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1.  Increased fibroblast functionality on CNN2-loaded titania nanotubes 
Infection and epithelial downgrowth are major problems associated with maxillofacial percutaneous implants. These complications are mainly due to the improper closure of the implant–skin interface. Therefore, designing a percutaneous implant that better promotes the formation of a stable soft tissue biologic seal around percutaneous sites is highly desirable. Additionally, the fibroblast has been proven to play an important role in the formation of biologic seals. In this study, titania nanotubes were filled with 11.2 kDa C-terminal CCN2 (connective tissue growth factor) fragment, which could exert full CCN2 activity to increase the biological functionality of fibroblasts. This drug delivery system was fabricated on a titanium implant surface. CCN2 was loaded into anodized titania nanotubes using a simplified lyophilization method and the loading efficiency was approximately 80%. Then, the release kinetics of CCN2 from these nanotubes was investigated. Furthermore, the influence of CCN2-loaded titania nanotubes on fibroblast functionality was examined. The results revealed increased fibroblast adhesion at 0.25, 0.5, 1, 2, 4, and 24 hours, increased fibroblast viability over the course of 5 days, as well as enhanced actin cytoskeleton organization on CCN2-loaded titania nanotubes surfaces compared to uncoated, unmodified counterparts. Therefore, the results from this in vitro study demonstrate that CCN2-loaded titania nanotubes have the ability to increase fibroblast functionality and should be further studied as a method of promoting the formation of a stable soft tissue biologic seal around percutaneous sites.
PMCID: PMC3292419  PMID: 22403489
anodization; titania nanotubes; adhesion; connective tissue growth factor; fibroblast
2.  Effects of titania nanotubes with or without bovine serum albumin loaded on human gingival fibroblasts 
Modifying the surface of the transmucosal area is a key research area because this process positively affects the three functions of implants: attachment to soft tissue, inhibiting bacterial biofilm adhesion, and the preservation of the crestal bone. To exploit the potential of titania nanotube arrays (TNTs) with or without using bovine serum albumin (BSA) to modify the surface of a dental implant in contact with the transmucosal area, BSA was loaded into TNTs that were fabricated by anodizing Ti sheets; the physical characteristics of these arrays, including their morphology, chemical composition, surface roughness, contact angle, and surface free energy (SFE), were assessed. The effect of Ti surfaces with TNTs or TNTs-BSA on human gingival fibroblasts (HGFs) was determined by analyzing cell morphology, early adhesion, proliferation, type I collagen (COL-1) gene expression, and the extracellular secretion of COL-1. The results indicate that early HGF adhesion and spreading behavior is positively correlated with surface characteristics, including hydrophilicity, SFE, and surface roughness. Additionally, TNT surfaces not only promoted early HGF adhesion, but also promoted COL-1 secretion. BSA-loaded TNT surfaces promoted early HGF adhesion, while suppressing late proliferation and COL-1 secretion. Therefore, TNT-modified smooth surfaces are expected to be applicable for uses involving the transmucosal area. Further study is required to determine whether BSA-loaded TNT surfaces actually affect closed loop formation of connective tissue because BSA coating actions in vivo are very rapid.
PMCID: PMC3949701  PMID: 24623977
titania nanotubes; bovine serum albumin; modified surface; transmucosal area; human gingival fibroblast
3.  Self-Assembled Antimicrobial and biocompatible copolymer films on Titanium 
Macromolecular bioscience  2011;11(11):1515-1525.
Biofilm formation on biomedical devices such as dental implants can result in serious infections and finally in device failure. Polymer coatings which provide antimicrobial action to surfaces without compromising the compatibility with human tissue are of great interest. Copolymers of 4-vinyl-N-hexylpyridinium bromide and dimethyl(2-methacryloyloxyethyl) phosphonate are interesting candidates in this respect. These copolymers form ultrathin polycationic layers on titanium surfaces. As the copolymerization reaction is almost ideal statistical, copolymers with varying compositions can be synthesized and immobilized onto titanium surfaces for comprehensive screening concerning antimicrobial activity and biocompatibility. Copolymer films on titanium were characterized by contact angle measurements, ellipsometry and X-ray photoelectron spectroscopy. Antibacterial properties were assessed by investigation of adherence of S. mutans which represents a strain found in the human oral cavity. Biocompatibility was rated based on human gingival fibroblast adhesion, proliferation and cell morphology. Depending on polymer composition the coatings displayed a behavior ranging from biocompatibility equal to titanium but no antibacterial action to highly antimicrobial activity but poor biocompatibility. By balancing these two opposing effects by tailoring chemical composition, copolymer coatings were fabricated, which were able to inhibit the growth of S. mutans on the surface significantly but still show a sufficient attachment of gingival fibroblasts.
