming a drop or spreading on the surface. Photos were taken through lenses (LEITZ IIA optical stage microscope equipped with LEICA camera) to record the shape of the drops and measure the contact angle. 2.4. Metal ion release Metal ion release tests were performed by immersing the samples in a standard SBF solution kept at 37 C in a BICASA Alpha incubator. The SBF used in this work was proposed by Kokubo and co-workers [911] to mimic the inorganic salt composition of human physiological uids. Small amounts of solution were withdrawn at different times, ranging from 3 h to 60 days, and replaced with fresh solution. The replacement of the withdrawn solution with fresh one leads to dilution effects; knowing exactly the amount of soaking solution and the withdrawn (and replaced) quantity it was possible to process the data taking into account the experimental procedure. An atomic absorption spectrophotometer (ICP-GFAA technique) was used to evaluate metal ions concentration in the withdrawn solution, focusing on titanium and aluminum content. Every different treatment tests were carried out on at least three samples. 2.5. Soaking in simulated body uids Samples immersed in SBF for up to 30 days were carefully dried and observed at SEM to investigate the possible precipitation of HA on their surface. Such phenomenon is often associated to bioactivity, and represents a simple in vitro test [911]. 2.6. In vitro biocompatibility tests At least 20 samples per treatment were prepared for in vitro tests involving cell cultures. Cytotoxicity tests consisted of the quantication of the activity of lactate dehydrogenase (LDH) in culture medium of cells in contact with samples. The activity of the LDH enzyme rises when cells are damaged: the LDH activity induced

Table 1 Composition of the Ti-6Al-7Nb alloy (wt% values) Ti Min Max Bal. Al 5.5 6.5 Nb 6.5 7.5 Ta 0.5 Fe 0.25 H 0.009 N 0.05 O 0.20 C 0.08

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by samples was compared to that induced by a toxic agent (Triton X100 0.05% in phosphate-buffer saline (PBS)) and to that induced by a non-toxic polymeric substrate (Thermanoxs slides, Nunc, Milano, Italy). Murine broblasts (NIH 3T3) and human primary osteoblast-like cells were used for adhesion and proliferation tests. Cells were cultured on the tested materials at 2 104 cells/cm2 for adhesion studies (6 h incubation) and at 1 104 cells/cm2 for proliferation studies (3 and 5 days). At the end of the incubation period samples with cells were xed and stained with a uorescent nuclear staining (Acridine Orange solution 0.025% in PBS) and cells counted with a computer assisted uorescence microscopy at 400 magnication. Cell count results were reported as cell number 7SD obtained in 20 random elds of the samples of 0.068 mm2. Samples carrying broblasts or osteoblastlike cells were also prepared for SEM observation; cells were xed and dehydrated, in order to investigate cell morphology at 6 h and 5 days cell cultures. Protein adsorption assays were performed using human platelet poor plasma (PPP) obtained by centrifugation at 2000 g for 15 min at 4 C of venous blood from four healthy donors collected in tubes containing 3.5% (w/v) sodium citrate (Becton Dickinson, Meylan, France). The materials were incubated with 200 ml/cm2 PPP diluted 1:4 in deionized water for 30 min at room temperature. Samples were washed three times in PBS and adsorbed proteins were measured using a commercial protein quantication kit (Pierce, Rockford, IL) based on bicinchoninic acid (BCA) colorimetric detection of the cuprous cation obtained by protein Cu2+ reduction in an alkaline medium. The optical densities were read at 562 nm against a calibration curve using a BSA working range of 50800 mg/ml. The results were expressed in micrograms protein per milliliter. We have used polystyrene plastic wells of 1.77 cm2 area as protein binding controls. After PPP incubation and PBS washings, other sets of materials were incubated at 90 C for 10 min in 200 ml of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (0.015 m Tris-HCl, pH 6.8, containing 2.5% (w/v) glycerol, 1.25% (v/v) bmercaptoethanol, 0.5% (w/v) SDS and 0.01% (w/v) bromophenol blue). Samples were denatured by boiling for 3 min, centrifuged at 12,000g; and then a 10 ml volume was loaded onto a 10% separating polyacrylamide gel with a 4% stacking gel. Electrophoresis was conducted at 100 V by an I electrophoresis system (Biorad Mini-Protean II, Milan, Italy). Proteins were stained by a BioRad Silver Stain Plus kit or blotted onto nitrocellulose membrane (Amersham, Milan, Italy) by Brunettes method in a Bio-Rad Mini Trans-Blot electrophoresis Transfer Cell. The membranes were treated with 5% (w/v) BSA (Sigma) in PBS pH 7.4 for 1 h at room temperature, and then after washings with

