In-vitro study of the cellular viability and nitric oxide production by J774 macrophages with ceramic, polycarbonate, and polyoxymethylene brackets.

INTRODUCTION: Studies show that ceramic brackets are chemically inert in the oral cavity, whereas polycarbonate and polyoxymethylene brackets can degrade, releasing bisphenol-A and formaldehyde, respectively. In addition to the traditional cytotoxicity tests, the study of nitric oxide cellular production stimulated by a specific material has been shown to be a reliable tool for evaluating its cytotoxic potential. METHODS: We aimed to assess cellular viability by MTT (Sigma, St. Louis, Mo): 3,(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide assay in a murine macrophage cell line J774 with esthetic brackets and quantify nitric oxide production by these macrophages. Cell cultures were evaluated at 3 times: 24, 48, and 72 hours. RESULTS: Cellular viability in all groups was higher at 72 hours compared with 24 hours. This increase was significant in the control and ceramic brackets groups. Final means in the bracket groups showed no significant differences compared with the control group. Nitric oxide production was significantly greater in all groups at final time. There was no significant difference between the final means of the bracket groups and the control group, although polyoxymethylene brackets showed significantly greater means at 24 and 48 hours. CONCLUSIONS: Final means in the bracket groups showed no significant differences compared with the control group.

Cytotoxic effects of dental prosthesis grinding dust on RAW264.7 cells.

Respiratory diseases, including pulmonary fibrosis, silicosis, and allergic pneumonia, can be caused by long-term exposure to dental prosthesis grinding dust. The extent of the toxicity and pathogenicity of exposure to PMMA dust, Vitallium dust, and dentin porcelain dust differs. The dust from grinding dental prosthesis made of these three materials was characterized in terms of morphology, particle size, and elemental composition. The adverse effects of different concentrations of grinding dust (50, 150, 300, 450, and 600 µg ml-l) on RAW264.7 macrophages were evaluated, including changes in cell morphology and the production of lactate dehydrogenase (LDH) and reactive oxygen species (ROS). The dust particles released by grinding dental prosthesis made of these materials had different morphologies, particle sizes, and elemental compositions. They also induced varying degrees of cytotoxicity in RAW264.7 macrophages. A possible cytotoxicity mechanism is the induction of lipid peroxidation and plasma membrane damage as the dust particles penetrate cells. Therefore, clinicians who regularly work with these materials should wear the appropriate personal protection equipment to minimize exposure and reduce the health risks caused by these particulates.

In vitro cytotoxic response to lithium disilicate dental ceramics.

OBJECTIVES: The use of lithium disilicate dental ceramics is increasing in dentistry and previous reports have suggested that they may have greater biological risks than previously thought. We tested a hypothesis that composition and processing influence the biological properties of these ceramics. METHODS: The cytotoxicity of two machined and three pressed lithium disilicate materials (n=6) were tested in vitro using mouse fibroblasts in direct contact with the materials for 72h. Cellular response was estimated by mitochondrial succinate dehydrogenase activity (MTT method). Mitochondrial activity was expressed as a percentage of Teflon controls, then compared to Teflon using 2-sided t-tests (alpha=0.05). Polished materials were aged in artificial saliva and tested for cytotoxicity periodically over 6 weeks, then were repolished (320grit SiC paper), aged and tested again for 4 weeks. RESULTS: All materials significantly (50-70%) suppressed cellular mitochondrial activity in the initial week, but suppression decreased by 25-30% over the next 2 weeks. In weeks 4 and 6 some materials exhibited a cytotoxic 'relapse' of 10-20%. The cytotoxic response was no different for machined or pressed materials, but the presence of ZnO had at least an association with longer-term cytotoxicity and relapse. Repolishing to 320grit did not increase cytotoxicity significantly. SIGNIFICANCE: Our results suggest that lithium disilicates are not biologically inert, and that many have a similar cytotoxicity dynamic regardless of small differences in composition or processing.

Toxicity of porcelain-fused-to-metal substrate to zebrafish (Danio rerio) embryos and larvae.