PMCID: PMC3784832  PMID: 21818855
antimicrobial polymer coatings; biocompatibility; copolymerization; medical implants; cell adhesion
4.  Regulation of Angiogenesis during Osseointegration by Titanium Surface Microstructure and Energy 
Biomaterials  2010;31(18):4909-4917.
Rough titanium (Ti) surface microarchitecture and high surface energy have been shown to increase osteoblast differentiation, and this response occurs through signaling via the α2β1 integrin. However, clinical success of implanted materials is dependent not only upon osseointegration but also on neovascularization in the peri-implant bone. Here we tested the hypothesis that Ti surface microtopography and energy interact via α2β1 signaling to regulate the expression of angiogenic growth factors. Primary human osteoblasts (HOB), MG63 cells and MG63 cells silenced for α2 integrin were cultured on Ti disks with different surface microtopographies and energies. Secreted levels of vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), and angiopoietin-1 (Ang-1) were measured. VEGF-A increased 170% and 250% in MG63 cultures, and 178% and 435% in HOB cultures on SLA and modSLA substrates, respectively. In MG63 cultures, FGF-2 levels increased 20 and 40-fold while EGF increased 4 and 6-fold on SLA and modSLA surfaces. These factors were undetectable in HOB cultures. Ang-1 levels were unchanged on all surfaces. Media from modSLA MG63 cultures induced more rapid differentiation of endothelial cells and this effect was inhibited by anti-VEGF-A antibodies. Treatment of MG63 cells with 1α,25(OH)2D3 enhanced levels of VEGF-A on SLA and modSLA. Silencing the α2 integrin subunit increased VEGF-A levels and decreased FGF-2 levels. These results show that Ti surface microtopography and energy modulate secretion of angiogenic growth factors by osteoblasts and that this regulation is mediated at least partially via α2β1 integrin signaling.
PMCID: PMC2896824  PMID: 20356623
Titanium; microstructure; surface energy; osteoblast; angiogenesis; VEGF
5.  Reduced adhesion of macrophages on anodized titanium with select nanotube surface features 
One of the important prerequisites for a successful orthopedic implant apart from being osteoconductive is the elicitation of a favorable immune response that does not lead to the rejection of the implant by the host tissue. Anodization is one of the simplest surface modification processes used to create nanotextured and nanotubular features on metal oxides which has been shown to improve bone formation. Anodization of titanium (Ti) leads to the formation of TiO2 nanotubes on the surface, and the presence of these nanotubes mimics the natural nanoscale features of bone, which in turn contributes to improved bone cell attachment, migration, and proliferation. However, inflammatory cell responses on anodized Ti remains to be tested. It is hypothesized that surface roughness and surface feature size on anodized Ti can be carefully manipulated to control immune cell (specifically, macrophages) responses. Here, when Ti samples were anodized at 10 V in the presence of 1% hydrofluoric acid (HF) for 1 minute, nanotextured (nonnanotube) surfaces were created. When anodization of Ti samples was carried out with 1% HF for 10 minutes at 15 V, nanotubes with 40–50 nm diameters were formed, whereas at 20 V with 1% HF for 10 minutes, nanotubes with 60–70 nm diameters were formed. In this study, a reduced density of macrophages was observed after 24 hours of culture on nanotextured and nanotubular Ti samples which were anodized at 10, 15, and 20 V, compared with conventional unmodified Ti samples. This in vitro study thus demonstrated a reduced density of macrophages on anodized Ti, thereby providing further evidence of the greater efficacy of anodized Ti for orthopedic applications.
PMCID: PMC3184936  PMID: 21980239
anodization; titanium implants; TiO2 nanotube
6.  Morphological alterations of T24 cells on flat and nanotubular TiO2 surfaces 
Croatian Medical Journal  2012;53(6):577-585.
To investigate morphological alterations of malignant cancer cells (T24) of urothelial origin seeded on flat titanium (Ti) and nanotubular titanium dioxide (TiO2) nanostructures.