PBS/Tween-20 0.1%, incubated with a mouse antihuman Fibronectin clone IST-3 (Sigma), rabbit antihuman albumin, IgG and IgA (Dako, Milan, Italy) 5 mg/ml in PBS overnight at 4 C. After washing procedures, the membrane was incubated with 1:10,000 diluted secondary antibody conjugated with horseradish peroxidase (Amersham) for 1 h at room temperature. Pico-chemiluminescence Western blotting detection reagents (Pierce) were used following the directions of the manufacturer for immunodetection of the eluted proteins.

3. Results 3.1. Surface characterization The blasted surface prior to any treatment is highly irregular and rough, as expected. After the passivation treatment (BP samples), the surface does not look like different in morphology from the blasted and untreated one. Alkaline and thermal treatments produce homogeneous layers consisting of sub-micrometric cylindrical or spherical particles, showing some differences depending on the treatment parameters (solution concentration, atmosphere employed during heat treatment) and on the previous mechanical state of the surface. Such layers can also be described as micro-porous or microtextured layers. SEM observations on BSA samples (Fig. 1a) show almost spherical and thickened particles, about 8090 nm in diameter. BPSA and BPSV samples show a less thickened layer, and while BPSA has again 8090 nm sized particles, BPSV has thinner ones, down to 60 nm (Fig. 1b and c). The behavior of Ti-6Al-7Nb thus appears different from what observed in the case of c.p. titanium. In fact commercially pure titanium samples were also treated and examined, and the surface layer observed can be described as less dense and with a trabecular aspect, according to what is reported in literature [911]. The thickness of the described surface layers can be estimated observing how they follow the irregular prole of the blasted substrate (Fig. 1d): the order of magnitude is 106 m or lower. The reduced thickness of surface layer suggested the use of parallel-beam conguration for X-ray diffractometry, which collects more information from the surface of the sample and is less inuenced by the substrate. No relevant diffraction peaks, apart from those due to the substrate, were detected on BP samples. Fig. 2 shows the PB-XRD patterns obtained for BPSA, BPSV and BSA samples. It can be noticed that a diffraction signal at ( was registered in the about 2y 25:5 (d 3:49 A) diffraction pattern of the alkali and air heat-treated (BSA) and of the passivated, alkali and vacuum/air heat-treated samples (BPSA and BPSV). A second

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Fig. 1. SEM images of the surface of treated samples: (a) BSA, (b) BPSA, (c) BPSV and (d) BPSA at higher magnication.

( was signal, centered at about 2y 27:5 (d 3:24 A), observed only on the air heat-treated samples (BSA and BPSA). The other signals were ascribed to the titanium substrate. The rst signal could be assigned to a titanium oxide, as well as Ti3O5 monoclinic phase or to a sodium-containing titanium oxide, as for instance Na2Ti3O7. According to the reduced thickness of the surface layer and to the difculty to detect it by XRD, even by using parallel-beam conguration, the awarding of the rst diffraction peak to a unique phase is quite dubious. By considering the absence of a relevant amount of Na on the surface registered by surface compositional analysis [12] (EDS, Auger Electron Spectroscopy) the rst option was selected. On the other hand, it is easier to ascribe the second signal to rutile. The surface porosity due to the formation of the modied layer was evaluated by mercury intrusion porosimetry. In the case of a BPSA sample a porosity of 1.15% was detected, with an average pore radius of 30 nm. Data are reported in Fig. 3, where a broad pore size distribution starting from 10 nm and up to 0.2 mm can be observed. In the case of a BPSV sample a total porosity of 0.13% was measured, with an average pore radius of 6 nm. 3.2. Wettability Chemical and heat treatments showed a great inuence on the wettability of the surfaces. Distilled

