Nanostructured Ti Surfaces Research Articles

Nanostructured Implant-Tissue Interface Assessment Using a Three-Dimensional Gingival Tissue Equivalent.

Improved soft tissue integration (STI) around dental implants is key for implant success. The formation of an early and long-lasting transmucosal seal around the implant abutment might help to prevent peri-implantitis, one of the major causes of late implant failure. In natural teeth, collagen fibers are firmly inserted and fixed in the cementum of the tooth and emerge perpendicular to the gingival tissue. In contrast, around dental implants, collagen fibers run predominantly parallel to the implant surface, allowing bacterial migration into the peri-implant interface that might lead to peri-implantitis. Previous studies have shown that nanostructured Ti surfaces improve gingival cell response in monolayer cell cultures. Here, we aimed at evaluating the implant-tissue interface using a 3D gingival tissue equivalent (GTE). First, we evaluated the GTE response to a nanostructured (NN) and machined Ti surface after the stimulation with Porphyromonas gingivalis lipopolysaccharide (LPS), to simulate peri-implantitis conditions. Thus, GTE viability, through MTT assay, the release of metalloproteinase-1 (MMP1) and its inhibitor (TIMP1) through ELISA, and the gene expression of extracellular matrix turnover genes by real-time RT-PCR were analyzed. Second, GTE-implant interaction was characterized by serial block face scanning electron microscopy, and collagen-1 orientation at the tissue-implant interface was analyzed by immunofluorescence. While a similar GTE response to LPS stimulation was found for both implant surfaces, a higher proportion of collagen oriented perpendicular to the implant was observed on the NN implant surface. Thus, our results indicate that the nanostructuration of titanium dental implant abutments could allow the correct orientation of collagen fibers and greater soft tissue sealing, while keeping biocompatibility levels and LPS response comparable.

Influence of a Physiologically Formed Blood Clot on Pre-Osteoblastic Cells Grown on a BMP-7-Coated Nanoporous Titanium Surface.

Titanium (Ti) nanotopography modulates the osteogenic response to exogenous bone morphogenetic protein 7 (BMP-7) in vitro, supporting enhanced alkaline phosphatase mRNA expression and activity, as well as higher osteopontin (OPN) mRNA and protein levels. As the biological effects of OPN protein are modulated by its proteolytic cleavage by serum proteases, this in vitro study evaluated the effects on osteogenic cells in the presence of a physiological blood clot previously formed on a BMP-7-coated nanostructured Ti surface obtained by chemical etching (Nano-Ti). Pre-osteoblastic MC3T3-E1 cells were cultured during 5 days on recombinant mouse (rm) BMP-7-coated Nano-Ti after it was implanted in adult female C57BI/6 mouse dorsal dermal tissue for 18 h. Nano-Ti without blood clot or with blood clot at time 0 were used as the controls. The presence of blood clots tended to inhibit the expression of key osteoblast markers, except for Opn, and rmBMP-7 functionalization resulted in a tendency towards relatively greater osteoblastic differentiation, which was corroborated by runt-related transcription factor 2 (RUNX2) amounts. Undetectable levels of OPN and phosphorylated suppressor of mothers against decapentaplegic (SMAD) 1/5/9 were noted in these groups, and the cleaved form of OPN was only detected in the blood clot immediately prior to cell plating. In conclusion, the strategy to mimic in vitro the initial interfacial in vivo events by forming a blood clot on a Ti nanoporous surface resulted in the inhibition of pre-osteoblastic differentiation, which was minimally reverted with an rmBMP-7 coating.

TiO2 nanostructured implant surface-mediated M2c polarization of inflammatory monocyte requiring intact cytoskeleton rearrangement

BackgroundMicrogravity directly disturbs the reorganization of the cytoskeleton, exerting profound effects on the physiological process of macrophages. Although it has been established that macrophage M1/M2 polarization could be manipulated by the surface nanostructure of biomaterial in our previous study under normal gravity, how will inflammatory monocytes (iMos)-derived macrophages respond to diverse nanostructured Ti surfaces under normal gravity or microgravity remains unrevealed.ResultsIn this study, Cytochalasin D, a cytoskeleton relaxant, was employed to establish the simulated microgravity (SMG) environment. Our results showed that human iMos polarized into M2c macrophages on NT5 surface but M1 type on NT20 surface with divergent inflammatory phenotypes according to the profile of macrophage polarization featured molecules under normal gravity. However, such manipulative effects of NTs surfaces on iMos-derived macrophages were strikingly weakened by SMG, characterized by the altered macrophage morphology, changed cytokine secretion profile, and decreased cell polarization capacity.ConclusionsTo our knowledge, this is the first metallic implantable material study focusing on the functions of specific monocyte subsets and its crucial role of the cytoskeleton in materials-mediated host immune response, which enriches our mechanism knowledge about the crosstalk between immunocytes and biomaterials. The results obtained in the present study may also provide potential targets and strategies for biomaterial development and clinical treatment via precise immune-regulation under normal gravity and microgravity.Graphic

