A qualitative method for testing the antimicrobial ability of osteosynthetic fixation material by simulating in vitro contamination by Staphylococcus aureus.

External fixators of serious fractures could be an attractive substrate on which microorganisms can accumulate. Therefore, this study aimed to develop a suitable method for enabling the simulation of a real situation when osteosynthetic fixation material is open for the potential threat of bacterial attack. Agar-based media represented human tissue, and the metallic pin characterized the screw in the fixation. Various types of agar, supplements, and contamination strategy by Staphylococcus aureus were tested. The influence of the initial bacterial concentration was also examined. Surfaces were observed by scanning electron microscopy (SEM), and all results were compared. Brain Heart Infusion Agar with the Egg Yolk Tellurite Emulsion was established in a transparent test tube as a suitable system for enabling the good interpretability of bacterial contamination in the pin's surroundings. Pin contamination has been found to be an appropriate approach for testing microbial growth, rather than agar surface contamination, which distorted obtained results. A lower initial colony forming units (CFU) provided better clarity of the test. SEM observation of the pin surface was comparable with the visual evaluations in the test tubes. Results were assembled for positive and negative control samples as well. Screening method for the most common bacteria S. aureus has been standardized and developed. This experimental setup could also be a useful tool for surface modification with antibacterial properties testing.

A two-phase gradual silver release mechanism from a nanostructured TiAlV surface as a possible antibacterial modification in implants.

Titanium biomaterials are widely used in the medical field due to their biocompatibility and excellent corrosion and mechanical resistance. However, these materials have no antibacterial properties. To obtain an antibacterial active surface, a nanostructure of Ti6Al4V alloy was created. This specific nanostructure contained nanotubes and micro-cavities and was used as a substrate for silver anchoring. The electrochemical approach to silver reduction was studied. It is a common approach for silver deposition and in this work, inhomogeneities in the nanostructure were used as a preferential area for silver localisation. The galvanostatic regimen of deposition allowed for a technically quantitative process and the required silver placement. The experimental conditions used enabled testing and silver dissolution rate evaluation within a reasonable time span. Based on the corrosion and analytical results (EDS, XPS and ICP-MS), a two-phase silver release mechanism was confirmed. The openings of the individual nanotubes were filled with silver nanoparticles, whose release was relatively fast. By contrast, the silver anchored inside the cavities allowed the silver to release gradually. Antibacterial efficiency against Staphylococcus aureus and Escherichia coli was successfully demonstrated. Cytotoxicity testing with murine fibroblasts showed cell metabolic activity far above the normative limit of 70%.

Different diameters of titanium dioxide nanotubes modulate Saos-2 osteoblast-like cell adhesion and osteogenic differentiation and nanomechanical properties of the surface.

The formation of nanostructures on titanium implant surfaces is a promising strategy to modulate cell adhesion and differentiation, which are crucial for future application in bone regeneration. The aim of this study was to investigate how the nanotube diameter and/or nanomechanical properties alter human osteoblast like cell (Saos-2) adhesion, growth and osteogenic differentiation in vitro. Nanotubes, with diameters ranging from 24 to 66 nm, were fabricated on a commercially pure titanium (cpTi) surface using anodic oxidation with selected end potentials of 10 V, 15 V and 20 V. The cell response was studied in vitro on untreated and nanostructured samples using a measurement of metabolic activity, cell proliferation, alkaline phosphatase activity and qRT-PCR, which was used for the evaluation of osteogenic marker expression (collagen type I, osteocalcin, RunX2). Early cell adhesion was investigated using SEM and ELISA. Adhesive molecules (vinculin, talin), collagen and osteocalcin were also visualized using confocal microscopy. Moreover, the reduced elastic modulus and indentation hardness of nanotubes were assessed using a TriboIndenter™. Smooth and nanostructured cpTi both supported cell adhesion, proliferation and bone-specific mRNA expression. The nanotubes enhanced collagen type I and osteocalcin synthesis, compared to untreated cpTi, and the highest synthesis was observed on samples modified with 20 V nanotubes. Significant differences were found in the cell adhesion, where the vinculin and talin showed a dot-like distribution. Both the lowest reduced elastic modulus and indentation hardness were assessed from 20 V samples. The nanotubes of mainly 20 V samples showed a high potential for their use in bone implantation.

