Toxicity of some dental cements in a cell culture system

abstract – A cell culture method has been used to study the effect of zinc phosphate cement (De Trey's Zink Zement Improved®), zinc silicophosphate cement (Fluoro-Thin®) and polycarboxylate cement (Durelon®) on animal cells. Disks (30 × 1 mm) of the materials were placed in the center of plastic Petri dishes and subsequently incubated with human epithelial cells. Cell multiplication, medium pH and the release of cement constituents were measured. All three cements exhibited a cytotoxic effect, which was most pronounced in the cultures with zinc silicophosphate cement and polycarboxylate cement. The results also indicated that cell growth on the surface of the disks is a more sensitive indicator of cytotoxicity than cell growth around the disks. pH of the medium was only slightly affected in cultures with polycarboxylate cement, whereas a decrease was found in cultures with zinc phosphate cement and especially with zinc silicophosphate cement. A rapid release of phosphate was found in cultures with zinc silicophosphate cement. Zinc was released into the medium from disks of zinc phosphate cement, zinc silicophosphate cement and polycarboxylate cement – exceeding the toxicity level for the present cell line after 24 h. In cultures with zinc silicophosphate cement and polycarboxylate cement the release of fluoride reached toxic levels within the same time interval.     

Toxicity of some dental cements in a cell culture system.

A cell culture method has been used to study the effect of zinc phosphate cement (De Trey's Zinc Zement Improved), zinc silicophosphate cement (Fluoro-Thin) and polycarboxylate cement (Durelon) on animal cells. Disks (20 x 1 mm) of the materials were placed in the center of plastic Petri dishes and subsequently incubated with human epithelial cells. Cell multiplication, medium pH and the release of cement constituents were measured. All three cements exhibited a cytotoxic effect, which was most pronounced in the cultures with zinc silicophosphate cement and polycarboxylate cement. The results also indicated that cell growth on the surface of the disks is a more sensitive indicator of cytotoxicity than cell growth around the disks. pH of the medium was only slightly affected in cultures with polycarboxylate cement, whereas a decrease was found in cultures with zinc phosphate cement and especially with zinc silicophosphate cement. A rapid release of phosphate was found in cultures with zinc silicophosphate cement. Zinc was released into the medium from disks of zinc phosphate cement, zinc silicophosphate cement and polycarboxylate cement--exceeding the toxicity level for the present cell line after 24 h. In cultures with zinc silicophosphate cement and polycarboxylate cement the release of fluoride reached toxic levels within the same time interval.     

Evaluation of biologic effects of dental materials using four different cell culture techniques

abstract – The cytotoxicity of fresh and 1-day-old silicate cement, composite restorative material and zinc oxide-eugenol cement (ZOE) was tested using human epithelial cells (NCTC 2544) in four different cell culture systems: (1) ^5.1Cr-release from prelabeled cells after incubation for 4 and 24 h in the presence of the materials, (2) Implanting the materials on an agar overlay and visualizing any cytotoxic effects after 24 h by neutral red vital stain. (3) Cell growth during 5 d in the presence of the materials7hellip; (4) Colony-forming ability after exposure of the cells for 30 min to medium previously incubated with the materials for 24 h. Freshly prepared and 1-day-old ZOE exhibited a prominent cytotoxic effect in all four systems. A less marked effect was found for the composite material in systems 2, 3, and 4, while silicate cement appeared to be the least toxic material in these three systems. Neither silicate cement nor composite showed any cytotoxic effect in system 1 based on⁵¹Cr-release. It is concluded that the effects obtained by the cell culture techniques did not mimic the reactions obtained when the materials are tested under conditions which reflect their clinical use.     

A further testing of the effect of dental materials on growth and adhesion of animal cells in vitro

abstract – Human epithelial cells (NCTC 2544) were cultivated in plastic Petri dishes containing disks of dental materials. The effect of the materials on cell growth around and on the surface of the disks was estimated by cell counts. The amount of glucose utilized and lactate formed in the culture media was measured and related to cell growth. In the presence of gold alloy no effect was found. By cell counts a toxic effect was observed on the surface of QC-20® and Biodent® (heat-cured acrylics). Around the disks, however, cell growth was not affected. In cultures with silicate cement, Addent 12®, silver amalgam, and copper amalgam, cell growth was inhibited both around and on the surface of the disks, the effect being most pronounced with the two amalgams. Discrepancies observed between cell counts and the metabolic parameters indicate that the latter should be used with caution when evaluating relative toxicities of dental materials.     

