Stress protein assay for the evaluation of cytotoxicity of dental amalgam

To evaluate the cytotoxicity of mercury in dental amalgams, a stress protein assay was performed and the results were compared with the cytotoxicity evaluated by a neutral red uptake assay. The induction of a major stress protein, hsp70, was analyzed at levels of mRNA, synthesis and accumulation in human HeLa cells treated with extracts from amalgam, metal mercury and mercuric chloride. Mercuric chloride induced an increase in the synthesis of hsp70 at concentrations of mercury half those used for the neutral red uptake assay. The extracts from dental amalgam and metal mercury induced an increase in hsp70 mRNA at concentrations of mercury half those causing the inhibition of neutral red uptake into cells. Furthermore, the extracts from dental amalgam or metal mercury increased the synthesis of hsp70 and inhibited the uptake of dye at concentrations of mercury 1/10-1/50 lower than those at which mercuric chloride acted. These results suggest that the stress protein assay is more sensitive than the conventional neutral red assay for the evaluation of the cytotoxicity of mercury in dental amalgams and that the methods used in the preparation of metal solutions seem to be critical to the evaluation of cytotoxicity of dental materials.     

Influence of thermal treatments on Ag Sn Cu powders in order to reduce mercury contents in dental amalgam

The mercury content of dental amalgams is a controversial subject with regard to the biological properties of these materials. The object of this study is to optimize the thermal treatments performed on an experimental powder in order to obtain a low mercury ratio (41% by weight) while preserving the desirable clinical qualities of the material. Using atomized powder, two types of thermal treatments are performed: A1, to obtain a partially annealed structure and A2, to obtain a complete homogenization. The kinetics of the amalgamation reaction is mainly evaluated by X-ray diffraction to identify the newly formed phases as a function of setting time. Mechanical properties are evaluated according to the ISO norms at 37 °C. Metallographical examination of the amalgams shows a «Ag-Hg» phase which acts as a matrix incorporating the «Cu-Sn» and «Ag-Sn» compounds. The setting kinetics of the A1 amalgams is linear and slightly more rapid than that of the A2 amalgam. The mechanical properties of the amalgams are significantly improved regarding the higher mercury content commercial amalgams. A specific thermal treatment permits us to slow down the diffusion of mercury between the different intermetallic compounds into the powder. The final amalgam composition, thus, most approaches the stoechiometric ratio calculated from a quaternary diagram.     

Effect of setting time and artificial saliva on the strength evaluated by different methods of dental silver amalgam: A comparative study

The aim of this study is to evaluate the effect of setting time and artificial saliva on the strength of dental amalgam by diametral compressive strength, Hertzian failure loading testing and compressive strength test methods. Nine dental amalgam products are evaluated using three strength measurements, compressive, diametral, and Hertzian loading testing at early (1 h or 2 h), 24 h and 30 days after setting at 37 °C. The trituted amalgams are used to prepare Hertzian, tapered disc by manual condensation according to the ISO standard procedure (ISO 17988: 2014). The compressive and diametral testing specimens are obtained using the mechanical condensation by two apparatus with different specimen dimensions of height and diameter, 8.0 mm×4.0 mm and 2.0 mm×8.0 mm, respectively. The apparatus and schedule specification for preparing compressive testing specimens are according to ISO 24234: 2015 while dimensions of some parts of diametral testing apparatus were modified and the schedule was optimized to provide sound disc test‐pieces by a one‐shot procedure. The diametral and Hertzian testing specimens are also evaluated after 30 days under dry storage conditions and after aging in the artificial saliva. The artificial saliva was prepared according to ISO17988: 2014 standard specifications. The single diametral or “triple‐cleft” fracture path, and radial cracking are observed in all products for diametral and Hertzian testing methods, respectively. The discriminatory power in early strength tests is in order to Hertzian < diametral < compressive. A significant correlation between diametral and compressive strength value is observed at early setting time. It is found a correlation between diametral and Hertzian strength mean value and variance ratio in immersion test.     

