Paul Johan Høl

Induction of cell death by TiO2 nanoparticles : Studies on a human monoblastoid cell line

The cellular responses to degradation products from titanium (Ti) implants are important indicators for the biocompatibility of these widely used implantable medical devices. The potential toxicity of nanoparticulate matter released from implants has been scarcely studied. The aim of this study was to investigate the potential of TiO2 nanoparticles to induce modifications characteristic for death by apoptosis and/or necrosis in U937 human monoblastoid cells. Suspensions of TiO2 nanoparticles with a diameter <100nm were prepared in RPMI cell culture medium at concentrations that covered a range (0.005-4mg/ml) corresponding to concentrations found in blood, plasma, or in tissues surrounding Ti implants. The cells were exposed to the nanoparticulate suspensions for 24 and 48h and the responses were evaluated by flow cytometry and transmission electron microscopy. TiO2 nanoparticles induced both apoptotic and necrotic modifications in U937 cells.

Role of physicochemical characteristics in the uptake of TiO2 nanoparticles by fibroblasts.

The relation between the physico-chemical properties of nanoparticles (NPs) and the degree of cellular uptake is incompletely elucidated. In this study, we investigated the influence on the cellular uptake of a wide range of fully characterized TiO2 NPs. L929 fibroblasts were exposed for 24 h to clinically relevant concentrations of nano-TiO2 and the degree of their association was assessed by ultrahigh resolution imaging microscopy (URI), scanning (SEM) and transmission (TEM) electron microscopy, as well as inductivity coupled plasma-mass spectroscopy (ICP-MS). The role of actin polymerization, a central feature of active internalization, was also studied and the results indicated that the internalization of TiO2 NPs involves a combination of actin-dependent uptake of large agglomerates as well as non actin-dependent uptake of small agglomerates. SEM and TEM revealed that the agglomerates of all NPs types were attached to the cellular membrane as well as internalized and confined inside cytoplasmic vesicles. URI and ICP-MS demonstrated that the particle association with cells was dose-dependent. The highest association was observed for spherical particles having mixed anatase-rutile crystallographic phase and the lowest for spindle-shaped rutile particles. ICP-MS revealed that the association was size-dependent in the order 5>10>40 nm for anatase spherical nanoparticles.

Formation of potential titanium antigens based on protein binding to titanium dioxide nanoparticles

Degradation products of titanium implants include free ions, organo-metallic complexes, and particles, ranging from nano to macro sizes. The biological effects, especially of nanoparticles, is yet unknown. The main objective of this study was to develop Ti-protein antigens in physiological solutions that can be used in testing of cellular responses. For this purpose, 0.1% TiO2 nanoparticles less than 100 nm were mixed with human serum albumin (HSA), 0.1% and 1%, in cell culture medium (DMEM, pH 7.2). The Ti concentrations in the resulting solutions were analyzed by inductively coupled plasma mass spectrometry. The stability of the nanoparticles in suspension was analyzed by UV-vis spectrophotometer and Dynamic Light Scattering. The concentration of Ti in suspension was dependent on the presence and concentration of HSA. Albumin prevented high aggregation rate of TiO2 nanoparticles in cell culture medium. It is shown that nano TiO2-protein stable aggregates can be produced under physiological conditions at high concentrations, and are candidates for use in cellular tests.

Mercury and silver in saliva from subjects with symptoms self-related to amalgam fillings.

Abstract The amount of mercury released into saliva from dental amalgam fillings is currently being debated. Mercury enters saliva as vapor, ions and particles of amalgam. The aim of the present study was to determine mercury and silver concentrations in saliva of persons with amalgam fillings. Moreover, it was the aim to investigate whether amalgam particles were present in samples of stimulated saliva in control subjects. In that case, we also wanted to determine the influence of these particles on the mercury concentrations found. Fifty-three patients with a wide range of complaints self-related to their amalgam fillings were examined by the Dental Biomaterials Adverse Reaction Unit of Norway. Among other tests, stimulated saliva was collected from each patient and analyzed for mercury and silver. Mercury and silver correlated with the amount of amalgam present. There was a strong correlation between mercury and silver concentrations. Amalgam particles were found by energy dispersive X-ray analysis. It appears that a considerable part of the mercury and silver were present as amalgam particles. The present study shows that amalgam particles in saliva have to be controlled for when analyzing mercury in saliva from subjects with amalgam fillings.