A methodologic study of the effect of dental materials on growth and adhesion of animal cells in vitro

abstract – An in vitro method was used to investigate the ability of animal cells to (a) grow in the proximity of various dental materials, (b) adhere to and grow on the surface of the same materials during long-term contact. Disks (30 × 1 mm), made from the different materials, were placed in the center of plastic Petri dishes and incubated with human epithelial cells (NCTC 2544) and mouse fibroblasts (L 929). The growth around and on the surface of the disks was followed by measuring the amount of deoxyribonucleic acid (DNA) and/or cell number. The cultures were stained with crystal violet to visualize the distribution of the cells. In the presence of gold alloy, growth was similar to that obtained in control cultures, whereas the other materials tested showed various degrees of a toxic effect. With the two heat-cured acrylics tested, a transient toxic effect was observed on the surface of the disks. In the presence of silicate cement, various degrees of growth inhibition were observed, both on the surface of and around the disks. A toxic effect of silver amalgam was evident on the surface of and in the close proximity of the disks, whereas Addent 12® and copper amalgam appeared to have a general toxic effect. The present method might thus be of value in the evaluation of the tissue compatibility of biomaterials.     

Evaluation of biologic effects of dental materials using four different cell culture techniques.

The cytotoxicity of fresh and 1-day-old silicate cement, composite restorative material and zinc oxide-eugenol cement (ZOE) was tested using human epithelial cells (NCTC 2544) in four different cell culture systems: (1) 51Cr-release from prelabeled cells after incubation for 4 and 24 h in the presence of the materials. (2) Implanting the materials on an agar everlay and visualizing any cytotoxic effects after 24 h by neutral red vital stain. (3) Cell growth during 5 d in the presence of the materials. (4) Colony-forming ability after exposure of the cells for 30 min to medium previously incubated with the materials for 24 h. Freshly prepared and 1-day-old ZOE exhibited a prominent cytotoxic effect in all four systems. A less marked effect was found for the composite material in systems 2, 3, and 4, while silicate cement appeared to be the least toxic material in these three systems. Neither silicate cement nor composite showed any cytotoxic effect in system 1 based on 51Cr-release. It is concluded that the effects obtained by the cell culture techniques did not mimic the reactions obtained when the materials are tested under conditions which reflect their clinical use.     

Characterization and hydration kinetics of tricalcium silicate cement for use as a dental biomaterial

Objectives

Investigation and characterization of the replacement of the Portland cement component in mineral trioxide aggregate (MTA) with tricalcium silicate cement which is manufactured using the sol-gel method from pure raw materials.

Methods

Tricalcium silicate and Portland cement were characterized by viewing under the scanning electron microscope (SEM) and surface imaging and elemental analysis with X-ray energy dispersive analysis (EDX), and by X-ray diffraction analysis with Rietveld refinement. In addition the hydration products of the material after 28 days of curing were evaluated by plotting atomic ratio plots from the EDX data. The cement leachate was evaluated for pH and chemical composition by inductively coupled plasma.

Results

Portland cement was composed of 68% tricalcium silicate. The tricalcium silicate cement was 99% pure. On hydration both cements produced calcium silicate hydrate and calcium hydroxide. The calcium hydroxide was leached in solution with higher leaching in HBSS. The leaching of calcium hydroxide in solution resulted in an alkaline pH. The reaction of calcium with the phosphorus present in HBSS resulting in the deposition of calcium phosphate on the cement surface.

Significance

Tricalcium silicate could prospectively replace the Portland cement component in MTA.     

