TiO2 nanotubes on Ti: Influence of nanoscale morphology on bone cell-materials interaction

Ti being bioinert shows poor bone cell adhesion with an intervening fibrous capsule. Ti could be made bioactive by several methods including growing in situ TiO2 layer on Ti-surface. TiO2 nanotubes were grown on Ti surface via anodization process and the bone cell-material interactions were evaluated. Human osteoblast cell attachment and growth behavior were studied using an osteoprecursor cell line for 3, 7, and 11 days. An abundant amount of extracellular matrix (ECM) between the neighboring cells was noticed on anodized nanotube surface with filopodia extensions coming out from cells to grasp the nanoporous surface of the nanotube for anchorage. To better understand and compare cell-materials interactions, anodized nanoporous sample surfaces were etched with different patterns. Preferential cell attachment was noticed on nanotube surface compare to almost no cells in etched Ti surface. Cell adhesion with vinculin adhesive protein showed higher intensity, positive contacts on nanoporous surface and thin focal contacts on the Ti-control. Immunochemistry study with alkaline phosphatase showed enhanced osteoblastic phenotype expressions in nanoporous surface. Osteoblast proliferation was significantly higher on anodized nanotube surface. Surface properties changed with the emergence of nanoscale morphology. Higher nanometer scale roughness, low contact angle and high surface energy in nanoporous surface enhanced the osteoblast-material interactions. Mineralization study was done under simulated body fluid (SBF) with ion concentration nearly equal to human blood plasma to understand biomimetic apatite deposition behavior. Although apatite layer formation was noticed on nanotube surface, but it was nonuniform even after 21 days in SBF.     

Bone cell-materials interactions and Ni ion release of anodized equiatomic NiTi alloy

A laser processed NiTi alloy was anodized for different times in H(2)SO(4) electrolyte with varying pH to create biocompatible surfaces with low Ni ion release as well as bioactive surfaces to enhance biocompatibility and bone cell-material interactions. The anodized surfaces were assessed for their in vitro cell-material interactions using human fetal osteoblast (hFOB) cells for 3, 7 and 11 days, and Ni ion release up to 8 weeks in simulated body fluids. The results were correlated with the surface morphologies of anodized surfaces characterized using field-emission scanning electron microscopy (FESEM). The results show that anodization creates a surface with nano/micro-roughness depending on the anodization conditions. The hydrophilicity of the NiTi surface was found to improve after anodization, as shown by the lower contact angles in cell medium, which dropped from 32° to <5°. The improved wettability of anodized surfaces is further corroborated by their high surface energy, comparable with that of commercially pure Ti. Relatively high surface energies, especially the polar component, and nano/micro surface features of anodized surfaces significantly increased the number of living cells and their adherence and growth on these surfaces. Finally, a significant drop in Ni ion release from 268±11 to 136±15 ppb was observed for NiTi surfaces after anodization. This work indicates that anodization of a NiTi alloy has a positive influence on the surface energy and surface morphology, which in turn improves bone cell-material interactions and reduces Ni ion release in vitro.     

Preparation, characterization, in vitro bioactivity, and osteoblast adhesion of multi-level porous titania layer on titanium by two-step anodization treatment

To combine the advantages of different electrolytes in anodic oxidation, pure titanium samples were anodized in CH(3) COOH electrolyte according to a novel anodizing treatment regime and then in H(2) SO(4) electrolyte in potentialstatic mode. The in vitro bioactivity of the as-prepared titanium samples was evaluated by simulated body fluid (SBF) test. In addition, MG63 osteoblast-like cells were cultured on surfaces of the as-prepared titanium samples to evaluate osteoblast adhesion ability. The titanium samples after the two-step anodization treatment were covered by titania layers of anatase and/or rutile with several micrometres thickness and presented a multi-level porous surface morphology consisting of interlaced grooves about 20-μm wide overlaid with submicron scale pores. The SBF test results showed that the crystal titania layers prepared at appropriate conditions were able to induce apatite-forming in 7 days, indicating that the abundance of surface Ti-OH groups and (101)-oriented rutile structure both played important roles in in vitro bioactivity of titania layers. The cell experiment results showed that the macroscopic grooves could effectively promote osteoblast adhesion and growth and submicron scale pores might be beneficial to osteoblast adhesion. The two-step anodization treatment might be a promising candidate for surface modification of titanium implant.     