How Streptococcus mutans Affects the Surface Topography and Electrochemical Behavior of Nanostructured Bulk Ti.

The metabolization of carbohydrates by Streptococcus mutans leads to the formation of lactic acid in the oral cavity, which can consequently accelerate the degradation of dental implants fabricated from commercially available microcrystalline Ti. Microstructure influences surface topography and hence interaction between bacteria cells and Ti surfaces. This work offers the first description of the effect of S. mutans on the surface topography and properties of nanostructured bulk Ti, which is a promising candidate for modern narrow dental implants owing to its superior mechanical strength. It was found that S. mutans incubation resulted in the slight, unexpected decrease of surface nanoroughness, which was previously developed owing to privileged oxidation in areas of closely spaced boundaries. However, despite the changes in nanoscale surface topography, bacteria incubation did not reduce the high level of protection afforded by the oxide layer formed on the nanostructured Ti surface. The results highlight the need–hitherto ignored–to consider Ti microstructure when analyzing its behavior in the presence of carbohydrate-metabolizing bacteria.

Improved osteogenic differentiation of human amniotic mesenchymal stem cells on gradient nanostructured Ti surface.

Titanium (Ti) and Ti-based alloys are widely used in the manufacture of dental and orthopedic implants. However, how to improve their osteogenic differentiation ability is still a key issue to be resolved. In this study, gradient nanostructured surface (GNS) samples were prepared by surface mechanical grinding treatment, and coarse-grained (CG) samples were obtained by recrystallization annealing, making sure that the two kinds of specimens had similar roughness. Then, human amniotic mesenchymal stem cells (hAMSCs) were cocultured with the two kinds of Ti to investigate the material effects on the cellular functions. The results demonstrated that the grains with size ~56 nm were formed on the surface of the GNS Ti, and the grain size gradually increases from the sample surface to interior. Compared to the CG samples, the GNS ones could make the adhesion effect of the hAMSCs better, and promote the cell proliferation and osteogenic differentiation more significantly, the preliminary mechanism of which might be due to their specific nanostructure, the thicker oxide layer formed on their surface and the enhanced hardness. Our results indicated that the gradient nanostructured Ti materials could enhance both osteogenic differentiation and mechanical properties, which may possess broader applications in bone tissue engineering and clinical implanting.

Azimuthal and polar anchoring energies of aligning layers structured by nonlinear laser lithography

ABSTRACT In spite of the fact that there are different techniques in the creation of the high-quality liquid crystals (LCs) alignment by means of various surfaces, the azimuthal and polar anchoring energies as well as the pre-tilt angle are important parameters to all of them. Here, the modified by a certain manner aligning layers, previously formed by nonlinear laser lithography (NLL), having high-quality nano-periodic grooves on Ti surfaces, recently proposed for LC alignment was studied. The change of the scanning speed of NLL in the process of nano-structured Ti surfaces and their further modification by means of ITO-coating, and deposition of polyimide film has enabled different aligning layers, whose main characteristics, namely azimuthal and polar anchoring energies, were measured. For the modified aligning layers, the dependencies of the twist and pre-tilt angles for LC cells filled by nematic E7 (Δε > 0) and MLC-6609 (Δε < 0) were obtained. Also, the contact angle for droplets of isotropic liquid (glycerol), and nematic LCs was measured for the various values of the scanning speed during the laser processing.

Oriented Cell Alignment Induced by a Nanostructured Titanium Surface Enhances Expression of Cell Differentiation Markers.

