Facets of protein assembly on nanostructured titanium oxide surfaces

One key for the successful integration of implants into the human body is the control of protein adsorption by adjusting the surface properties at different length scales. This is particularly important for titanium oxide, one of the most common biomedical interfaces. As for titania (TiO₂) the interface is largely defined by its crystal surface structure, it is crucial to understand how the surface crystallinity affects the structure, properties and function of protein layers mediating subsequent biological reactions. For rutile TiO₂ we demonstrate that the conformation and relative amount of human plasma fibrinogen (HPF) and the structure of adsorbed HPF layers depend on the crystal surface nanostructure by employing thermally etched multi-faceted TiO₂ surfaces. Thermal etching of polycrystalline TiO₂ facilitates a nanoscale crystal faceting and, thus, the creation of different surface nanostructures on a single specimen surface. Atomic force microscopy shows that HPF arranges into networks and thin globular layers on flat and irregular crystal grain surfaces, respectively. On a third, faceted category we observed an alternating conformation of HPF on neighboring facets. The bulk grain orientation obtained from electron backscatter diffraction and thermodynamic mechanisms of surface reconstruction during thermal etching suggest that the grain and facet surface-specific arrangement and relative amount of adsorbed proteins depend on the associated free crystal surface energy. The implications for potentially favorable TiO₂ crystal facets regarding the inflammatory response and hemostasis are discussed with a view to the advanced surface design of future implants.     

Osteoconductivity of hydrophilic microstructured titanium implants with phosphate ion chemistry

This study investigated the surface characteristics and bone response of titanium implants produced by hydrothermal treatment using H₃PO₄, and compared them with those of implants produced by commercial surface treatment methods – machining, acid etching, grit blasting, grit blasting/acid etching or spark anodization. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, contact angle measurement and stylus profilometry. The osteoconductivity of experimental implants was evaluated by removal torque testing and histomorphometric analysis after 6 weeks of implantation in rabbit tibiae. Hydrothermal treatment with H₃PO₄ and subsequent heat treatment produced a crystalline phosphate ion-incorporated oxide (titanium oxide phosphate hydrate, Ti₂O(PO₄)₂(H₂O)₂; TiP) surface approximately 5 μm in thickness, which had needle-like surface microstructures and superior wettability compared with the control surfaces. Significant increases in removal torque forces and bone-to-implant contact values were observed for TiP implants compared with those of the control implants (p < 0.001). After thorough cleaning of the implants removed during the removal torque testing, a considerable quantity of attached bone was observed on the surfaces of the TiP implants.     

XPS,AES and SEM analysis of recent dental implants

Today, surface chemistry modifications of titanium implants have become a development strategy for dental implants. The present study investigated the chemistry and morphology of commercially available dental implants (Nobel biocare TiUnite, Astra AB OsseoSpeed, 3i Osseotite, ITI-SLA). X-ray photoelectron spectroscopy (XPS) and auger electron spectroscopy were employed for the analysis of surface chemistry. The morphology was investigated by scanning electron microscopy. The present study demonstrated the major differences of surface properties, mainly dependent on the surface treatment used. The blasting and acid etching technique for the OsseoSpeed, Osseotite and SLA surfaces generally showed mainly TiO₂, but a varying surface morphology. In contrast, the electrochemical oxidation process for TiUnite implants not only produces microporous surface (pore size: 0.5–3.0 μm), but also changes surface chemistry due to incorporation of anions of the used electrolyte. As a result, TiUnite implants contain more than 7 at.% of P in oxide layer and higher amounts of hydroxides compared to the other implants in XPS analysis. F in OsseoSpeed implants was detected at 0.3% before as well as after sputter cleaning.     

