The growth and structure of human oral keratinocytes in culture.

Human keratinocytes derived from explants of cheek (buccal) mucosa grow vigorously in culture and can be subcultivated twice. The structure of the oral keratinocytes in vitro is the same in primary cultures and subcultures. The cells stratify, are characterized by well-developed tonofibrillar-desmosomal complexes, and rarely exhibit signs of terminal differentiation. Unique features of the culture system that favor keratinocyte growth are: incubation at 34 degrees C, inclusion of 0.5% dimethyl sulfoxide in the culture medium, and initiating subcultures as 5.0 mm colonies containing 100,000/20 microliter of medium. One primary culture can yield 6 first-passage subcultures, which subsequently achieve confluence in 10-12 days. Such cultures are a useful source of human keratinocytes that stratify but generally do not undergo terminal differentiation.

Formaldehyde cytotoxicity in three human cell types assessed in three different assays.

International standards for preclinical screening of the cytotoxicity of dental materials so far recommend the use of established cell lines. The aim of this study was to assess the relative susceptibility of human dental pulp fibroblasts (HPF), human buccal epithelial cells (HBE) and HeLa cervix cancer cells exposed to identical cytotoxic challenges. Formaldehyde, which may be released from dental materials such as dental composites, glassionomer cements, and endodontic sealers, was used as test chemical. Cytotoxicity data including dose-response relations and TC(50) values were assessed in three different assays: BrdU incorporation, neutral red uptake and MTT assays. HBE and HPF demonstrated statistically significant lower TC(50) values in both the neutral red and the BrdU assay in comparison to HeLa cells. In the MTT assay no statistically significant differences were observed between the cell types. In the two target-tissue cell types (HPF and HBE) the Neutral Red assay revealed lower TC(50) values in comparison to the BrdU assay. In HeLa cells no statistically significant differences were observed between the assays. In conclusion, the present study confirms that cytotoxicity data obtained by cell culture studies are influenced by both cell culture model and choice of assay. Under identical experimental conditions, human target tissue cells appeared to be more sensitive to formaldehyde toxicity than human HeLa cancer cells.

Chelate root filling cements: biological properties.

The purpose of this study was to test in vivo and in vitro the toxicity and the antibacterial activity of an experimental chelate cement (HN cement) using zinc oxide-eugenol cement as a reference. After subcutaneous injection of the spatulated HN cement paste in rats, it induced markedly less tissue injuries than did the zinc oxide-eugenol cement. In toxicity tests using cultures of human fibroblasts, the HN cement was found to be less toxic than the reference cement. Bacteriologically, blood agar plates inoculated with Streptococcus sanguis, Staphylococcus aureus, and the anaerobic Prevotella intermedia developed inhibition zones between 3 and 12 mm upon application of both cements. Both demonstrated marked bacteriostatic and bactericidal properties.

A methodical study of shape changes in human oral cells perturbed by a simulated orthodontic strain in vitro.

Cells are known to alter their shape as a response to physical and chemical changes. Mechanical loads applied to teeth produced cellular perturbations resulting in orthodontic movement. An in vitro model was developed to simulate the in vivo strain of orthodontic movement. Calibrated forces were applied to human periodontal ligament cells and buccal mucosal fibroblasts (controls). A biaxial strain-producing device was used to stretch vital cells growth on flexible polytetrafluorethylene membranes. In addition, a new cell adhesive, Cell Tak, was employed to examine the effect of an adhesive substrate on the cellular response to two known loads. The shape changes of unstrained (control) and strained cells were evaluated by time-lapse telemicroscopy, and plots of time-dependent alterations in area and shape were recorded. The fusiform cells became more rounded over a given time of up to 1400 s. The responses appeared to be independent of cell type, the strain employed, and the presence of cell adhesive. Scanning electron microscopy demonstrated, irrespective of cell type, that the surface of stressed cells produced a striking number of microvilli as compared with the relatively smooth-surfaced controls.

Antagonistic effect of selenite on tumor promoter induced cell proliferation in cultures of rat tongue epithelium.

In cultures of rat tongue epithelial cells, cell proliferation following incubation with different doses of the potent tumor promoter TPA has been studied by using a stathmokinetic method counting colchicine arrested metaphases. It was demonstrated that 24 h incubation with concentrations higher than 5 ng TPA/mL medium caused inhibition, whereas below 5 ng TPA/mL medium caused stimulation of the mitotic activity reaching a maximum around 30 h from the start of the incubation period. Based on the evidence of the anticarcinogenic effect of selenium in several animal models, experiments have been performed elucidating the influence of an atoxic dose (1/1.000.000M) of selenite on the observed TPA-induced cell proliferation. Our results indicate that addition to the culture medium of an atoxic dose of selenite, not affecting the mitotic activity of control cultures, inhibits the TPA-induced stimulation of cell proliferation.