A key factor for dental implant success is a good sealing between the implant surface and both soft (gum) and hard (bone) tissues. Surface nanotopography can modulate cell response through mechanotransduction. The main objective of this research was the development of nanostructured titanium (Ti) surfaces that promote both soft and hard tissue integration with potential application in dental implants. Nanostructured Ti surfaces were developed by electrochemical anodization—nanopores (NPs) and nanonets (NNs)—and characterized by atomic force microscopy, scanning electronic microscopy, and contact angle analysis. In addition, nanoparticle release and apoptosis activation were analyzed on cell culture. NP surfaces showed nanoparticle release, which increased in vitro cell apoptosis. Primary human gingival fibroblasts (hGFs) and human bone marrow mesenchymal stem cells (hBM-MSCs) were used to test cell adhesion, cytotoxicity, metabolic activity, and differentiation markers. Finally, cell orientation on the different surfaces was analyzed using a phalloidin staining. NN surfaces induced an oriented alignment of both cell types, leading in turn to an improved expression of differentiation markers. Our results suggest that NN structuration of Ti surfaces has great potential to be used for dental implant abutments to improve both soft and hard tissue integration.

Surface and Bulk Modification of Titanium Grade 2 Substrates for Enhanced Biological Activity

The cellular response on the surface of equal-channel angular pressing (ECAP)-processed nanostructured titanium grade 2 substrates after plasma electrolytic oxidation (PEO) has been investigated. The results demonstrate that, after four passes of the ECAP process, the mechanical properties of the nanostructured sample were significantly enhanced compared with the coarse-grained sample. The results of x-ray diffractometry and energy-dispersive x-ray spectroscopy (EDS) showed that hydroxyapatite particles formed on the nanostructured Ti surface modified by PEO, indicating enhanced bioactivity. The growth rate and degree of cell attachment were notably increased, while the number of necrotic and apoptotic cells was negligible on the surface of the PEO-coated nanostructured titanium. This enhanced cell activity is related to the ultrafine grains with a distinct surface oxide layer having specific morphology and composition. As both the surface and bulk properties of the PEO-processed nanostructured titanium samples were improved, these samples could be excellent candidates for use as bone substitute materials.

Highly Adherent Parylene-C Coatings With Nanostructuring for Enhanced Cell Adhesion and Growth.

Parylene-C is a frequently used polymeric thin-film coating in medical applications and is known for its excellent biocompatibility and flexible deposition process. However, its use in long-term implants is limited due to its poor adhesion to metals in liquid environments. In this paper, we present a strategy to anchor Parylene-C to medical grade titanium (Ti) by means of nanostructuring the Ti substrates surface prior to Parylene coating. We observe that, after aging in the physiological salt solution for three days, Parylene coating lose their adhesion to bare titanium surfaces. However, the Parylene films deposited on nanostructured Ti surfaces retain full adhesion, even after aging them in the same solution for ten days. In addition, we demonstrate that combining nanostructured surfaces with very thin Parylene coatings provides the additional benefit of accelerating cell proliferation. Nanostructured surfaces showed cell proliferation without the typically required oxygen plasma treatment. Combining plasma treatment and nanostructuring further improved proliferation performance over smooth Parylene surfaces.

Effects of Surface Nanotopography and Calcium Chemistry of Titanium Bone Implants on Early Blood Platelet and Macrophage Cell Function

Early responses of blood platelets and immunoinflammatory cells (macrophages) to titanium (Ti) bone implants affect the subsequent biological healing of implants by modulating early tissue healing-microenvironments via the formation of temporary fibrin matrix scaffolds for stem cell migration and production of growth factors and cytokines. This study investigated the effects of nanoscale surface topography and calcium ion (Ca2+) modification of Ti surfaces on biocompatibility regulated by blood platelets and macrophages, for the future surface design of Ti bone implants with enhanced early osteogenic capacity. A nanostructured Ti surface with or without Ca2+ enrichment was prepared using the hydrothermal treatment. Immediate and early functions of platelets and macrophages modulated by modified Ti surfaces were investigated by morphological observation of platelet spreading and fibrin matrix formation, platelet growth factor release, immunostaining of macrophage phenotypes, and macrophage inflammatory cytokine production. The results showed that surface nanoscale topographical modification of Ti promotes blood platelet activation and suppresses the inflammatory response of macrophages. In addition, surface chemistry modifications with Ca2+ enhanced the platelet response-modulating function of the nanostructured Ti surface, which accelerated immediate fibrin matrix formation and platelet-derived growth factor-AB release. Thus, nanotopographical and Ca2+ modifications of implant surfaces are expected to be effective approaches that favor the initial phase of wound healing around the Ti bone implants through positive modulation of immediate blood platelet function and early macrophage immunoinflammatory response.

Nanostructured Ti surfaces and retinoic acid/dexamethasone present a spatial framework for the maturation and amelogenesis of LS-8 cells.

