N-acetyl cysteine protects pulp cells from resin toxins in vivo.

Potential risks of the use of resin-based restorative materials include direct damage to the pulp cells and the induction of hypersensitivity reactions in patients. In this study, we tested the hypothesis that N-acetyl cysteine (NAC) inhibits resin toxicity and restores the function of pulp cells. Analysis of our data demonstrates toxicity of composite resins on pulp cells in both an in vivo rat and an ex vivo human model system. Moreover, cells that survive after the placement of composites are weaker, and they are induced to undergo cell death when exposed to 2-hydroxyethyl methacrylate (HEMA). The toxic effect of composites on pulp cells is neutralized by NAC. Therefore, NAC protects the cells from damage induced by clinically relevant levels of restorative materials, in both rat and human model systems. The addition of N-acetyl cysteine prior to or concomitant with the application of restorative materials may be beneficial for the health and safety of dental patients.

A proposal for a new classification of lesions of exposed tooth surfaces.

In the presence of improved methods of identification and treatment of lesions on the exposed surfaces of teeth, it should now be acknowledged that the GV Black "classification of carious cavities" is out of date. This paper describes a new system, proposed in 1997, discussed broadly throughout the profession, and eventually modified. The system has been adopted in several regions around the world as being a useful corollary to the current developing concept of minimal intervention dentistry. It is now desirable to adopt a new approach to the identification and recording of the lesions caused by both caries and non-carious tooth loss. A major advantage arising from its adoption would be that it would encourage the profession to minimise the amount of normal healthy tooth structure that is often sacrificed in pursuit of the cavity designs as suggested by Black. The authors are members of a Project Group of the FDI Science Committee, and this paper explains the concept and offers justification for the adoption of the system.

Resin monomer 2-hydroxyethyl methacrylate (HEMA) is a potent inducer of apoptotic cell death in human and mouse cells.

Mechanisms by which the resin monomer 2-hydroxyethyl methacrylate (HEMA) induces hypersensitivity reactions in humans are not well-established, nor have the direct effects of HEMA on cell death been fully characterized. The objective of this study was to establish whether HEMA is capable of inducing apoptotic cell death, and whether differences exist in the levels of apoptotic death induced by HEMA in cells obtained from healthy individuals and from patients with established HEMA hypersensitivity. HEMA induced apoptotic death in Peripheral Blood Mononuclear Cells (PBMCs) obtained from both healthy and HEMA-sensitized patients and in the murine RAW cells in a dose-dependent manner. However, induction of cell death by HEMA was lower in PBMCs obtained from patients in comparison with healthy individuals. Studies reported in this paper demonstrate that HEMA induces apoptotic death, and that decreased susceptibility of lymphocytes to HEMA-mediated death might be an important mechanism for the generation and persistence of hypersensitivity reactions in patients.

Proline and thymidine uptake in rabbit ear artery segments in vitro increased by chronic tangential load.

Indices of structural change were examined in blood vessel walls subjected to increased tangential load in a new, in vitro model system. Ring segments of rabbit ear artery were maintained in organ culture medium for times up to 9 days. Tangential load was chronically applied with small, intraluminal springs made of 0.010 in. diameter stainless steel wire. the applied load was considered to produce levels of circumferential wall tension corresponding to those induced by a range of levels of blood pressure. The indices of structural change examined were the uptake of radioactively-labelled proline and thymidine, which indicate protein synthesis and cell division respectively. Increased uptake of both proline and thymidine was noted in artery segments under elevated mechanical tension after a latency of 3 to 4 days. The degree of uptake was related to the degree of calculated wall tension elevation. The work indicated that cell division and protein synthesis can be induced in the blood vessel wall by increased wall tension alone, in vitro.

Transient and persistent changes in rabbit blood vessels associated with maintained elevation in arterial pressure.

Arteries and veins of hypertensive rabbits were examined 8 weeks after partially constricting the abdominal aorta above both kidneys, and compared with those from sham-operated animals. Structural and functional changes in blood vessels after 2 weeks, when the arterial pressure first attained a new elevated level, have been described previously, and are now compared with changes 6 weeks later. The increase in blood vessel mass could be correlated with an increase in deoxyribonucleic acid (DNA) content. In contrast to the status at 2 weeks postoperatively, there was no increased uptake of 3H-thymidine, 3H-proline, or 3H-lysine at 2 months. Furthermore, at this time cell nuclei labeled with 3H-thymidine were infrequent. Some vessels showed evidence of change in the physical characteristics of their wall. Only minimal changes were observed in those parameters of adrenergic nerve function measured -- neuronal 3H-norepinephrine uptake and vessel wall catecholamine content -- that had been markedly changed at 2 weeks. The results of this work, together with those of other studies of this model, suggest two phases of response of the arterial wall to pressure rise: an initial dynamic proliferative cellular response mainly of vascular smooth muscle associated with changes in adrenergic neuronal parameters, and a subsequent equilibrium phase characterized by an increased number of smooth muscle cells, some changes in the extracellular components, and minimal changes in the adrenergic innervation.

