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.     

Toxic effects of various retrograde root filling materials on gingival fibroblasts and rat sarcoma cells.

The aim of this in vitro study was to evaluate the effect of amalgam, glass ionomer, composite and titanium on the growth of gingival fibroblasts (GF) and rat sarcoma cells (UMR) in vitro. The cells were either obtained from gingival biopsies taken during deliberation of unerupted canines (GF) or were of commercial origin (UMR). Equal numbers of cells were placed on culture dishes and incubated for a period of two weeks with the freshly prepared test materials. The cultures were photographed through a light microscope after 7 days incubation and finally counted after 14 days. It was shown that the proliferation of gingival fibroblasts was less disturbed by titanium, being approximately 96% of the control value (cell cultures without test particles), followed by composite, amalgam and glass ionomer (61%, 49% and 35% of the control value respectively). The number of UMR cells after 14 days incubation with the various materials was 76% of the control value with titanium, 12% with composite and 5% with both amalgam and glass ionomer. Inhibition of cell growth (UMR) around the test particles was most prominent around amalgam and glass ionomer, followed by composite and titanium. These effects were noted only with freshly prepared components however, so that the toxic reaction was less pronounced or minimal in a second incubation using the same particles sterilized in between. The results demonstrated that potential retrograde root filling materials have a variable toxic effect on gingival fibroblasts and rat sarcoma cells. The fact that the influence on proliferation disappeared when the test was performed with materials already tested once may be of clinical importance when estimating the biocompatibility in vivo.     

Toxic effects of various retrograde root filling materials on gingival fibroblasts and rat sarcoma cells

Abstract The aim of this in vitro study was to evaluate the effect of amalgam, glass ionomer, composite and titanium on the growth of gingival fibroblasts (GF) and rat sarcoma cells (UMR) in vitro. The cells were either obtained from gingival biopsies taken during deliberation of unerupted canines (GF) or were of commercial origin (UMR). Equal numbers of cells were placed on culture dishes and incubated for a period of two weeks with the freshly prepared test materials. The cultures were photographed through a light microscope after 7 days incubation and finally counted after 14 days. It was shown that the proliferation of gingival fibroblasts was less disturbed by titanium, being approximately 96% of the control value (cell cultures without lest particles), followed by composite, amalgam and glass ionomer (61%, 49% and 35% of the control value respectively). The number of UMR cells after 14 days incubation with the various materials was 76% of the control value with titanium, 12% with composite and 5% with both amalgam and glass ionomer. Inhibition of cell growth (UMR) around the test particles was most prominent around amalgam and glass ionomer, followed by composite and titanium. These effects were noted only with freshly prepared components however, so that the toxic reaction was less pronounced or minimal in a second incubation using the same particles sterilized in between. The results demonstrated that potential retrograde root filling materials have a variable toxic effect on gingival fibroblasts and rat sarcoma cells. The fact that the influence on proliferation disappeared when the test was performed with materials already tested once may be of clinical importance when estimating the biocompatibility in vivo.     

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.     

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.     

Adhesion of human osteoblasts on root-end filling materials

Adhesion of human osteoblasts to root-end filling materials (mineral trioxide aggregate (MTA), IRM, composite, and amalgam) was observed by scanning electron microscopy. Root-end filling materials were inserted into 96-well flat-bottomed plates and condensed to disks of approximately 1 mm thick and the same diameter as the wells. After the disks were set, they were placed in the bottom of Nunc four-well culture plates at one disk per well. Then human osteoblasts were seeded into the wells at 1.5 x 10(5) cells per well. After 1 day in culture the disks of root-end filling materials along with cells grown on their surface were examined with a scanning electron microscopy. Results showed that osteoblasts attached and spread on MTA and composite by forming a monolayer. Osteoblasts also attached on amalgam, but with few cells spreading. In the presence of IRM, osteoblasts appeared rounded with no spreading. These results indicate that osteoblasts have a favorable response to MTA and composite resin compared with IRM and amalgam.     