The removal torque of titanium screw inserted in rabbit tibia treated by dual acid etching

Chemical acid etching alone of the titanium implant surface have the potential to greatly enhance osseointegration without adding particulate matter (e.g. TPS or hydroxyapatite) or embedding surface contaminants (e.g. grit particles). The aims of the present study were to evaluate any differences between the machined and dual acid etching implants with the removal torque as well as topographic analysis. A total of 40 custom-made, screw-shaped, commercially pure titanium implants with length of 5 mm and an outer diameter of 3.75 mm were divided into 4 groups, 10 screws in each, and chemical modification of the titanium implant surfaces were achieved using HF and HCl/H(2)SO(4) dual acid etching. The first exposure was to hydrofluoric acid and the second was to a combination of hydrochloric acid and sulfuric acid. The tibia metaphysics was exposed by incisions through the skin, fascia, and periosteum. One implant of each group was inserted in every rabbit, 2 in each proximal tibia metaphysics. Every rabbit received 3 implants with acid etched surfaces and 1 implant with a machined surface. Twelve weeks post-surgically, 7 rabbits were sacrificed, Subsequently, the leg was stabilized and the implant was removed under reverse torque rotation with a digital torque gauge (Mark-10 Corporation, USA) (Fig. 1). Twelve weeks after implant placement, the removal torque mean values were the dual acid etched implants (24%HF+HCl/H(2)SO(4), group C) required a higher average force (34.7 Ncm), than the machined surface implants (group A) (p=0.045) (Mann-Whiteney test). Scanning electron micrographs of acid etching of the titanium surface created an even distribution of very small (1-2 microm) peaks and valleys, while machining of the titanium surface created typical microscopically grooved surface characteristics. Nonetheless, there was no difference in surface topography between each acid etched implant groups. Therefore, chemically acid etching implant surfaces have higher strengths of osseointegration than machined implant surfaces. There is less correlation between removal torque and the difference in HF volume%.     

Simultaneous interaction of bacteria and tissue cells with photocatalytically activated,anodized titanium surfaces

Photocatalytic-activation of anodized TiO₂-surfaces has been demonstrated to yield antibacterial and tissue integrating effects, but effects on simultaneous growth of tissue cells and bacteria in co-culture have never been studied. Moreover, it is unknown how human-bone-marrow-mesenchymal-stem (hBMMS) cells, laying the groundwork for integration of titanium implants in bone, respond to photocatalytic activation of anodized TiO₂-surfaces. Photocatalytically-activated, anodized titanium and titanium-alloy surfaces achieved 99.99% killing of adhering Staphylococcus epidermidis and Staphylococcus aureus, an effect that lasted for 30 days of storage in air. Surface coverage by osteoblasts was not affected by photocatalytic activation of anodized TiO₂-surfaces. Co-cultures of osteoblasts with contaminating S. epidermidis however, enhanced surface coverage on photocatalytically-activated, anodized titanium-alloy surfaces. hBMMS cells grew less on photocatalytically-activated, anodized titanium surfaces, while not at all on photocatalytically-activated, anodized titanium-alloy surfaces and did not survive the presence of contaminating staphylococci. This reduced surface coverage by hBMMS cells disappeared when photocatalytically-activated, anodized titanium-alloy surfaces were exposed to buffer for 60 min, both in absence or presence of contaminating S. aureus. Consequently, it is concluded that photocatalytically-activated, anodized titanium and titanium-alloy surfaces will effectively kill peri-operatively introduced staphylococci contaminating an implant surface and constitute an effective means for antibiotic prophylaxis in cementless fixation of orthopaedic hardware.     

An increase in mouse tumor growth by an in vivo immunomodulating effect of titanium dioxide nanoparticles

