Mukta Kulkarni

Titanium nanostructures for biomedical applications

Titanium and titanium alloys exhibit a unique combination of strength and biocompatibility, which enables their use in medical applications and accounts for their extensive use as implant materials in the last 50 years. Currently, a large amount of research is being carried out in order to determine the optimal surface topography for use in bioapplications, and thus the emphasis is on nanotechnology for biomedical applications. It was recently shown that titanium implants with rough surface topography and free energy increase osteoblast adhesion, maturation and subsequent bone formation. Furthermore, the adhesion of different cell lines to the surface of titanium implants is influenced by the surface characteristics of titanium; namely topography, charge distribution and chemistry. The present review article focuses on the specific nanotopography of titanium, i.e. titanium dioxide (TiO2) nanotubes, using a simple electrochemical anodisation method of the metallic substrate and other processes such as the hydrothermal or sol-gel template. One key advantage of using TiO2 nanotubes in cell interactions is based on the fact that TiO2 nanotube morphology is correlated with cell adhesion, spreading, growth and differentiation of mesenchymal stem cells, which were shown to be maximally induced on smaller diameter nanotubes (15 nm), but hindered on larger diameter (100 nm) tubes, leading to cell death and apoptosis. Research has supported the significance of nanotopography (TiO2 nanotube diameter) in cell adhesion and cell growth, and suggests that the mechanics of focal adhesion formation are similar among different cell types. As such, the present review will focus on perhaps the most spectacular and surprising one-dimensional structures and their unique biomedical applications for increased osseointegration, protein interaction and antibacterial properties.

Wettability studies of topologically distinct titanium surfaces

Biomedical implants made of titanium-based materials are expected to have certain essential features including high bone-to-implant contact and optimum osteointegration, which are often influenced by the surface topography and physicochemical properties of titanium surfaces. The surface structure in the nanoscale regime is presumed to alter/facilitate the protein binding, cell adhesion and proliferation, thereby reducing post-operative complications with increased lifespan of biomedical implants. The novelty of our TiO2 nanostructures lies mainly in the high level control over their morphology and roughness by mere compositional change and optimisation of the experimental parameters. The present work focuses on the wetting behaviour of various nanostructured titanium surfaces towards water. Kinetics of contact area of water droplet on macroscopically flat, nanoporous and nanotubular titanium surface topologies was monitored under similar evaporation conditions. The contact area of the water droplet on hydrophobic titanium planar surface (foil) was found to decrease during evaporation, whereas the contact area of the droplet on hydrophobic nanorough titanium surfaces practically remained unaffected until the complete evaporation. This demonstrates that the surface morphology and roughness at the nanoscale level substantially affect the titanium dioxide surface-water droplet interaction, opposing to previous observations for microscale structured surfaces. The difference in surface topographic nanofeatures of nanostructured titanium surfaces could be correlated not only with the time-dependency of the contact area, but also with time-dependency of the contact angle and electrochemical properties of these surfaces.

Binding of plasma proteins to titanium dioxide nanotubes with different diameters

Titanium and titanium alloys are considered to be one of the most applicable materials in medical devices because of their suitable properties, most importantly high corrosion resistance and the specific combination of strength with biocompatibility. In order to improve the biocompatibility of titanium surfaces, the current report initially focuses on specifying the topography of titanium dioxide (TiO2) nanotubes (NTs) by electrochemical anodization. The zeta potential (ζ-potential) of NTs showed a negative value and confirmed the agreement between the measured and theoretically predicted dependence of ζ-potential on salt concentration, whereby the absolute value of ζ-potential diminished with increasing salt concentrations. We investigated binding of various plasma proteins with different sizes and charges using the bicinchoninic acid assay and immunofluorescence microscopy. Results showed effective and comparatively higher protein binding to NTs with 100 nm diameters (compared to 50 or 15 nm). We also showed a dose-dependent effect of serum amyloid A protein binding to NTs. These results and theoretical calculations of total available surface area for binding of proteins indicate that the largest surface area (also considering the NT lengths) is available for 100 nm NTs, with decreasing surface area for 50 and 15 nm NTs. These current investigations will have an impact on increasing the binding ability of biomedical devices in the body leading to increased durability of biomedical devices.

Adherence of oral streptococci to nanostructured titanium surfaces.

