C. T. Hanks

Cytotoxic Effects of Resin Components on Cultured Mammalian Fibroblasts

The objectives of this study were to determine the cytotoxic concentrations of 11 components of resin composites on monolayers of cultured Balb/c 3T3 fibroblasts, to study the inhibitory effects of these components on DNA synthesis, total protein content, and protein synthesis, and to determine whether effects were reversible when the components were withdrawn from the medium. These data were reported as concentrations which inhibited 10% (ID10) and 50% (ID50) of a particular metabolic process as well as the range of concentrations over which cell metabolism was irreversibly inhibited. For any individual component, the ID50 values for all three metabolic parameters were of the same magnitude. The same was true for the ranges of irreversibility. Ethoxylated Bis-phenol A dimethacrylate (E-BPA) was the most toxic molecule of the group (ID50 being between 1 and 10 μmol/L). The ID50 concentrations for three of the components, including Bis-GMA, UDMA, TEGDMA, and Bis-phenol A, ranged between 10 and 100 μmol/L, while the ID 50 values of three components (N,N dihydroxyethyl-p-toluidine, camphoroquinone, and N,N dimethylaminoethyl methacrylate) were above 100 μmol/L. The concentrations to which the cells and tissues are exposed in uiuo are not known. This study should help to identify the concentrations of organic composite components which pose clinical cytotoxic hazards.

Cytotoxic Interactive Effects of Dentin Bonding Components on Mouse Fibroblasts

Previous studies have shown a wide range of pulpal reactions to dentin bonding systems and a poor correlation between in vitro and in vivo toxicity of dentin bonding agents. Because dentin bonding agents are composed of multiple components which may diffuse through dentin, we hypothesized that these components may cause cytotoxicity through interactive (synergistic) effects. We investigated the cytotoxicities of four dentin bonding components-HEMA, Bis-GMA, TEGDMA, and UDMA-and interactive effects for three binary combinations of the dentin bonding components-HEMA and Bis-GMA, Bis-GMA and TEGDMA, and TEGDMA and UDMA. Cytotoxicities to Balb/c 3T3 mouse fibroblasts were measured by the MTT assay. Concentrations which caused 50% toxicity compared with controls (TC50 values) were compared, and the interactive effects were determined by evaluation of the differences between observed and expected MTT activities of the cells. The ranks of toxicity of the dentin bonding components in terms of TC50 values were as follows: Bis-GMA > UDMA > TEGDMA >>> HEMA (least toxic) after 24- and 72-hour exposures. As binary combinations, the three combinations of dentin bonding components interacted in three ways—synergism, additivism, and antagonism-which were influenced by the concentrations of both components. The longer period of exposure resulted in a significant increase in the cytotoxicity of the dentin bonding components and combinations. The findings indicate that both exposure time and the interactions between the dentin bonding components may be important parameters in determining the cytotoxicity of dentin bonding agents in vivo.

In vitro models of biocompatibility: A review

The objectives of this paper were to define in vitro biocompatibility of materials, to discuss some of the issues concerning why conclusions from tissue culture are sometimes different from in vivo biocompatibility, to give highlights of the sequence of the development of these in vitro assays from the early 1950s to their present state of development, and to discuss possible future trends for in vitro testing. In vitro biocompatibility tests were developed to simulate and predict biological reactions to materials when placed into or on tissues in the body. Traditional assays have measured cytotoxicity by means of either an end-stage event, (i.e., permeability of cytoplasmic membranes of dead and dying cells, or some metabolic parameter such as cell division or an enzymatic reaction). In vitro assays for initiation of inflammatory and immune reactions to materials have also begun to appear in the literature. More recently, the concept of dentin barrier tests has been introduced for dental restorative materials. Four models which measure both permeability and biological effects of materials are compared and discussed. Future efforts may be directed toward development of materials which will allow or promote function and differentiation of tissues associated with materials. New analytical procedures and understanding of optimal characteristics of materials should improve our ability to develop more biocompatible materials. Both molecular biology techniques, and altered design of material surfaces may make the materials either more or less reactive to the biological milieu. These trends suggest a greater future role of the biological sciences in the development of biomaterials.