Phenomenological study of elastic and anelastic properties of dental alloys

Measurements of Young's modulus, mechanical damping, stress and unit damping energy have been made as a function of temperature on five dental amalgams with the PUCOT (piezoelectric ultrasonic composite oscillator technique). In Dispersalloy amalgam the mechanical damping changed by a factor of 4.3 and Young's modulus was reduced by 0.5% as the temperature increased from 20 to 80° C. The effective activation energy for the change in damping was 0.42 eV. For the five amalgams the curves of unit damping energy against stress, damping against modulus, and (damping/modulus) against modulus had a variety of forms: one, two, three or four straight lines, with or without hysteresis effects. All the curves for Tytin amalgam showed a sharp change near 68° C. The data are examined phenomenologically to show that they lend themselves well to the detection and monitoring of transformations taking place in dental alloys.     

Fracture toughness and critical strain energy release rate of dental amalgam

Fracture toughness, critical strain energy release rate and critical stress intensity factor were determined for lathe-cut, spherical, admixed, and two atomized high-copper dental amalgams. At a loading rate of 0.005cm min^–1 for 24-hour samples, the spherical amalgam had the highest resistance to unstable crack propagation. At a loading rate of 0.05cm min^–1 for both 24-hour and one-month samples, the lathe-cut amalgam had the highest resistance to unstable crack propagation. One of the atomized high copper amalgams showed the lowest resistance to crack propagation. The values were consistent with data obtained in single-pass wear studies.     

The matrix phase of a ductile dental amalgam

In this study, the ternary HgAgSn-₁ matrix of a ductile dental amalgam has been characterized and compared with that of a brittle amalgam. Specimens of both amalgams were prepared form commercial alloy powders according to the ADA specification 1, stored at 37°C and studied by differential scanning calorimetry technique at timed intervals up to 1 year. Experimental amalgams of AgCu eutectic admixed with varying concentrations of Sn were used as standards. The matrix of the ductile amalgam was found to contain two substructures initially. One was saturated with Sn and the other contained lesser amounts of Sn. These substructures disappeared with time leading to a matrix in which Sn was well below its saturation limit but uniformly distributed. The matrix of the brittle amalgam, on the other hand, was saturated with Sn during the entire period of this study.     

Corrosion and bone response of magnesium implants after surface modification by heat-self-assembled monolayer

The aim of the present study is to find the correlation between the Mg²⁺ concentration degraded from pure magnesium material and the biocompatibility of the material. Hemolysis ratio (HR) of the extracts of pure magnesium with different Mg²⁺ concentration were measured according to ISO 10993.4 standard. The cytotoxicity tests were carried out by both indirect contact with fibroblast L929 and preosteoblasts MC3T3-E1, and MTT tests were used. Cytotoxicity of the pure magnesium with and without surface modification was further evaluated by direct contact method. Samples were cultured with Osteoblast MC3T3-E1 and the effects of the material on viability and activity of cells were discussed. The results showed that the hemolysis rate and cytotoxicity of the modified Mg could meet the requirement for biomaterials. In our test, the hemolysis rate of the extracts was qualified when the concentration of Mg²⁺ ⩽ 42 mg/L; the extracts with 202 mg/L Mg²⁺ met the cytotoxicity requirement, and the extracts with 156 mg/L Mg²⁺ promoted cell proliferation. Therefore, the biocompatibility of magnesium-based materials can be improved by suitable surface modification.     

Structure and strength of low-mercury dental amalgams prepared with liquid Hg–47.4% In alloy