Here, we investigated whether titanium dioxide (TiO₂) nanoparticles affect in vivo tumor growth through the modulation of mononuclear leukocytes. In vitro lymphocyte proliferation by lipopolysaccharide (LPS) or concanavalin A (ConA) was reduced by < 25?nm TiO₂ with a dose-dependent manner. Similarly, TiO₂ nanoparticles inhibited nitric oxide (NO) production from bone marrow-derived macrophages obtained from naïve mice. When mice were intraperitoneally (IP) injected with < 25 or < 100?nm TiO₂ once a day for 7 days, total cell number of splenocytes was reduced in the spleen of TiO₂ nanoparticle-exposed mice. Both CD4⁺ and CD8⁺ T-lymphocyte numbers were significantly decreased and B-lymphocyte development was retarded by host exposure to the TiO₂ nanoparticles. LPS-stimulated spleen cell proliferation was significantly reduced by host exposure to < 25 or < 100?nm TiO₂, but no changes were detected in ConA-stimulated spleen cell proliferation. Further, LPS-stimulated cytokine production by peritoneal macrophages and the percentage of NK1.1⁺ natural killer cells among splenocytes was reduced by the host exposures to the TiO₂ nanoparticles. When mice were IP injected with TiO₂ nanoparticles once a day for 28 days prior to the subcutaneous implantation of B16F10 melanoma cells, tumor growth was subsequently significantly increased. Collectively, these results show that TiO₂ nanoparticles may damage the development and proliferation of B- and T-lymphocytes, reduce the activity of macrophages, and decrease natural killer (NK) cell population levels, outcomes that appear to lead to an increase in tumor growth in situ. These studies allow us to suggest that TiO₂ nanoparticles might have the potential to enhance tumor growth through immunomodulation of B- and T-lymphocytes, macrophages, and NK cells.     

Bone growth enhancement in vivo on press-fit titanium alloy implants with acid etched microtexture

Early bone ongrowth secures long-term fixation of primary implants inserted without cement. Implant surfaces roughened with a texture on the micrometer scale are known to be osseoconductive. The aim of this study was to evaluate the bone formation at the surface of acid etched implants modified on the micro-scale. We compared implants with a nonparticulate texture made by chemical milling (hydrofluoric acid, nitric acid) (control) with implants that had a dual acid etched (hydrofluoric acid, hydrochloric acid) microtexture surface superimposed on the primary chemically milled texture. We used an experimental joint replacement model with cylindrical titanium implants (Ti-6Al-4V) inserted paired and press-fit in cancellous tibia metaphyseal bone of eight canines for 4 weeks and evaluated by histomorphometric quantification. A significant twofold median increase was seen for bone ongrowth on the acid etched surface [median, 36.1% (interquartile range, 24.3-44.6%)] compared to the control [18.4% (15.6-20.4%)]. The percentage of fibrous tissue at the implant surface and adjacent bone was significantly less for dual acid textured implants compared with control implants. These results show that secondary roughening of titanium alloy implant surface by dual acid etching increases bone formation at the implant bone interface.     

Titanium implant surface modification by cathodic reduction in hydrofluoric acid: surface characterization and in vivo performance

Etching is used for the surface modification of titanium to improve the implant performance in bone. In this study, pure titanium implants were surface modified by a cathodic reduction process by using hydrofluoric acid (HF) at various concentrations (0.001, 0.01, and 0.1 vol %) and a constant current of 1 mA/cm(2). The resulting surface microtopographies were analyzed by atomic force microscopy, scanning electron microscopy, and profilometry, while the surface chemical contents were evaluated by time of flight secondary ion mass spectrometry. The competitive forces between ionic surface implementation induced by the current direction and the HF etching effect on titanium were highlighted. The implant performance was evaluated in an in vivo rabbit model by using a pull-out test method. The group of implants modified with 0.01% HF showed the highest retention in bone. Fluoride and hydride amounts measured in the surfaces, as well as surface skewness (S(sk)), kurtosis (S(ku)), and core fluid retention (S(ci)) were positively correlated to the implant's retention in bone in vivo. Frequently used parameters for characterizing the implant, such as oxide content and the average height deviation from the mean plane (S(a)), were not correlated to implant performance, suggesting that these parameters are not the most important in predicting the implant performance.     

Electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy analysis of titanium surfaces cultured with osteoblast-like cells derived from human mandibular bone.