Environmental issues in dentistry--mercury. FDI Commission.

One of the consequences of placing amalgam restorations is that mercury is required for the trituration process. In turn, this raises the issue of the possible environmental impact of mercury. This report considers ways in which any impact can be modified and reduced by careful attention to mercury usage and hygiene in the dental practice, the use of filters and separators in waste water pipes and the appropriate disposal of waste contaminated with amalgam. The total amount of mercury discharged into the environment varies considerably in different parts of the world due to both natural and human activities. The extent to which dentistry adds to this total also varies according to local circumstances and requirements. Recommendations are given for further development of ways to reduce mercury discharge and for further research into the environmental impact of the metal.

Dental amalgam--environmental aspects.

Increasing knowledge about the risk of toxic effects caused by anthropogenic mercury accumulation in ecosystems has resulted in a growing pressure for reduction of the discharge of mercury waste. Consequently, the mercury waste problems of dental clinics have been given increased attention, and restrictions on handling and discharge of contaminated waste have been established in several countries. Major amalgam particles from trituration surplus of those produced during the carving and burnishing of new amalgam restorations are generally collected in coarse filters and sold for refinement. Minor amalgam particles released by production of new fillings or by removal of old restorations partly sediment in tubes and drains. The remaining particles are carried with the waste water stream to the local purifying plant. In Scandinavia, the industrial discharge of mercury-contaminated waste water has been reduced to a minimum. According to recent investigations, dental clinics appear to be responsible for the major amount of mercury collected in the sludge generated in purifying plants. If threshold values for heavy metal content, including mercury, are exceeded, the sludge is not allowed to be recycled as fertilizer. Installation of an approved amalgam-separating apparatus in dental clinics is now mandatory in several countries--for example, Switzerland, Germany, Sweden, and Denmark. Approval of amalgam separators is based on national testing programs, including clinical or laboratory tests demanding 95-99% separating efficiency.

Environmental aspects of dental filling materials.

In recent years, the possible environmental impact caused by certain routines in dental practice has attracted attention among regulators. As part of point source reduction strategies, the discharge of mercury/amalgam-contaminated wastes has been regulated in a number of countries, even though it has been documented that by adopting appropriate mercury hygiene measures, including installation of amalgam-separating devices, the environmental impact of amalgam use in dentistry is minimal. There are, so far, no data indicating the environmental impact of methacrylate-based dental filling materials. As to the occupational environment, recent reports have stated that when normal occupational recommendations for proper mercury hygiene routines are followed (e.g., water spray coolant and high vacuum suction during removal of amalgam restorations), no occupational health risk can be assumed. An increasing number of reports on occupational allergic reactions to components of polymer-based dental filling materials call for attention to the sensitizing potential of certain ingredients in these products.

Effect of low level diode laser irradiation of human oral mucosa fibroblasts in vitro.

The effects of low level laser (LLL) irradiation on the proliferation of human buccal fibroblasts were studied. A standardized LLL set-up was developed (812 nm, 4.5 +/- 0.5 mW/cm2). Cultures in petridishes were divided into eight groups (1 group served as control). On day 6 after seeding, routine growth medium was replaced with PBS for 1/2 hour. At the beginning of this period, LLL irradiation was performed for 0, 1, 3, 10, 32, 100, 316, or 1,000 seconds, respectively--corresponding to the radiant exposures 0, 4.5, 13.5, 45, 144, 450, 1,422, 4,500 mJ/cm2. Subsequently the cells received 3H-dT in fresh medium for 16 hours DNA-incorporation. Scintillations from tritium and total protein concentration per culture dish were determined. The individual 3H-cpm/protein-concentration ratios were calculated in % of control. Three experiments were performed (N = 151). Following LLL exposure the 3H-cpm/protein ratio was increased with maximum cpm/protein ratio (132.5% +/- 10.6% SEM) in the group receiving 450 mJ/cm2 (P < 0.03 nonparametric Kruskal Wallis one-way ANOVA-test). This study demonstrated an increased incorporation on tritiated thymidine in cultured human oral fibroblasts following LLL exposure and suggests that LLL irradiation can induce increased DNA synthesis.

Characterization of two types of human oral fibroblast with a potential application to cellular toxicity studies: tooth pulp fibroblasts and buccal mucosa fibroblasts.

Cultures of human oral buccal mucosa fibroblasts and human tooth pulp fibroblasts were established and grown under standard routine conditions. The biological characteristics of cell proliferation, growth pattern, cell morphology and enzyme release (lactate dehydrogenase and acid phosphatase) were studied. Under the same standard in vitro conditions the two cell types demonstrated different growth patterns and different levels of enzyme activity. It is suggested that differences in biological characteristics should be considered when selecting appropriate cells for toxicological studies of dental materials.