Two low-mercury amalgams: (1) low-copper lathe-cut and (2) high-copper (Tytin) were prepared by amalgamation with liquid Hg–47.4% In alloy. The strength–structure relationship of these amalgams was investigated and compared with standard amalgams (i.e. amalgams prepared with the same powders and pure mercury). The matrix phase of the low-mercury amalgam was found to be depleted of mercury and may be thought of as In4Ag9 compound with some mercury dissolved, indicating that less mercury (compared with standard amalgam) combines with silver, thus producing a strong amalgam matrix. On the other hand, an increase was observed in the consumption of the initial (Ag3Sn)-phase, leading to an increase of the tin released. As a result, the potential of [HgSn]-phase formation in low-mercury amalgams increases. The observed increase in the quantity of 2(Sn7–8Hg)-phase in low-copper amalgam, or its appearance in high-copper amalgam (where it is normally absent), contributes to a deterioration in the strength of the investigated amalgam. The conclusion drawn was that low-mercury amalgam may be prepared with liquid Hg–47.4%In alloy but, in order to eliminate 2-phase formation, novel and possibly tin-free amalgamable alloys should be developed. © 1998 Chapman & Hall     

Effect of Mg<Superscript>2+</Superscript> concentration on biocompatibility of pure magnesium

The aim of the present study is to find the correlation between the Mg²⁺ concentration degraded from pure magnesium material and the biocompatibility of the material. Hemolysis ratio (HR) of the extracts of pure magnesium with different Mg²⁺ concentration were measured according to ISO 10993.4 standard. The cytotoxicity tests were carried out by both indirect contact with fibroblast L929 and preosteoblasts MC3T3-E1, and MTT tests were used. Cytotoxicity of the pure magnesium with and without surface modification was further evaluated by direct contact method. Samples were cultured with Osteoblast MC3T3-E1 and the effects of the material on viability and activity of cells were discussed. The results showed that the hemolysis rate and cytotoxicity of the modified Mg could meet the requirement for biomaterials. In our test, the hemolysis rate of the extracts was qualified when the concentration of Mg²⁺ ? 42 mg/L; the extracts with 202 mg/L Mg²⁺ met the cytotoxicity requirement, and the extracts with 156 mg/L Mg²⁺ promoted cell proliferation. Therefore, the biocompatibility of magnesium-based materials can be improved by suitable surface modification.     

Chemotaxis assays of mouse sperm on microfluidic devices

Sperm chemotaxis is an area of significant interest to scientists involved in reproductive science. Understanding how and when sperm cells are attracted to the egg could have profound effects on reproduction and contraception. In an effort to systematically study this problem, we have fabricated and evaluated a microfluidic device to measure sperm chemotaxis. The device was designed with a flow-through configuration using a spatially and temporally stable chemical gradient. Mouse sperm cells were introduced into the chemotaxis chamber between confluent flows of mouse ovary extract and buffer. The sperm experiencing chemotaxis swam toward the extract and were counted relative to those that swam toward the buffer. The ovary extracts were diluted from 10(2) to 10(7) times, and each extract dilution was screened for chemotaxis. Four out of six ovaries showed a strong chemotactic response at extract dilutions of 10(-3) to 10(-5). This device provided a convenient, disposable platform on which to conduct chemotaxis assays, and the flow-through design overcomes difficulties associated with distinguishing chemotaxis from trapping.     

Testing the cytotoxicity of metal alloys used as magnetic prosthetic devices

Technical magnetic materials are increasingly used for the development of magnetic retained dental prosthetic and orofacial epithetic devices. Since most of the magnets based on rare earth metals, such as samarium–cobalt based alloys have a high tendency for corrosion they were first coated by tin and then encapsulated by titanium. However, the high mechanical load particularly on dental devices may cause a rupture of the titanium capsule and the alloys contact directly biological fluids. Hence, it is important to know the cytotoxicity of these magnets to assess their potential effects on the surrounding tissue. In this study, the cytotoxicity of neodymium–iron–boron and samarium–cobalt (plain, tin and titanium coated) magnets was tested. First, magnets were incubated up to 7 days in culture medium to prepare extracts for cytotoxicity measurements. Changes in the surface morphology due to corrosion were visualized by scanning electron microscopy and analysis of the elemental composition. 3T3 mouse fibroblasts were cultured in the presence of extracts and their viability measured by neutral red and metabolic assays. To learn more about a possible toxic activity of the main components of magnets, salt solutions of different concentrations resembling those elements, which are main constituents of the magnets, were used. 3T3 fibroblasts were also cultured in direct contact with the materials and material induced effects on cell morphology and growth monitored by microscopy. As a result of this study it was found that samarium–cobalt magnets have a strong tendency for corrosion and exert a considerable cytotoxicity. Neodymium–iron–boron magnets have a lesser tendency for corrosion and are only moderate cytotoxic. Coating of samarium–cobalt magnets with tin or titanium makes the materials non-toxic. Application of salt solutions shows that cobalt has a tendency to be cytotoxic at higher concentrations, but enhances cell metabolism and proliferation at lower concentrations while the other magnet constituents had a lower or negligible cytotoxic potential.     