Effects of metal ions on osteoblast-like cell metabolism and differentiation

The objective of this study was to evaluate the effects of metal ions, which may be released from orthopedic or dental implants, on osteoblast metabolism and differentiation. ROS 17/2.8 cells were cultured in F-12 medium for 7 days. Then Al+3, Co+2, Cr+3, Ni+2, Ti+4, and V+3 were added at concentrations less than their cytotoxic concentrations. After 3 days, DNA synthesis, succinate dehydrogenase activity, alkaline phosphatase (ALP) activity, and culture calcification were assessed. Northern blots were performed for ALP, osteocalcin (OCN), and osteopontin (OPN) mRNA transcription. The data indicated that Cr+3 and A1+3 had few inhibitory effects on ROS cell metabolism below their cytotoxic concentrations, Ni+2, Co+2, Ti+4, and V+3 affected all these parameters of ROS cell metabolism at concentrations below cytotoxic levels. For RNA analysis, A1+3 significantly suppressed the expression of ALP, OCN, and OPN at both cytotoxic and noncytoxic concentrations. Co+2 specifically suppressed ALP expression at cytotoxic concentrations. Cr+3 and Ni+2 inhibited OCN, OPN, and ALP gene expression only at cytotoxic concentrations. For Ti+4 and V+3 ions, gene expression at cytotoxic levels was not significantly affected as compared with the effects at noncytotoxic level. These results show that metal ions may alter osteoblast behavior even at subtoxic concentrations, but do not always affect the expression of all genes similarly.

In vitro cytotoxicity and dentin permeability of HEMA

An in vitro diffusion chamber was used to measure the diffusion of 2-hydroxyethyl methacrylate (HEMA) through etched human dentin disks. Concentrations of HEMA, which diffused through dentin, were measured by ultraviolet spectroscopy, and the effect of initial HEMA concentration, dentin thickness, and back pressure on diffusion were assessed. The cytotoxicity of HEMA was determined using BALB/c 3T3 mouse fibroblasts in direct contact with HEMA for 12 or 24 h. HEMA diffused rapidly through dentin under all conditions, but increased thickness, back pressure, or decreased initial concentration all reduced diffusion. The permeability coefficient of HEMA was approximately 0.0003 cm/min, and diffusion through 0.5 mm of dentin reduced the HEMA concentration by a factor of approximately 6,000 (with 10 cm of H2O back pressure). It was concluded that the risk of acute cytotoxicity to HEMA through dentin was probably low, but that decreased dentin thickness, lack of polymerization, or extended exposure times might increase the risk significantly.

CLONED 3T6 CELL LINE FROM CD-1 MOUSE FETAL MOLAR DENTAL PAPILLAE

Only primary pulpal cell cultures and one virally transformed mouse cell culture have been formally reported in the literature to synthesize proteins such as phosphophoryn which are unique to dentin matrix. In the present study, a mixed culture was derived from dental papilla cells of 18-19 fetal day CD-1 mouse mandibular first molars, maintained on a 3T6 plating regimen, and subsequently cloned after 28 passages. This cloned cell line (MDPC-23) exhibited several unique features, some of which were characteristic of odontoblasts in vivo. The features of this cell line included (1) epithelioid morphology of all cells with multiple cell membrane processes, (2) high alkaline phosphatase activity in all cells, (3) formation of multilayered nodules and multilayered cultures when maintained in ascorbic acid and beta-glycerophosphate, and (4) expression of two markers for odontoblast differentiation, i.e. dentin phosphoprotein and dentin sialoprotein.

Current status of pulp capping with dentin adhesive systems: a review

UNLABELLED Several studies have assessed the morphology and thickness of hybrid layer, the dentin bond strengths as well as sealing ability of dentin adhesive systems. However, few in vivo studies have evaluated the biocompatibility of the adhesive systems following application to deep dentin or directly to the pulp of human teeth. Many studies performed in non-human primate teeth or teeth of rats have reported pulp healing and dentin bridging following pulp capping with bonding agents. In addition, a few clinical and radiographical reports of the success of resin pulp capping have been described in the dental literature. OBJECTIVES The aim of this review was to evaluate the literature on pulp responses following total acid etching and application of adhesive resins on deep cavities or pulp exposures. In addition, the clinical/radiographical evidence for the apparent success of vital pulp therapy and results obtained from animal and human studies were compared and discussed. SIGNIFICANCE AND CONCLUSIONS The self-etching adhesive systems may be useful and safe when applied on dentin. In contrast, persistent inflammatory reactions as well as delay in pulpal healing and failure of dentin bridging were seen in human pulps capped with bonding agents. The results observed in animal teeth cannot be directly extrapolated to human clinical conditions. Consequently, vital pulp therapy using acidic agents and adhesive resins seems to be contraindicated.