Effects of growth factors on dental pulp cell sensitivity to amalgam toxicity.

OBJECTIVES: The objective was to determine the effects of growth factor treatment on dental pulp cell sensitivity to toxicity. METHODS: The toxicity of zinc-containing and zinc-free dental amalgam was tested on four types of cells; glia, neurons, embryonic stem cells, and dental pulp cells. The effects of six different growth factors were tested on dental pulp cell sensitivity to amalgam toxicity. RESULTS: Zinc-containing amalgam was highly toxic to all cell types tested. Zinc-free amalgam was less toxic, but it was most toxic to dental pulp cells. Exposure of dental pulp cells to the growth factors IGF-I or BMP-7 had no effect on their morphology or rate of cell division, and did not alter their sensitivity to zinc-free amalgam toxicity. Exposure to EGF, bFGF, or TGF-beta altered the morphology and decreased the rate of cell division, and the cells were no longer sensitive to zinc-free amalgam toxicity. Exposure to BMP-2 also altered the morphology and decreased the rate of cell division, but the cells remained sensitive to zinc-free amalgam toxicity. SIGNIFICANCE: The results indicate that untreated dental pulp cells are highly sensitive to amalgam toxicity, but that sensitivity can be decreased by exposure to certain growth factors. Therefore, for treatment of conditions in which pulp cells may come in contact with substances released from dental materials, such as pulp capping, the use of amalgam should be avoided unless the pulp cells are first treated with the proper growth factors.     

Surface treatment of mercury-free alloys.

Finishing and polishing methods were examined for two metallic direct restorative materials being proposed as possible alternatives to amalgam, namely a gallium alloy and a consolidated silver alloy. The polished surfaces were compared to a conventional spherical amalgam (Tytin). After initial surface treatment with a 12-fluted tungsten carbide bur in a high-speed dental hand-piece, three polishing methods were evaluated: slow-speed polishing burs, rubber polishing points, and polishing disks (Sof-Lex). Each of these methods was followed by an additional surface treatment in which a pumice-flour/water slurry was applied with a rotary brush and a final surface treatment with a zinc-oxide/ethanol slurry that was applied with rotary rubber cups. The surface roughness was evaluated by profilometric measurements and light microscopy. The results showed that the smoothest surfaces for all metals were achieved with rotary finishing and polishing disks. Using the rubber points resulted in surfaces that were statistically similar to the disk-polished surfaces on all three materials. The polished surface of gallium alloy was consistently slightly rougher than that of amalgam. The consolidated silver also presented a consistently rougher surface than did amalgam, although these differences were not statistically significant. The additional polishing with pumice and zinc oxide improved the luster, but did not significantly improve the measured surface smoothness in any of the restorative materials studied.     

Silicate cement in a cell culture system

abstract — Human epithelial cells (NCTG 2544) were grown as monolayer cultures in the presence of silicate cement disks (Bio-trey 9®). A cytotoxic effect was found on the surface of the disks after 24 h, whereas a corresponding effect was obvious around the disks after incubation for 3 d. In the silicate cement cultures more glucose was utilized and more lactate formed per cell than in control cultures. In the presence of silicate cement, pH of the culture medium decreased during incubation, reaching 6.3–6.4 after 6 d. Phosphate, silicon, zinc, and fluoride were released into the medium from the silicate cement disks. The medium concentration of sodium, however, remained constant, and aluminum was not detected. The concentrations of calcium and magnesium decreased, and experiments with ⁴⁵Ca showed that calcium was bound to the silicate cement disks.     

Cytotoxic evaluation of root-end filling materials in cultures of human osteoblast-like cells and periodontal ligament cells.

