New insight into interference-free and highly sensitive dopamine electroanalysis.

Early diagnosis of Parkinson's disease and hyperprolactinemia based on electrochemical dopamine (DA) sensing appears as an efficient and promising practical diagnostic method. However, the coexistence of DA in real samples with ascorbic acid (AA) and uric acid (UA), which oxidize at potentials close to its own, prevents the accurate electrochemical DA sensing and therefore, hinders the effective diagnosis of these diseases. In this work, we successfully combined the electrostatic proprieties of GO, the electron transfer properties of an AuNPs@MWCNTs nanocomposite and the ability of thiol group of the amino acid l-cysteine to react chemically with carbonyl groups of UA, to develop a novel approach that enabled complete suppression of interference from AA and UA and hence, accurate DA electroanalysis in the conditions close to those of human blood serum. The chemical reaction between l-cysteine and UA was evidenced by monitoring the DPV responses of UA under different conditions. XRD, Raman spectroscopy, XPS and FE-SEM revealed the successful synthesis of GO and AuNPs@MWCNTs. The study of the electrode material (GO-AuNPs@MWCNTs) morphology via FE-SEM and HR-TEM showed that AuNPs@MWCNTs are distributed throughout the exfoliated GO layers. The fabricated sensor was calibrated in the concentration range of 0.5-5 µM, in the presence of the highest blood concentrations of AA and UA for healthy individuals. A linear relationship was observed and the LOD was found to be 1.31 nM (S/N = 3). Furthermore, the sensor showed good electron transfer kinetics, good repeatability and reproducibility, satisfactory long-term stability, and recoveries in human blood serum.

Development of bioactive and antimicrobial nano-topography over selective laser melted Ti6Al4V implant and its in-vitro corrosion behavior.

Additive manufacturing (AM) or 3D printing of bone defect models is gaining much attention in the biomedical field as it could significantly facilitate the development of customized implants with a high degree of dimensional accuracy. Due to their satisfactory biocompatibility and minimal stress shielding effect, Ti6Al4V (Ti64) alloys are increasingly preferred in the development of such implants. However, their poor osseointegration abilities and lack of antibacterial properties often cause implant loosening and microbial infections, leading to implant failure. To address these drawbacks, we propose in this work a simple surface modification approach of customized Ti64 alloys (3D printed Ti6Al4V) that enables the formation of porous calcium titanate (CT) over their surface as well as the incorporation of silver nanoparticles (AgNPs) into the thus formed porous network. The successful CT formation with the incorporation of AgNPs throughout the 3D printed Ti64 surface and their influence in changing the morphological and mechanical behaviour were studied by Raman spectroscopy, SEM, AFM, Contact angle measurement, XPS, HR-TEM and nano-indentation. Antibacterial studies using Staphylococcus aureus and Escherichia coli, and in-vitro cell studies using MG-63 cell lines showed that surface modified samples resulting from the proposed method exhibit satisfactory antimicrobial property and are highly biocompatible. The obtained surface modified samples also showed a significant improvement in corrosion resistance as compared to unmodified 3D printed Ti64 alloys. The improvement in corrosion resistance was revealed by electrochemical impedance Spectroscopy (EIS). Obtained results emphasis that thus surface modified 3D printed Ti64 alloys are promising candidates for hard tissue implant applications.

Cerium Ion-Incorporated Titanium Metal Implants of Enhanced Bioactivity for Biomedical Applications.