Mapping of titanium particles in peri-implant oral mucosa by laser ablation inductively coupled plasma mass spectrometry and high-resolution optical darkfield microscopy.

The present study examines the quantity, size, element signatures and distribution of titanium particles in normal oral mucosal tissue and in oral mucosa exposed to a titanium implant. Tissue samples from six healthy patients were obtained by a full thickness biopsy taken from the edge of the oral mucosa when inserting a titanium dental implant. At the abutment insertion 6 months later, a punch test biopsy of oral mucosa was taken over the implant site. Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) is a sensitive and specific multi-element microanalytical technique that demonstrated the presence of Ti particles in the tissue adjacent to implant cover-screws. The epithelial part of the control samples revealed more particles than the corresponding area of the test samples, consisting partly of newly formed scar tissue. High-Resolution Optical Darkfield Microscope (HR-ODM) confirmed the presence of particles in both the control and the test samples. The combination of LA-ICP-MS and HR-ODM appears to be a powerful combination for detection of particles in oral tissues; optical microscopy provides an overview with histological references, whereas LA-ICP-MS identifies the chemical composition.

Wear particles and ions from cemented and uncemented titanium-based hip prostheses—A histological and chemical analysis of retrieval material

Wear debris-induced inflammation is considered to be the main cause for periprosthetic osteolysis in total hip replacements (THR). The objective of this retrieval study was to examine the tissue reactions and exposure to metal ions and wear particles in periprosthetic tissues and blood samples from patients with titanium (Ti)-based hip prostheses that were revised due to wear, osteolysis, and/or aseptic loosening. Semiquantitative, histological tissue evaluations in 30 THR-patients revealed numerous wear debris-loaded macrophages, inflammatory cells, and necrosis in both groups. Particle load was highest in tissues adjacent to loosened cemented Ti stems that contained mainly submicron zirconium (Zr) dioxide particles. Particles containing pure Ti and Ti alloy elements were most abundant in tissues near retrieved uncemented cups. Polyethylene particles were also detected, but accounted only for a small portion of the total particle number. The blood concentrations of Ti and Zr were highly elevated in cases with high abrasive wear and osteolysis. Our findings indicate that wear particles of different chemical composition induced similar inflammatory responses, which suggests that particle size and load might be more important than the wear particle composition in periprosthetic inflammation and osteolysis.

Is there still a place for the cemented titanium femoral stem? 10,108 cases from the Norwegian Arthroplasty Register.

Background and purpose Despite the fact that there have been some reports on poor performance, titanium femoral stems intended for cemented fixation are still used at some centers in Europe. In this population-based registry study, we examined the results of the most frequently used cemented titanium stem in Norway. Patients and methods 11,876 cases implanted with the cemented Titan stem were identified for the period 1987–2008. Hybrid arthroplasties were excluded, leaving 10,108 cases for this study. Stem survival and the influence of age, sex, stem offset and size, and femoral head size were evaluated using Cox regression analyses. Questionnaires were sent to the hospitals to determine the surgical technique used. Results Male sex, high stem offset, and small stem size were found to be risk factors for stem revision, (adjusted RR = 2.5 (1.9–3.4), 3.3 (2.3–4.8), and 2.2 (1.4–3.5), respectively). Patients operated in the period 2001–2008 had an adjusted relative risk (RR) of 4.7 (95% CI: 3.0–7.4) for stem revision due to aseptic stem loosening compared to the period 1996–2000. Changes in broaching technique and cementing technique coincided with deterioration of the results in some hospitals. Interpretation The increased use of small stem sizes and high-offset stems could only explain the deterioration of results to a certain degree since the year 2000. The influence of discrete changes in surgical technique over time could not be fully evaluated in this registry study. We suggest that this cemented titanium stem should be abandoned. The results of similar implants should be carefully evaluated.