Variations in the oxide films on titanium surfaces blasted with TiO(2) particles of various sizes were analyzed after cultures with cells derived from human mandibular bone. Turned titanium surfaces and surfaces blasted with 63-90-, 106-180-, and 180-300-microm TiO(2) particles were cultured with osteoblast-like cells. The surfaces were characterized before and after the cell culture with electrochemical impedance spectroscopy (EIS). The surface chemical composition of selected samples was analyzed with X-ray photoelectron spectroscopy (XPS). EIS revealed that with respect to the turned surfaces, the effective surface area was about 5, 6, and 4 times larger on the surfaces blasted with 63-90-, 106-180-, and 180-300-microm particles, respectively. After 28 days of the cell culture, the corrosion resistance on all sample types was unaffected. The impedance characteristics suggest a considerable effect of ion incorporation and precipitation during culturing. XPS revealed that before the cell culture, a typical surface layer consisted of TiO(2). After the culture, the surface oxide film contained both phosphorus and calcium, along with large amounts of oxidized carbon (carbonate) and nitrogen. There were lower concentrations of carbon and nitrogen on the blasted surfaces. We concluded that the effective surface area was several times higher on blasted surfaces than on turned surfaces. Cells derived from human mandibular bone affected ion incorporation into the implant surface.     

Lung exposure of titanium dioxide nanoparticles induces innate immune activation and long-lasting lymphocyte response in the Dark Agouti rat

Nanomaterial of titanium dioxide (TiO₂) is manufactured in large-scale production plants, resulting in risks for accidental high exposures of humans. Inhalation of metal oxide nanoparticles in high doses may lead to both acute and long-standing adverse effects. By using the Dark Agouti (DA) rat, a strain disposed to develop chronic inflammation following exposure to immunoactivating adjuvants, we investigated local and systemic inflammatory responses after lung exposure of nanosized TiO₂ particles up to 90 days after intratracheal instillation. TiO₂ induced a transient response of proinflammatory and T-cell-activating cytokines (interleukin [IL]-1α, IL-1β, IL-6, cytokine-induced neutrophil chemoattractant [CINC]-1, granulocyte–macrophage colony-stimulating factor [GM-CSF], and IL-2) in airways 1–2 days after exposure, accompanied by an influx of eosinophils and neutrophils. Neutrophil numbers remained elevated for 30 days, whereas the eosinophils declined to baseline levels at Day 8, simultaneously with an increase of dendritic cells and natural killer (NK) cells. The innate immune activation was followed by a lymphocyte expansion that persisted throughout the 90-day study. Lymphocytes recruited to the lungs were predominantly CD4⁺ helper T-cells, but we also demonstrated presence of CD8⁺ T-cells, B-cells, and CD25⁺ T-cells. In serum, we detected both an early cytokine expression at Days 1–2 (IL-2, IL-4, IL-6, CINC-1, IL-10, and interferon-gamma [IFN-γ] and a second response at Day 16 of tumor necrosis factor-alpha (TNF-α), indicating systemic late-phase effects in addition to the local response in airways. In summary, these data demonstrate a dynamic response to TiO₂ nanoparticles in the lungs of DA rats, beginning with an innate immune activation of eosinophils, neutrophils, dendritic cells, and NK cells, followed by a long-lasting activation of lymphocytes involved in adaptive immunity. The results have implications for the assessment of risks for adverse and persistent immune stimulation following nanoparticle exposures in sensitive populations.     

The effect of hydrofluoric acid treatment of titanium surface on nanostructural and chemical changes and the growth of MC3T3-E1 cells

Fluoride-modification of dental titanium (Ti) implants is used to improve peri-implant bone growth and bone-to-implant contact and adhesion strength. In this study, the surface topography, chemistry and biocompatibility of polished Ti surfaces treated with hydrofluoric acid solution (HF) were studied. Murine osteoblasts (MC3T3-E1) were cultured on the different groups of Ti surfaces. Surfaces treated with HF had higher roughness, lower cytotoxicity level and better biocompatibility than controls. For short treatment times (40 and 90 s), fluorine was detected only within the first 5 nm of the surface layer (X-ray Photoemission Spectroscopy, XPS), whereas longer treatment time (120 and 150 s) caused fluoride ions to penetrate deeper (Secondary Ion Mass Spectrometry, SIMS). These results suggest that submerging Ti implants in a weak HF solution instigate time-dependant specific surface changes that are linked to the improved biocompatibility of these surfaces.     