water put on untreated samples formed a regular drop, with a contact angle of about 55 . After passivation (BP samples), the contact angle decreases to less than 50 . After any kind of subsequent chemical and heat treatment, the surface is much more easily wetted, resulting in a very low to null contact angle. In detail, water spreads completely on the surface of BSA and BPSA samples (contact angle 0 ). BPSV samples show a contact angle of less than 15 , but it is difcult to observe regular-shaped drops since water begins to spread in some directions. 3.3. Metal ions release A rst campaign covered 1 month as immersing time and was aimed to compare the behavior of samples treated by passivation (BP) or by the new treatment (BPSA and BPSV) to some data found in literature [11]. As shown in Fig. 4, all kinds of samples induce a rapid increase in metal ion concentration during the rst 3 days. After longer time exposures, a slow increase of metal ion concentration was found in SBF. Samples that were only passivated with HNO3 induced a concentration of about 7 mg/l for Al ions after 28 days. With BPSA samples we obtained about 6 mg/l for Al ions. In the case of BPSV sample, a maximum value of about 3 mg/l for Al ions was detected. Due to the quite low level of metallic ions in the solution, the results are affected by moderate statistical scattering. Values for Ti ions were negligible in all cases.

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Fig. 2. Parallel-beam XRD patterns for BSA, BPSA and BPSV samples.

LIT REF

BP

BPSA

BPSV

Al ions release in SBF 100

concentration [ug/L]
Fig. 3. Porosimetry test results for BPSA and BPSV samples.

10

0.1 0 5 10 15 20 25 30 time [days]

Fig. 4. Metal ions release tests results. Literature reference (LIT REF) curve is related to data reported in [11].

A second campaign covered 2 months as immersing time and was aimed to conrm the low metal ion release data over a prolonged time and to compare the air and vacuum treatments. Al ions concentration stays under 10 mg/l even after 2 months, and Ti ions concentration reaches a maximum of 2 mg/l. No signicant differences between air and vacuum treatments were observed. Literature data [11] refer to Ti-6Al-2Nb-Ta samples with grinded surfaces, thermochemically treated as BSA. Al ions concentration is more than 50 mg/l after 28 days. Values for Ti ions are negligible. Our values for Al ions release are about one order of magnitude lower than literature ones, so the passivation stage introduced by the new process seems to be effective against Al ions release. The passivation alone seems to be effective as well, but electrochemical tests [12] have demonstrated that only BPSA samples have a good corrosion behavior also in aggressive environments.

3.4. Soaking in simulated body uids Aggregates, which prove to be rich in calcium on EDS analysis, appear on the surface of the samples after a few days in SBF, without signicant differences among the various treatments. After longer time exposure to SBF (1430 days), groups of spherical aggregates appear on the surface of BPSA and BPSV samples, their composition was closed to the standard Ca/P ratio of bone-like apatite. 3.5. In vitro biocompatibility The LDH activity in culture media obtained from cells cultured on all the tested materials was found to be

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not signicantly different from the negative control and no time-dependent increase at long time evaluation was found for all the materials (Fig. 5). Fibroblasts adhesion on all the materials was similar to that observed on positive control with the exception of BPSV that evidenced statistically lower cell number. Fibroblast proliferation at 3 and 5 days evidenced good proliferation on B samples, similar to that of control; broblasts on BPSA, BPSV and BSA evidenced similar low proliferation with no differences between them and lower proliferation respect to B and positive Thermanoxs proliferation control (Fig. 6a). Osteoblast-like cell adhesion evidenced higher cell number on B respect to the other materials with cell number comparable to control. When evaluating osteoblast-like cells proliferation at 3 and 5 days also cells on B evidenced lower number respect to cells proliferating on control polystyrene. Between the four kinds of samples, BPSA evidenced the lowest cell number with results statistically lower respect to B (Fig. 6b). SEM of the broblasts and of the osteoblast-like cells adhering and proliferating on the surface of the four tested materials showed a good cell spreading with abundant cell lopodes forming bridges between the holes of the material (Fig. 7). Protein adsorption assays evidenced that BPSA and BPSV induced statistically higher total protein binding respect to B. These results are quantitatively represented in Fig. 8. Fig. 8b shows the SDS-PAGE proles of the proteins eluted from the tested materials. All the materials showed a main band at about 66 kDa and a band in the range 6645 kDa particularly evident in BPSA and BSA. Fig. 9a shows by Western blot the identication of albumin, a main adhesion plasma protein; it was found on all the samples with no