Choline acetyltransferase activity in the sympathetic nerves of the rabbit ear artery.

1 No statistically significant difference in the activity of choline acetyltransferase (ChAT) was detected between sympathetically denervated and control rabbit ear artery (REA) tissue. This was interpreted as evidence against the hypothesis that endogenous acetylcholine plays an obligatory role in sympathetic postganglionic neurotransmission. 2 The values obtained for ChAT activity in the extraneuronal REA tissue were very low, but were greater than the boiled blank values. 3 Treatment with a specific inhibitor of ChAT did not reduce the REA values, while it did for rabbit iris. Addition of acetylcholinesterase to the REA assay reduced the activity of the collectable product to a markedly lesser degree than was observed with other tissues. 4 The specificity of the enzyme assay at the very low yield levels observed in the extraneuronal REA tissue was therefore questioned.

Biological clearance of HEMA in guinea pigs.

No toxicokinetic data are available about the dental composite component 2-hydroxyethylmethacrylate (HEMA) in vivo in the literature. Therefore, the excretion of HEMA in feces and urine in vivo and, using the pendular perfusion technique with segments of jejunum and colon, in the biliary and enteric excretion in situ were investigated in anesthetized guinea pigs. In the in situ experiments, guinea pigs (n = 4) received HEMA (0.02 mmol/kgbw labelled with a tracer dose 14C-HEMA 0.3 kBq/gbw) injected into the jugular vein. In the in vivo experiments, guinea pigs (n = 4) received HEMA (+ 14C-HEMA, same dose as above) via gastric tube. Urine and feces were collected for 24h. In the in situ experiments, organs from guinea pigs were removed 60 min after the beginning of the experiment, and then the 14C-radioactivity was measured. During the 60 min perfusion period the calculated amount of 14C-activity excreted into the total jejunum and colon was 6.0 +/- 1.0% and 2.7 +/- 0.7% of the dose administered, respectively (mean +/- sem). Of the 14C-HEMA dose, 5.3 +/- 0.3% was found in the bile. Significantly (p < 0.05) higher bile/blood concentration ratios were found at 10-40 min after the injection of HEMA, as compared to the ratio at 60 min. The total 14C-recovery in all organs tested was 20.0 +/- 2.6%. During 24h the amounts of 14C-activity excreted in the feces and urine were 1.1 +/- 0.1% or 17.1 +/- 1.50% of the dose administered, respectively (mean +/- sem). The total 14C-recovery in all organs tested was 11.6 +/- 0.6%. In a second series of in vivo experiments, exhaled air from the animals was captured during the 24h experimental period. 14C was exhaled to 63.6 +/- 2.11% of the administered 14C-HEMA dose (mean +/- sem; n = 4) as 14C-carbondioxide. The results indicate a rapid clearance of 14C-HEMA and/or 14C-HEMA metabolite(s) from the organism, exhalation being the major route of elimination.

Effect of eugenol on constrictor responses in blood vessels of the rabbit ear.

Aqueous solutions of eugenol depressed vasoconstrictor responses to exogenous norepinephrine (NE), histamine, and peri-arterial sympathetic nerve stimulation in the isolated central artery of the rabbit ear and in the isolated perfused whole rabbit ear. The depression was dose- and time-dependent, and was slowly reversible. Eugenol appeared to act as an inhibitor of smooth muscle contractility.

An analysis of the release and the diffusion through dentin of eugenol from zinc oxide-eugenol mixtures.

Tritium-labeled eugenol was released from mixtures of zinc oxide eugenol (ZOE) into aqueous solution at rates which declined exponentially with time, and which were directly proportional to the liquid-powder ratio. The release pattern was consistent with a model of progressive hydrolysis of zinc eugenolate in a limited-thickness ZOE surface layer. Intervening dentin had a profound effect on this pattern of release. In human teeth in vitro containing ZOE as a base or temporary filling, peak eugenol release at the pulpal surface of dentin was of the order of a thousand-fold less than that at the salivary surface. In such teeth, eugenol reached concentrations in excess of 10(-2) M in dentin just beneath ZOE, and 10(-4) M or less adjacent to the pulp space. Both pulpal outflow and dentin concentrations of eugenol remained relatively constant for more than a week, unlike release into aqueous solution. While these data were derived from studies on human teeth in vitro, they give a strong indication of probable events in vivo, and appear to provide a basis for the explanation of the paradox of the therapeutic and toxic actions of ZOE.