OBJECTIVES Peri-implantitis and peri-mucositis pose a severe threat to the success of dental implants. Current research focuses on the development of surfaces that inhibit biofilm formation while not inferring with tissue integration. This study compared the adherence of two oral bacterial species, Streptococcus sanguinis and Streptococcus mutans to nanostructured titanium surfaces. METHODS The samples included TiO2 nanotubes formed by anodization of titanium foil of 100, 50 and 15nm diameter (NT15, NT50, NT100), a nanoporous (15nm pore diameter) surface and compact TiO2 control. Adherent surviving bacteria were enumerated after 1h in an artificial saliva medium containing bovine mucin. RESULTS Lowest numbers of adherent bacteria of both species were recovered from the original titanium foil and nanoporous surface and highest numbers from the Ti100 nanotubes. Numbers of attached S. sanguinis increased in the order (NT15<NT50<NT100), correlated with increasing percentage of surface fluoride. The lowest adhesion of S. sanguinis and S. mutans on TiO2 nanostructured surfaces was observed for small diameter nanoporous surfaces which coincides with the highest osteoblast adhesion on small diameter nanotubular/nanoporous surfaces shown in previous work. SIGNIFICANCE This study indicates that the adherence of oral streptococci can be modified by titanium anodization and nanotube diameter.

Influence of nanoparticle-membrane electrostatic interactions on membrane fluidity and bending elasticity

The aim of this work is to investigate the effect of electrostatic interactions between the nanoparticles and the membrane lipids on altering the physical properties of the liposomal membrane such as fluidity and bending elasticity. For this purpose, we have used nanoparticles and lipids with different surface charges. Positively charged iron oxide (γ-Fe2O3) nanoparticles, neutral and negatively charged cobalt ferrite (CoFe2O4) nanoparticles were encapsulated in neutral lipid 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine lipid mixture. Membrane fluidity was assessed through the anisotropy measurements using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Though the interaction of both the types of nanoparticles reduced the membrane fluidity, the results were more pronounced in the negatively charged liposomes encapsulated with positively charged iron oxide nanoparticles due to strong electrostatic attractions. X-ray photoelectron spectroscopy results also confirmed the presence of significant quantity of positively charged iron oxide nanoparticles in negatively charged liposomes. Through thermally induced shape fluctuation measurements of the giant liposomes, a considerable reduction in the bending elasticity modulus was observed for cobalt ferrite nanoparticles. The experimental results were supported by the simulation studies using modified Langevin-Poisson-Boltzmann model.

Interaction between Dipolar Lipid Headgroups and Charged Nanoparticles Mediated by Water Dipoles and Ions

In this work, a theoretical model describing the interaction between a positively or negatively charged nanoparticle and neutral zwitterionic lipid bilayers is presented. It is shown that in the close vicinity of the positively charged nanoparticle, the zwitterionic lipid headgroups are less extended in the direction perpendicular to the membrane surface, while in the vicinity of the negatively charged nanoparticle, the headgroups are more extended. This result coincides with the calculated increase in the osmotic pressure between the zwitterionic lipid surface and positively charged nanoparticle and the decrease of osmotic pressure between the zwitterionic lipid surface and the negatively charged nanoparticle. Our theoretical predictions agree well with the experimentally determined fluidity of a lipid bilayer membrane in contact with positively or negatively charged nanoparticles. The prospective significance of the present work is mainly to contribute to better understanding of the interactions of charged nanoparticles with a zwitterionic lipid bilayer, which may be important in the efficient design of the lipid/nanoparticle nanostructures (like liposomes with encapsulated nanoparticles), which have diverse biomedical applications, including targeted therapy (drug delivery) and imaging of cancer cells.

Influence of various sterilization procedures on TiO2 nanotubes used for biomedical devices

Sterilization is the final surface treatment procedure of all implantable devices and is one of the key factors which have to be considered before implementation. Since different sterilization procedures for all implantable devices influence mechanical properties as well as biological response, the influence of different sterilization techniques on titanium nanotubes was studied. Commonly used sterilization techniques such as autoclaving, ultra-violet light sterilization, hydrogen peroxide plasma sterilization as well as the not so frequently used gaseous oxygen plasma sterilization were used. Three different nanotube diameters; 15 nm, 50 nm and 100 nm were employed to study the effects of various sterilization techniques. It was observed that autoclave sterilization resulted in destruction of nanotubular features on all three studied nanotube diameters, while UV-light and both kinds of plasma sterilization did not cause any significant morphological changes on the surfaces. Differences between the sterilization techniques employed influenced cytocompatibility, especially in the case of nanotubes with 100 nm diameter.