The Release of Elements of Dental Casting Alloys into Cell-culture Medium

Ten dental casting alloys were tested for alloy-element release into cell-culture medium, and this release was related to alloy composition, alloy microstructure, and alloy cytotoxicity (previously determined). Cell-culture medium was analyzed for alloy elements by flame atomic absorption. Concentrations of elements in the medium were normalized by dividing them by their atomic abundance in the alloy, giving element medium-alloy ratios (EMA ratios). Results showed that Au, In, and Pd generally did not dissolve into the medium, but that Ag, Cd, Cu, Ga, Ni, and Zn frequently dissolved. Comparison of EMA ratios for Ag, Cu, and Zn showed that each element retained a behavioral identity in diverse metallurgical environments, but that these environments influenced the release behavior to some degree. Some EMA ratios in multiphase alloys were greater than those in solid solutions, and EMA ratios showed great diversity within all the alloys. Nominal composition seemed to be of little value in the prediction of metal release unless the composition supported multiple-phase formation. In addition, release of alloy elements did not, in itself, completely predict alloy cytotoxicity measured previously. However, cytotoxicity was associated with metal release in each case. The commercial alloys used in this study exhibited more complex and less predictable release behavior than did the simpler ternary alloy systems used by previous investigators. It is believed that the use of commercial preparations is necessary for their in vivo behavior to be modeled.

Effect of cell line on in vitro metal ion cytotoxicity

OBJECTIVES The choice of cell line for in vitro biological tests which assess the cytotoxicity of dental materials remains controversial, yet this issue is important because these tests are widely used to rate the biocompatibility of new and existing materials, and many different cell lines are commonly used. The purpose of the current study was to quantify the responses of four cell lines (Balb/c 3T3, L929, ROS 17/2.8 and WI-38) to 14 metal ions which are released from dental materials, and relate these responses to the metabolic activity and population doubling times of these cells. METHODS Succinic dehydrogenase (SDH) activity was used to monitor metabolic activity and cytotoxic response. RESULTS The cell lines responded differently to most metal ions. In general, the Balb/c 3T3 line was the most sensitive, and the WI-38 line was the least sensitive. However, there were many exceptions depending on the metal ion. The passage number of the cells also affected the cytotoxic response. It was concluded that the cytotoxicity of materials which release metal ions will be significantly different depending on which cell line is selected and its passage number. SIGNIFICANCE Based on the findings that cell lines ranked the toxicities of the metal ions similarly, it seems reasonable to use these types of in vitro tests to rank the cytotoxicities of materials. However, if these types of tests are used to predict in vivo cytotoxicity, care should be taken to choose conditions and cells which are relevant.

Cytotoxic effects of current dental adhesive systems on immortalized odontoblast cell line MDPC-23

OBJECTIVES Evaluate the cytotoxic effect of the three dental adhesive systems. METHODS The immortalized mouse odontoblast cell line (MDPC-23) was plated (30,000 cell/cm2) in 24 well dishes, allowed to grow for 72 h, and counted under inverted light microscopy. Uncured fresh adhesives were added to culture medium to simulate effects of unset adhesive. Three adhesives systems were applied for 120 min to cells in six wells for each group: Group 1) Single Bond (3M), Group 2) Prime & Bond 2.1 (Dentsply), and Group 3) Syntac Sprint (Vivadent). In the control group, PBS was added to fresh medium. The cell number was counted again and the cell morphology was assessed under SEM. In addition, the adhesive systems were applied to circles of filter paper, light-cured for 20 s, and placed in the bottom of 24 wells (six wells for each experimental materials and control group). MDPC-23 cells were plated (30,000 cell/cm2) in the wells and allowed to incubate for 72 h. The zone of inhibition around the filter papers was measured under inverted light microscopy; cell morphology was evaluated under SEM; and the MTT assay was performed for mitochondrial respiration. RESULTS The fresh adhesives exhibited more toxic (cytopathic effects) to MDPC-23 cells than polymerized adhesives on filter papers, and as compared to the control group. The cytopathic effect of the adhesive systems occurred in the inhibition zone around the filter papers, which was confirmed by the MTT assay and statistical analysis (ANOVA) combined with Fishers PLSD test. In the control group, MDPC-23 cells were dense on the plastic substrate and were in contact with the filter paper. In the experimental groups, when acid in the adhesive systems was removed by changing the culture medium, or when the adhesives were light-cured, some cells grew in the wells in spite of the persistent cytotoxic effect. SIGNIFICANCE All dentin adhesive systems were cytotoxic odontoblast-like cells. Both acidity and non-acidic components of these systems were responsible for the high cytopathic effect of those dental materials.