Dental plaque species, Streptococcus sanguis and Capnocytophaga gingivalis, were grown in continuous culture with progressively increasing concentrations of triclosan or its phosphorylated derivative, triclosan monophosphate (TMP). For both organisms, the maximum specific growth rates decreased with increasing concentrations of triclosan or TMP until complete inhibition of growth occurred, which for S. sanguis was at 20 mg/L and 50 mg/L, and for C. gingivalis was at 10 mg/L and 5 mg/L for triclosan and TMP respectively. For both species, biomass levels remained approximately constant or, in some cases, increased slightly at low levels of triclosan or TMP. However, biomass levels then decreased significantly as the triclosan or TMP concentrations approached lethal levels. For S. sanguis, levels of hydrolytic enzymes (acid phosphatase, leucine aminopeptidase and esterase) generally remained approximately constant or increased with increasing concentrations of triclosan or TMP until close to inhibitory levels where enzyme levels were reduced. The ratio of extracellular soluble enzymes to cell-bound enzymes remained constant or increased slightly with increasing levels of triclosan or TMP. For C. gingivalis, production of hydrolytic enzymes (neutral phosphatase, leucine aminopeptidase and trypsin-like protease) remained constant or were reduced when grown with low levels of triclosan and TMP but in some cases increased with higher levels of agents. The proportion of extracellular soluble activity increased significantly when concentrations of agent neared inhibitory levels. The results taken together show that the physiology of cells is significantly altered and that hydrolytic enzymes are released from the cells when these are grown in the presence of increasing concentrations of triclosan or TMP. Enzyme release is more pronounced in the Gram-negative C. gingivalis and indicates that triclosan or TMP can cause membrane perturbation with subsequent release of membrane-located (S. sanguis) or periplasmic (C. gingivalis) hydrolytic enzymes. S. sanguis was more sensitive to triclosan than TMP while C. gingivalis was more sensitive to TMP. This suggests that, in C. gingivalis, TMP may diffuse into the cell wall more easily than triclosan and then be converted to triclosan by phosphatase activity within the cell wall complex, where it may give rise to high localized concentrations and subsequent cell damage.

Triclosan monophosphate is a phosphorylated derivative of the antimicrobial agent, triclosan. In comparison with triclosan, it is highly soluble in aqueous solutions. It is hypothesized that, within the oral environment, triclosan monophosphate (which may be devoid of antimicrobial activity) will be hydrolyzed into triclosan by the action of microbial phosphatases. The liberated triclosan may then exert antimicrobial activity. To test this hypothesis, we designed experiments to measure the phosphatase activity of plaque and selected species of oral micro-organisms and to demonstrate hydrolysis of triclosan monophosphate. Tests comparing the minimal inhibitory concentration and minimal bactericidal concentration of triclosan and triclosan monophosphate were also undertaken. Dental plaque and the majority of the bacterial strains tested showed phosphatase activity against p-nitrophenyl phosphate which peaked below neutral pH (acid phosphatases) or above neutral pH (alkaline phosphatases). Dental plaque showed the highest levels of alkaline phosphatase (optimum at pH 9.0) and relatively high levels of acid phosphatase (optimum at pH 6.0 to 6.5). Dental plaque and selected species of micro-organisms were all capable of hydrolyzing triclosan monophosphate, albeit at different rates. The minimal inhibitory concentration and minimal bactericidal concentration values for triclosan monophosphate against eight bacterial strains were always considerably higher than the corresponding values for triclosan. Addition of triclosan monophosphate to an established culture (ca. 10(9) cfu/mL) of Capnocytophaga gingivalis growing continuously showed that triclosan monophosphate was rapidly hydrolyzed into triclosan with concomitant loss of total bacterial viability. It is therefore likely that triclosan monophosphate will be broken down into triclosan within the oral environment with concomitant antimicrobial activity.

A pH cycling model which incorporated a severe demineralization component was used to evaluate fluoride uptake and lesion progression for each of a NaF-based dentifrice, NaF-based mouthrinse and a monofluorophosphate (MFP)-based dentifrice. Simultaneous transverse fluoride and calcium profiles across the resulting artificial lesions were obtained using a proton microprobe technique with a resolution of approximately 10 microns. Longitudinal microhardness testing and proton microprobe calcium profiles were used to determine the extent of lesion progression (delta Z) with respect to untreated controls. Under the pH cycling conditions of the present study, the NaF dentifrice and mouthwash were observed to have a considerably higher uptake of fluoride in the lesion than the MFP dentifrice. Although the mineral content profiles of the lesions differed for treatments with each of the fluoride products, the differences were not significantly different in this model.

Coating hydroxyapatite (HA) onto metal implant surfaces using the plasma-spraying technique has been investigated in several laboratories as a means of improving the mechanical properties of the bulk ceramic. This study describes crystallographic changes which can occur during the plasma-spraying of calcium phosphate powders. A precipitated calcium-deficient apatite and a high temperature near-stoichiometric HA were each sprayed onto metal substrate in an argon plasma using several hydrogen gas flow conditions at various temperatures. The surfaces were examined by X-ray diffraction and scanning electron microscopy. The plasma-sprayed products were identified as a mixture of calcium phosphates including HA, beta-tricalcium phosphate (beta-TCP) and calcium oxide. Stoichiometric HA when plasma-sprayed showed the least (5%) degradation. Since beta-TCP is more resorbable than HA in vivo, varying the HA/beta-TCP ratio on the plasma-sprayed surface may provide a method to control surface dissolution of the coating.