A new technique for screening chemical toxicity to the pulp from dental restorative materials and procedures.

An in vitro test system is described which allows for quick and relatively inexpensive examination of the potential for chemical toxicity to the pulp of materials and procedures used in the restoration of single teeth. The test system consisted of two sequential steps. First, a restorative procedure was carried out on a freshly-extracted human tooth crown, to the pulpal surface of which had been attached a chamber filled with sterile tissue-culture medium. The preparation was kept at 37 degrees C. The culture medium was removed at day one and replaced with fresh medium, which was removed at day 3. In the second step, we used a standard tissue-culture toxicity assessment technique to examine both culture medium samples for the presence of chemical toxins. In use, this system gave results which correlated well with the known clinical potential for pulpal toxicity of various dental materials and techniques. For example, zinc oxide-eugenol used as temporary filling or base had no apparent potential for toxicity. Sealing a cotton pellet containing phenol into a cavity was of high apparent potential toxicity. Acrylic resin as intracoronal or extracoronal fillings showed potential for toxicity; this potential was decreased by lining with calcium hydroxide cement. Composite resin placed onto etched dentin had apparent toxic potential, but had less such potential when placed onto unetched dentin. The technique had some advantages over previously described in vitro toxicity test for restorative materials, because it included a step requiring diffusion of potential toxins into and through human dentin, and because it allowed for examination of variations in technique which mimic clinical behavior, and of materials used in sequence or in combination.

Effect of eugenol on respiration and division in human pulp, mouse fibroblasts, and liver cells in vitro.

Eugenol depressed cell respiration in homogenates of human dental pulp and in mouse fibroblast monolayers. The depression was concentration-dependent, with a threshold at about 10(-4)M and a maximum at 10(-3)M in both preparations. Onset of the depression appeared to be rapid. The effects of variation in both duration and concentration of eugenol exposure on subsequent uptake of 3H-thymidine were examined in mouse fibroblast monolayers and human pulp explants. Fibroblasts survived short-term (up to 12 hr) exposure to 10(-3)M eugenol or less, but died after exposure to 10(-3)M for one day or more. The cells survived exposure to 10(-4)M for ten days, the longest period examined. Human pulp maintained in tissue culture medium showed similar eugenol susceptibility. Analysis of these data, when coupled with those of previous studies on eugenol release from ZOE and diffusion through dentin, gives strong support for the concepts that: the blandness of ZOE when applied to intact dentin is due to eugenol reaching the pulp in sub-toxic concentrations, and the irritant effect of ZOE when applied directly to soft tissue is due to the development of concentrations of eugenol in tissue adjacent to ZOE sufficient to inhibit respiration and thus kill cells.

Effect of hydrostatic pressure on the diffusion of monomers through dentin in vitro.

In previous work, the diffusion of monomers from composite and bonding resins through dentin was demonstrated in vitro. The monomers triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) were identified in samples from the pulp space. In the current study, we examined the effects of two levels of positive hydrostatic pressure on the passage of resin monomers through dentin in vitro from a composite-resin/bonding-resin combination to test the hypothesis that monomer diffusion is prevented by such pressure. An occlusal cavity prepared in the tooth crown was restored with the resins. Distilled water samples from the pulpal space were removed over time and analyzed for monomer content by high-performance liquid chromatography and mass spectrometry. Positive pulpal pressure reduced but did not prevent pulpward movement of diluent monomers that leach from bonding agents and from resin composites through dentin in vitro. The degree of reduction of diffusion was greater with TEGDMA than with the lower-molecular-weight monomer HEMA.

In vitro studies on the potential for pulpal cytotoxicity of glass-ionomer cements.

Elution samples of glass-ionomer cement were prepared in sterile tissue culture medium either by direct contact between the fluid and standard cement samples or through a layer of human dentin, and then tested for toxicity to cultured mouse fibroblasts (L929). The directly-prepared eluates of the cements were highly cytotoxic, but those prepared through dentin were of either limited or no cytotoxicity. The degree of toxicity of some directly-prepared eluates was reduced by adjustment of the pH to neutrality. It was apparent that dentin reduced the potential for cytotoxicity of glass-ionomer cements to a large degree. Proposed mechanisms for the reduction were limited availability of water at the dentin-cement interface and thus limited dissolution of components, buffering of acid components of the cements by dentin, or other chemical interactions with dentin.