The cytotoxicity of three root-end filling materials (amalgam, IRM, and Super-EBA) was evaluated in cultures of human periodontal ligament cells and human osteoblast-like cells. Ten-millimeter-long plastic test tubes were filled with 3 mm of freshly mixed root-end filling materials at one end (1.5 mm diameter). The opposite end was sealed and attached by heat to a 35-mm cell culture dish. Human periodontal ligament cells and human osteoblast-like cells were seeded in the dishes. The size of cell-free zones around the root-end filling materials and the total cell number per dish were calculated after 3 and 7 days. Empty test tubes used as controls did not influence the growth and distribution of the cultured cells. Cell density increased in all groups in the test period. Amalgam had a larger cell-free zone, compared with IRM and Super-EBA and showed a reduction in total cell number per dish for both tested cell types. IRM and Super-EBA also had a cell-free inhibition zone for both cell types, but no significant reduction in total cell number per dish. This study showed that amalgam had a higher cell toxicity to human periodontal ligament cells and human osteoblast-like cells than IRM and Super-EBA.     

Studies on the marginal leakage around various restorative materials. Part 1: Testing method and condition (author's transl)]

Standardized cavities prepared in bovine teeth were restored with 4 types of filling materials, lathe-cut amalgam, spherical amalgam, silicate cement and composite resin. Restored teeth were immersed in 4 degrees C and 60 degrees C dye solution alternately by automatic device which makes it possible to change the immersion interval and number optionally. By means of measuring the depth of dye penetration, the marginal leakage induced during the thermal change was evaluated and the effects of the immersion interval and number were investigated. Results are as follows. 1) It is possible to obtain the marginal leakage quantitatively by means of measuring the depth of dye penetration. 2) The marginal leakage around various restorations are significantly influenced by the immersion interval and number. 3) The depth of dye penetration varies with types of filling materials. 4) The optimum immersion interval and number are 2 minutes and 60-120 times to evaluate the marginal leakage around various restorative materials.     

The effect of the surface morphology of Ti-implants on the proliferation activity of fibroblasts and osteoblasts

It is known that surface morphology greatly influences the osseointegration of dental implants. The goal of the experiments conducted by the authors was to study in vitro the effect of various surface modifications on the activity of bone and connective tissue cells. In the experiments they introduced NIH3T3 fibroblast and MCH3T3 osteoblast cells were cultured the surface of ten titanium disks with various morphology in 24-multiwell plates. The cells were let grow on the surface of the disks for two days in a culture medium. One group of the disks underwent scanning electronmicroscopy and the changes in the number and form of cells on the surfaces were studied under various magnifications. From the surface of the second group the cells were lysed and were counted in a Bürker's chamber. After counting the cells they were lysed in a lysis buffer and their protein concentrations were measured with the help of a spectrophotometer. The authors found that only a morphological study of the cells is possible under SEM, they could not detect a quantitative difference in the number or activity of the cells. Upon counting the cells and determining their protein concentration the best result were yielded by the surface roughened by aluminium oxide. In growing fibroblasts the titanium oxide treated surfaces provided good results, while in the case of the osteoblasts the laser treated disks were more successful.     

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.     

Effects of root-end filling materials on fibroblasts and macrophages in vitro

OBJECTIVE: The aim of this study was to investigate the effects of 4 root-end filling materials (mineral trioxide aggregate [MTA], intermediate restorative material [IRM], amalgam, and Retroplast) on cell growth, cell morphology, and cytokine (interleukin [IL]1beta and IL-6) production in mouse fibroblasts and macrophages. STUDY DESIGN: Millipore culture plate inserts with freshly mixed or set root-end filling material were placed into 6-well cell culture plates with already attached mouse fibroblasts or macrophages. Cells cultured with only the Millipore culture plate inserts served as a control. After a 3-day incubation, cell morphology was examined, and the total cell number per well was counted and analyzed by using 1-way analysis of variance. For cytokine assay, mouse macrophages were incubated in 24-well flat-bottom plates with set root-end filling material disks in the bottom. Cells cultured without the material disks served as negative controls, and cells cultured with lipopolysaccharides served as positive controls. After 24-hour incubation, culture media were collected for cytokine assay by using enzyme-linked immunosorbent assay. RESULTS: All root-end filling materials inhibited the cell growth of mouse fibroblasts and macrophages. There was no growth in the originally seeded cells in the fresh IRM, the fresh Retroplast, and the set IRM group. There was no difference between MTA and amalgam for cell growth either in the fresh material groups or in the set material groups. The total cell number in the set Retroplast group was significantly less than that in the set MTA group. Morphologically, MTA was characterized by denatured medium proteins and dead cells adjacent to the material, which were observed only in the fresh MTA group. There was no detected cytokine production in any of the tested material groups. CONCLUSION: All root-end filling materials inhibited cell growth, and none induced IL-1beta and IL-6 production.     