PurposeTo investigate the amelogenesis-inductive effects of surface structures at the nanoscale. For this purpose, variable nanostructured titanium dioxide (TiO2) surfaces were used as a framework to regulate the amelogenic behaviors of ameloblasts with the administration of retinoic acid (RA)/dexamethasone (DEX).Materials and methodsTiO2 nanotubular (NT) surfaces were fabricated via anodization. Mouse ameloblast-like LS-8 cells were seeded and cultured on NT surfaces in the presence or absence of RA/DEX for 48 h. The amelogenic behaviors and extracellular matrix (ECM) mineralization of LS-8 cells on nanostructured Ti surfaces were characterized using field emission scanning electron microscope, laser scanning confocal microscope, quantitative polymerase chain reaction, MTT assay, and flow cytometry.ResultsTiO2 NT surfaces (tube size ~30 and ~80 nm) were constructed via anodization at 5 or 20 V and denoted as NT5 and NT20, respectively. LS-8 cells exhibited significantly increased spread and proliferation, and lower rates of apoptosis and necrosis on NT surfaces. The amelogenic gene expression and ECM mineralization differed significantly on the NT20 and the NT5 and polished Ti sample surfaces in standard medium. The amelogenic behaviors of LS-8 cells were further changed by RA/DEX pretreatment, which directly drove maturation of LS-8 cells.ConclusionControlling the amelogenic behaviors of ameloblast-like LS-8 cells by manipulating the nanostructure of biomaterials surfaces represents an effective tool for the establishment of a systemic framework for supporting enamel regeneration. The administration of RA/DEX is an effective approach for driving the amelogenesis and maturation of ameloblasts.

Antibacterial and bioactive properties of stabilized silver on titanium with a nanostructured surface for dental applications

Titanium (Ti) is used in dental applications owing to their excellent mechanical properties, corrosion resistance, and biocompatibility. However, postoperative bacterial infection may cause serious complications and remains one of the most difficult challenges hindering the development of long term Ti dental implants. Therefore, the desire for high quality dental care has led to significant interest in designing implant surfaces that offer stable antibacterial activity with excellent cellular response. In this study, we propose a simple and efficient approach for fabricating an antibacterial stabilized Ag nanostructure on a Ti surface, which is based on a two-step process involving target-ion induced plasma sputtering (TIPS) and Ag sputtering. The TIPS process generates a nanostructured Ti surface that provides a nanotemplate on which the Ag nanostructure may be deposited through Ag sputtering. The Ag nanoclusters adhere tightly to the TIPS-treated Ti (TIPS-Ti) nanostructured surface with no noticeable defects, and the amount of stabilized Ag deposited may be controlled by simply adjusting the Ag-sputtering time. The silver ion is released continuously from the Ag-TIPS-Ti surface for 7 d. The Ag nanostructured TIPS-Ti (Ag-TIPS-Ti) surface not only offers outstanding antibacterial activity toward Escherichia coli and Staphylococcus aureus over 12 h of culturing but also exhibits no severe cytotoxicity for fibroblast cells for up to 10 days. In particular, Ag stabilization by 10 s Ag-sputtering on TIPS-Ti provides the best balance between antibacterial activity and cellular performances, and the resulting fibroblast cell-attachment morphology and proliferation level are similar to those for a polished Ti surface. Therefore, the controllable antibacterial activity and fibroblast tissue affinity of the Ag-TIPS-Ti present a promising avenue for producing reliable, long term dental implants.

Ultrashort-pulse laser structured titanium surfaces with sputter-coated platinum catalyst as hydrogen evolution electrodes for alkaline water electrolysis

For the first time, we report on micro- and nanostructured Ti surfaces produced by ultrashort-pulse laser processing followed by sputter deposition of Pt aiming at efficient cathode electrodes for alkaline water electrolysis. We studied the laser processing-induced surface morphology, the elemental composition of the surface, the specific surface increase, the wetting behavior as well as the activity of the hydrogen evolution reaction. It is demonstrated that ultrashort-pulse laser structuring in combination with thin layer catalyst deposition can dramatically boost the performance of cathodes for the hydrogen evolution reaction due to the enormous increase in specific surface in combination with superhydrophilic and superwetting properties leading to a rapid gas bubble detachment.