Binding of human coronary artery endothelial cells to plasma‐treated titanium dioxide nanotubes of different diameters

Nanoscale topography in improving vascular response in vitro was established previously on various titanium surfaces. In the present study different surface nanotopographies that is different diameters of titanium dioxide (TiO2 ) nanotubes (NTs) were fabricated by electrochemical anodization and conditioned with highly reactive gaseous oxygen plasma. The morphology of different diameter NTs was studied by scanning electron microscopy and atomic force microscopy, while changes in chemical composition on the surface before and after plasma treatment were determined by X-ray photoelectron spectroscopy. Performance of human coronary artery endothelial cells (HCAEC) on those conditioned surfaces was studied in regard to cell proliferation, released IL-6 protein and immunofluorescence microscopy (IFM). We show that HCAEC function is dependent on the diameter of the TiO2 NTs, functioning far less optimally when bound to 100 nm TiO2 NTs as compared to Ti foil, 15 nm NTs or 50 nm NTs. There were improved, morphological cell shape changes, observed with IFM, between HCAEC growing on oxygen-rich plasma-treated versus nontreated 100 nm NTs. These endothelialized conditioned Ti nanosurfaces could elucidate optimization conditions necessary for vascular implants in coronary arteries.

Enhanced biocompatibility of TiO2 surfaces by highly reactive plasma

In the present study the biological response to various nanotopographic features after gaseous plasma treatment were studied. The usefulness of nanostructured surfaces for implantable materials has already been acknowledged, while less is known on the combined effect of nanostructured plasma modified surfaces. In the present work the influence of oxygen plasma treatment on nanostructured titanium oxide (TiO2) surfaces was studied. Characterization of the TiO2 surface chemical composition and morphological features was analyzed after plasma modification by x-ray photoelectron spectroscopy and by scanning electron microscopy while surface wettability was studied with measuring the water contact angle. Cell adhesion and morphology was assessed from images taken with scanning electron microscopy, whereas cell viability was measured with a calorimetric assay. The obtained results showed that oxygen plasma treatment of TiO2 nanotube surfaces significantly influences the adhesion and morphology of osteoblast-like cells in comparison to untreated nanostructured surfaces. Marked changes in surface composition of plasma treated surfaces were observed, as plasma treatment removed hydrocarbon contamination and removed fluorine impurities, which were present due to the electrochemical anodization process. However no differences in wettability of untreated and plasma treated surfaces were noticed. Treatment with oxygen plasma stimulated osteoblast-like cell adhesion and spreading on the nanostructured surface, suggesting the possible use of oxygen plasma surface treatment to enhance osteoblast-like cell response.

Interaction of nanostructured TiO2 biointerfaces with stem cells and biofilm-forming bacteria

Nanostructured TiO2 nanotubes (NTs) of diameters from 15 to 100nm were fabricated by an electrochemical anodization process. Biofilm-positive strains of Bacillus cereus and Pseudomonas aeruginosa behaved similarly on all TiO2 NTs as well as on native titanium (Ti) foil. The adhesion and growth of mesenchymal stem cells (MSc), embryonic stem cells (ESc), and pure cardiomyocytes derived from ESc exhibited significant differences. MSc as well as ESc were, in contrast to cardiomyocytes, able to adhere, and grow on TiO2 NTs. A correlation between NTs diameter and cell behaviour was however observed in the case of MSc and ESc. MSc were not in a physiological state in the case of 100nm TiO2 NTs, while ESc were not able to grow on 15nm TiO2 NTs. It can be stated that these differences can be assigned to different diameters of the NTs but not to the chemistry of the surface. This is the first study describing the comprehensive behaviour of both eukaryotic and prokaryotic cells on TiO2 NTs. On the basis of obtained results, it can be concluded that new generation of medical devices providing selective cell behaviour can be fabricated by optimizing the nanoscale morphology of TiO2.