A simple model for evaluating relative toxicity of root filling materials in cultures of human oral fibroblasts.

Standardized test tubes filled with freshly mixed root filling materials (AH26, CRCS, N2, Kloroperka NO, ZOE cement and 2 experimental cements, ECI and ECII) were transferred into tissue culture flasks. Normal human oral fibroblasts were seeded in the flasks. Morphological cell changes were studied up to 15 days after seeding. The size of cell-free zones around the test tubes and the total cell number per culture flask were calculated after 5, 10 and 15 days. The findings showed N2 cement to be by far the most toxic material at all observation periods, whereas no toxic reactions could be seen in relation to tubes filled with Kloroperka NO. Compared with the 5-day observation period, some cell recovery was observed around test tubes with AH26 and ECII, whereas almost full cell recovery was found around test tubes with CRCS, ZOE and ECI. It was concluded that the present model, which allows long-term observations of human cellular reactions to dental materials, can be used as a simple and relatively cheap screening test for initial toxicity testing of dental materials.

Angiogenic activity of malignant oral epithelial cells: a preliminary study.

We investigated the angiogenetic capacity of normal and malignant oral epithelial cells by intradermal injection of cells into nude mice. Cells investigated: 1) a tumorigenic cell line from rat palate; 2) a non-tumorigenic epithelial cell line from rat palate; 3) cells from second passage of rat palate epithelium explant cultures; 4) cells from epidermis of neonatal mice. A 0.1 ml suspension containing 10(5) epithelial cells was injected into the flank region. Mice were killed 8 days later to evaluate angiogenesis. Under a dissection microscope, the number of vessels in the skin adjacent to the injection site was counted. The tumorigenic cell line demonstrated the strongest angiogenetic capacity and the non-tumorigenic cell line was more angiogenic than any of the other non-tumorigenic cell types. The greater angiogenic activity of neoplastic oral epithelial cells may be a useful feature in the evaluation of oral malignant development.

Toxic effects of two dental materials on human buccal epithelium in vitro and monkey buccal mucosa in vivo.

Confluent cultures of human buccal epithelial cells were exposed to graded dilutions of Gluma Bond or 3M Etching Liquid for 5 min. The cytotoxic effects induced by this treatment were observed (epithelial cell damage, growth inhibition). In vivo, monkey buccal mucosa was exposed to Gluma Bond or 3M Etching Liquid for 5 min. Biopsies were taken after 24 h, and the buccal epithelium processed for light microscopic examination. The toxic reactions to Gluma Bond were far more pronounced compared with the toxic reactions to 3M Etching Liquid in both models. Data obtained suggest that the in vitro model may be useful in assessing mucosal toxicity and in studying mechanisms of toxic action.

Behavior of in vitro grown normal human mucosal epithelial cells and tumorigenic rat cells inoculated into nude mice.

The present study describes the behavior of in vitro grown normal human oral mucosal epithelial cells and that of a tumorigenic epithelial cell line following subcutaneous inoculation into nude mice. A successful recovery of viable human epithelial cell inocula was seen in 25-90% of mice and there was no improvement in recovery rates after addition of fibroblasts. These inocula resulted in cyst formation lined by a 2-6 cell layer unkeratinized squamous epithelium without rete ridges. There was no increase in recovery rate or size of cysts when coinoculated with fibroblasts. The tumorigenic cell inocula were successfully recovered in all cases. Tumors established from these inocula had a low grade of differentiation and were without signs of metastasis. Inocula of tumorigenic cells showed an increased size after addition of fibroblasts to the inocula. The model may be useful in studies of interactions between inoculations of heterologous normal and pathologic cells as well as in studies of differentiation of carcinogen-treated epithelial cells.

Bisphenol-A content of resin monomers and related degradation products.

Recently, it was reported that Bisphenol-A (BPA) was released from one fissure sealant (Delton) into saliva causing estrogenic activity in vitro. The aim of this study was to chemically analyze the BPA content of different fissure sealant resin monomers and their release of BPA under hydrolytic conditions. BPA content was first measured in commercially available monomers of bisphenol-A glycidyldimethacrylate (Bis-GMA), bisphenol-A dimethacrylate (Bis-DMA) and bisphenol-A diglycidylether (BADGE). Then, Bis-GMA-monomer and Bis-DMA-monomer in methanol were subjected to pH values of 0 to 11 for 30 minutes at 50 degrees C, to porcine liver esterase, and to pooled saliva for up to 24 hours. The BPA-content was determined by high-performance liquid chromatography (HPLC). Bis-GMA-monomer and BADGE-monomer from one manufacturer did not contain any detectable amounts of BPA (< or = 2 ppm); Bis-DMA and BADGE-monomer from a second manufacturer contained BPA quantities of 4-155 ppm. For Bis-GMA-monomer, no BPA could be detected under any hydrolytic conditions chosen (detection limit: < or = 1%). For Bis-DMA-monomer an increase of BPA was observed at pH 11, resulting in a conversion of approx. 100% Bis-DMA to BPA. When Bis-DMA was subjected to esterase, a conversion of 82.5% resulted after 24 h; saliva led to an 81.4% conversion of Bis-DMA after 24 h. Hence, we conclude that the results reported in the literature may be attributed to the Bis-DMA-content of the fissure sealant tested (Delton). No BPA-release is expected under physiologic conditions from fissure sealants based on Bis-GMA if pure base monomers are used.