Plaque accumulation on different dental filling materials

abstract — The purpose of the present study was to investigate whether some dental filling materials collect more plaque than others under standardized conditions in vitro and in vivo . Round disks of the filling materials of silicate, composite and amalgam were prepared and placed on an agar/sucrose medium or hung in liquid medium containing 5% sucrose. The mediae were inoculated with Streptococcus mutans OMZ 176 and OMZ 52–3. After 6 d the disks were rinsed in distilled water and the plaque that had accumulated on the surfaces was scraped off and measured by the orcinol method. The materials were also fitted to acrylic plates and introduced into the mouth. Six patients wore two plates each for comparison of the materials and were told to rinse their mouth every hour with 15% sucrose solution. After 8 h the plaque was scraped off and measured. Silicate disks on agar medium invariably produced inhibition zones of about 4 mm. Composites and amalgam showed no such zones. In liquid medium an insignificant amount of plaque was absorbed to the silicate disks, whereas abundant amounts were found on composite materials and some on amalgam disks. The same trend could be demonstrated in the clinical experiments.     

On the mechanism of cytotoxicity of silver and copper amalgams in a cell culture system

ABSTRACT – Silver and copper amalgams show a pronounced cytotoxic effect on monolayer cultures of human epithelial cells (NCTG 2544). Analyses of the medium from silver amalgam cultures showed that, zinc was released in substantial amounts (14 μg/ml after incubation for 24 h). Small amounts of mercury and possibly also some silver were released, whereas release of copper and tin could not be. detected. Toxicity tests showed that 10 μg Zn²+/ml reduced the number of cells by 96% after incubation for 24 h. In the copper amalgam cultures about. 100 μ g copper and 5 μg, cadmium per ml medium were found after 24 h. Only small amounts of mercury were released, Toxicity tests showed an increasing effect of Cu²+ and Cd²+ with tune. With 50 μg Cu²+/ml the number of cells was reduced by 73% after incubation for 24 h, and after 3 d a similar effect was found with 25 μg/ml. With 10 μg Cd²+/ml no cells were left after 24 h, whereas after 3 d 1μg/ml reduced cell growth by more than 80 %.     

Discoloration of dental cements and composites in a sulfide solution

In an early study the discoloration of certain hardened silicate cements, after exposure to an atmosphere of hydrogen sulfide (H2S) for 24 h at room temperature, was ascribed to the formation of dark-colored sulfides of base metal impurities (Paffenbarger et al. JADA 25,32,1938). A recent study noted that, in general, silicate and glass ionomer cements were more prone to color shifts than composites after exposure to H2S for 9 weeks (Sugawara, Ph. D. Thesis, Nihon Univ.). The aim of the present study was to devise a simple, aqueous sulfide exposure test for esthetic restorative materials. The general procedure was to expose specimen disks to a 0.1% (w/v) sodium sulfide solution, adjusted to pH 9, for 1-7 days at 37 degrees or 55 degrees C. The 55 degrees C-Na2S exposure was designed as an accelerated test. Materials studied included: 1 silicate and 2 silicophosphate cements of known lead content, a glass ionomer cement (FIIF), several commercial composites and an experimental, hydrophilic composite. Known amounts of base metal contaminants in the form of appropriate salt solutions were added to the liquid components of FIIF and the composites. Specimens exposed to distilled water under the same conditions served as controls. Exposure to the aqueous sulfide medium resulted in the following ranking in order of decreasing discoloration: Glass ionomer cement greater than silicophosphate cement greater than silicate cement greater than hydrophilic composite greater than hydrophobic composite. Generally, the results of the aq. Na2S test paralleled those obtained with H2S. The degree of discoloration is dependent on a number of factors: the nature, concentration and leachability of the metal impurities, and the hydrophilicity and permeability to sulfide of the esthetic restoratives.     

Three-dimensional culture of dental pulp stem cells in direct contact to tricalcium silicate cements

Objectives

Calcium silicate cements are biocompatible dental materials applicable in contact with vital tissue. The novel tricalcium silicate cement Biodentine? offers properties superior to commonly used mineral trioxide aggregate (MTA). Objective of this study was to evaluate its cytocompatibility and ability to induce differentiation and mineralization in three-dimensional cultures of dental pulp stem cells after direct contact with the material.

