W.R. Hume

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.

Basic Biological Sciences 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 -4M and a maximum at 10-3M 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 10-3M eugenol or less, but died after exposure to 10-3M for one day or more. The cells survived exposure to 10-4M 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: (1) the blandness of ZOE when applied to intact dentin is due to eugenol reaching the pulp in sub-toxic concentrations, and (2) 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.

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 cytotaxicity. 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.

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°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 tests 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 acetylcholine on the response of the isolated rabbit ear artery to stimulation of the perivascular sympathetic nerves

Abstract In the isolated rabbit ear artery, low concentrations of exogenous acetylcholine inhibited the response of the artery to periarterial electrical stimulation, while much greater concentrations of acetylcholine were required to inhibit the response to exogenous noradrenaline. This action of acetylcholine on periarterial stimulation was suppressed by atropine and potentiated by cholinesterase inhibitors. Neither atropine nor cholinesterase inhibitors, in concentrations which modified the effects of exogenous acetylcholine, altered the response of the artery to periarterial stimulation. The study failed to confirm that either a cholinergic element or endogenous acetylcholine has an effect in the arterial sympathetic nerve response.

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.

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-wtl3, 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. Also, when compared with similarly-derived indices of susceptibility of mammalian cells to CX, the data obtained give rise to new possibilities for therapeutic use of CX or other bisguanides in dentistry.