Using anodization method, TiO2 surfaces composed of vertically aligned nanotubes of 50-100 nm diameters were produced. The flat Ti surface was used as a reference. The alteration in the morphology of cancer cells was evaluated using scanning electron microscopy (SEM). A computational model, based on the theory of membrane elasticity, was constructed to shed light on the biophysical mechanisms responsible for the observed changes in the contact area of adhesion.
Large diameter TiO2 nanotubes exhibited a significantly smaller contact area of adhesion (P < 0.0001) and had more membrane protrusions (eg, microvilli and intercellular membrane nanotubes) than on flat Ti surface. Numerical membrane dynamics simulations revealed that the low adhesion energy per unit area would hinder the cell spreading on the large diameter TiO2 nanotubular surface, thus explaining the small contact area.
The reduction in the cell contact area in the case of large diameter TiO2 nanotube surface, which does not enable formation of the large enough number of the focal adhesion points, prevents spreading of urothelial cells.
PMCID: PMC3541584  PMID: 23275323
7.  Focal adhesion linker proteins expression of fibroblast related to adhesion in response to different transmucosal abutment surfaces 
To evaluate adherence of human gingival fibroblasts (HGFs) to transmucosal abutment of dental implant with different surface conditions with time and to investigate the roles of focal adhesion linker proteins (FALPs) involved in HGFs adhesion to abutment surfaces.
Morphologies of cultured HGFs on titanium and ceramic discs with different surface were observed by scanning electron microscopy. Biocompatibility and focal adhesion were evaluated by ultrasonic wave application and cell viability assay. FALPs expression levels were assessed by RT-PCR and western blot.
There seemed to be little difference in biocompatibility and adhesion strength of HGFs depending on the surface conditions and materials. In all experimental groups, the number of cells remaining on the disc surface after ultrasonic wave application increased more than 2 times at 3 days after seeding compared to 1-day cultured cells and this continued until 7 days of culture. FALPs expression levels, especially of vinculin and paxillin, also increased in 5-day cultured cells compared to 1-day cultured fibroblasts on the disc surface.
These results might suggest that the strength of adhesion of fibroblasts to transmucosal abutment surfaces increases with time and it seemed to be related to expressions of FALPs.
PMCID: PMC3774950  PMID: 24049577
Transmucosal abutment; Focal adhesion linker proteins; Gingival fibroblast; Adhesion
8.  Inhibited bacterial biofilm formation and improved osteogenic activity on gentamicin-loaded titania nanotubes with various diameters 
Titania nanotubes loaded with antibiotics can deliver a high concentration of antibiotics locally at a specific site, thereby providing a promising strategy to prevent implant-associated infections. In this study we have fabricated titania nanotubes with various diameters (80, 120, 160, and 200 nm) and 200 nm length via electrochemical anodization. These nanotubes were loaded with 2 mg of gentamicin using a lyophilization method and vacuum drying. A standard strain, Staphylococcus epidermidis (American Type Culture Collection 35984), and two clinical isolates, S. aureus 376 and S. epidermidis 389, were selected to investigate the anti-infective ability of the gentamicin-loaded nanotubes (NT-G). Flat titanium (FlatTi) and nanotubes with no drug loading (NT) were also investigated and compared. We found that NT-G could significantly inhibit bacterial adhesion and biofilm formation compared to FlatTi or NT, and the NT-G with 160 nm and 200 nm diameters had stronger antibacterial activity because of the extended drug release time of NT-G with larger diameters. The NT also exhibited greater antibacterial ability than the FlatTi, while nanotubes with 80 nm or 120 nm diameters had better effects. Furthermore, human marrow derived mesenchymal stem cells were used to evaluate the effect of nanotubular topographies on the osteogenic differentiation of mesenchymal stem cells. Our results showed that NT-G and NT, especially those with 80 nm diameters, significantly promoted cell attachment, proliferation, spreading, and osteogenic differentiation when compared to FlatTi, and there was no significant difference between NT-G and NT with the same diameter. Therefore, nanotube modification and gentamicin loading can significantly improve the antibacterial ability and osteogenic activity of orthopedic implants.