Effect of heat treatment on structure and properties of dispersed-type dental amalgam

Effect of heat treatment of Ag–Cu–Pd dispersant particles on the structure, mechanical properties and mercury vapor release rate of an Ag–Cu–Sn/Ag–Cu–Pd-based dental amalgam has been investigated. Experimental results indicate that crystallinity of dispersant Ag-Cu–Pd alloy increases with increasing HTT, with most notable increase occurring between 100 and 200 °C. Increasing HTT of Ag–Cu–Pd alloy does not change much of the mercury/alloy ratio for amalgamation, but largely reduces working/setting time of the amalgam. The Ag–Cu–Pd particles in 7 d-aged amalgam are comprised primarily of an outer Sn/Cu/Pd-rich zone and an inner Ag/Cu/Pd-rich zone with eutectic-type morphology and chemical distribution. The annealing-enhanced Pd segregation effect is most significantly observed in the amalgam derived from 300 °C-annealed Ag–Cu–Pd dispersant. This amalgam also has the highest compressive strength, highest DTS, and lowest creep rate. Higher annealing temperature causes mechanical property of the amalgam to deteriorate. The initial mercury vapor release rates of amalgams derived from 100, 200 and 300 °C-annealed Ag–Cu–Pd dispersant are significantly lower than that derived from 400 °C-annealed dispersant.     

Classification of simple amalgams

Taking into account the type of phase diagram of metal-mercury, solubility, heat of dissolution process, activity and diffusion coefficient of a metal in mercury as well as the kinetics of electroreduction of metallic aquo-cation on a mercury electrode with an amalgam formation, a general classification of simple amalgams into four groups is proposed. On this basis some experimentally unknown amalgam properties may be predicted which have significant meaning in technical and chemical applications.     

Functionalized bridged silsesquioxane-based nanostructured microspheres: ultrasound-assisted synthesis and in vitro cytotoxicity characterization

Different kinds of polymers have been employed in medicine as biomaterials for different purposes. In recent years, considerable attention has been focused on the development of new drug-delivery systems in order to increase bio-availability, sustain, localize and target drug action in the human body. The versatility of the sol–gel processing to synthesize nanostructured materials and the possibility of incorporating organic molecules into the matrix of the final hybrid material, represent a novel and attractive path to the synthesis of new functionalized hybrid biomaterials with advanced properties. In this work, acetylsalicylic acid (ASA)-functionalized hybrid microspheres based on bridged silsesquioxanes synthesized via ultrasound-assisted sol–gel processing, were characterized. An investigation concerning the cytotoxic response of these new microspheres on CHO-K1 cells was accomplished based on ISO 10993-5 standard (Biological Evaluation of Medical Devices). Microspheres incorporating ASA showed a cytotoxic effect when pure extracts of the microspheres were analyzed, however, they strongly diminished their cytotoxicity as the extracts were diluted. When a 10% concentration extract was employed, hybrid microspheres were shown to be non cytotoxic. These results are promising for considering these novel functionalized organic–inorganic microspheres as potential drug-carriers to be employed in drug delivery-related applications.     