Corrosion behaviour and cell interaction of Ti-6Al-4V alloy prepared by two techniques of 3D printing.

3D printing seems to be the technology of the future for the preparation of metallic implants. For such applications, corrosion behaviour is pivotal. However, little is published on this topic and with inconsistent results. Therefore, we carried out a complex study in which we compared two techniques of the 3D printing technology - selective laser melting and electron beam melting. The corrosion behaviour was studied in physiological solution by standard electrochemical techniques and susceptibility to localised corrosion was estimated too. All samples showed typical passive behaviour. Localised corrosion was shown to be possible on the original as-printed surfaces. Corrosion experiments were repeated tree times. To reveal possible negative effects of 3D printing on cytocompatibility, direct in vitro tests were performed with U-2 OS cells. The cells showed good viability and proliferation, but their growth was impeded by surface unevenness. Our results suggest that both techniques are suitable for implants production. Statistical evaluation was performed by ANOVA followed by Tukey's test.

Cell interaction with modified nanotubes formed on titanium alloy Ti-6Al-4V.

Nanotubes with diameters ranging from 40 to 60nm were prepared by electrochemical oxidation of the Ti-6Al-4V alloy in electrolyte containing ammonium sulphate and ammonium fluoride. The nanotubes were further modified with calcium and phosphate ions or were heat treated. Polished Ti-6Al-4V alloy served as a reference sample. The spreading of human osteoblast-like cells was similar on all nanotube samples but lower than on polished samples. The number of initially adhered cells was higher on non-modified nanotubes, but the final cell number was the highest on Ca-enriched nanotubes and the lowest on heat-treated nanotubes. However, these differences were relatively small and less pronounced than the differences in the concentration of specific molecular markers of cell adhesion and differentiation, estimated by their intensity of immunofluorescence staining. The concentration of vinculin, i.e. a protein of focal adhesion plaques, was the lowest on nanotubes modified with calcium. Collagen I, an early marker of osteogenic cell differentiation, was also the lowest on samples modified with calcium and was highest on polished samples. Alkaline phosphatase, a middle marker of osteogenic differentiation, was observed in lowest concentration on nanotubes modified with phosphorus and the highest on heat-treated samples. Osteocalcin concentrations, a late marker of osteogenic cell differentiation, were similar on all tested samples, although they tended to be the highest on heat-treated samples. Thus, osteogenic differentiation can be modulated by various additional treatments of nanotube coatings on Ti-6Al-4V implants.

The diameter of nanotubes formed on Ti-6Al-4V alloy controls the adhesion and differentiation of Saos-2 cells.

Ti-6Al-4V-based nanotubes were prepared on a Ti-6Al-4V surface by anodic oxidation on 10 V, 20 V, and 30 V samples. The 10 V, 20 V, and 30 V samples and a control smooth Ti-6Al-4V sample were evaluated in terms of their chemical composition, diameter distribution, and cellular response. The surfaces of the 10 V, 20 V, and 30 V samples consisted of nanotubes of a relatively wide range of diameters that increased with the voltage. Saos-2 cells had a similar initial adhesion on all nanotube samples to the control Ti-6Al-4V sample, but it was lower than on glass. On day 3, the highest concentrations of both vinculin and talin measured by enzyme-linked immunosorbent assay and intensity of immunofluorescence staining were on 30 V nanotubes. On the other hand, the highest concentrations of ALP, type I collagen, and osteopontin were found on 10 V and 20 V samples. The final cellular densities on 10 V, 20 V, and 30 V samples were higher than on glass. Therefore, the controlled anodization of Ti-6Al-4V seems to be a useful tool for preparing nanostructured materials with desirable biological properties.

Corrosion behavior of Ti-39Nb alloy for dentistry.