PMCID: PMC3952900  PMID: 24634583
titania nanotubes; gentamicin; bacteria adhesion; biofilm formation; osteogenic activity
9.  Immobilization of pamidronic acids on the nanotube surface of titanium discs and their interaction with bone cells 
Nanoscale Research Letters  2013;8(1):124.
Self-assembled layers of vertically aligned titanium nanotubes were fabricated on a Ti disc by anodization. Pamidronic acids (PDAs) were then immobilized on the nanotube surface to improve osseointegration. Wide-angle X-ray diffraction, X-ray photoelectron microscopy, and scanning electron microscopy were employed to characterize the structure and morphology of the PDA-immobilized TiO2 nanotubes. The in vitro behavior of osteoblast and osteoclast cells cultured on an unmodified and surface-modified Ti disc was examined in terms of cell adhesion, proliferation, and differentiation. Osteoblast adhesion, proliferation, and differentiation were improved substantially by the topography of the TiO2 nanotubes, producing an interlocked cell structure. PDA immobilized on the TiO2 nanotube surface suppressed the viability of the osteoclasts and reduced their bone resorption activity.
PMCID: PMC3602675  PMID: 23497321
Pamidronic acid; TiO2 nanotubes; Immobilization; Surface modification; Bone cell
10.  Antibacterial titanium plate deposited by silver nanoparticles exhibits cell compatibility 
Microbial colonization and biofilm formation on the surface of implant devices may cause peri-implantitis and lead to bone loss. The aim of this study was to develop a novel antibacterial titanium implant surface and to test its biological performance. In a previous study, we demonstrated that titanium plates deposited by nanosilver acquired antibacterial activity to Staphylococcus aureus and Escherichia coli. While antibacterial activity is important, biomaterial surfaces should be modified to achieve excellent cell compatibility as well. In the present study, using the MTT assay, fluorescence microscopy, and scanning electron microscopy, we assessed cell viability, cytoskeletal architecture and cell attachment, respectively, on our silver nanoparticle-modified titanium (Ti-nAg) plate. The results demonstrate that the Ti-nAg do not show any cytotoxicity to the human gingival fibroblasts. Our data indicate that Ti-nAg is a novel material with both good antibacterial properties and uncompromised cytocompatibility, which can be used as an implanted biomaterial.
PMCID: PMC2875727  PMID: 20517478
nanosilver; titanium; antibacterial activity; cytocompatibility
11.  Enhanced osteoblast adhesion to drug-coated anodized nanotubular titanium surfaces 
Current orthopedic implants have functional lifetimes of only 10–15 years due to a variety of reasons including infection, extensive inflammation, and overall poor osseointegration (or a lack of prolonged bonding of the implant to juxtaposed bone). To improve properties of titanium for orthopedic applications, this study anodized and subsequently coated titanium with drugs known to reduce infection (penicillin/streptomycin) and inflammation (dexamethasone) using simple physical adsorption and the deposition of such drugs from simulated body fluid (SBF). Results showed improved drug elution from anodized nanotubular titanium when drugs were coated in the presence of SBF for up to 3 days. For the first time, results also showed that the simple physical adsorption of both penicillin/streptomycin and dexamethasone on anodized nanotubular titanium improved osteoblast numbers after 2 days of culture compared to uncoated unanodized titanium. In addition, results showed that depositing such drugs in SBF on anodized titanium was a more efficient method to promote osteoblast numbers compared to physical adsorption for up to 2 days of culture. In addition, osteoblast numbers increased on anodized titanium coated with drugs in SBF for up to 2 days of culture compared to unanodized titanium. In summary, compared to unanodized titanium, this preliminary study provided unexpected evidence of greater osteoblast numbers on anodized titanium coated with either penicillin/streptomycin or dexamethasone using simple physical adsorption or when coated with SBF; results which suggest the need for further research on anodized titanium orthopedic implants possessing drug-eluting nanotubes.
PMCID: PMC2527662  PMID: 18686785
anodization; titanium; adhesion; simulated body fluid; nanotubes; osteoblasts
12.  Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation 
The Journal of Cell Biology  1988;107(2):743-751.
Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.
PMCID: PMC2115214  PMID: 2971068
13.  Osseointegration of zirconia implants compared with titanium: an in vivo study 
Head & Face Medicine  2008;4:30.