Ni–Cr based dental alloys; Ni release,corrosion and biological evaluation

In the last years the dental alloy market has undergone dramatic changes for reasons of economy and biocompatibility. Nickel based alloys have become widely used substitute for the much more expensive precious metal alloys.In Europe the prevalence of nickel allergy is 10–15% for female adults and 1–3% for male adults. Despite the restrictions imposed by the EU for the protection of the general population in contact dermatitis, the use of Ni–Cr dental alloys is on the increase. Some questions have to be faced regarding the safety risk of nickel contained in dental alloys.We have collected based on many EU markets, 8 Ni–Cr dental alloys. Microstructure characterization, corrosion resistance (generalized, crevice and pitting) in saliva and the quantities of cations released in particular nickel and CrVI have been evaluated. We have applied non parametric classification tests (Kendall rank correlation) for all chemical results. Also cytotoxicity tests and an evaluation specific to TNF-alpha have been conducted.According to the obtained results, it was found that their behavior to corrosion was weak but that nickel release was high. The quantities of nickel released are higher than the limits imposed in the EU concerning contact with the skin or piercing.Surprisingly the biological tests did not show any cytotoxic effect on Hela and L929 cells or any change in TNF-alpha expression in monocytic cells. The alloys did not show any proinflammatory response in endothelial cells as demonstrated by the absence of ICAM-1 induction.We note therefore that there is really no direct relationship between the in vitro biological evaluation tests and the physico-chemical characterization of these dental alloys. Clinical and epidemiological studies are required to clarify these aspects.     

Methylcellulose cell culture as a new cytotoxicity test system for biomaterials

The cytotoxicity of biomaterials can be testedin vitro using various culture systems. Liquid culture systems may detect cytotoxicity of a material either by culture of cells with extracts or with the material itself. In the latter instance, renewing the medium will remove possible released cytotoxic products. The agar-overlay test is a short term semi-solid culture system in which the possible cytotoxicity of biomaterials is identified only by the presence of cell free zones. The aim of this study was to develop a more sensitive cytotoxicity test system for biomaterials, using methylcellulose as a culture gel, mixed with human fibroblasts. The main advantage of the test system is the possibility of evaluating cytotoxicity for a period of up to seven days without renewal of the culture gel. Furthermore it is possible to both quantitatively evaluate by counting absolute cell numbers and to qualitatively evaluate by studying cell morphology with light- and/or electron microscopy. Processed dermal sheep collagen was selected as test material, since contradictory results concerning the cytotoxicity of its extracts have been reported by others [2, 15, 18, 19]. Using our test system, both primary and secondary cytotoxic effects were found. Primary cytotoxicity is due to direct leakage of products from the material, detected by testing, extracts of the collagen or the collagen itself. Secondary cytotoxicity is due to release of cytotoxic products resulting from cell-biomaterial interactions. We conclude that our test system is extremely useful to test materials which are suspected of primary and/or secondary cytotoxicity, either with slow release of cytotoxic products or release of products with late cytotoxic effects.     

Cytotoxicity of some catalysts commonly used in the synthesis of copolymers for biomedical use

The cytotoxicity of four catalysts commonly used for the synthesis of copolymers for biomedical use, such as segmented polyurethanes, was evaluated towards two types of cells, the first being the well-characterized cell line Swiss 3T3 mouse fibroblasts, the second the actual living system that faces any device in contact with blood, i.e. human endothelial cells (HEC). The catalysts were two tertiary aliphatic amines: TMBDA (tetramethylbutanediamine), and DABCO (1–4 diazo (2,2,2) octane); two alkyl tin compounds: DBTDL (dibutyl-tin-dilaurate), and SnOct (stannous octoate). Cytotoxicity tests were carried out by adding to the culture medium, after cell adhesion, different concentrations of each catalyst in dimethylsulphoxide, and keeping them in contact with the monolayer for 72 h. All the catalysts proved to be cytotoxic, although at different extent (in the order: DABCO < TMBDA < SnOct < DBTDL); their dose inhibiting 50% of cell growth (IC50) came out to be lower for 3T3 fibroblasts than for HEC, with the exception of DBTDL, which showed a similar toxicity for both the cell lines. As an example, the cytotoxicity of a polyurethane-amide, laboratory synthesized with DBTDL as catalysts, was checked with fibroblasts. By using both the method of the extract, and that of the direct contact (through a microporous membrane), a moderate to severe cell growth inhibition, related to the Sn content in the material, was observed.     