Materials and methods

Test materials included a new tricalcium silicate (Biodentine?, Septodont, Saint-Maur-des-Fossés, France), MTA (ProRoot® MTA, DENSPLY Tulsa Dental Specialities, Johnson City, TN, USA), glass ionomer (Ketac? Molar Aplicap?, 3M ESPE, Seefeld, Germany), human dentin disks and polystyrene. Magnetic activated cell sorting for to the surface antigen STRO-1 was performed to gain a fraction enriched with mesenchymal stem cells. Samples were allowed to set and dental pulp stem cells in collagen carriers were placed on top. Scanning electron microscopy of tricalcium silicate cement surfaces with and without cells was conducted. Cell viability was measured for 14 days by MTT assay. Alkaline phosphatase activity was evaluated (days 3, 7, and 14) and expression of mineralization-associated genes (COL1A1, ALP, DSPP, and RUNX2) was quantified by real-time quantitative PCR. Nonparametric statistical analysis for cell viability and alkaline phosphatase data was performed to compare different materials as well as time points (Mann-Whitney U test, α?=?0.05).

Results

Cell viability was highest on tricalcium silicate cement, followed by MTA. Viability on glass ionomer cement and dentin disks was significantly lower. Alkaline phosphatase activity was lower in cells on new tricalcium silicate cement compared to MTA, whereas expression patterns of marker genes were alike.

Conclusions

Increased cell viability and similar levels of mineralization-associated gene expression in three-dimensional cell cultures on the novel tricalcium silicate cement and mineral trioxide aggregate indicate that the material is cytocompatible and bioactive.

Clinical relevance

The tested new tricalcium silicate cement confirms its suitability as an alternative to MTA in vital pulp therapy.

     

pH and the cytotoxicity of fluoride in an animal cell culture system

abstract — To investigate the mechanism for the toxicity of silicate cement as observed in a cell culture system, the effects of pH and fluoride were tested on human epithelial cells (NCTC 2544). At pH 7.3, fluoride concentrations from 15 to 25 μg/ml (0.79 to 1.3 mM) had a growth inhibitory effect. When pH of the incubation medium was lowered in the range 7.0 to 6.4, an enhanced cytotoxic effect of fluoride was found, and even at 5 to 10 μg/ml growth inhibition occurred. Concomitant with the enhanced cytotoxicity of fluoride at low pH, there was an increased utilization of glucose and formation of lactate. Upon lowering the pH of the incubation medium from 7.4 to 6.7 a twofold increase in the intra-cellular concentration of fluoride was found.     

Volume of Voids in Retrograde Filling: Comparison between Calcium Silicate Cement Alone and Combined with a Calcium Silicate–based Sealer

IntroductionThe aim of this study was to compare the volume of voids between retrograde filling using calcium silicate cement alone and retrograde filling using a combination of calcium silicate cement with a calcium silicate–based sealer.MethodsTwenty single-rooted, extracted human teeth were instrumented with nickel-titanium files and obturated with gutta-percha. We resected the roots at a point 3 mm from the apex, prepared the root ends, and filled the root-end cavities with Endocem Zr (Maruchi, Wonju, Korea) or Endoseal MTA (Maruchi) plus Endocem Zr. Then, we scanned the prepared samples using micro–computed tomographic imaging and performed 3-dimensional reconstruction. The percentage volume of the gap between the canal wall and root-end filling was calculated along with the percentage volume of voids in the filling materials. All data were analyzed using the Mann-Whitney U test. Selected specimens were further observed using scanning electron microscopy.ResultsThere were no significant differences in the percentage volumes of the gaps and internal voids between the 2 groups. Both calcium silicate and the calcium silicate–based sealer exhibited good adaptation to the cavity wall.ConclusionsThe findings of our micro–computed tomographic and scanning electron microscopic analyses suggested that the volume of voids in retrograde filling using a combination of calcium silicate cement and calcium silicate–based sealer was comparable with that in retrograde filling using calcium silicate cement only. Calcium silicate–based sealers could be used in retrograde filling for clinical convenience without harmful effects on the quality of filling.     