The enhancement in the performance of metallic bone implants based on commercially pure titanium (CP-Ti) by incorporation of cerium (Ce) ions onto the surface was evaluated. The incorporation of Ce ions onto the CP-Ti surface was carried out by a simple two-step chemical treatment method, where an initial NaOH treatment and then a subsequent treatment with different molar concentrations of ceric nitrate solution followed by heat treatment at 600 °C were carried out. The modified surfaces were observed using field emission scanning electron microscopy (FE-SEM), scanning electron microscopy-energy dispersive X-ray analysis (SEM-EDX), X-ray photoelectron spectroscopy (XPS), the laser Raman spectroscopic technique, high-resolution transmission electron microscopy (HR-TEM), and atomic force microscopy (AFM). The formation of a nanonetwork structure by the initial NaOH treatment and the replacement of Na ions with Ce ions along with different phases of TiO2 was evident from the surface characterization results. The transition of rutile TiO2 to anatase TiO2 in the modified surface is evident from the Raman spectra with respect to the treatment of higher to lower concentrations of ceric nitrate solution. The presence of two different oxidation states of Ce (Ce3+ and Ce4+) and improvement in the surface wettability were also distinct in the modified samples. Thus, the incorporated Ce ions over the nanostructured titania network showed low cytotoxicity, good cell adhesion, and enhanced extracellular mineralization on MG-63 cells with better protein adsorption in BSA medium. Taken together, the thus-improved nanostructured surface morphology with the anatase TiO2 phase and distinct extracellular mineralization in the Ce-incorporated Ti metal with good biocompatibility make it a promising candidate for bone implant applications.

Bioactive and antimicrobial macro-/micro-nanoporous selective laser melted Ti-6Al-4V alloy for biomedical applications.

Metal Additive Manufacturing (AM) technology is an emerging technology in biomedical field due to its unique ability to manufacture customized implants [Patients-specific Implants (PSIs)] replicating the complex bone structure from the relevant metal powders. PSIs could be developed through any AM technology, but the ultimate challenge lies in integrating the metallic implant with the living bone. Considering this aspect, in the present study, Ti alloy (Ti-6Al-4V) powder has been used to fabricate scaffolds of channel type macropores with 0-60% porosity using selective laser melting (SLM) and subsequent post-treatments paving way for surface microporosities. Surface chemical and subsequent heat treatments were carried out on thus developed Ti alloy scaffolds to improve its bioactivity, antibacterial activity and osteoblastic cell compatibility. NaOH and subsequent Ca(NO3)2/AgNO3 treatment induced the formation of a nanoporous network structure decorated with Ca-Ag ions. Ag nanoparticles covering the entire scaffold provided antibacterial activity and the presence of Ca2+ ions with anatase TiO2 layer further improved the bioactivity and osteoblastic cell compatibility of the scaffold. Therefore, SLM technology combined with heat treatment and surface modification could be effectively utilized to create macro-micro-nano structure scaffolds of Ti alloy that are bioactive, antibacterial, and cytocompatible.

Mechanistic studies of biomineralisation on silver incorporated anatase TiO2.

Here we report silver incorporated anatase TiO2 developed on Ti metal by H2O2-AgNO3 and heat treatment to have faster biomineralisation or apatite-forming ability in simulated body fluid (SBF). Apatite-forming ability has been investigated concerning heat treatment temperatures ranges, 400-800 °C and duration of soaking period in SBF. The apatite formation showed an increasing trend with increase in the heat treatment temperatures up to 600 °C and beyond that the Ti metal lost this ability. XRD as wells as Raman results of such chemical and heat-treated Ti metal at different temperatures further correlates the apatite nucleation directly in relation with that of anatase to rutile TiO2 formation. Further, a time dependent apatite mineralisation study by XPS revealed simultaneous calcium and phosphate deposition at the early stage of soaking in SBF. Therefore, the apatite nucleation in the present chemically treated Ti metal depends on the crystalline phase of TiO2 formed by H2O2 and heat treatment along with Ag+ ion release.

Ca-Ag coexisting nano-structured titania layer on Ti metal surface with enhanced bioactivity, antibacterial and cell compatibility.

A nano-structured titanate layer encapsulated with Ca2+ and Ag+ ions was successfully grown over commercially pure (CP) Ti metal by chemical treatment with H2O2 and subsequent treatment with Ca (NO3)2/AgNO3 solutions. Heat treatment at 600 °C, further transformed this nano-structured titanate layer into titania containing Ca2+ and Ag+ ions. Thus modified Ti metal showed significant enhancement in apatite-forming ability when soaked in simulated body fluid (SBF). Presence of Ag+ ions showed good antimicrobial activity against pathogenic Staphylococcus aureus, and, Ca2+ ions being a major component of bone mineral accelerated the apatite-forming ability over Ti metal in SBF. Further, Ca2+and Ag+ ions proportion over Ti metal surface could be optimised in order to have minimum Ag concentration that can have not only antibacterial activity and also cell compatibility against MG 63 osteoblast-like cells. Therefore, the proposed surface modification approach presented here is expected to be useful in orthopaedic implants that necessitate enhanced bioactivity, antibacterial activity and cell compatibility.