Time-related bisphenol-A content and estrogenic activity in saliva samples collected in relation to placement of fissure sealants.

It was recently reported that estrogenic activity was detected in saliva samples collected during 1 h after placement of one fissure sealant (Delton) and this related to Bisphenol-A (BPA) content. The aim of the present study was to determine the time-related BPA content and estrogenic activity in saliva samples collected before and after placement of two fissure sealants each with a different monomer composition. Eight healthy male volunteers with no history of prior placement of fissure sealants or composite resin fillings had four molars sealed with either Delton LC (four people) or Visio-Seal (four people). Base-line saliva samples were collected preexperimentally, in the morning when fasting. Fissure sealants were placed and saliva samples collected immediately, 1 h and 24 hs after placement of the fissure sealant. BPA was found in saliva samples collected immediately after placement of Delton LC (range 0.3-2.8 ppm). No detectable amounts of BPA were determined 1 h and 24 h after Delton treatment (detection limit < or = 0.1 ppm). In base-line samples and in all samples collected from Visio-Seal treated individuals, no BPA was detected. In a recombinant yeast cell assay, significantly increased estrogenic activity was found in saliva samples collected immediately after placement of Delton LC sealant (P < 0.05; ANOVA) whereas no statistically significant estrogenic activity was observed in the remaining groups. In conclusion, minute amounts of BPA, however considerably lower than previously reported, were detected in saliva samples collected immediately after but not 1 and 24 h(s) after placement of Delton LC fissure sealant. BPA was not detected after placement of Visio-Seal fissure sealant.

Epithelium-fibroblast co-culture for assessing mucosal irritancy of metals used in dentistry.

No valid animal or in vitro model exists to assess the potential mucosal irritancy of dental materials. However, recently, a commercially available model system based on a recombined co-culture of human fibroblasts and human epithelial cells has been introduced for evaluating the time-dependent irritancy of cosmetic products. Cell viability and prostaglandin E2 (PGE2) release from the cells were used as markers for the irritative potential of test materials. The objective of the present study was to evaluate the suitability of this model for monitoring the irritative potential of metals and cast alloys used in dentistry. The human fibroblast-keratinocyte co-cultures were exposed to test specimens fabricated from copper, zinc, palladium, nickel, tin, cobalt, indium, a high noble cast alloy, and from a dental ceramic. Cell survival rates decreased after exposure to copper (14-25%), cobalt (60%), zinc (63%), indium (85%), nickel (87%), and the non-oxidized and oxidized high noble cast alloy (87%/90%) compared to untreated control cultures. Dental ceramic, palladium and tin did not influence cell viability. In parallel, the PGE2 release was continuously monitored up to 24 h using a competitive displacement enzyme immunoassay. PGE2 release increased most highly in the cultures exposed to copper (6-25 fold), cobalt (7 fold), indium (4 fold), and zinc (2 fold) compared to untreated control cultures. The PGE2 determination proved to be a non-destructive method for continuous monitoring of cell reactions in the same culture. The model used seems promising for evaluating the time-dependent mucosal irritancy of dental cast alloys.

Effect of folic acid on human oral epithelium in vitro.

The cytomorphological effects of folic acid were studied using in vitro established human oral epithelium. It was demonstrated that a concentration twice that used clinically (200 micrograms/ml) did not induce marked cytotoxic reaction in the cultured cells. The most pronounced changes were observed in cultures exposed to 200 micrograms/ml folic acid both in primary culture and subculture. The cultures displayed areas of degenerating cells showing oedema and increased translucency of the cytoplasm, flattened cells with distinct tonofilaments and atypical mitotic figures. Identical changes were found in cultures exposed to 50 and 100 micrograms/ml folic acid but to a lesser extent than in 200 micrograms/ml. These changes indicated that folic acid increased the number of cells undergoing terminal differentiation. From this study we suggested that folic acid when applied topically may play a role in local stimulation of epithelial cell differentiation leading to enhanced healing of oral ulcers.