Antimicrobial effect of 2% sodium hypochlorite and 2% chlorhexidine tested by different methods

The objective of this study was to analyze the antimicrobial effect of 2% sodium hypochlorite (NaOCl) and 2% chlorhexidine (CHX) by agar diffusion test and by direct exposure test. Five microorganisms: Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aernginosa, Bacillus subtilis, Candida albicans, and one mixture of these were used. These strains were inoculated in brain heart infusion (BHI) and incubated at 37 degrees C for 24 h. For the agar diffusion test (ADT), 18 Petri plates with 20 ml of BHI agar were inoculated with 0.1 ml of the microbial suspensions, using sterile swabs that were spread on the medium, obtaining growth injunction. Fifty-four paper disks (9 mm in diameter) were immersed in the experimental solutions for 1 min. Subsequently, three papers disks containing one of the substances were placed on the BHI agar surface in each agar plate. The plates were maintained for 1 h at room temperature, and then incubated at 37 degrees C for 48 h. The diameter of microbial inhibition was measured around the papers disks containing the substances. For the direct exposure test, 162#50 sterile absorbent paper points were immersed in the experimental suspensions for 5 min, and were then placed on Petri plates and covered with one of the irrigant solutions, or with sterile distilled water (control group). After intervals of 5, 1 0 and 30 min, the paper points were removed from contact with the solutions and individually immersed in 7 ml of Letheen Broth, followed by incubation at 37 degrees C for 48 h. Microbial growth was evaluated by turbidity of the culture medium. A 0.1 ml inoculum obtained from the Letheen Broth was transferred to 7 ml of BHI, and incubated at 37 degrees C for 48 h. Bacterial growth was again evaluated by turbidity of the culture medium. Gram stain of BHI cultures was used for verification of contamination and growth was determined by macroscopic and microscopic examination. The best performance of antimicrobial effectiveness of NaOCI was observed in the direct exposure test, and of CHX was observed in the agar diffusion test. The magnitude of antimicrobial effect was influenced by the experimental methods, biological indicators and exposure time.     

Cytotoxicity of corroded and non-corroded dental silver amalgams

The cytotoxicity of one conventional and four non-gamma 2-amalgams was studied in a cell culture system, using the Millipore filter method. Before testing set amalgam specimens were kept in distilled water or in artificial saliva at pH 4, 5, or 7 for up to 28 wk to produce a corrosion layer on the test surface. Non-corroded set amalgam specimens was also tested. None of the noncorroded, set amalgams showed any sign of surface accumulation of cytotoxic products whereas the corroded amalgams showed varying degrees of cytotoxicity. Generally, the non-gamma 2-amalgams gave a more pronounced cytotoxic effect than the conventional amalgam. When the corrosion procedure was carried out at pH 7, the various non-gamma 2-amalgams showed different degrees of cytotoxicity. It appears that the difference in cytotoxic effect between the non-gamma 2-amalgams and the conventional amalgam as well as the differences among the various non-gamma 2-amalgams could be related to variation in the retention of corrosion products deposited on the amalgam surface.     