Titanium and titanium alloys are widely used for fabrication of dental implants. Since the material composition and the surface topography of a biomaterial play a fundamental role in osseointegration, various chemical and physical surface modifications have been developed to improve osseous healing. Zirconia-based implants were introduced into dental implantology as an altenative to titanium implants. Zirconia seems to be a suitable implant material because of its tooth-like colour, its mechanical properties and its biocompatibility. As the osseointegration of zirconia implants has not been extensively investigated, the aim of this study was to compare the osseous healing of zirconia implants with titanium implants which have a roughened surface but otherwise similar implant geometries.
Forty-eight zirconia and titanium implants were introduced into the tibia of 12 minipigs. After 1, 4 or 12 weeks, animals were sacrificed and specimens containing the implants were examined in terms of histological and ultrastructural techniques.
Histological results showed direct bone contact on the zirconia and titanium surfaces. Bone implant contact as measured by histomorphometry was slightly better on titanium than on zirconia surfaces. However, a statistically significant difference between the two groups was not observed.
The results demonstrated that zirconia implants with modified surfaces result in an osseointegration which is comparable with that of titanium implants.
PMCID: PMC2614983  PMID: 19077228
14.  Homodimerization Controls the Fibroblast Growth Factor 9 Subfamily's Receptor Binding and Heparan Sulfate-Dependent Diffusion in the Extracellular Matrix▿ §  
Molecular and Cellular Biology  2009;29(17):4663-4678.
Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20's receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.
PMCID: PMC2725704  PMID: 19564416
15.  Greater osteoblast proliferation on anodized nanotubular titanium upon electrical stimulation 
Currently used orthopedic implants composed of titanium have a limited functional lifetime of only 10–15 years. One of the reasons for this persistent problem is the poor prolonged ability of titanium to remain bonded to juxtaposed bone. It has been proposed to modify titanium through anodization to create a novel nanotubular topography in order to improve cytocompatibility properties necessary for the prolonged attachment of orthopedic implants to surrounding bone. Additionally, electrical stimulation has been used in orthopedics to heal bone non-unions and fractures in anatomically difficult to operate sites (such as the spine). In this study, these two approaches were combined as the efficacy of electrical stimulation to promote osteoblast (bone forming cell) density on anodized titanium was investigated. To do this, osteoblast proliferation experiments lasting up to 5 days were conducted as cells were stimulated with constant bipolar pulses at a frequency of 20 Hz and a pulse duration of 0.4 ms each day for 1 hour. The stimulation voltages were 1 V, 5 V, 10 V, and 15 V. Results showed for the first time that under electrical stimulation, osteoblast proliferation on anodized titanium was enhanced at lower voltages compared to what was observed on conventional (nonanodized) titanium. In addition, compared to nonstimulated conventional titanium, osteoblast proliferation was enhanced 72% after 5 days of culture on anodized nanotubular titanium at 15 V of electrical stimulation. Thus, results of this study suggest that coupling the positive influences of electrical stimulation and nanotubular features on anodized titanium may improve osteoblast responses necessary for enhanced orthopedic implant efficacy.
PMCID: PMC2636582  PMID: 19337416
titanium; anodization; nanotubular; electrical stimulation; osteoblast; proliferation
16.  Biomechanical evaluation of dental implants with different surfaces: Removal torque and resonance frequency analysis in rabbits 
Macroscopic and especially microscopic properties of implant surfaces play a major role in the osseous healing of dental implants. Dental implants with modified surfaces have shown stronger osseointegration than implants which are only turned (machined). Advanced surface modification techniques such as anodic oxidation and Ca-P application have been developed to achieve faster and stronger bonding between the host bone and the implant.
The purpose of this study was to investigate the effect of surface treatment of titanium dental implant on implant stability after insertion using the rabbit tibia model.
Three test groups were prepared: sandblasted, large-grit and acid-etched (SLA) implants, anodic oxidized implants, and anodized implants with Ca-P immersion. The turned implants served as control. Twenty rabbits received 80 implants in the tibia. Resonance frequencies were measured at the time of implant insertion, 2 weeks and 4 weeks of healing. Removal torque values (RTV) were measured 2 and 4 weeks after insertion.