Effects of topography and composition of titanium surface oxides on osteoblast responses

To investigate the roles of composition and characteristics of titanium surface oxides in cellular behaviour of osteoblasts, the surface oxides of titanium were modified in composition and topography by anodic oxidation in two kinds of electrolytes, (a) 0.2 M H(3)PO(4), and (b) 0.03 M calcium glycerophosphate (Ca-GP) and 0.15 M calcium acetate (CA), respectively. Phosphorus (P: ca.10at%) or both calcium (Ca: 1-6at%) and phosphorus (P: 3-6at%) were incorporated into the anodized surfaces in the form of phosphate and calcium phosphate. Surface roughness was slightly decreased or enhanced (R(a) in the range of 0.1-0.5 microm) on the anodized surfaces. The geometry of the micro-pores in the anodized surfaces varied with diameters up to 0.5 microm in 0.2 M H(3)PO(4) and to 2 microm in 0.03 M Ca-GP and 0.15 M CA, depending on voltages and electrolyte. Contact angles of all the anodic oxides were in the range of 60-90 degrees. Cell culture experiments demonstrated absence of cytotoxicity and an increase of osteoblast adhesion and proliferation by the anodic oxides. Cells on the surfaces with micro-pores showed an irregular and polygonal growth and more lamellipodia, while osteoblasts on the titanium surface used as a control or on anodic oxides formed at low voltages showed many thick stress fibres and intense focal contacts. Alkaline phosphatase (ALP) activity of the cells did not show any correlation with surface characteristics of anodic oxides.     

TiO2 nanotubes functionalized with regions of bone morphogenetic protein-2 increases osteoblast adhesion

Titanium (Ti) and its alloys are widely used in orthopedic and dental applications. However, the native TiO2 layer is not bioactive enough to form a direct bond with bone, which sometimes translates into a lack of osseointegration into juxtaposed bone that might lead to long term implant failure. In this study, the 20 amino acid peptide sequence (the so-called "knuckle epitope") of bone morphogenetic protein-2 (BMP-2) was immobilized onto Ti nanotubes created by electrochemical anodization. Further, human osteoblast (bone-forming cell) responses to such anodic Ti oxides functionalized with the BMP-2 knuckle epitope was examined in vitro. Materials were characterized by scanning electron and atomic force microscopy. Results of this in vitro study continued to provide evidence of increased osteoblast adhesion on Ti anodized to possess nanotubes compared to unanodized Ti. However, for the first time, results also showed that the immobilization of the BMP-2 knuckle epitope onto Ti anodized to possess nanotubes increased osteoblast adhesion compared to non-functionalized anodized Ti, anodized Ti functionalized with amine (NH2) groups, and unanodized Ti after 4 h. Results also showed increased osteoblast adhesion on amine terminated anodized Ti compared to respective non-functionalized anodized Ti and unanodized Ti. In summary, results of this in vitro study provided evidence that Ti anodized to possess nanotubes and then further functionalized with the BMP-2 knuckle epitope should be further studied for improved orthopedic applications.     

In vitro osteoblast response to anodized titanium and anodized titanium followed by hydrothermal treatment

In this study, Titanium (Ti) surfaces were modified using anodization. The electrolyte used for anodization was a mixture of calcium glycerophosphate and calcium acetate. The anodized surfaces were divided into three groups. Hydrothermal treatments were performed on two of the anodized groups for either 2 or 4 h. In vitro osteoblast response to anodized oxide and the hydrothermal treated oxide after anodization was evaluated in this study. Calcium and phosphorus ions were deposited on the Ti oxide during anodization. Anodized surfaces following a 4-h hydrothermal treatment were observed to promote the growth apatite-like crystals as compared with anodized surfaces after a 2-h hydrothermal treatment. Cellular function and onset of mineralization, as indicated by protein production and osteocalcin production, respectively, also were observed as enhanced on hydrothermal-treated surfaces. It was thus concluded from this study that calcium phosphate and apatite-like crystals could be deposited on Ti surfaces using anodization and a combination of anodization and hydrothermal treatment. It was also concluded that the phenotypic expression of osteoblast was enhanced by the presence of calcium phosphate or apatite-like crystals on anodized or hydrothermally treated Ti surfaces.     