AIMS: Porcelain-fused-to-metal (PFM) crowns are a standard restoration technique in dentistry, but toxicity of PFM in vivo has not been systematically evaluated. The present study evaluated the effects of various metal alloy shells of PFM crowns on the development of zebrafish embryos and larvae in order to determine the safety of these materials. MAIN METHODS: Gold palladium (Au-Pd), silver palladium (Ag-Pd), Nickel chromium (Ni-Cr), cobalt chromium (Co-Cr), titanium (Ti) alloy porcelain crowns were immersed in artificial saliva for 1, 4, and 7 weeks, and the leach solution was collected and used to treat zebrafish embryos at 4-144 h PFM. Toxicity was assessed based on mortality, spontaneous movement, heart rate, hatchability, malformation, and swimming behavior. KEY FINDINGS: The 1-week leachates of the five PFMs were not toxic to zebrafish. The rates of mortality and malformation of zebrafish in the Ni-Cr alloy group were increased whereas spontaneous movement, heart rate, and swimming behavior were decreased for 4- and 7-week leachates. SIGNIFICANCE: Among metal substrates commonly used in dental work, Ni-Cr alloy was most toxic, followed by Co-Cr and Ag-Pd alloys. Ti and Au-Pd alloys showed good biocompatibility and are therefore the most suitable materials for clinical applications.

Effect of hydrolyzed surface layer on the cytotoxicity and chemical resistance of a low fusing porcelain.

OBJECTIVE: The objectives of this study were to verify the formation of a hydrolyzed surface layer on Duceram LFC and to determine its effect on the cytotoxicity of the porcelain as measured by cellular activity and concentrations of leached ionic species. METHODS: Specimens were fabricated from dentin porcelain by a vibration blotting technique. Half of the specimens underwent accelerated aging by subjecting them to the standard test for hydrolytic resistance (ISO 6872). Fibroblast cell cultures were placed in direct contact with specimens. Cell viability was assessed using succinic dehydrogenase activity. Chemical resistance was determined by leaching specimens with a continuous flow of deionized water and measuring the concentrations of soluble ions in the leachates. Porcelain surface topography was examined using atomic force microscopy, and Fourier transform infrared spectroscopy was used to detect the composition of the surface layer. RESULTS: Hydrolyzation treatment created a smooth texture on the porcelain surfaces but did not result in a hydrolyzed surface layer with increased hydroxyl content. There was a decreased alkali ion content in the surface layer of hydrolyzed specimens. Mean cellular SDH activities for non-hydrolyzed and hydrolyzed porcelain were 75+/-7 and 80+/-5% of Teflon controls, respectively. Only sodium ions were present in significant concentrations in collected leachates. The sodium concentration decreased over the initial 4.5h of leaching. SIGNIFICANCE: The changes in surface layer texture and composition as the result of the aging treatment had little effect on the cytotoxicity and chemical resistance of Duceram LFC. The results suggest that Duceram LFC would pose no biocompatibility risk even after extended exposure to the oral environment. These data also provide baseline material properties to be used in future studies of the effects of porcelain additives.

Cytotoxicity and bonding property of dental ceramics.

OBJECTIVES: Yttria partially stabilized zirconia (YPSZ) ceramic is suitable for dental and medical use because of its high fracture toughness and chemical durability. The purpose of this study was to estimate the cytotoxicity and bonding property of zirconia ceramic compared to other dental ceramics. METHODS: Eight commercial dental ceramics including Denzir (YPSZ) are used in this cytotoxicity test. The human gingival fibroblast (GF) cells were cultured using extraction solutions of ceramics. The cytotoxicity was estimated by two different methods. The bonding strength of Denzir was compared to Empress2 using zinc phosphate, glass ionomer, and adhesive resin cements. A brass plate was prepared with drilled tapered holes and ceramic specimens were prepared to fit the holes and bonded. The bonding strength was estimated by the punching test. RESULTS: No significant (p>0.05) cytotoxicity was observed in all ceramic extractions. The two evaluation methods showed no significant differences. Denzir and Empress2 showed similar bonding strength with zinc phosphate or glass ionomer cement bonding. For both Empress2 and Denzir the glass ionomer cement showed significantly (p<0.001) higher bonding strength compared to the zinc phosphate cement. Empress2 showed significantly higher bonding strength with adhesive resin cement. However, the Denzir showed lower bonding strength with adhesive resin cement. SIGNIFICANCE: No ceramic extractions showed any evidence of cytotoxicity. Therefore, the low in vitro cytotoxicity of ceramic extractions including Denzir was confirmed. Denzir showed a similar bonding strength to Empress2 with zinc phosphate or glass ionomer cement bonding with this testing method and lower bonding strength with adhesive resin cement than with Empress2.

Occupational dermatoses among workers in a porcelain manufacturing factory.