Role of calcium ions in defining the bioactivity of surface modified Ti metal.

Nano-structured hydrogen titanate and sodium hydrogen titanate layers were formed when Ti metal was treated with H2O2 and NaOH solutions, respectively. The chemically treated Ti metals upon subsequent treatment with Ca(NO3)2 and CaCl2 solutions, resulted in incorporation of Ca2+ ions into the nano-structured titanate layer. Thus formed nano-structured titanate layers containing Ca2+ ions when subjected to heat treatment, forms anatase and calcium titanate-rutile phases, respectively. In vitro apatite-forming ability in simulated body fluid (SBF) was positive for H2O2-Ca and heat-treated Ti metal in contrast to NaOH-Ca and heat treatment. Formation of anatase phase together with Ca2+ ion release into SBF was found to be the key driving force for such a high bioactivity of Ca2+ containing H2O2 treated Ti metal on contrary to NaOH and heat treatment. This study provides a new insight into the factors accelerating the bioactivity of Ti metals during various chemical and thermal treatments, which further aid and abet to design dental and orthopaedic implants with high bone-bonding ability.

Effect of Silver-Containing Titania Layers for Bioactivity, Antibacterial Activity, and Osteogenic Differentiation of Human Mesenchymal Stem Cells on Ti Metal.

Biomaterials with better osteogenic capacity, rapid osteo-integration, and higher mechanical strength are undoubtedly preferred for successful bone implant development. A porous sodium hydrogen titanate layer was formed on Ti metal by NaOH treatment, and the Na+ ions were replaced by Ag+ ions by subsequent AgNO3 treatment that formed silver-containing hydrogen titanate. Heat treatment at 600 °C transformed sodium hydrogen titanate into sodium titanate with sheet-like morphology, whereas silver-containing hydrogen titanate was converted to anatase TiO2 with an elongated rod-like structure. Further increment in temperature lead to the formation of rutile TiO2 with distracted network morphology. Between these two, the anatase TiO2 was ascertained to be bioactive by being capable of forming bonelike apatite in simulated body fluid within a period of 12 h. The concentration of silver on Ti metal was further optimized for better antibacterial activity against S. aureus and biocompatibility toward bone cells. A detailed investigation of thus optimized silver-containing Ti metal on the proliferation and differentiation of multipotent human mesenchymal stem cells further proved their biocompatibility nature and facilitation of osteogenic differentiation, thereby conferring those as ideally suited materials for bioimplant development in bone tissue engineering.

Comparative study on Ti-Nb binary alloys fabricated through spark plasma sintering and conventional P/M routes for biomedical application.

The main purpose of this work is to obtain homogenous, single ß phase in binary Ti-xNb (x = 18.75, 25, and 31.25 at.%) alloys by simple mixing of pure elemental powders using different sintering techniques such as spark plasma sintering (pressure-assisted sintering) and conventional powder metallurgy (pressure-less sintering). Synthesis parameters such as sintering temperature and holding time etc. are optimized in both techniques in order to get homogenous microstructure. In spark plasma sintering (SPS), complete homogeneous ß phase is achieved in Ti25at.%Nb using 1300 °C sintering temperature with 60 min holding time under 50 MPa pressure. On the other hand, complete ß phase is obtained in Ti25at.%Nb through conventional powder metallurgy (P/M) route using sintering temperature of 1400 °C for 120 min holding time which are adopted from the dilatometry studies. Nano-indentation is carried out for mechanical properties such as Young's modulus and nano-hardness. Elastic properties of binary Ti-xNb compositions are fallen within the range of 80-90 GPa. Cytotoxicity as well as cell adhesion studies carried out using MG63, osteoblast-like cells showed excellent biocompatibility of thus developed Ti25at.%Nb surface irrespective of fabrication route.

Influence of pH on wet-synthesis of silver decorated hydroxyapatite nanopowder.