Characterization and chemical activity of Portland cement and two experimental cements with potential for use in dentistry

Aim  To evaluate the chemical activity of Portland cement and two other cement types with similar chemical composition to mineral trioxide aggregate with the aim of developing these cements for further applications in dentistry.
Methodology  The chemical composition of the three cement types namely Portland cement, calcium sulpho-aluminate cement and calcium fluoro-aluminate cement was evaluated by elemental analysis using energy dispersive analysis with X-ray under the scanning electron microscope and by X-ray diffraction analysis (XRD) to determine the phases. The constituents of the hydration reaction by-products were evaluated by XRD analysis of the set cements at 1, 7, 28 and 56 days and by analysis of the leachate by ion chromatography. The pH of both cements and leachate was determined at different time intervals. Cements admixed with micro-silica were also tested to determine the effect of micro-silica on the reaction by-products.
Results  All three cement types were composed of tricalcium silicate as the main constituent phase. The hydration reaction of Portland cement produced calcium hydroxide. However, this was not present in the other cements tested at all ages. Admixed micro-silica had little or no effect on the cements with regard to reaction by-products. The pH of all cements tested was alkaline.
Conclusions  Both the experimental calcium sulpho-aluminate cement and calcium flouro-aluminate cement had different hydration reactions to that of Portland cement even though calcium silicate was the major constituent element of both cement types. No calcium hydroxide was produced as a by-product to cement hydration. Micro-silica addition to the cement had no effect on the hydration reaction.     

Characterization of hydration products of mineral trioxide aggregate

Objective To characterize the hydration products of white mineral trioxide aggregate (MTA). Methodology Mineral trioxide aggregate, white Portland cement and bismuth oxide were evaluated using X‐ray diffraction (XRD) analysis and Rietveld XRD. The cements were tested un‐hydrated and after hydration and curing for 30 days at 37 °C. Analysis of hydrated cement leachate was performed weekly for five consecutive weeks from mixing using inductively coupled plasma atomic emission spectroscopy after which the cements were viewed under the scanning electron microscope to evaluate the cement microstructure. Quantitative energy dispersive analysis with X‐ray was performed and atomic ratios were plotted. Results Both Portland cement and MTA produced calcium silicate hydrate (C‐S‐H) and calcium hydroxide (CH) on hydration. The tricalcium aluminate levels were low for MTA which resulted in reduced production of ettringite and monosulphate. On hydration the bismuth level in the hydrated MTA decreased; bismuth oxide replaced the silica in the C‐S‐H and was leached out once the C‐S‐H decomposed with time. Both MTA and Portland cement released a high amount of calcium ions which decreased in amount over the 5‐week period. Conclusions The hydration mechanism of MTA is different to that of Portland cement. In MTA the bismuth oxide is bound to the C‐S‐H and is leached out from the cement with time as the C‐S‐H decomposes. MTA produces a high proportion of calcium ions from CH a by‐product of hydration and also by decomposition of C‐S‐H. The release of calcium ions reduces with time.     

pH and the cytotoxicity of fluoride in an animal cell culture system.

To investigate the mechanism for the toxicity of silicate cement as observed in a cell culture system, the effects of pH and fluoride were tested on human epithelial cells (NCTC 2544). At pH 7.3, fluoride concentrations from 15 to 25 mug/ml (0.79 to 1.3 mM) had a growth inhibitory effect. When pH of the incubation medium was lowered in the range 7.0 to 6.4, an enhanced cytoxic effect of fluoride was found, and even at 5 to 10 mug/ml growth inhibition occurred. Concomitant with the enhanced cytotoxicity of fluoride at low pH, there was an increased utilization of glucose and formation of lactate. Upon lowering the pH of the incubation medium from 7.4 to 6.7 a twofold increase in the intracellular concentration of fluoride was found.     