differences between the various treatments and this band was assigned to that found at about 66 kDa in the SDS-PAGE. Fig. 9b and c shows the identication of, respectively, IgA and IgG eluted from the tested materials. Western blot prole evidenced IgG and IgA binding to all the four materials and seem to bind in few manner to BPSV. Fig. 9d shows Western blot analysis of C3 fragments present on the surface of the tested biomaterials incubated with human plasma. C3 fragments bound on the surface of all the materials; the predominant polypeptides had molecular weights of B70 and 40 kDa, representing the b-chain of C3/C3b and fragments of iC3b, respectively. Fibronectin evidenced very low quantity of protein eluted from the materials with no differences between them.

4. Discussion During the present research work, two new processes (BPAA and BPAV) were proposed in order to obtain titanium implants presenting good osteointegrability. The process parameters were optimized and different alkali solution concentrations were tested in a previous work [12]. The main differences between these innovative processes and those previously proposed in literature [48,14] are the mechanical pre-treatment (blasting), the use of a passivation step before alkali etching and the vacuum heat treatment performed in the case of BPAV process. Furthermore, no data were published up to date about the eventual bioactive behavior of the Ti-6Al-7Nb treated alloy. The use of a passivation treatment before the alkali one changes the crystallographic structure of the surface, causes the

Fig. 5. Results provided by assessment of cell membrane damage are expressed as LDH activity (U/L)7standard deviation at 340 nm.

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Fig. 6. Fibroblasts (a) and osteoblast-like cells (b) quantication on the four substrates studied and on control Thermanoxs slide. Cell number was the mean of 20 measurements in three experiments (n 60) and was referred to a surface area of 0.068 mm2. po0:05 respect to control and  po0:05 respect to A.

formation of a diffusion border region between the surface and the bulk, increases the mechanical adhesion of the surface layer to the substrate and reduce metal ion release [12]. The passivation treatment alone was widely discussed in literature and in conclusion it seems to cause pitting corrosion and higher metal ion release [3,15,16], but it represents in our innovative processes an important step without disadvantages. The main surface characteristics of the two kinds of treated samples (BPSA and BPSV) will be briey summarized in the following, with the aim of discussing the inuence of the surface state respect to cell interaction. Both the treated samples present an oxide layer thicker than the native one and presenting different crystallographic structure. In the BPSV case, it is mainly formed by Ti3O5 monoclinic phase while on the BPSA surface rutile is also present. The increase of thickness of the oxide layer, due to the thermochemical treatments, could be potentially benecial considering

that quantitative histomorphometrical data in literature indicated stronger bone tissue reactions to implants with a thick oxide (6001000 nm) compared to implants with a reduced oxide thickness (17 nm) [10,1720]. The inuence of the surface crystallographic structure on implantcell interaction is not clear, but the absence of rutile in the BPSV case could be interesting from the fatigue resistance point of view. The morphology of the treated surface can be described as macroscopically rough due to blasting and porous on a sub-micrometric scale due to thermochemical treatment. It is covered by a homogeneous layer consisting of almost spherical and thickened particles (about 80 nm in diameter). The treated materials show a quite good wettability while the untreated one does not. The marked lowering of contact angle respect to the sandblasted and untreated surface can be ascribed to the increased lowscale roughness and porosity [21]. The good wettability

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Fig. 7. Morphology of broblasts and osteoblast-like cells adhering on cell culture dish. Samples were examined by SEM: (a) osteoblast-like cells at 6 h adhesion, (b) osteoblast-like cells at 5 days proliferation, (c) broblasts cells at 6 h adhesion and (d) broblasts cells at 5 days proliferation.