Here, effect of solution pH on precipitation of silver decorated hydroxyapatite (Ag-HAp) nano powder during its wet-synthesis was systematically studied. XRD pattern of Ag-HAp nano powder synthesised at pH ranging from 5 to 10 shows that calcium hydrogen phosphate was formed as dominating phase when the solution pH was between 5 and 9 and this phase was gradually transformed into a stable HAp above pH 9. A quantitative analysis of silver amount in Ag-HAp nano powder synthesised at different pH showed that silver can be precipitated to its maximum amount at pH 8 and the further addition of ammonia leads to the formation of a silver-ammonium complex, thereby remaining in the solution. HR-TEM and XPS analysis further confirmed the presence of silver in HAp nanocrystals, synthesised at an optimum pH 9. This trace amount of silver in HAp nano powder showed effective antibacterial activity against Staphylococcus aureus and Escherichia coli. In addition, the cytocompatibility studies carried out on MG63 cells further confirmed the present optimised silver concentration of the Ag-HAp nano powder is well within the toxic limit to be useful in various biomedical applications.

Topotactic transition of α-Co(OH)2 to ß-Co(OH)2 anchored on CoO nanoparticles during electrochemical water oxidation: synergistic electrocatalytic effects.

Herein, we report a single step, anionic surfactant-assisted, low temperature-hydrothermal synthetic strategy of CoO nanoparticles anchored on ß-Co(OH)2 nanosheets which show a low overpotential (295 mV @ 10 mA cm-2) for the oxygen evolution reaction (OER). They also demonstrate much better kinetic parameters compared to the state-of-the-art RuO2. Interestingly, under the OER operational conditions (in alkaline medium), the topotactic transformation of α-Co(OH)2 to a stable Brucite-like ß-Co(OH)2 phase leads to a synergistic interaction between the ß-Co(OH)2 sheets on the CoO nanoparticles for enhancing the OER electrocatalytic activity.

Divalent ion encapsulated nano titania on Ti metal as a bioactive surface with enhanced protein adsorption.

A novel approach on incorporation of divalent species such as Mg, Ca and Sr into the titania nanostructures formed on Ti metal surface and their comparative study on enhancement of bioactivity, protein adsorption and cell compatibility is reported. When treated with hydrogen peroxide, Ti metal forms hydrogen titanate. On subsequent treatment with Mg or Ca or Sr nitrate solutions, respective ions are incorporated into hydrogen titanate layer, and heat treatment leads to titania decorated with these ions. The resultant heat-treated samples when soaked in simulated body fluid form bone-like apatite which indicates the present surface modification enhances the bioactivity. Further, enhanced protein adsorption in bovine serum albumin is an indication of suitability of these divalent species to form chelate compounds with amino acids, and Ca containing titania nanostructure favours more protein adsorption compared to the others. Cytocompatibility studies using MG-63, human osteosarcoma cell lines shows these divalent ion containing titania nanostructure favours the cell attachment and did not show any cytotoxicity. Bioactivity, enhanced protein adsorption along with cytocompatibility clearly indicates such surface modification approach to be useful to design hard tissue replacement materials in orthopaedic and dental field.

Osteoinduction on acid and heat treated porous Ti metal samples in canine muscle.

Samples of porous Ti metal were subjected to different acid and heat treatments. Ectopic bone formation on specimens embedded in dog muscle was compared with the surface characteristics of the specimen. Treatment of the specimens by H2SO4/HCl and heating at 600 °C produced micrometer-scale roughness with surface layers composed of rutile phase of titanium dioxide. The acid- and heat-treated specimens induced ectopic bone formation within 6 months of implantation. A specimen treated using NaOH followed by HCl acid and then heat treatment produced nanometer-scale surface roughness with a surface layer composed of both rutile and anatase phases of titanium dioxide. These specimens also induced bone formation after 6 months of implantation. Both these specimens featured positive surface charge and good apatite-forming abilities in a simulated body fluid. The amount of the bone induced in the porous structure increased with apatite-forming ability and higher positive surface charge. Untreated porous Ti metal samples showed no bone formation even after 12 months. Specimens that were only heat treated featured a smooth surface composed of rutile. A mixed acid treatment produced specimens with micrometer-scale rough surfaces composed of titanium hydride. Both of them also showed no bone formation after 12 months. The specimens that showed no bone formation also featured almost zero surface charge and no apatite-forming ability. These results indicate that osteoinduction of these porous Ti metal samples is directly related to positive surface charge that facilitates formation of apatite on the metal surfaces in vitro.