The objectives of the present work are to determine the prevalence and risk factors of occurrence of occupational dermatoses among workers in a porcelain manufacturing factory. The study included 235 workers (132 males and 103 females). After history taking, dermatological examination was performed at the work place. Cases of contact dermatitis (CD) were patch tested using potassium dichromate 0.5%, Cobalt chloride 1% and Nickel sulphate 5% in petrolatum. Results showed that the prevalence of occupational dermatoses (OD) was 26.8% among exposed workers. The highest percentage of OD was found among workers in the decoration department. It represented 30.2% of all cases. CD was the commonest presentation among the studied group, it represented 27% of all cases of OD. Results of patch testing indicated that out of 17 cases of CD 12 cases (70%) were of the irritant variety and 5 cases (30%) were of the allergic type. The prevalence of OD was found to be affected by age, duration of work, type of exposure and daily bathing after work.

In vitro evaluation of cytotoxicity of permanent prosthetic materials.

OBJECTIVES: To assess qualitative and quantitative cytotoxicity effect on permanent prosthetic materials to human gingival fibroblasts. METHODS: Human gingival tissues were collected (with informed consent) from patients undergoing periodontal surgical procedures and fibroblasts were cultured in vitro. Cell type was determined by performing proteomic analysis. Selected prosthetic materials including titanium, feldspathic ceramic, gold and chrome-cobalt alloy specimens (5×2 mm) were fabricated. The toxicity of prepared specimens was tested by exposing them to cell culture medium for 48, 72, 96 and 120 hours at 37°C under sterile conditions. Cell viability was estimated using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. The data concerning cell viability were statistically analyzed using two-way ANOVA test and Tukey multiple comparison test. RESULTS: Results obtained after 48 hours showed no toxic effect of titanium compared to control group. Cytotoxic effect was observed in gold alloy and feldspathic ceramic, however, it was not significant compared to control group. Chrome-cobalt alloy significantly reduced cell viability compared to control group (p≤0.001). Cytotoxicity diminished with increasing incubation time of specimens. After 120 hours of incubation all tested materials, except chrome-cobalt alloy, had no cytotoxicity. CONCLUSIONS: Titanium proved to be non-toxic. Gold alloy and feldspathic ceramic had short-term cytotoxic effect. Chrome-cobalt alloy had highest cytotoxic effect on fibroblast cells.

Cytotoxicity evaluation of endosequence root repair material.

OBJECTIVE: The purpose of this study was to evaluate the cytotoxicity of EndoSequence Root Repair Material (Brasseler USA, Savannah, GA) and compare it with gray and white MTA. STUDY DESIGN: Samples of 2 mg freshly mixed or set gray MTA (GMTA), white MTA (WMTA), EndoSequence Root Repair Material (ERRM), and AH26 were eluted with 300, 600, and 1,000 microL cell culture medium for 24 and 72 hours. L929 cells were seeded into 96-well plates at 3 x 10(4) cells/well and incubated with 100 microL elute from each elute group. Cells cultured only with culture medium served as negative control. AH26 was used as positive control. After 24 hours' incubation, cell cytotoxicity was evaluated by MTT assay. Cell viability was calculated as percentage of the control group. The results were analyzed with 1-way analysis of variance. RESULTS: For both set and fresh samples, there were no significant cell viability differences among GMTA, WMTA, and ERRM. Cell viability in the AH26 group was less than in all of the other 3 materials. CONCLUSION: This study suggests that ERRM may have cell viability similar to GMTA and WMTA in both set and fresh conditions.

In vitro cytotoxicity evaluation of elemental ions released from different prosthodontic materials.

OBJECTIVES: This study investigated the cytotoxicity of elemental ions contained in four fixed prosthodontic materials (gold, nickel-chromium, stainless-steel alloys and CAD-CAM ceramics). MATERIALS AND METHODS: According to the determination of elements released from prosthodontic materials by using inductively coupled plasma mass spectroscopy, similar amounts of elements Pd, Ag, Zn, Cu, Ni, Cr, Mo, Be, Fe, Al, and K were prepared as salt solutions. Wells with a tenfold higher concentration of the tested elements were used as positive controls, while a well without any tested element was used as a negative control. These salt solutions were tested for cytotoxicity by culturing mouse L-929 fibroblasts in the salt solutions for a 7-day period of incubation. Then, the percentage of viable cells for each element was measured using trypan blue exclusion assay. The data (n=5) were statistically analyzed by ANOVA/Tukey test (p<0.05). RESULTS: The results showed a statistically significant difference for the cytotoxic effect of the tested elements salt solutions. For the released element concentrations the lowest percentage of viable cells (mean+/-SD) was evident with Zn, Cu or Ni indicating that they are the highly toxic elements. Be and Ag were found to be intermediate in cytotoxic effect. Fe, Cr, Mo, Al, Pd or K were found to be the least cytotoxic elements. SIGNIFICANCE: Zn and Cu released from gold alloys, and Ni released from nickel-chromium alloys, which are commonly used as fixed prosthodontic restorations, show evidence of a high cytotoxic effect on fibroblast cell cultures.