From cytotoxicity to biocompatibility testing in vitro: cell adhesion molecule expression defines a new set of parameters

Determination of potential cytotoxicity is a central issue in current biocompatibility testing standards such as ISO and ASTM. Most of these tests do not assess biocompatibility of a biomaterial with regard to cell function. This study was aimed at screening a number of potential parameters that could be included in assessment of cell functional aspects of biocompatibility. Human umbilical vein endothelial cells (HUVEC) were seeded directly on titanium, NiCr alloy, CoCr alloy, PMMA, PE, PU, PVC, and silicone, or were exposed to the material extracts. Cytotoxicity was assessed for these materials through MTT conversion, crystal violet protein determination and Ki67 expression. In addition, expression of the cell adhesion molecules E-selectin, cadherin-5 and PECAM, as well as of the adhesion-associated proteins fibronectin and vinculin (focal adhesions), was determined by immunocytochemistry and western blotting. Cytotoxicity was not detected with the material extracts. Cells were able to adhere to bare metals, but not polymers. Fibronectin preadsorption resulted in adhesion and spreading also on the polymers. Cells were able to establish cell–cell contacts and focal adhesions. Western blotting, in combination with differential detergent extraction, indicated that linkage of cell–cell adhesion markers to the cytoskeleton may be used as an additional parameter relevant to cell function.     

Effect of starch-based biomaterials on the <Emphasis Type="Italic">in vitro</Emphasis> proliferation and viability of osteoblast-like cells

The cytotoxicity of starch-based polymers was investigated using different methodologies. Poly-L-lactic acid (PLLA) was used as a control for comparison purposes. Extracts of four different starch-based blends (corn starch and ethylene vinyl alcohol (SEVA-C), corn starch and cellulose acetate (SCA), corn starch and polycaprolactone (SPCL) and starch and poly-lactic acid (SPLA70) were prepared in culture medium and their toxicity was analysed. Osteoblast-like cells (SaOs-2) were incubated with the extracts and cell viability was assessed using the MTT test and a lactate dehydrogenase (LDH) assay. In addition DNA and total protein were quantified in order to evaluate cell proliferation. Cells were also cultured in direct contact with the polymers for 3 and 7 days and observed in light and scanning electron microscopy (SEM). LDH and DNA quantification revealed to be the most sensitive tests to assess respectively cell viability and cell proliferation after incubation with starch-based materials and PLLA. SCA was the starch blend with higher cytotoxicity index although similar to PLLA polymer. Cell adhesion tests confirmed the worst performance of the blend of starch with cellulose acetate but also showed that SPCL does not perform as well as it could be expected. All the other materials were shown to present a comparable behaviour in terms of cell adhesion showing slight differences in morphology that seem to disappear for longer culture times.The results of this study suggest that not only the extract of the materials but also their three-dimensional form has to be biologically tested in order to analyse material-associated parameters that are not possible to consider within the degradation extract. In this study, the majority of the starch-based biomaterials presented very promising results in terms of cytotoxicity, comparable to the currently used biodegradable PLLA which might lead the biocompatibility evaluation of those novel biomaterials to other studies.     

Intergranular structure in dental amalgams

The existence of an intergranular precipitate that provides a structural basis of creep, intergranular corrosion and thermal instability in dental amalgams has been in dispute. Also in dispute is a related matter concerning the solid solubility of Sn in ₁. Both these aspects have been examined in this differential scanning calorimetry (DSC) study using the following materials: (a) a conventional ₂–containing amalgam, (b) six ₂–free amalgams, and (c) a series of ₁ specimens with up to 4 wt% Sn. It has been observed that the solid solubility of Sn in ₁ is about 1.0 wt%. Above this Sn concentration, an intergranular precipitate, identified as the ₂ phase of the SnHg system, appears in ₁. This phase is characterized by a low melting temperature (90°C). The existence of this phase in the conventional dental amalgam has been confirmed. It has been observed further that the ₂–free dental amalgams, depending on their nature and Hg contents, may contain ₂ either as a transient or a permanent phase.