The implant stability quotient (ISQ) values of implants for resonance frequency analysis (RFA) increased significantly (P < .05) during 2 weeks of healing period although there were no significant differences among the test and control groups (P > .05). The test and control implants also showed significantly higher ISQ values during 4 weeks of healing period (P < .05). No significant differences, however, were found among all the groups. All the groups showed no significant differences in ISQ values between 2 and 4 weeks after implant insertion (P > .05). The SLA, anodized and Ca-P immersed implants showed higher RTVs at 2 and 4 weeks of healing than the machined one (P < .05). However, there was no significant difference among the experimental groups.
The surface-modified implants appear to provide superior implant stability to the turned one. Under the limitation of this study, however, we suggest that neither anodic oxidation nor Ca-P immersion techniques have any advantage over the conventional SLA technique with respect to implant stability.
PMCID: PMC2994679  PMID: 21165264
surface treatment; bone to implant contact; removal torque; dental implant
17.  Photokilling of T-24 human bladder cancer cells with titanium dioxide. 
British Journal of Cancer  1994;70(6):1107-1111.
A photoexcited titanium dioxide surface has a strong ability to decompose water into hydrogen and oxygen. We have studied this effect in order to use it to kill cancer cells in vitro and in vivo. A distinct cell killing effect was observed on cultured T-24 human bladder cancer cells treated with titanium dioxide particles and 300-400 nm UV light irradiation. Titanium dioxide plus UV light also dramatically suppressed the tumour growth of T-24 cells that were implanted in nude mice. Cells cultured on the titanium dioxide electrode were also killed under UV irradiation when the electrode was anodically polarised, suggesting that photogenerated holes are involved in the cell killing. The cell killing effect caused by titanium dioxide particles plus UV light irradiation was significantly hampered in the presence of L-cysteine and catalase, scavengers of hydroxyl radicals and hydrogen peroxide respectively. Transmission electron microscopic observations showed the titanium dioxide particles to be distributed on the cell surface and inside the cells. These results suggest that titanium dioxide particles under UV light irradiation produced photogenerated holes on the surface yielding hydroxyl radicals and hydrogen peroxide inside or outside the cells and the cells were then killed by the action of these highly oxidising molecules. The possible application of photoexcited titanium dioxide particles to cancer treatment as a new anti-cancer modality is discussed.
PMCID: PMC2033697  PMID: 7981061
18.  Anodizing color coded anodized Ti6Al4V medical devices for increasing bone cell functions 
Current titanium-based implants are often anodized in sulfuric acid (H2SO4) for color coding purposes. However, a crucial parameter in selecting the material for an orthopedic implant is the degree to which it will integrate into the surrounding bone. Loosening at the bone–implant interface can cause catastrophic failure when motion occurs between the implant and the surrounding bone. Recently, a different anodization process using hydrofluoric acid has been shown to increase bone growth on commercially pure titanium and titanium alloys through the creation of nanotubes. The objective of this study was to compare, for the first time, the influence of anodizing a titanium alloy medical device in sulfuric acid for color coding purposes, as is done in the orthopedic implant industry, followed by anodizing the device in hydrofluoric acid to implement nanotubes. Specifically, Ti6Al4V model implant samples were anodized first with sulfuric acid to create color-coding features, and then with hydrofluoric acid to implement surface features to enhance osteoblast functions. The material surfaces were characterized by visual inspection, scanning electron microscopy, contact angle measurements, and energy dispersive spectroscopy. Human osteoblasts were seeded onto the samples for a series of time points and were measured for adhesion and proliferation. After 1 and 2 weeks, the levels of alkaline phosphatase activity and calcium deposition were measured to assess the long-term differentiation of osteoblasts into the calcium depositing cells. The results showed that anodizing in hydrofluoric acid after anodizing in sulfuric acid partially retains color coding and creates unique surface features to increase osteoblast adhesion, proliferation, alkaline phosphatase activity, and calcium deposition. In this manner, this study provides a viable method to anodize an already color coded, anodized titanium alloy to potentially increase bone growth for numerous implant applications.
PMCID: PMC3540959  PMID: 23319862
nanotechnology; titanium; osteoblasts; anodization
19.  αvβ3 Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells 
Molecular Biology of the Cell  1997;8(12):2449-2461.
Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38–61 and 82–101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-αvβ3 antibodies prevent cell adhesion to FGF-2. Also, purified human αvβ3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-αvβ3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with αvβ3 integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.