Attachment of human periodontal ligament fibroblasts to 3 different root-end filling materials: Scanning electron microscope observation

OBJECTIVE: The attachment behavior of the human periodontal ligament (PDL) fibroblasts to root-end filling materials (amalgam and Super-EBA) was compared in vitro to gutta-percha by means of scanning electron microscope. STUDY DESIGN: Amalgam and Super-EBA were placed in a prepared cavity of root slices of freshly extracted human teeth and evaluated freshly prepared. Root slices of teeth with cold-burnished gutta-percha filling with AH26 sealer were used for comparison. The root slices were placed in tissue culture cluster, and 1 mL of cell suspension was added carefully over the root slice. They were incubated at 37 degrees C and 100% humidity for 4, 24, and 72 hours. RESULTS: Results showed that the cold-burnished gutta-percha provides a better substrate than amalgam and Super-EBA for cell growth and attachment. Amalgam was the most toxic material, showing early manifestation of cell injury. CONCLUSION: It was concluded that the composition and surface texture of the substrate have an influence on the morphology and the attachment of the PDL fibroblasts. It is suggested that cell attachment and morphology might reflect the biocompatibility of the substratum.     

Accumulation of Streptococcus mutans on Light-Cured Composites and Amalgam: An In Vitro Study

Abstract: The aim of the in vitro study was to examine the accumulation of Streptococcus mutans on light-cured composite materials and amalgam. Bacteria cultures were grown in a brain heart infusion medium, and their growth rate was determined through turbidity measurements. The data, so obtained, were evaluated statistically by analysis of variance (ANOVA) followed by Scheffe test. Experiments on amalgam showed better results compared to those on composite materials. There were no statistically significant differences in plaque accumulation on different composite materials after finishing and polishing procedures, compared to plaque accumulation on composite materials against a Mylar strip.     

Cytotoxicity of corroded and non-corroded dental silver amalgams

The cytotoxicity of one conventional and four non-γ₂-amalgams was studied in a cell culture system, using the Millipore filter method. Before testing set amalgam specimens were kept in distilled water or in artificial saliva at pH 4, 5 or 7 for up to 28 wk to produce a corrosion layer on the test surface. Non-corroded set amalgam specimens were also tested. None of the non-corroded, set amalgams showed any sign of surface accumulation of cytotoxic products whereas the corroded amalgams showed varying degrees of cytotoxicity. Generally, the non-γ₂-amalgams gave a more pronounced cytotoxic effect than the conventional amalgam. When the corrosion procedure was carried out at pH 7, the various non-γ₂-amalgams showed different degrees of cytotoxicity. It appears that the difference in cytotoxic effect between the non-γ₂-amalgams and the conventional amalgam as well as the differences among the various non-γ₂-amalgams could be related to variation in the retention of corrosion products deposited on the amalgam surface.     

Effect of various zinc oxide materials as root-end fillings on healing after replantation

This study examined the effect of various zinc oxide materials as root-end fillings of teeth in a replantation model. A total of 35 molar teeth were used from 19 monkeys. After extraction, root ends were resected, the canals contaminated with oral bacteria, root-end cavities prepared and fillings placed prior to replantation. After 8 weeks the teeth and surrounding jaw were removed and prepared for histological examination. Twelve roots were filled with IRM plus dentine chips, and six with Cavit; the tissue response around root ends filled with these materials as assessed by inflammation was similar to that previously reported to IRM and Super EBA cement and was characterized by little or no inflammation of limited extent. In contrast, more severe inflammation was observed around root ends filled with plain zinc oxide–eugenol or Kalzinol; however, the reaction was neither as severe nor as extensive as that to amalgam root–end fillings. Giant cells were observed most often on the surface of fillings with Cavit and zinc oxide–1eugenol. It is concluded that the tissue response to IRM with or without added dentine, Super EBA and Cavit was similar and mild; it was less severe than that to zinc oxide-eugenol and Kalzinol. All these materials had a much more favourable response than amalgam.