Bone mesenchymal stem cell secretion of sRANKL/OPG/M-CSF in response to macrophage-mediated inflammatory response influences osteogenesis on nanostructured Ti surfaces

Although it has been well established that osteogenic differentiation of bone mesenchymal stem cells (bMSCs) as well as osteoclastic differentiation of macrophages can be manipulated by the nanostructure of biomaterial surfaces, the interactions among the effects of the surface on immune cells and bMSCs remained unknown. Therefore, in this study, the osteogenic behaviors and secretion of osteoclastogenesis-related cytokines of human bMSCs on TiO2 nanotubular (NT) surfaces in conditioned medium (CM) generated by macrophages cultured on the respective NT surfaces (NT-CM) were analyzed. Although bMSCs showed consistent osteogenic behaviors on the NT5 and NT20 surfaces in both standard culture medium and both types of NT-CM, collagen synthesis and extracellular matrix mineralization were partially impeded on the NT20 surface in NT20-CM and bMSC cytokine secretions on the NT20 surface in NT20-CM elicited remarkable multinuclear giant cell and osteoclast formation compared with that observed on the NT5 surface in NT5-CM. After implantation in vivo, mineralized bone formation was significantly delayed around the NT20 implant compared with the NT5 implant, but both surfaces contributed to good bone formation after 12 weeks. The results obtained in this study advance our understanding of the confounding influence of the implant surface nanostructure, macrophage inflammatory response, and osteogenic differentiation of bMSCs as well as the retro-regulative effects of bMSCs on the osteoclastic differentiation of macrophages, and the culture system based on different NT surfaces and CM generated on the respective surfaces may provide a systematic research model for evaluating the performance of endosseous implants as well as a prospective approach for improving implant osseointegration via immune-regulation.

Corrosion resistance and biological properties of a micro–nano structured Ti surface consisting of TiO2 and hydroxyapatite

A micro–nanostructured Ti surface consisting of TiO2 and HA was prepared by MAO, and showed improved corrosion resistance and biological properties.

Modulating macrophage polarization with divalent cations in nanostructured titanium implant surfaces

Nanoscale topographical modification and surface chemistry alteration using bioactive ions are centrally important processes in the current design of the surface of titanium (Ti) bone implants with enhanced bone healing capacity. Macrophages play a central role in the early tissue healing stage and their activity in response to the implant surface is known to affect the subsequent healing outcome. Thus, the positive modulation of macrophage phenotype polarization (i.e. towards the regenerative M2 rather than the inflammatory M1 phenotype) with a modified surface is essential for the osteogenesis funtion of Ti bone implants. However, relatively few advances have been made in terms of modulating the macrophage-centered early healing capacity in the surface design of Ti bone implants for the two important surface properties of nanotopography and and bioactive ion chemistry. We investigated whether surface bioactive ion modification exerts a definite beneficial effect on inducing regenerative M2 macrophage polarization when combined with the surface nanotopography of Ti. Our results indicate that nanoscale topographical modification and surface bioactive ion chemistry can positively modulate the macrophage phenotype in a Ti implant surface. To the best of our knowledge, this is the first demonstration that chemical surface modification using divalent cations (Ca and Sr) dramatically induces the regenerative M2 macrophage phenotype of J774.A1 cells in nanostructured Ti surfaces. In this study, divalent cation chemistry regulated the cell shape of adherent macrophages and markedly up-regulated M2 macrophage phenotype expression when combined with the nanostructured Ti surface. These results provide insight into the surface engineering of future Ti bone implants that are harmonized between the macrophage-governed early wound healing process and subsequent mesenchymal stem cell-centered osteogenesis function.

Ti surface modification with a natural antioxidant and antimicrobial agent

Ti surface was modified with a natural antioxidant and antimicrobial agent, torularhodin, by its incorporation onto a Ti/TiO2 substrate via anodization process. This natural antioxidant and antimicrobial agent can be produced from waste materials, as an alternative to synthetic antimicrobial compounds.Scanning electron microscopy (SEM), Structural Fourier Transform Infrared (FTIR) spectroscopy, contact angle (CA) measurements, and atomic force microscopy (AFM) are employed to assess the surface properties of resulting coatings.Surface and electrochemical analyses highlighted that torularhodin was embedded in the TiO2 shielding layer, improving the antibacterial activity with about 65% compared to Ti/TiO2. Several species of microorganisms: Staphylococcus aureus, Candida albicans and Aspergillus fumigatus were utilized to test the antimicrobial effect. Hemolysis and cytotoxicity tests were additionally performed, showing good results.This nanostructured Ti surface based on a natural antioxidant and antibacterial agent is a promising biomaterial for medical device purposes.