PMCID: PMC25719  PMID: 9398667
20.  Dual effects and mechanism of TiO2 nanotube arrays in reducing bacterial colonization and enhancing C3H10T1/2 cell adhesion 
Competition occurs between the osteoblasts in regional microenvironments and pathogens introduced during surgery, on the surface of bone implants, such as joint prostheses. The aim of this study was to modulate bacterial and osteoblast adhesion on implant surfaces by using a nanotube array. Titanium oxide (TiO2) nanotube arrays, 30 nm or 80 nm in diameter, were prepared by a two-step anodization on titanium substrates. Mechanically polished and acid-etched titanium samples were also prepared to serve as control groups. The standard strains of Staphylococcus epidermidis (S. epidermidis, American Type Culture Collection [ATCC]35984) and mouse C3H10T1/2 cell lines with osteogenic potential were used to evaluate the different responses to the nanotube arrays, in bacteria and eukaryotic cells. We found that the initial adhesion and colonization of S. epidermidis on the surface of the TiO2 nanotube arrays were significantly reduced and that the adhesion of C3H10T1/2 cells on the surface of the TiO2 nanotube arrays was significantly enhanced when compared with the control samples. Based on a surface analysis of all four groups, we observed increased surface roughness, decreased water contact angles, and an enhanced concentration of oxygen and fluorine atoms on the TiO2 nanotube surface. We conclude that the TiO2 nanotube surface can reduce bacterial colonization and enhance C3H10T1/2 cell adhesion; multiple physical and chemical properties of the TiO2 nanotube surface may contribute to these dual effects.
PMCID: PMC3747852  PMID: 23983463
bacterial adhesion; titanium implant; surface modification
21.  Development of Anodic Titania Nanotubes for Application in High Sensitivity Amperometric Glucose and Uric Acid Biosensors 
Sensors (Basel, Switzerland)  2013;13(10):14161-14174.
The purpose of this study was to develop novel nanoscale biosensors using titania nanotubes (TNTs) made by anodization. Titania nanotubes were produced on pure titanium sheets by anodization at room temperature. In this research, the electrolyte composition ethylene glycol 250 mL/NH4F 1.5 g/DI water 20 mL was found to produce the best titania nanotubes array films for application in amperometric biosensors. The amperometric results exhibit an excellent linearity for uric acid (UA) concentrations in the range between 2 and 14 mg/dL, with 23.3 (μA·cm−2)·(mg/dL)−1 UA sensitivity, and a correlation coefficient of 0.993. The glucose biosensor presented a good linear relationship in the lower glucose concentration range between 50 and 125 mg/dL, and the corresponding sensitivity was approximately 249.6 (μA·cm−2)·(100 mg/dL)−1 glucose, with a correlation coefficient of 0.973.
PMCID: PMC3859114  PMID: 24152934
titania nanotubes (TNTs); amperometry; glucose; uric acid (UA)
22.  Characterization of drug-release kinetics in trabecular bone from titania nanotube implants 
The aim of this study was to investigate the application of the three-dimensional bone bioreactor for studying drug-release kinetics and distribution of drugs in the ex vivo cancellous bone environment, and to demonstrate the application of nanoengineered titanium (Ti) wires generated with titania nanotube (TNT) arrays as drug-releasing implants for local drug delivery
Nanoengineered Ti wires covered with a layer of TNT arrays implanted in bone were used as a drug-releasing implant. Viable bovine trabecular bone was used as the ex vivo bone substrate embedded with the implants and placed in the bone reactor. A hydrophilic fluorescent dye (rhodamine B) was used as the model drug, loaded inside the TNT–Ti implants, to monitor drug release and transport in trabecular bone. The distribution of released model drug in the bone was monitored throughout the bone structure, and concentration profiles at different vertical (0–5 mm) and horizontal (0–10 mm) distances from the implant surface were obtained at a range of release times from 1 hour to 5 days.
Scanning electron microscopy confirmed that well-ordered, vertically aligned nanotube arrays were formed on the surface of prepared TNT–Ti wires. Thermogravimetric analysis proved loading of the model drug and fluorescence spectroscopy was used to show drug-release characteristics in-vitro. The drug release from implants inserted into bone ex vivo showed a consistent gradual release of model drug from the TNT–Ti implants, with a characteristic three-dimensional distribution into the surrounding bone, over a period of 5 days. The parameters including the flow rate of bone culture medium, differences in trabecular microarchitecture between bone samples, and mechanical loading were found to have the most significant influence on drug distribution in the bone.