Immobilization of oligonucleotides on titanium based materials by partial incorporation in anodic oxide layers

This paper describes the immobilization of bioactive molecules on titanium based surfaces through a combination of nano-mechanical fixation of nucleic acid anchor strands (ASs) by partial and regioselective incorporation within an anodic oxide layer and their hybridization with complementary strands (CSs) intended to be conjugated to bioactive molecules. We focus on the interaction between the substrate surface and the anchor strands, the integrity of ASs and their hybridization ability. The observed dependence of adsorption on pH suggests that initial interaction of terminally phosphorylated ASs with the substrate surface is mediated by electrostatic interaction. Using ASs labelled with ³²P at different termini, it could be shown that strand breaks occur, which are attributed (i) to the formation of reactive oxygen species during anodic polarization, (ii) the photocatalytic activity of the titanium oxide and (iii) drying effects. Damage to AS could be considerably reduced if the electrolyte contained 5 mol l^?1 ethanol, light was excluded during the experimental procedure, and the number of drying and rewetting steps was minimized. A total surface density of AS of 4.5 pmol cm^?2 was reached and could be hybridized to CS with an efficiency of up to 100%. A non-complementary strand (NS) bound with less than 0.5% of the amount of CS under similar conditions. Therefore, non-specific binding of CS is considered as negligible.     

Fabrication and characterization of oxygen-diffused titanium for biomedical applications

Although titanium has been successful as an orthopedic or dental implant material, performance problems still persist concerning implant–bone interfacial bonding strength. In this study a novel oxygen-diffused titanium (ODTi), fabricated by introducing oxygen into the titanium crystal lattice by thermal treatment, was investigated. The fabricated material is the result of a surface modification made on commercially pure titanium (cp Ti) previously coated with poly(vinyl alcohol) (PVA) by means of a thermal treatment performed at 700 °C in an ultra-pure argon atmosphere. The thermal treatment at 700 °C led to the formation of an anatase TiO₂ film on the cp Ti surface and a concentration gradient of oxygen into titanium. The surface of the fabricated ODTi consisted of an outer nanometric layer of anatase TiO₂ and an inner nanometric layer of Ti₂O_x (x < 1) in which the oxygen is in solid solution with the titanium metal. It was found that ODTi possesses in vitro apatite formation ability after being soaked into simulated body fluid (SBF) solution. This apatite formation ability is attributed to the presence of the anatase TiO₂ outermost surface layer and to abundant hydroxyl groups (–OH) formed on the ODTi surface after immersion in SBF.     

In vitro assessment of Staphylococcus epidermidis and Staphylococcus aureus adhesion on TiO₂ nanotubes on Ti-6Al-4V alloy

The aim of this study was to evaluate Staphylococcus sp. adhesion to modified surfaces of titanium alloy (Ti-6Al-4V). Specimens of Ti-6Al-4V alloy 6-4 ELI-grade 23 that meets the requirements of ASTM F136 2002A (AMS 2631B class A1) were anodized in a mixture of sulfuric/hydrofluoric acid at 20 V for 5 and 60 min to form nanoporous (NP) and nanotubular (NT) oxide layers with pore diameter of 20 and 100 nm, respectively. The amount of fluorine incorporated in the oxide films from the electrolyte was 6 and 4 wt %, respectively. Bacterial adherence was studied using laboratory strains and six clinical strains each of Staphylococcus aureus and Staphylococcus epidermidis. Lower adherence of laboratory strains was demonstrated on fluoride nanostructured surfaces in comparison with the fluoride-free surfaces. Significant differences between clinical strains and laboratory strains were also found (p < 0.0001, Kruskal-Wallis test) when NP and NT specimens were compared with chemically polished (CP) surfaces. The results of the tests using multiple clinical strains confirmed a decrease in bacterial adherence on F-containing titanium oxide surfaces, suggesting a potential applicability of this surface, with a confirmed added value of decreasing clinical staphylococci adherence, for medical prosthetic devices.     