Effects of extracellular plaque components on the chlorhexidine sensitivity of strains of Streptococcus mutans and human dental plaque.

An in vitro study was undertaken to determine the effects of sucrose-derived extracellular plaque components on the sensitivity of selected oral bacteria to chlorhexidine (CX). Cultures of Streptococcus mutans HS-6, OMZ-176, Ingbritt C, 6715-wt13, and pooled human plaque were grown in trypticase soy media with or without 1% sucrose. The sensitivity to CX of bacteria grown in each medium was determined by fixed-time exposure to CX and subsequent measurement of 3H-thymidine uptake. One-hour exposure to CX at concentrations of 10(-4) M (0.01% w/v) or greater substantially inhibited subsequent cellular division among all the S. mutans strains and human plaque samples tested. An IC50 (the CX concentration which depressed 3H-thymidine incorporation to 50% of control level) of close to 10(-4) M was noted for S. mutans strains HS-6, OMZ-176, and 6715-wt13 when grown in the presence of sucrose. The same strains grown in cultures without added sucrose showed about a ten-fold greater sensitivity to CX (IC50 close to 10(-5) M). A three-fold difference was noted for S. mutans Ingbritt C. Only a slight increase in the IC50 was noted for the plaque samples cultured in sucrose-containing media, but their threshold for depression of 3H-thymidine uptake by CX was lower than that for the sucrose-free plaque samples. The study showed that extracellular products confer some protection against CX to the bacteria examined, and provided an explanation for the disparity between clinically-recommended concentrations for plaque suppression and data on in vitro susceptibility.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution and excretion of TEGDMA in guinea pigs and mice.

The monomer triethyleneglycoldimethacrylate (TEGDMA) is used as a diluent in many resin-based dental materials. It was previously shown in vitro that TEGDMA was released into the adjacent biophase from such materials during the first days after placement. In this study, the uptake, distribution, and excretion of 14C-TEGDMA applied via gastric, intradermal, and intravenous administration at dose levels well above those encountered in dental care were examined in vivo in guinea pigs and mice as a test of the hypothesis that TEGDMA reaches cytotoxic levels in mammalian tissues. 14C-TEGDMA was taken up rapidly from the stomach and small intestine after gastric administration in both species and was widely distributed in the body following administration by each route. Most 14C was excreted within one day as 14CO2. The peak equivalent TEGDMA levels in all mouse and guinea pig tissues examined were at least 1000-fold less than known toxic levels. The study therefore did not support the hypothesis.

Movement of resin cement components through acid-treated dentin during crown cementation in vitro.

This study examined the hypothesis that components of crown cements may be forced through acid-treated dentin during cementation. Freshly extracted, human third molar teeth were prepared to accept full crowns. Roots were removed to allow irrigation of the pulp chamber with saline before, during, and after crown placement with resin dentin bond and resin composite cement. Saline samples were collected and analyzed by high performance liquid chromatography to identify and quantify resin components arriving in the pulp space. Two components of the bond-cement system used were identified in the pulp space samples immediately after crown cementation. These were 2-hydroxyethylmethacrylate and 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane. The amounts of these components in the pulp space decreased when the bonding agent was cured prior to crown placement. The results of this study supported the hypothesis that crown cementing components may flow through acid-treated dentin during crown cementation.

A study of component release from resin pit and fissure sealants in vitro.

OBJECTIVE: A recent study reported that an estrogenic chemical, bisphenol-A, was released from a fissure sealant. The aim of this study was to identify and quantify the major (or detectable) components released from any of seven commercially-available, light-cured pit and fissure sealants in vitro. METHODS: The fissure systems of ten extracted, third molar teeth were filled with sealant, light-activated and immersed in separate containers of distilled water. Separate, cylindrical stainless steel molds were filled with sealant which was then light-activated and immersed. Each mold or tooth with sealant was moved to a new container of water at defined times and each remaining water sample (eluate) then analyzed by high performance liquid chromatography (HPLC). RESULTS: Triethylene glycol dimethacrylate (TEGDMA) was present in all eluates from each of the sealants tested. 2,2-bis[4'-(2'-hydroxy-3'-methacryloyloxy)phenyl]propane (BisGMA) was detected at much lower levels (about one thousand-fold less) in eluates from one sealant only. Bisphenol-A was not detected in any eluates. The rates of TEGDMA and BisGMA release were highest on first immersion and decreased thereafter. The total amount of TEGDMA released was on the order of 0.25 mg per tooth. Most release occurred during the first day. SIGNIFICANCE: Because bisphenol-A release could not be detected from any of the seven sealants tested, these results call into question earlier concerns expressed about possible adverse effects of bisphenol-A released from resin sealants.