To increase an orthopedic implant's lifetime, researchers are now concerned on the development of new titanium alloys with suitable mechanical properties (low elastic modulus-high fatigue strength), corrosion resistance and good workability. Corrosion resistance of the newly developed titanium alloys should be comparable with that of pure titanium. The effect of medical preparations containing fluoride ions represents a specific problem related to the use of titanium based materials in dentistry. The aim of this study was to determine the corrosion behavior of ß titanium alloy Ti-39Nb in physiological saline solution and in physiological solution containing fluoride ions. Corrosion behavior was studied using standard electrochemical techniques and X-ray photoelectron spectroscopy. It was found that corrosion properties of the studied alloy were comparable with the properties of titanium grade 2. The passive layer was based on the oxides of titanium and niobium in several oxidation states. Alloying with niobium, which was the important part of the alloy passive layer, resulted in no significant changes of corrosion behavior. In the presence of fluoride ions, the corrosion resistance was higher than the resistance of titanium.

Properties of titanium-alloyed DLC layers for medical applications.

DLC-type layers offer a good potential for application in medicine, due to their excellent tribological properties, chemical resistance, and bio-inert character. The presented study has verified the possibility of alloying DLC layers with titanium, with coatings containing three levels of titanium concentration prepared. Titanium was present on the surface mainly in the form of oxides. Its increasing concentration led to increased presence of titanium carbide as well. The behavior of the studied systems was stable during exposure in a physiological saline solution. Electrochemical impedance spectra practically did not change with time. Alloying, however, changed the electrochemical behavior of coated systems in a significant way: from inert surface mediating only exchange reactions of the environment in the case of unalloyed DLC layers to a response corresponding rather to a passive surface in the case of alloyed specimens. The effect of DLC layers alloying with titanium was tested by the interaction with a simulated body fluid, during which precipitation of a compound containing calcium and phosphorus--basic components of the bone apatite--occurred on all doped specimens, in contrast to pure DLC. The results of the specimens' surface colonization with cells test proved the positive effect of titanium in the case of specimens with a medium and highest content of this element.

Influence of porosity on corrosion behaviour of Ti-39Nb alloy for dental applications.

Porous materials allow for easier osseointegration of implants and their firmer connection with the bone. The presence of pores in a material may become a source of both mechanical and corrosion problems. The presented study explored a Ti-39Nb alloy with a porosity of 0-33%. Specimens were exposed in the physiological solution of two pH values. In view of this material's possible use in dental applications, the effect of fluoride ions on its corrosion behaviour was studied. The open circuit potential and polarization resistance were measured. Data concerning susceptibility to crevice corrosion were obtained from potentiostatic measurements based on the ASTM F746 standard. In terms of corrosion behaviour, specimens with a lower porosity were not much different from the non-porous material. Porosity produced its effect at the level of 24 and 33%. It is obvious that porosity affects corrosion behaviour of this type of material. This conclusion was confirmed by measurements of susceptibility to crevice corrosion which grew with the specimens' increasing porosity. Corrosion resistance of the Ti-39Nb alloy was comparable with that of the compact material, but the presence of pores initiated a local attack of the material.

Monitoring of selenium in oral cavity argyria - a clinical and microscopic study.

OBJECTIVE: Argyria is generally classified as localized or generalized condition. Distinct pigmentation of the oral mucosa in the vicinity of amalgam fillings is often referred to as amalgam tattoos. Pigmented areas can also be associated with silver-containing corrosion products of dental alloys used for prosthetic restorations. Silver-containing electron dense particles (Ag-EDPs) are frequently found in pigmented areas. We attempted to correlate results of the elemental composition of Ag-EDPs with excerpts from health profiles of our study paticipants. DESIGN/SETTING: Eight patients with diagnosed signs of localized argyria were investigated in this study. Biopsies from distinctly pigmented gingival areas were subjected to histological examination, electron microscopy and x-ray microanalysis. RESULTS: Elemental composition of Ag-EDPs determined by x-ray microanalysis showed mainly silver in combination with sulfur or selenium or a combination of both chalcogens. Elemental analyzes results of Ag-EDPs were analyzed along with excerpts from the patient's clinical records. Two patients with low or undetectable selenium in the Ag-EDPs suffered from autoimmune thyroiditis, Parkinson's disease, bronchial asthma, and allergies to molds, pollen and dust. CONCLUSIONS: We suggest that selenium in Ag-EDPs is a product of the detoxification process for Ag(+) ions in gingival tissue and that it may reflect the availability of endogenous selenium for physiological processes in the human body. Its presence or absence might thus be used as another marker of a patient's health status.