High surface energy enhances cell response to titanium substrate microstructure

Titanium (Ti) is used for implantable devices because of its biocompatible oxide surface layer. TiO2 surfaces that have a complex microtopography increase bone-to-implant contact and removal torque forces in vivo and induce osteoblast differentiation in vitro. Studies examining osteoblast response to controlled surface chemistries indicate that hydrophilic surfaces are osteogenic, but TiO2 surfaces produced until now exhibit low surface energy because of adsorbed hydrocarbons and carbonates from the ambient atmosphere or roughness induced hydrophobicity. Novel hydroxylated/hydrated Ti surfaces were used to retain high surface energy of TiO2. Osteoblasts grown on this modified surface exhibited a more differentiated phenotype characterized by increased alkaline phosphatase activity and osteocalcin and generated an osteogenic microenvironment through higher production of PGE2 and TGF-beta1. Moreover, 1alpha,25OH2D3 increased these effects in a manner that was synergistic with high surface energy. This suggests that increased bone formation observed on modified Ti surfaces in vivo is due in part to stimulatory effects of high surface energy on osteoblasts.     

Suppressed primary osteoblast functions on nanoporous titania surface

Titiania nanotubes have large potential in medical implant applications but their tissue compatibility is still controversial. Considering that the biological behavior of primary osteoblasts is closer to the in vivo situation than other common cell lines, we investigate the response of primary osteoblasts on anodized nanotextured titania surfaces. Two nanotextured surface morphologies, namely the 5 V anodized surface with a pore diameter of 25 nm and the 20 V anodized surface with a tube diameter of 80 nm are chosen for this study. Initial cell adhesion is not obviously affected by the anodized surfaces. With the exception of slightly higher intracellular alkaline phosphatase activity and more extracellular matrix deposition, cell growth, and cell differentiation represented by the expressions of osteogenesis-related genes are impaired on both anodized surfaces. This may be attributed to the compromised focal contact formation on the anodized surfaces. The difference in the phenotypes of the primary osteoblasts and the osteoblastic cell lines may partly account for the controversy in osteoblast cytocompatibility on titania nanotubes.     

Initial responses of human osteoblasts to sol-gel modified titanium with hydroxyapatite and titania composition

Sol-gel thin films of hydroxyapatite (HA) and titania (TiO(2)) have received a great deal of attention in the area of bioactive surface modification of titanium (Ti) implants. Sol-gel coatings were developed on Ti substrates of pure HA and TiO(2) and two composite forms, HA+10% TiO(2) and HA+20% TiO(2), and the biological properties of the coatings were evaluated. All the coating layers exhibited thin and homogeneous structures and phase-pure compositions (either HA or TiO(2)). Primary human osteoblast cells showed good attachment, spreading and proliferation on all the sol-gel coated surfaces, with enhanced cell numbers on all the coated surfaces relative to uncoated Ti control at day 1, as observed by MTT assay and scanning electron microscopy. Cell attachment rates were also enhanced on the pure HA coating relative to control Ti. The pure HA and HA+10% TiO(2) composite coating furthermore enhanced proliferation of osteoblasts at 4 days. Moreover, the gene expression level of several osteogenic markers including bone sialoprotein and osteopontin, as measured by RT-PCR at 24h, was shown to vary according to coating composition. These findings suggest that human primary bone cells show marked and rapid early functional changes in response to HA and TiO(2) sol-gel coatings on Ti.     

Molecular plasma deposited peptides on anodized nanotubular titanium: an osteoblast density study

A large amount of work is currently being conducted to design, fabricate, and characterize materials coated or immobilized with bioactive molecules for tissue engineering applications. Here, a novel method, molecular plasma deposition (MPD), is introduced with can efficiently coat materials with numerous bioactive peptides. Specifically, here, RGDS (arginine-glycine-aspartic acid-serine), KRSR (lysine-arginine-serine-arginine), and IKVAV (isoleucine-lysine-valine-alanine-valine) were coated on anodized nanotubular titanium using MPD. The anodized nanotubular titanium surfaces were characterized using scanning electron microscopy (SEM), atomic force microscopy (AFM), and water contact angle measurements. Peptide coatings were examined by X-ray photoelectron spectroscopy (XPS) and an amine reactive fluorescence molecule, 3-(4 carboxybenzoyl)quinoline 2-carboxaldehyde (CBQCA). Electrospray ionization (ESI) was used to confirm peptide integrity. Osteoblast (bone-forming cell) density was determined on the materials of interest. Results confirmed peptide coatings and showed that the MPD RGDS and KRSR coatings on anodized nanotubular titanium increased osteoblast density compared with uncoated substrates and those coated with IKVAV and a control peptide (RGES) after 4 h and 7 days. SEM confirmed differences in the morphology of the attached cells. These results, to the best of our knowledge, are the first reports using MPD to efficiently create peptide coatings to increase osteoblast density on metals commonly used in orthopedics. Since MPD represents a quick, inexpensive, and versatile technique to coat implants with peptides, it should be further studied for numerous implant applications.