Structure-related antibacterial activity of a titanium nanostructured surface fabricated by glancing angle sputter deposition

The aim of this study was to reproduce the physico-mechanical antibacterial effect of the nanocolumnar cicada wing surface for metallic biomaterials by fabrication of titanium (Ti) nanocolumnar surfaces using glancing angle sputter deposition (GLAD). Nanocolumnar Ti thin films were fabricated by GLAD on silicon substrates. S. aureus as well as E. coli were incubated with nanostructured or reference dense Ti thin film test samples for one or three hours at 37 °C. Bacterial adherence, morphology, and viability were analyzed by fluorescence staining and scanning electron microscopy and compared to human mesenchymal stem cells (hMSCs).Bacterial adherence was not significantly different after short (1 h) incubation on the dense or the nanostructured Ti surface. In contrast to S. aureus the viability of E. coli was significantly decreased after 3 h on the nanostructured film compared to the dense film and was accompanied by an irregular morphology and a cell wall deformation. Cell adherence, spreading and viability of hMSCs were not altered on the nanostructured surface. The results show that the selective antibacterial effect of the cicada wing could be transferred to a nanostructured metallic biomaterial by mimicking the natural nanocolumnar topography.

Oxidative nanopatterning of titanium surfaces promotes production and extracellular accumulation of osteopontin

The bone-biomaterial interface has been characterized by layers of afibrillar extracellular matrix (ECM) enriched in non collagenous proteins, including osteopontin (OPN), a multifunctional protein that in bone controls cell adhesion and ECM mineralization. Physical and chemical aspects of biomaterial surfaces have been demonstrated to affect cell-ECM-substrate interactions. The present paper described the ability of oxidative nanopatterning of titanium (Ti) surfaces to control extracellular OPN deposition in vitro. Ti discs were chemically treated by a mixture of H2SO4/H2O2 for either 30 min [Nano(30') Ti] or 4 h [Nano(4h) Ti]. Non-etched Ti discs were used as control. Primary osteogenic cells derived from newborn rat calvarial bone were plated on control and etched Ti and grown under osteogenic conditions up to 7 days. High resolution scanning electron microscopy revealed that treated Ti discs exhibited a nanoporous surface and that areas of larger nanopits were noticed only for Nano(4h) Ti. Large extracellular OPN accumulation were detectable only for Nano(4h) Ti, which was associated with OPN-positive cells with typical aspects of migrating cells. At day 3, quantitative results in terms of areas of OPN labeling were as follows: Nano(4h) Ti > Nano(30') Ti > Control Ti. In conclusion, chemically nanostructured Ti surfaces may support the enhancement of endogenous extracellular OPN deposition by osteogenic cells in vitro depending on the etching time, a finding that should be taken into consideration in strategies to biofunctionalize implant surfaces with molecules with cell adhesion capacity.

Oxidative nanopatterning of titanium surfaces promotes production and extracellular accumulation of osteopontin

The bone-biomaterial interface has been characterized by layers of afibrillar extracellular matrix (ECM) enriched in non collagenous proteins, including osteopontin (OPN), a multifunctional protein that in bone controls cell adhesion and ECM mineralization. Physical and chemical aspects of biomaterial surfaces have been demonstrated to affect cell-ECM-substrate interactions. The present paper described the ability of oxidative nanopatterning of titanium (Ti) surfaces to control extracellular OPN deposition in vitro. Ti discs were chemically treated by a mixture of H2SO4/H2O2 for either 30 min [Nano(30') Ti] or 4 h [Nano(4h) Ti]. Non-etched Ti discs were used as control. Primary osteogenic cells derived from newborn rat calvarial bone were plated on control and etched Ti and grown under osteogenic conditions up to 7 days. High resolution scanning electron microscopy revealed that treated Ti discs exhibited a nanoporous surface and that areas of larger nanopits were noticed only for Nano(4h) Ti. Large extracellular OPN accumulation were detectable only for Nano(4h) Ti, which was associated with OPN-positive cells with typical aspects of migrating cells. At day 3, quantitative results in terms of areas of OPN labeling were as follows: Nano(4h) Ti > Nano(30') Ti > Control Ti. In conclusion, chemically nanostructured Ti surfaces may support the enhancement of endogenous extracellular OPN deposition by osteogenic cells in vitro depending on the etching time, a finding that should be taken into consideration in strategies to biofunctionalize implant surfaces with molecules with cell adhesion capacity.