These results demonstrate the utility of the Zetos™ system for ex vivo drug-release studies in bone, which can be applied to optimize the delivery of specific therapies and to assist in the design of new drug delivery systems. This method has the potential to provide new knowledge to understand drug distribution in the bone environment and to considerably improve existing technologies for local administration in bone, including solving some critical problems in bone therapy and orthopedic implants.
PMCID: PMC3446838  PMID: 23028217
local drug delivery; Zetos bone bioreactor; drug-releasing implant; drug diffusion
23.  Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease 
Journal of Biochemistry  2010;149(2):121-130.
Fibroblast growth factors (FGFs) are a family of structurally related polypeptides that are essential for embryonic development and that function postnatally as homoeostatic factors, in the response to injury, in the regulation of electrical excitability of cells and as hormones that regulate metabolism. In humans, FGF signalling is involved in developmental, neoplastic, metabolic and neurological diseases. Fgfs have been identified in metazoans but not in unicellular organisms. In vertebrates, FGFs can be classified as having intracrine, paracrine and endocrine functions. Paracrine and endocrine FGFs act via cell-surface FGF receptors (FGFRs); while, intracrine FGFs act independent of FGFRs. The evolutionary history of the Fgf family indicates that an intracrine Fgf is the likely ancestor of the Fgf family. During metazoan evolution, the Fgf family expanded in two phases, after the separation of protostomes and deuterostomes and in the evolution of early vertebrates. These expansions enabled FGFs to acquire diverse actions and functions.
PMCID: PMC3106964  PMID: 20940169
Development; disease; evolution; FGF; metabolism
24.  Bacterial adhesion and colonization differences between zirconia and titanium implant abutments: an in vivo human study 
Several parameters have been described for determining the success or failure of dental implants. The surface properties of transgingival implant components have had a great impact on the long-term success of dental implants. The purpose of this study was to compare the tendency of two periodontal pathogens to adhere to and colonize zirconia abutments and titanium alloys both in hard surfaces and soft tissues.
Twelve patients participated in this study. Three months after implant placement, the abutments were connected. Five weeks following the abutment connections, the abutments were removed, probing depth measurements were recorded, and gingival biopsies were performed. The abutments and gingival biopsies taken from the buccal gingiva were analyzed using real-time polymerase chain reaction to compare the DNA copy numbers of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and total bacteria. The surface free energy of the abutments was calculated using the sessile water drop method before replacement. Data analyses used the Mann Whitney U-test, and P-values below 0.05 find statistical significance.
The present study showed no statistically significant differences between the DNA copy numbers of A. actinomycetemcomitans, P. gingivalis, and total bacteria for both the titanium and zirconia abutments and the biopsies taken from their buccal gingiva. The differences between the free surface energy of the abutments had no influence on the microbiological findings.
Zirconia surfaces have comparable properties to titanium alloy surfaces and may be suitable and safe materials for the long-term success of dental implants.
PMCID: PMC3543937  PMID: 23346465
Bacterial adhesion; Dental abutments
25.  Decreased cervical cancer cell adhesion on nanotubular titanium for the treatment of cervical cancer 
Cervical cancer can be treated by surgical resection, chemotherapy, and/or radiation. Titanium biomaterials have been suggested as a tool to help in the local delivery of chemotherapeutic agents and/or radiation to cervical cancer sites. However, current titanium medical devices used for treating cervical cancer do not by themselves possess any anticancer properties; such devices act as carriers for pharmaceutical agents or radiation sources and may even allow for the growth of cancer cells. Based on studies, which have demonstrated decreased lung, breast, and bone cancer cell functions on nanostructured compared to nanosmooth polymers, the objective of the present in vitro study was to modify titanium to possess nanotubular surface features and determine cervical cancer cell adhesion after 4 hours. Here, titanium was anodized to possess nanotubular surface features. Results demonstrated the ability to decrease cervical cancer cell adhesion by about a half on nanotubular compared to currently used nanosmooth titanium (without the use of chemotherapeutics or radiation), opening up numerous possibilities for the use of nanotubular titanium in local drug delivery or radiation treatment of cervical cancer.
PMCID: PMC3593771  PMID: 23493522
cervical cancer; titanium; nanotubular; cell density

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