Corrosion behaviour of TiN and ZrN in the environment containing fluoride ions.

Nowadays, a wide range of materials for human implants is used. To reach the required properties of implants, coatings are applied in some cases. This contribution is focused on the corrosion properties of TiN and ZrN layers on cp-titanium (commercially pure titanium) under environment modelling conditions in an oral cavity. Measurements were done in artificial saliva and a physiological solution unbuffered and buffered to a pH value of 4.2 with the addition of fluoride ions up to 4000 ppm. Standard corrosion electrochemical techniques were applied. Both types of layers were stable in both model saliva and physiological solution with non-adjusted pH. The decrease in pH to 4.2 resulted in a minor decrease of corrosion resistance in all cases, but polarization resistance was still in the order of 10(5) Ω cm². An important change in a specimens' behaviour was noticed in the presence of fluoride ions. TiN was stable in the highest concentration of fluorides used. The ZrN layers were destabilized in an environment containing a few hundred ppm of fluoride ions. As for TiN, the decisive factor is the influence of porosity; the corrosion resistance of ZrN is limited. From the corrosion point of view, the application of the TiN-based barrier layers in dental implantology is more advisable than the use of ZrN, provided that the application of a barrier is inevitable.

Corrosion behaviour of titanium after short-term exposure to an acidic environment containing fluoride ions.

The negative effect of fluoride ions on titanium has been known in dentistry for a long time. The presented work was aimed at the interaction between titanium and model saliva following a short-term exposure of a specimen to a model medical preparation rich in fluoride ions. The experimental work was carried out using titanium grade 2 in a physiological solution (pH non-adjusted, 5.8, 4.2; 5000 ppm F-) and in model saliva. Electrochemical measurement techniques were supplemented with XPS analysis. The presence of fluoride ions resulted in partial degradation of the passive layer even in a slightly acidic environment. The decrease of pH to the value of 4.2 and the presence of 5000 ppm F- caused titanium activation followed by a slow repassivation in model saliva. Formation of low soluble compound rich in fluorine explains experimental data. Short medical treatment can result in relatively long period of increased titanium corrosion.

The mechanism of gingiva metallic pigmentations formation.

The occurrence of blue-grey areas in the soft tissue represents one of the problems affecting patients whose teeth have been restored using metallic materials. It is generally accepted that it is caused by mechanical penetration of a metallic material into the soft tissue. Several facts indicate that this mechanism is not general. The aim of the study was to determine an alternative mechanism of the origin of pigmentations, based on the corrosion interaction of metallic materials used in prosthodontics with the oral environment. The study was comprised of an analysis of pigment particles, determination of exposure conditions of metallic materials in vivo and laboratory evaluation of corrosion properties of the studied materials. Particles containing silver, sulphur and/or selenium could be seen in ultra-thin sections in the lamina propria gingivae. Comparison of the corrosion laboratory results with the results of in vivo measurements indicated the intensification of corrosion under these conditions. Amalgams and silver-containing alloys used for teeth restorations may release silver under the conditions of the oral cavity. The formation of soluble silver compounds in the sulcular area or in a crevice between the crown and the cast post-and-core reconstruction facilitates their transport to the soft tissue and subsequent deposition.

Corrosion behavior of palladium-silver-copper alloys in model saliva.

OBJECTIVES: Palladium-silver system alloyed with other metals represents one of possible material choices in prosthetics. Its corrosion properties are influenced by minority components added in order to obtain the properties required for stomatological purposes. The objective of this work was to ascertain the influence of copper on the corrosion mechanism of palladium-silver alloys. METHODS: Corrosion properties of four palladium-silver-copper alloys were compared with the behavior of the palladium-silver binary system. Standard electrochemical measurements in a model saliva solution were complemented with an XPS analysis of the specimens surface. Experimental data were compared with the results of thermodynamic analysis. RESULTS: The foregoing study revealed formation of a saline layer of insoluble silver compounds as the dominant feature of the corrosion mechanism in a binary system. This process is suppressed in ternary alloys where electrochemical reactions of copper take place on the alloy-electrolyte phase boundary leading to the formation of a layer based on copper oxides. SIGNIFICANCE: The alloying of the palladium-silver binary system with copper results in an important change in the corrosion behavior of ternary alloys. A change in the mechanism of interaction with the environment leads to susceptibility to non-uniform corrosion.