Genotoxicity evaluation of locally produced dental porcelain--an in vitro study using the Ames and Comet assays.

The aim of this study was to determine the genotoxicity of a locally produced dental porcelain (Universiti Sains Malaysia, Malaysia) using the Ames and Comet assays. In the Ames assay, four genotypic variants of the Salmonella strains (TA98, TA100, TA1537 and TA1535) carrying mutations in several genes were used. The dental porcelain was incubated with these four strains in five different doses both in the presence and absence of metabolic activation (S9) and the result was assessed based on the number of revertant colonies. Concurrently, appropriate positive controls were used so as to validate the test. The average number of revertant colonies per plate treated with locally produced dental porcelain was less than double as compared to that of negative control. In the Comet assay, L929 (CCL-1 ATCC, USA) mouse fibroblast cells were treated with the dental porcelain in three different concentrations along with concurrent negative and positive controls. The tail moment which was used as a measurement of DNA damage was almost equal to that of the negative control, suggesting that the locally produced dental porcelain did not induce any DNA damage. The results indicated that the locally produced dental porcelain is non-genotoxic under the present test conditions.

[Biological evaluation of In-Ceram-ceramics compared to cobalt-base-alloys and the metals titanium, tantalum and niobium in animal experiments]. / Biologische Prüfung der In-Ceram-Keramik im Vergleich mit Kobaltbasis-Legierungen und den Metallen Titan, Tantal und Niob im Tierexperiment.

The aim of the study was to evaluate the biocompatibility of the in-cream-ceramic-system in several technological phases by animal experiments. The comparative materials were the Co-Cr-alloys Remanium CD and Wirobond and the implant materials Titanium, Tantalum and Columbium and the high biocompatible material Teflon. The experiments were carried out in accordance with the DIN-standard 13,930 using subcutaneous implantation to verify the toxicity of the materials. 20 cylindrical test pieces were implanted into the subcutaneous connective tissue of the neck in rats. After 12 weeks the animals were sacrificed and the implants were removed with the surrounding tissues. The thickness of the fibrous capsule and the number of cells within the capsule were measured microscopically. The in-ceram-ceramic-system showed the same results like approved Co-Cr-alloys. The results of the implant materials were not attained.

Cytotoxicity of dental alloys, metals, and ceramics assessed by millipore filter, agar overlay, and MTT tests.

STATEMENT OF PROBLEM: Biocompatibility of dental materials is dependent on the release of elements from the materials. In addition, the composition, pretreatment, and handling of the materials influence the element release. PURPOSE: This study evaluated the cytotoxicity of dental alloys, metals, and ceramics, with specific emphasis on the effects of altering the composition and the pretreatment. MATERIAL AND METHODS: By using cells from a mouse fibroblast cell line and the agar overlay test, Millipore filter test, and MTT test, cytotoxicity of various metals, metal alloys, and ceramics for dental restoration were studied. Effects of altering the composition of a high noble gold alloy and of pretreatment of a ceramic-bonding alloy were also studied. In addition, the release of elements into the cell culture medium by the materials studied was measured using an inductively coupled plasma optical emission spectrophotometer. The results of the MTT test were analyzed statistically using ANOVA and Scheffé test at a significance level of P <.05. RESULTS: Specimens manufactured from materials intended for dental restorations and handled in accordance with the manufacturers' instructions were ranked from "noncytotoxic" to "mildly cytotoxic" according to the agar overlay and Millipore filter tests. For the MTT test, no significant differences were observed between these materials and controls, with the exception of JS C-gold and unalloyed titanium. The modified materials were ranked from "mildly cytotoxic" to "moderately cytotoxic" in the agar overlay and Millipore filter tests and from "noncytotoxic" to "moderately cytotoxic" in the MTT test. Thus, cytotoxicity was related to the alloy composition and treatment. The release of Cu and Zn seemed to be important for the cytotoxic effect. CONCLUSION: Alterations in the composition and the pretreatment can greatly influence the cytotoxicity, and the results stress the importance of carefully following the manufacturers' instructions when handling dental materials.

In vitro cytotoxicity of resin-containing restorative materials after aging in artificial saliva.