of the treated surfaces is an interesting aspect in order to obtain chemical interaction with the physiological uids [14]. Good wettability could be advantageous also by considering that the hydrophile surfaces avoid protein denaturation [22]. It has already been reported [14] that low contact angles (below 30 ) are promising for a successful osteointegration. One of the aims of the research was to obtain surfaces presenting low metal ion release. The hazardous physiological and cellular effects, due to metal ion release from implants, cover a wide and complex series of topics. Authors often underline disagreement between literature data and the issue is not fully understood [3,23,24]. Local effects, as accumulation of ions in the tissue adjacent to the implant or local inammatory events, are often described, for instance, during retrieval

studies. On the other hand, metal ions diffusion in the body and related accumulation in lymph nodes and organs such as spleen, liver and lungs must be also considered. In fact while the bone growth probably freezes the ion release through the committed osteoblasts and connes it to the areas adjacent to the implant, a different question is represented by titanium/ aluminum diffusion through the blood cells of the brous tissue and the related transport to the different organs. Furthermore, systemic damages were sometimes reported as well as allergies and long-term hypersensitivity [23]. Titanium contents as high as three times respect to the usual serum values were registered in the case of patients with titanium hip prostheses. At this concern, even the superiority of titanium respect to steel, concerning biocompatibility, was recently questioned

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Fig. 9. Immunoblotting of plasma proteins eluted from the materials with rabbit anti-human albumin antiserum (a), rabbit anti-human IgA antiserum (b), rabbit anti-human IgG antiserum (c) and rabbit antihuman C3 antiserum (d). Lane 1: proteins eluted from B; lane 2: proteins eluted from BPSA; lane 3: proteins eluted from BPSV; lane 4: proteins eluted from BSA.

Fig. 8. (a) BCA results of protein adsorption to the materials incubated 30 min at room temperature with 1:4 diluted PPP. Optical densities read at 562 nm against a calibration curve using BSA. Results expressed as micrograms protein7SD per milliliter; n 5: (b) Silverstained SDS-PAGE patterns of protein layers adhered to biomaterials, after in vitro incubation with 1:4 PPP.

[23], due to deep corrosion evidence and immuneinammatory reaction observed after explantation of plates used for internal xation of long bone fractures. In the light of these observations, surface treatments able to decrease metal ion release and surface corrosion acquire more importance. Considering the mechanical state of the implants, it was assessed that blasted materials show an increase in metal ion release, despite their good osteointegration ability. At this concern, it was also underlined that an eventual coating of plasmasprayed HA is not sufcient to reduce signicatively the metal corrosion [18]. Contrasting data were reported about the effect of passivation on metal ion release [3,15,18]. In Al-containing alloys, a signicant Al ion release was registered because of a great driving force for ion migration combined with smaller, more mobile aluminum ions [15].

By comparing our data with the literature, it can be concluded that both BPAV and BPAA processes are of interest at this regard. In fact in spite of some potential negative factors, as macro-roughness due to sandblasting, the small-scale porosity and the Al content of the alloy, the metal ion release is quite low. Moreover, it is lower than what reported in literature [11] in the case of samples treated by analogous process and lower than the hazardous metal ion levels reported in literature [13,25,26]. Our data evidenced a reduced in vitro bioactivity respect to what is reported in literature on analogous materials. Some authors previously underlined that the in vitro apatite precipitation was not completely reproducible. In vivo tests will be probably useful in order to clarify this aspect. It must be also considered that the bioactive behavior of treated titanium or titanium alloys surfaces was rather widely treated in literature during the last 67 years [4], but its mechanism as well as the relevance of the different factors involved is still a discussed topic. The determining step is probably the formation of negatively charged Ti-OH groups. Otherwise the Na content seems to be not so decisive [6] and the formation of a titanate phase during alkali etching is not a univocal effect [14]. Also