Effect of a bonding agent on in vitro biochemical activities of remineralizing resin-based calcium phosphate cements

OBJECTIVES: To test whether fluoride in a resin-based Ca-PO4 ion releasing cement or coating with an acidic bonding agent for improved adhesion compromised the cement remineralization potential. METHODS: Cements were formulated without fluoride (Cement A) or with fluoride (Cement B). The treatment groups were A=Cement A; A2=Cement A+bonding agent; B=Cement B; B2=Cement B+bonding agent. The calcium, phosphate, and fluoride ion release in saliva-like solution (SLS) was determined from hardened cement disks without or with a coating of bonding agent. For the remineralization, two cavities were prepared in dentin of extracted human molars and demineralized. One cavity received composite resin (control); the other received treatment A, A2, B or B2. After 6 week incubation in SLS, 180 microm cross-sections were cut. The percentage remineralization was determined by transverse microradiography comparing the dentin mineral density under the cement to that under the control. RESULTS: The percentage of remineralization (mean+/-S.D.) was A (39+/-14)=B (37+/-18), A2 (23+/-13), B2 (14+/-7). Two-way analysis of variance (ANOVA) and Holm-Sidak test showed a significant effect from the presence of bonding agent (p<0.05), but not from fluoride (p>0.05). The ion solution concentrations of all groups showed undersaturation with respect to dicalcium phosphate dihydrate and calcium fluoride and supersaturation for fluorapatite and hydroxyapatite suggesting a positive remineralization potential. SIGNIFICANCE: Compared to the control all treatments resulted in mineral increase. The remineralization was negatively affected by the presence of the bonding agent.     

Calcium silicate coating derived from Portland cement as treatment for hypersensitive dentine

OBJECTIVES: To evaluate the in vitro effectiveness on dentine permeability and dentine morphology of a calcium silicate treatment based on Portland cement (DSC). METHODS: The experimental treatment consisted of a calcium silicate paste based on Portland cement that was applied on dentine surface for 3 min. A professional re-mineralizing treatment (GC Tooth Mousse), two in office desensitizing agents (D/Sense Crystal, and By Sealant) and a commercial toothpaste Dentosan S were studied as comparison materials. All materials were applied accordingly with manufacturer directions on wet dentine. The quantitative changes in the hydraulic conductance i.e., permeability through tubules of dentine discs samples produced by treatment were quantified in vitro using a hydrostatic device working at 6.9 kPa. SEM/EDX analyses of dentine were carried out to obtain qualitative information on dentine morphology and surface deposits and to study their relationship with the hydraulic conductance. After treatment, each dentine sample was immersed in artificial saliva and permeability re-evaluated. Finally, each sample was exposed to 0.02 M citric acid solution and the final permeability was assessed. RESULTS: The experimental treatment and both oxalate-based products (D/Sense Crystal and By Sealant) significantly decreased dentine permeability and created crystals and precipitates on the dentine surface that reduced the diameter of dentinal tubules. Artificial saliva immersion and citric acid challenge increased dentine permeability and partially modified the treated surfaces. Dentosan S and GC Tooth Mousse treatments partially reduced dentine permeability and created small amount of precipitates that were removed by saliva immersion and citric acid exposure. EDX revealed the presence of calcium-rich layer after DSC experimental treatment. CONCLUSIONS: The application of the experimental calcium silicate paste and oxalate-based treatments was determined to be effective on dentine permeability reduction and tubules occlusion. The clinical use as desensitizing agent of materials derived from Portland cement as desensitizing agent should be considered for dentine hypersensitivity treatment.     

Response of rat parotid acini to barium.

The effect of barium on isolated acini was tested. Barium in the 0.1-10 mM concentration range non-competitively inhibited the efflux of 86Rb+ stimulated by carbamylcholine or substance P. This inhibition was independent of the presence of calcium in the extracellular medium. In the same preparation, barium did not affect the efflux of 45Ca2+ but, at a 10 mM concentration, it increased amylase release by 70%. Removal of extracellular calcium decreased basal amylase release and the response to carbamylcholine. Adding back calcium or barium to the incubation medium increased basal and carbamylcholine-stimulated amylase secretion, but calcium was more effective than barium. These results suggest that barium has two opposite effects on calcium-regulated processes in rat parotid gland: (1) it is an inhibitor of calcium-activated potassium channels; (2) it is a partial agonist of calcium-activated amylase secretion.