Surface hardness change of restorative filling materials stored in saliva.

OBJECTIVES: This study was to investigate the effect of saliva used as storage liquid and the length of storage effect on surface hardnesses of Fuji IX (GP) (FIX), Dyract (DR), Z-100 and Estio LC (ELC). METHODS: The materials were mixed according to the manufacturers' instructions and immersed in distilled water or human parotid saliva. Vickers hardness number (HVN) was measured 1, 7, 20 and 40 days after the materials were mixed. HVN was calculated from the indentation diameter after 100 or 300g loading on their surface for 15s. The two methods of characterization used in this work were X-ray photoelectron spectroscopy (XPS) for surface chemical composition and electron probe microanalysis (EPMA) for depth profile analysis. RESULTS: Only in FIX, did HVN increase with time at both storage conditions, distilled water and saliva. The increase rate of the value was higher when stored in saliva than distilled water. After 40 days storage in saliva, the HVN value of FIX increased by 39%. The increase for storage in saliva for DR was 22%, ELC 16%, and Z100 3%, compared to 1 day storage in distilled water. Ca and P peaks caused by saliva were detected by XPS and EPMA analysis, but these peaks did not exist in either composite resin or polyacid-modified composite resin by EPMA analysis. SIGNIFICANCE: Saliva has the remarkable effect of increasing surface hardness of Fuji IX (GP).

Cytotoxicity of dental composite components and mercury compounds in lung cells.

OBJECTIVE: The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA), as well as mercuric chloride (HgCl2) and methylmercury chloride (MeHgCl) was investigated on the release of lactatedehydrogenase (LDH) from alveolar epithelial lung cell lines in vitro. METHODS: The confluent cell layers from the A549 (human, malignant) and the L2 cells (rat) were incubated with various concentrations of HEMA, TEGDMA, MeHgCl and HgCl2 at 37 degrees C in 2% (v/v) CO2 atmosphere for 8h. In further experiments the L2 cells were incubated with the same compounds for 6-48 h. LDH release was measured and the values were expressed as percentage of the LDH content. The values were plotted on a concentration log-scale and the substance concentration at the maximum slope was assessed as effective concentration (EC50). RESULTS: A significant (p<0.05) increase in the LDH release was found in the L2 cells after 8-h incubation with HEMA (4 mmol/l), TEGDMA (2 mmol/l), MeHgCl (0.01 mmol/l) and HgCl2 (0.015 mmol/l), and in A549 cells with HEMA (14 mmol/l), TEGDMA (15 mmol/l), MeHgCl (0.15 mmol/l) and HgCl2 (0.05 mmol/l), compared to controls. The EC50 values from compounds in the L2 cells are shown in the following table (mean; sem in parentheses; n=3-6; #n=1): [see text]. SIGNIFICANCE: The toxic effect of HgCl2 and MeHgCl from the L2 cells was about 100-700-fold higher than of the dental composite components. A significant (p<0.05) time dependent increase of toxicity was observed with TEGDMA, HEMA and MeHgCl.

Diffusion of monomers from bonding resin-resin composite combinations through dentine in vitro.

OBJECTIVES: Previous work has demonstrated diffusion of the monomer triethylene glycol dimethacrylate (TEGDMA) from resin composite through dentine in vitro. The objective of the present work was to examine monomer diffusion from bonding resins and resin composites used in combination. METHODS: Occlusal cavities were prepared in tooth crowns and restored with bonding resin-resin composite combinations. Aqueous samples from the 'pulpal chamber' of each tooth were removed at timed intervals for analysis by reversed phase-high performance liquid chromatography and mass spectrometry. RESULTS: Bonding resins contributed to monomer diffusion. A TEDGMA-containing bonding resin used in combination with a TEGDMA-containing resin composite hastened and increased TEGDMA diffusion through dentine. A 2-hydroxyethyl methacrylate (HEMA)-containing bonding resin used in combination with a TEGDMA-containing resin composite reduced TEGDMA diffusion only slightly compared with the resin composite alone and added substantial diffusion of HEMA. CONCLUSION: The bonding resins tested contributed to monomer passage to the pulp space and did not prevent movement of monomer from resin composites to the pulp.