Enhancing surface free energy and hydrophilicity through chemical modification of microstructured titanium implant surfaces

Roughness-induced hydrophobicity, well-known from natural plant surfaces and intensively studied toward superhydrophobic surfaces, has currently been identified on microstructured titanium implant surfaces. Studies indicate that microstructuring by sandblasting and acid etching (SLA) enhances the osteogenic properties of titanium. The undesired initial hydrophobicity, however, presumably decelerates primary interactions with the aqueous biosystem. To improve the initial wettability and to retain SLA microstructure, a novel surface modification was tested. This modification differs from SLA by its preparation after acid etching, which was done under protective gas conditions following liquid instead of dry storage. We hypothesized that this modification should have increased wettability due to the prevention of contaminations that occurs during air contact. The main outcome of dynamic wettability measurements was that the novel modification shows increased surface free energy (SFE) and increased hydrophilicity with initial water contact angles of 0 degrees compared to 139.9 degrees for SLA. This hydrophilization was kept even after any drying. Reduced hydrocarbon contaminations were identified to play a possible role in altered surface thermodynamics. Such surfaces aim to retain the hydrophilicity and natural high surface energy of the Ti dioxide surface until surgical implants' insertion and are compared in this in vitro study with structural surface variants of titanium to compare roughness and chemically induced wettability.     

The effect of ultraviolet functionalization of titanium on integration with bone

Titanium implants are used as a reconstructive anchor in orthopedic and dental diseases and problems. Recently, ultraviolet (UV) light-induced photocatalytic activity of titanium has earned considerable and broad interest in environmental and clean-energy sciences. This study determines whether UV treatment of titanium enhances its osteoconductive capacity. Machined and acid-etched titanium samples were treated with UV for various time periods up to 48 h. For both surfaces, UV treatment increased the rates of attachment, spread, proliferation and differentiation of rat bone marrow-derived osteoblasts, as well as the capacity of protein adsorption, by up to threefold. In vivo histomorphometry in the rat model revealed that new bone formation occurred extensively on UV-treated implants with virtually no intervention by soft tissue, maximizing bone–implant contact up to nearly 100% at week 4 of healing. An implant biomechanical test revealed that UV treatment accelerated the establishment of implant fixation 4 times. The rates of protein adsorption and cell attachment strongly correlated with the UV dose-responsive atomic percentage of carbon on TiO₂, but not with the hydrophilic status. The data indicated that UV light pretreatment of titanium substantially enhances its osteoconductive capacity, in association with UV-catalytic progressive removal of hydrocarbons from the TiO₂ surface, suggesting a photofunctionalization of titanium enabling more rapid and complete establishment of bone–titanium integration.     

Biomimetic apatite formation on chemically treated titanium

Titanium treated in NaOH can form hydroxycarbonated apatite (HCA) after exposition in simulated body fluid (SBF). Generally, titanium is covered with a passive oxide layer. In NaOH this passive film dissolves and an amorphous layer containing alkali ions is formed on the surface. When exposed to SBF, the alkali ions are released from the amorphous layer and hydronium ions enter into the surface layer, resulting in the formation of Ti-OH groups in the surface. The released Na(+) ions increase the degree of supersaturation of the soaking solution with respect to apatite by increasing pH, and Ti-OH groups induce apatite nucleation on the titanium surface. The acid etching of titanium in HCl under inert atmosphere was examined as a pretreatment to obtain a uniform initial titanium surface before alkali treatment. Acid etching in HCl leads to the formation of a micro-roughened surface, which remains after alkali treatment in NaOH. It was shown by SEM, gravimetric and solution analysis that the apatite nucleation was uniform and the thickness of precipitated HCA layer increased continuously with time. The treatment of titanium by acid etching in HCl and subsequently in NaOH is a suitable method for providing the metal implant with bone-bonding ability.     