The effect of blood protein adsorption on cellular uptake of anatase TiO2 nanoparticles

Protein adsorption onto nanoparticles (NPs) in biological fluids has emerged as an important factor when testing biological responses to NPs, as this may influence both uptake and subsequent toxicity. The aim of the present study was to quantify the adsorption of proteins onto TiO2 NPs and to test the influence on cellular uptake. The surface composition of the particles was characterized by thermal analysis and by X-ray photoelectron spectroscopy. The adsorption of three blood proteins, ie, human serum albumin (HSA), γ-globulins (Glbs), and fibrinogen (Fib), onto three types of anatase NPs of different sizes was quantified for each protein. The concentration of the adsorbed protein was measured by ultraviolet-visible spectrophotometry using the Bradford method. The degree of cellular uptake was quantified by inductivity coupled plasma mass spectroscopy, and visualized by an ultra-high resolution imaging system. The proteins were adsorbed onto all of the anatase NPs. The quantity adsorbed increased with time and was higher for the smaller particles. Fib and Glbs showed the highest affinity to TiO2 NPs, while the lowest was seen for HSA. The adsorption of proteins affected the surface charge and the hydrodynamic diameter of the NPs in cell culture medium. The degree of particle uptake was highest in protein-free medium and in the presence HSA, followed by culture medium supplemented with Glbs, and lowest in the presence of Fib. The results indicate that the uptake of anatase NPs by fibroblasts is influenced by the identity of the adsorbed protein.

Blood mercury following DMPS administration to subjects with and without dental amalgam

The use of DMPS as a diagnostic tool in patients with symptoms allegedly caused by mercury from dental amalgam fillings is disputed. We have previously shown that the mercury concentrations in urine cannot be used in such a way. In the present study, we wished to evaluate the effect on blood mercury levels (B-Hg) following intravenously injected DMPS in four groups of subjects: 19 controls without amalgam experience; 21 healthy controls with amalgam fillings; 20 patients with self-reported symptoms from existing dental amalgams; and 20 patients who had removed amalgam fillings. A single dose of DMPS (2 mg/kg) was injected. Blood samples were collected prior to the injection and after 15, 30, 120 min, and after 24 h, and mercury was analyzed by cold vapor atomic absorption spectrophotometry. All groups showed an initial drop of 24 to 30% in the blood levels, approaching baseline values (2.5-5.5 microg/l) after 2 h. The subjects with no amalgam experience had the lowest mercury values. There was no significant difference between the three groups with such experience. There were no significant differences between the two groups with amalgam fillings present. Patients with symptoms allegedly caused by amalgam were not different from the control groups. There were indications that part of the urinary mercury excreted during the first 30 min originated from blood.

Dental amalgam affects urinary selenium excretion.

Selenium may have a protective effect against mercury toxicity. The aim of the present study was to investigate if selenium excretion in urine was affected in persons with dental amalgam fillings. The reason for this study is that dental amalgam is the most important source of inorganic mercury exposure in the general population, although the potential toxic effects of this exposure remain a subject for debate.The chelating agent 2,3 dimercaptopropane-1-sulfonate (DMPS) was injected intravenously (2 mg/kg) to provoke metal excretion. Urine samples were subsequently collected at intervals over a 24-h period. Selenium concentration was determined by hydride-generation atomic absorption spectrometry. The study was comprised of 20 persons who claimed symptoms from dental amalgam and 21 healthy persons with amalgam fillings. There were two control groups without amalgam. One control group had amalgam replaced because of concern about illness resulting from mercury release (n=20), whereas the other control group never had amalgam (n=19).Individuals with amalgam excreted less selenium (36.4 µg, median value) over 24 hours than those without amalgam (47.5 µg) (p=0.016). There was no difference in selenium excretion between groups with (42.4 µg) and without (39.4 µg) amalgam-related symptoms (p=0.15).The findings indicate that individuals exposed to low levels of elemental mercury from dental amalgam excrete less selenium to urine than unexposed individuals.