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the morphology of the bioactive layer was probably not so determinant, in fact either smooth or sub-micrometric porous titanium oxides were reported as bioactive according to different cases [27]. Different authors reported also bioactive behavior of different crystallographic structure as well as anatase [2830] or thermally prepared rutile [31]. We did not observed the formation of a sodium titanate phase on the surfaces of the samples investigated during this work and we registered Na content only in a very thin surface layer, quite thinner than the surface oxide [12]. As underlined by the previous literature data, this fact could be not essential in order to obtain bioactivity. The morphology of the investigated samples was slightly different from that reported in literature, but a sub-micrometric porosity was present so also topographical effect cannot be considered as the fundamental matter. Future work will be performed in order to quantify the Ti-OH groups present on the treated surfaces after immersion in physiological solution, because of the importance, as previously reported, of this topic respect to bioactive ability of a material. At this regard, the vacuum heat treatment could be favorable due to the increase of density of defective sites in the oxide under low oxygen atmosphere. As previously described, the novel processes proposed deeply modify the surfaces and so the cellbiomaterial interaction is quite interesting and complex to be analyzed because of the large number of variables involved. The absence of toxicity evidences (LDH test) conrmed the low metal ion release data. So the modied surfaces can be described as no toxic. By considering the osteoblast-like cell adhesion to the tested materials, it must be evidenced a lower number of cells on all the treated materials respect to the untreated one (B). On the contrary, broblast adhesion on all the treated materials was good and comparable to what observed on the untreated one, with a slight decrease only in the case of vacuum heat-treated one (BPSV). Furthermore, the osteoblast-like cell proliferation data seem to evidence a difference between the behavior of the two innovative thermochemical treatments tested. In fact in the case of the vacuum heat-treated material (BPSV) a good osteoblast-like cell proliferation, closed to what observed on the untreated blasted one (B), was observed, while in the case of the air heat-treated material (BPSA) it was lower. On the other hand, all the treated materials exhibit a lower proliferation degree in the case of broblast cells, respect to the untreated one (B). There could be different possible explanations for this complex behavior. The increase in roughness and wettability could cause an increase in surface energy and so it is potentially benecial, considering that surfaces with energy higher than 40 erg/cm2 generally have a good cellular interaction. On the other hand, it must be considered that osteoblasts often show a higher adhe-

sion degree on hydrophobic surfaces [32], while the treated materials (BPSV and BPSA) present high wettability. It must also be considered that these biomaterials present a small-scale porosity that is rather uncommon for prosthetic implants [33]. This is a rather unexplored eld, but some literature data show that when the topography is considered below the cell scale, cells appreciate smooth surface although when the topography is considered above the cell scale, they appreciate a rough isotropic landscape [34]. From this point of view our data do not reveal the same trend. In fact both broblasts and osteoblast-like cells revealed a positive interaction with the treated biomaterials by considering cell morphology. In fact cells presented very fusiform aspect with many lamentous extensions and branches bridging across surfaces discontinuities. Finally, the chemical interaction of the surface with the culture medium must be considered. Physicochemical features of the metallic surface can affect the reorganization of proteins on the specimens and change the prole of adsorbed proteins. The differences in protein adsorption could result in very different initial cellular behavior [35,36]. This investigation is intended to identify some of the proteins that may be involved in modulating cellular behavior. Our data evidenced that treated samples (BPSA and BPSV) induced higher total protein binding respect to the untreated one (B). The immunolocalization study demonstrated that no detectable bronectin binds to the materials, explaining the low cell adhesion found on all the different surfaces; so even if albumin binds to all the different titanium treated surfaces. Multi-step chemical and thermal treatments of titanium alloy did not inuence adsorption of serum proteins responsible for human macrophage behavior. In fact no complement activation was found (see C3) and a high presence IgA and IgG were bound to treated titanium and to untreated one. No modication in macrophage complement-mediated local inammatory reaction induced by the surface treatments could be deducted.

5. Conclusions The surfaces treated by the innovative multi-step processes proposed (BPSA and BPSV) can be described as constituted by a micro-textured oxide presenting high wettability and low metal ion release as well as moderate bone-like apatite induction ability. Its interaction with broblast and osteoblast-like cell is complex, but a good cell spreading and no cytotoxicity evidence can be underlined. At the same time, a good chemical interaction of the surfaces with the culture medium, with a higher total protein binding than on the untreated one, was observed. For the above-mentioned reasons the multi-step thermochemical treatment described in this

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work could be reasonably proposed as an alternative to traditional coatings in order to prepare low metal release prostheses with enhanced osteointegrability.

Acknowledgements The Synos Foundation is kindly acknowledged for the research grant supplied. The Centerpulse Orthopedics is kindly acknowledged for the material support. References
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