Studies have reported that dental resin-based materials release substances which have biological liabilities. However, some current methods for detecting these substances may not be adequate to detect biologically relevant concentrations. In the current study, we hypothesized that resin-based materials exhibit cytotoxic effects and alter cellular function in vitro when high-pressure liquid chromatography (HPLC-UV detection) cannot detect any release of substances. We further hypothesized that this release continues even after aging the samples in artificial saliva. Five types of composite or compomer materials (Z-100, Tetric Ceram, Dyract AP, Solitaire, and Clearfil AP-X) and one organically modified ceramic material (Definite) were tested after aging in artificial saliva for 0, 7, or 14 days. Cytotoxicity was assessed using direct contact with fibroblasts and measurement of succinic dehydrogenase activity after 48 h of exposure post aging. Release of substances from the materials was assessed using HPLC with UV detection. Altered cellular function was estimated by measuring proliferation of MCF-7 cells with sulforhodamine staining. HPLC showed that whereas initial release of substances was higher without aging, this release dropped significantly after 7 or 14 days of aging, and was equivalent to the Teflon controls after 14 days for four of the materials (Tetric Ceram, Definite, Solitaire, and Clearfil AP-X). Without aging in saliva, all materials had cytotoxicities > 50% of the Teflon negative controls. After 14 days of aging, all materials except the Definite continued to show severe cytotoxicity. Only the Definite could be tested for its ability to alter cellular function because of the continuing toxicity of the other materials. This modified ceramic material caused a significant proliferative effect on the MCF-7 cells indicating that sufficient substances were released to alter cellular function. We concluded that all of these commercially available resin-based dental materials continue to release sufficient components to cause lethal effects or alter cellular function in vitro even after 2 weeks of aging in artificial saliva.

In vitro cytotoxicity of traditional versus contemporary dental ceramics.

STATEMENT OF PROBLEM: The biocompatibility of new dental ceramics has not been assessed with the same scrutiny as has been applied to alloys and composites. Yet, the biocompatibility of ceramics is critical to the long-term success of dental prostheses because ceramics are in close contact with oral tissues for extended periods. MATERIAL AND METHODS: Five dental ceramics (2 traditional feldspathic veneer porcelains [Vita Omega and Duceragold], 2 lithium disilicate pressable materials [Stylepress and Empress-2], and a pressable leucite-based material [Empress-1]) were tested for their ability to alter cellular mitochondrial dehydrogenase activity after fabrication using a tetrazolium assay, after aging for 2 weeks in a biologic solution and after post-aging polishing with either a fine diamond or diamond polishing paste. Cellular responses were compared with polytetrafluoroethylene controls (analysis of variance, Tukey pairwise post-hoc comparison, alpha=.05). RESULTS: The feldspathic porcelains caused only mild (<25% of controls) mitochondrial suppression regardless of aging or polishing. The pressable leucite-based material initially caused a 5% stimulation (not significant) of mitochondrial activity, which decreased significantly (P<.05) by 30% with aging to levels comparable to the feldspathic porcelains, and did not change with polishing. Both lithium disilicate materials caused an initial suppression of mitochondrial activity that decreased significantly with aging, but Empress-2 was severely cytotoxic initially (<20% of controls, P<.01), and became more cytotoxic again after polishing. Stylepress was less cytotoxic initially (85% of controls, not significant) and did not become cytotoxic again after polishing. CONCLUSIONS: Dental ceramics are not equivalent in their in vitro biologic effects, even within the same class of material, and biologic safety should not be assumed. Most ceramics caused only mild in vitro suppression of cell function to levels that would be acceptable on the basis of standards used to evaluate alloys and composites. However, 1 Li-disilicate material (Empress-2) exhibited cytotoxicity that would not be deemed biologically acceptable on the basis of prevailing empirical standards for dental alloys and composites.

Interfacial reactions of osteoblasts to dental and implant materials.

Scanning electron microscopy was used to study the response of osteoblasts to various surfaces including ceramics and glasses as well as steel and titanium. Hydroxylapatite, tricalcium phosphate, bioglass, steel, and titanium supported cell adhesion. However, the toxic effects of the In-Ceram (Vita, Bad Säckingen, Germany), and feldspar ceramic and glaze were severe enough to cause verrucous necrosis that was identifiable after 2 days of culture. After 10 days in culture only the peripheral portions of these specimens were still occupied by cells; the cells in the central portion of the circular specimens had succumbed to necrosis. It was concluded that scanning electron microscopy is useful in identifying the response of cells to materials. Pathologic changes are not recognizable if they are limited to the internal structure of the cell, but readily discernible when they impinge on the morphologic integrity of the cell surface.