Evaluation of bioactivity of alkali- and heat-treated titanium using fluorescent mouse osteoblasts.

Stimulation of osteoblast proliferation and differentiation is important for the in vivo bone-bonding ability of biomaterials. Previous in vitro studies have used biochemical assays to analyze osteoblast-specific gene expression in cultured osteoblasts. In this study, we generated transgenic mice harboring a monomeric red fluorescent protein 1 transgene under the control of a 2.3-kb fragment of the Col1a1 promoter, which is active specifically in osteoblasts and osteocytes. We established a fluorescent primary osteoblast culture system to allow noninvasive observation of osteoblast proliferation and differentiation on opaque materials in vitro. We used this system to evaluate alkali- and heat-treated titanium, which has a strong bone-bonding ability in vivo, and we observed a rapid increase in fluorescence intensity and characteristic multifocal nodule formation. A cell proliferation assay and RT-PCR to examine osteoblast-specific gene expression showed increased osteoblast proliferation and differentiation consistent with the fluorescence observations. This mouse model allowed us to use fluorescence intensity to visualize and quantify in vivo newly formed bone around implanted materials in femurs. The use of these fluorescent osteoblasts is a promising method for simple screening of the bone-bonding ability of new materials.

Osteoconduction of porous Ti metal enhanced by acid and heat treatments.

Bone ingrowth into porous Ti metal is important for stable fixation of Ti metal implants to surrounding bone. However, without surface treatment this is limited to only a thin region of the outer surface of the Ti metal. In the present study, a porous Ti metal with a porosity of ~60 % and interpore connections of 70-200 micrometers in diameter was investigated in terms of its chemical and heat treatments, by implanting it into rabbit femur for periods varying from 3 to 12 weeks. The porous Ti metal subjected to heat treatment at 600 °C after H2SO4/HCl mixed acid treatment showed the largest bone ingrowth in comparison with those subjected to no treatment, only acid treatment, and only heat treatment even at an early stage after implantation, and remained as such even 12 weeks after implantation. Their bone ingrowths were well interpreted in terms of apatite-forming abilities of the Ti metals in body environment. Their apatite-forming abilities did not depend upon their surface roughness nor type of crystalline phase, but upon the positive surface charge.

Bone-bonding properties of Ti metal subjected to acid and heat treatments.

The effects of surface treatment on the bone-bonding properties of Ti metal were examined by both mechanical detaching test and histological observation after implantation into rabbit tibiae for various periods ranging from 4 to 26 weeks. The bone-bonding ability of Ti metal, which is extremely low as it is abraded, was hardly increased by simple heat treatment at 600 °C or treatment with H(2)SO(4)/HCl mixed acid alone, but was markedly increased by the heat treatment after the acid treatment. Even Ti metal that had been previously subjected to NaOH treatment showed considerably high bone-bonding ability after acid and heat treatments. Such high bonding abilities were attributed to their high apatite-forming ability in the body environment. Their high apatite-forming abilities were attributed to a high positive surface charge, and not to the type of crystalline phase or specific roughness of their surfaces. The present study has demonstrated that acid and subsequent heat treatments are effective for conferring stable fixation properties on Ti metal implants.

Apatite-forming ability of titanium in terms of pH of the exposed solution.

In order to elucidate the main factor governing the capacity for apatite formation of titanium (Ti), Ti was exposed to HCl or NaOH solutions with different pH values ranging from approximately 0 to 14 and then heat-treated at 600°C. Apatite formed on the metal surface in a simulated body fluid, when Ti was exposed to solutions with a pH less than 1.1 or higher than 13.6, while no apatite formed upon exposure to solutions with an intermediate pH value. The apatite formation on Ti exposed to strongly acidic or alkaline solutions is attributed to the magnitude of the positive or negative surface charge, respectively, while the absence of apatite formation at an intermediate pH is attributed to its neutral surface charge. The positive or negative surface charge was produced by the effect of either the acidic or alkaline ions on Ti, respectively. It is predicted from the present results that the bone bonding of Ti depends upon the pH of the solution to which it is exposed, i.e. Ti forms a bone-like apatite on its surface in the living body and bonds to living bone through the apatite layer upon heat treatment after exposure to a strongly acidic or alkaline solution.