Improved implant osseointegration of a nanostructured titanium surface via mediation of macrophage polarization

The use of endosseous implanted materials is often limited by undesirable effects that may be due to macrophage-related inflammation. The purpose of this study was to fabricate a nanostructured surface on a titanium implant to regulate the macrophage inflammatory response and improve the performance of the implant. Anodization at 5 and 20 V as well as UV irradiation were used to generate hydrophilic, nanostructured TiO₂ surfaces (denoted as NT5 and NT20, respectively). Their surface characteristics and in vivo osseointegration as well as the inflammatory response they elicit were analyzed. In addition, the behavior of macrophages in vitro was evaluated. Although the in vitro osteogenic activity on the two surfaces was similar, the NT5 surface was associated with more bone formation, less inflammation, and a reduced CD68⁺ macrophage distribution in vivo compared to the NT20 and polished Ti surfaces. Consistently, further experiments revealed that the NT5 surface induced healing-associated M2 polarization in vitro and in vivo. By contrast, the NT20 surface promoted the pro-inflammatory M1 polarization, which could further impair bone regeneration. The results demonstrate the dominant role of macrophage-related inflammation in bone healing around implants and that surface nanotopography can be designed to have an immune-regulating effect in support of the success of implants.     

Formation of hydroxyapatite within porous TiO2 layer by micro-arc oxidation coupled with electrophoretic deposition

Micro-arc oxidation (MAO) is commonly used to modify the surface of Ti-based medical implants with a bioactive and porous titanium oxide (TiO₂) layer. This study reports a novel method of incorporating hydroxyapatite (HA) within the TiO₂ layer by coupling MAO with an electrophoretic deposition (EPD) process. A HA-incorporated, porous TiO₂ layer was produced successfully on the Ti substrate using the EPD-coupled MAO treatment, as confirmed by electron microscopy observations. Addition of ethanol to the electrolyte solution containing the fine HA particles was essential to reduce the level of gaseous emission on the anode, which obstructs the attachment of HA particles. In vitro cellular assays showed that the incorporation of HA significantly improved the osteoblastic activity on the coating layer.     

Bioactive polymer grafting onto titanium alloy surfaces

Bioactive polymers bearing sulfonate (styrene sodium sulfonate, NaSS) and carboxylate (methylacrylic acid, MA) groups were grafted onto Ti6Al4V alloy surfaces by a two-step procedure. The Ti alloy surfaces were first chemically oxidized in a piranha solution and then directly subjected to radical polymerization at 70 °C in the absence of oxygen. The grafted surfaces were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and the toluidine blue colorimetric method. Toluidine blue results showed 1–5 μg cm^?2 of polymer was grafted onto the oxidized Ti surfaces. Grafting resulted in a decrease in the XPS Ti and O signals from the underlying Ti substrate and a corresponding increase in the XPS C and S signals from the polymer layer. The ToF-SIMS intensities of the S^? and SO^? ions correlated linearly with the XPS atomic percent S concentrations and the ToF-SIMS intensity of the TiO₃H₂^? ion correlated linearly with the XPS atomic per cent Ti concentration. Thus, the ToF-SIMS S^?, SO^? and TiO₃H₂^? intensities can be used to quantify the composition and amount of grafted polymer. ToF-SIMS also detected ions that were more characteristic of the polymer molecular structure (C₆H₄SO₃^? and C₈H₇SO₃^? from NaSS, C₄H₅O₂^? from MA), but the intensity of these peaks depended on the polymer thickness and composition. An in vitro cell culture test was carried out with human osteoblast-like cells to assess the influence of the grafted polymers on cell response. Cell adhesion after 30 min of incubation showed significant differences between the grafted and ungrafted surfaces. The NaSS grafted surfaces showed the highest degree of cell adhesion while the MA-NaSS grafted surfaces showed the lowest degree of cell adhesion. After 4 weeks in vivo in rabbit femoral bones, bone was observed to be in direct contact with all implants. The percentage of mineralized tissue around the implants was similar for NaSS grafted and non-grafted implants (59% and 57%). The MA-NaSS grafted implant exhibited a lower amount of mineralized tissue (47%).