Metallic pigmentation of human teeth and gingiva: morphological and immunological aspects.

The composition of metallic pigmentations in gingiva and dental roots was determined by means of transmission electron microscopy with energy dispersive x-ray microanalysis. The systemic immune response to the metals found in the oral cavity was evaluated in 10 patients by using a modified lymphocyte proliferation test. Immunological results were compared with a group of five controls without metallic materials and pigmentation. Dense particles of various shapes and sizes, as well as of diverse extracellular and intracellular localization patterns, were detected in the pigmented lamina propria gingivae. Metallic deposits consisted predominantly of silver accompanied by selenium or sulfur or both. Besides, Ag, Au, Cr, Ni, Fe, Hg, Cu, and Ti were identified in dentinal tubules of teeth reconstructed with dental alloys. Nine patients with metallic pigmentations had a positive lymphocyte proliferative response to one or more metals present in their own metal reconstructions. Results of this study thus indicated that dental alloys-by virtue of their corrosion process-might pose a significant risk to immunologically susceptible patients.

In vivo effects of dental casting alloys.

OBJECTIVE: Corrosion products of different metallic alloys used in prosthetic dentistry often cause the development of a bluish-grey pigmentation of gingiva and oral mucosa. The aim of this study was to determine the content of metals in metallic pigmentations and evaluate the immune response to metals found in the oral cavity. MATERIAL AND METHODS: The local tissue reactions were investigated clinically by electron microscopy and by energy dispersive x-ray microanalysis. An extensive anamnesis of the patients was recorded as well as earlier contacts with health care institutions. The immunological response to metallic components of dental alloys was evaluated in 34 patients by MELISA, a modified test for lymphocyte proliferation. In addition, cytokines in culture media were determined in 10 persons by the Human Inflammation Antibody Array. RESULTS: Dense particles containing metals were found in the matrix among collagen fibrils and in close proximity of the lamina basalis of the gingival epithelium. Particles were also localized intracellularly in fibroblasts, macrophages, and endothelial cells. Metallic depositions consisted predominantly of silver accompanied by selenium and sulphur. Twenty five out of 34 patients revealed high lymphocyte reactivity (positive MELISA test) to one or more metal components of dental restorations. A correlation between the positivity in MELISA test and number of dental alloys in the oral cavity was also found. Twenty MELISA positive patients suffered from serious health problems (various allergies, autoimmune diseases, Parkinson's syndrome etc.). Nickel and inorganic mercury were the most common sensitizers in vitro. The cytokine assay revealed that mercury chloride activated predominantly TH2 lymphocytes, while nickel chloride activated mainly TH1 lymphocytes. CONCLUSIONS: Metallic pigmentations in the oral cavity demonstrate a corrosion process and may pose a risk in immunologically susceptible patients.

Passivation of dental amalgams and mercury release.

OBJECTIVES: In this study the rate of dissolution of mercury from two dental amalgams with different compositions and structures was determined in vitro under different oxidation and abrasion conditions, and the results were correlated with the electrochemical characteristics. METHODS: A spherical high copper and a lathe-cut very high-copper amalgam were tested in aerated and deaerated artificial saliva. The electrochemical characteristics were determined by potential-time, anodic polarization, polarization resistance and cathodic stripping measurements. Mercury release tests were performed after either stabilization in the solution, or abrasion using SiC papers or rotary toothbrush, with or without toothpaste. Dissolved mercury was determined by atomic absorption spectrophotometry. RESULTS: Both amalgams exhibited passivation, the amalgam with the higher copper content passivating spontaneously even when the oxygen content in the solution was minimized. At a higher oxygen content in the solution the rate of mercury release from the amalgams was lower than when the oxygen content was minimized, and decreased further after a pre-exposure. Brushing generally increased the release. SIGNIFICANCE: The results show the importance of the oxidation conditions and passivation characteristics of dental amalgams for mercury release, especially in the transient state after abrasion by chewing or tooth brushing.