Nanostructured positively charged bioactive TiO2 layer formed on Ti metal by NaOH, acid and heat treatments.

Nanometer-scale roughness was generated on the surface of titanium (Ti) metal by NaOH treatment and remained after subsequent acid treatment with HCl, HNO(3) or H(2)SO(4) solution, as long as the acid concentration was not high. It also remained after heat treatment. Sodium hydrogen titanate produced by NaOH treatment was transformed into hydrogen titanate after subsequent acid treatment as long as the acid concentration was not high. The hydrogen titanate was then transformed into titanium oxide (TiO(2)) of anatase and rutile by heat treatment. Treated Ti metals exhibited high apatite-forming abilities in a simulated body fluid especially when the acid concentration was greater than 10 mM, irrespective of the type of acid solutions used. This high apatite-forming ability was maintained in humid environments for long periods. The high apatite-forming ability was attributed to the positive surface charge that formed on the TiO(2) layer and not to the surface roughness or a specific crystalline phase. This positively charged TiO(2) induced apatite formation by first selectively adsorbing negatively charged phosphate ions followed by positively charged calcium ions. Apatite formation is expected on the surfaces of such treated Ti metals after short periods, even in living systems. The bonding of metal to living bone is also expected to take place through this apatite layer.

Osteoinduction of porous Ti implants with a channel structure fabricated by selective laser melting.

Many studies have shown that certain biomaterials with specific porous structures can induce bone formation in non-osseous sites without the need for osteoinductive biomolecules, however, the mechanisms responsible for this phenomenon (intrinsic osteoinduction of biomaterials) remain unclear. In particular, to our knowledge the type of pore structure suitable for osteoinduction has not been reported in detail. In the present study we investigated the effects of interconnective pore size on osteoinductivity and the bone formation processes during osteoinduction. Selective laser melting was employed to fabricate porous Ti implants (diameter 3.3mm, length 15 mm) with a channel structure comprising four longitudinal square channels, representing pores, of different diagonal widths, 500, 600, 900, and 1200 µm (termed p500, p600, p900, and p1200, respectively). These were then subjected to chemical and heat treatments to induce bioactivity. Significant osteoinduction was observed in p500 and p600, with the highest observed osteoinduction occurring at 5mm from the end of the implants. A distance of 5mm probably provides a favorable balance between blood circulation and fluid movement. Thus, the simple architecture of the implants allowed effective investigation of the influence of the interconnective pore size on osteoinduction, as well as the relationship between bone quantity and its location for different pore sizes.

Bioactive Ti metal analogous to human cancellous bone: Fabrication by selective laser melting and chemical treatments.

Selective laser melting (SLM) is a useful technique for preparing three-dimensional porous bodies with complicated internal structures directly from titanium (Ti) powders without any intermediate processing steps, with the products being expected to be useful as a bone substitute. In this study the necessary SLM processing conditions to obtain a dense product, such as the laser power, scanning speed, and hatching pattern, were investigated using a Ti powder of less than 45 µm particle size. The results show that a fully dense plate thinner than 1.8 mm was obtained when the laser power to scanning speed ratio was greater than 0.5 and the hatch spacing was less than the laser diameter, with a 30 µm thick powder layer. Porous Ti metals with structures analogous to human cancellous bone were fabricated and the compressive strength measured. The compressive strength was in the range 35-120 MPa when the porosity was in the range 75-55%. Porous Ti metals fabricated by SLM were heat-treated at 1300 °C for 1h in an argon gas atmosphere to smooth the surface. Such prepared specimens were subjected to NaOH, HCl, and heat treatment to provide bioactivity. Field emission scanning electron micrographs showed that fine networks of titanium oxide were formed over the whole surface of the porous body. These treated porous bodies formed bone-like apatite on their surfaces in a simulated body fluid within 3 days. In vivo studies showed that new bone penetrated into the pores and directly bonded to the walls within 12 weeks after implantation into the femur of Japanese white rabbits. The percentage bone affinity indices of the chemical- and heat-treated porous bodies were significantly higher than that of untreated implants.