The high-resolution 19F NMR of solid inorganic fluorides and fluoridated apatitic materials is reported. Experimental data collected at spinning speeds approaching 16 kHz are described which demonstrate that fast magic-angle spinning (MAS) alone represents a viable alternative to multipulse techniques for effectively averaging the 19F homonuclear dipolar interactions which typically dominate the static spectra of these solids. The significant potential of fast-MAS 19F NMR as a spectroscopic tool for studying the surface chemistry associated with the fluoridation of apatitic surfaces is also demonstrated.

Backscattered electron (BE) microscopy is being used increasingly as a technique to study the dissolution of dental enamel because of its high resolution and relatively easy sample preparation. Subsurface details such as striae of Retzius, cross-striations and prism microstructure have been observed with a resolution better than 0.1 micrometers using this technique. Since BE images of demineralized enamel appear very similar to microradiography images, it is tempting to interpret them in a similar fashion. We attempt to show that the interpretation of BE images is not straightforward because enamel is not a homogeneous one-phase material, but a two-component composite material consisting of variable amounts of apatite mineral and organic matter. During re- and demineralization, other calcium phosphate phases may precipitate to further complicate the interpretation of the images. BE images of partially demineralized enamel are affected by local variations in the protein/mineral ratio and also by the reprecipitation of other calcium phosphate phases. BE images are not mineral density maps, but are mean atomic number maps.

Studies which used scanning electron microscopy (SEM) to investigate subsurface demineralization of dental enamel have recently been well reviewed. The purpose of the present paper was to review several studies, carried out in our laboratories, which have used electron microscopy to examine physicochemical properties of synthetic and biological apatites, to relate these results to previous studies, and to present new data. Aspects of the ultrastructure of hydroxyapatite and carbonated-apatites have been observed by high resolution transmission electron microscopy, and related to shape and growth of these crystals. Surface morphologies of discs prepared from precipitated carbonated-apatites and from ceramic carbonated-apatites were examined by SEM and the information was used in the interpretation of apatite dissolution studies relevant to dental caries. Improvements in the technique of backscattered electron imaging of demineralized enamel have enabled better interpretation of enamel caries experiments. SEM examination of enamel and dentin treated by low energy lasers of specific wavelengths have shown that lasing conditions can be chosen that produce surface fusion of the apatite which inhibits caries-like lesion progression. SEM examination of crystals formed on and in enamel during high concentration fluoride treatments implies that calcium fluoride-like crystals are formed and they may act as a slow-release fluoride reservoir in the mouth.

The application of high resolution electron microscopy, computer image processing, and image simulation techniques to the investigation of synthetic nonstoichiometric apatites has provided new details of apatite crystal growth mechanisms. Under certain precipitation conditions, calcium‐deficient apatites with distinct octacalcium phosphate (OCP)‐apatite intergrowths have been observed. Apatite crystals with unit‐cell thick overgrowths of OCP on their surfaces confirmed the stepwise hydrolysis crystal growth mechanism initially proposed by Brown ( Nature 196:1048–1050). However, many crystals also contained a central two‐dimensional OCP inclusion one to two unit cells thick, embedded in an apatite matrix. Similar planar defects have been observed in dental enamel, dentin, and bone apatite crystals. We have developed a modified version of Brown's stepwise OCP hydrolysis apatite crystal growth mechanism to explain the formation of crystals with OCP central planar defects. The mechanism involves the nucleation of an OCP seed that grows until it reaches a critical size, r h , before OCP hydrolysis occurs. Apatite subsequently grows epitaxially on the OCP seed, thereby embedding it in the center of an apatite crystal. Apatite growth is facilitated by partial screw dislocations emanating from the planar defect.

Developmental defects in incisors were induced by daily oral ingestion of sodium fluoride solutions. Teeth extracted at eruption from sheep that had been subjected to four different fluoride regimens—0.2 or 0.5 mg F/kg body weight daily for 6 months, 2 or 6 mg F/kg body weight daily for 21 days—were analysed for fluorine by gamma emission using a proton microprobe. Calcium and zinc profiles were also measured using proton-induced X-ray emission. Diffuse opacities, similar in appearance to mild human fluorosis, were produced by the first two regimens, whereas the last two produced hypoplastic lesions. Different distributions of fluoride were found in the unerupted enamel and dentine, and these patterns reflected variations in both the duration and concentration of the fluoride dose used to induce the fluorotic lesions.

We have used back-scattered electrons (BE) in the scanning electron microscope to produce mineral density images of enamel. Flat surfaces of artificially-carious enamel, softened in an intra-oral experiment, and naturally-carious (white spot) enamel were polished to a high gloss with diamond lapping compound, rendering them almost featureless by secondary electron scanning electron microscopy. They were then examined at 10 to 30 kV in a Philips 505 instrument fitted with a 4-quadrant BE detector. Study of surfaces prepared approximately parallel to the natural surface showed that mineral was lost from both prism core and the interprismatic region, leaving a thin mineral-rich rim at the prism periphery. The same lesions viewed longitudinally on a surface prepared perpendicular to the natural surface showed mineral-rich bands at the prism margins in the outer enamel. Near the advancing front of the lesion, the prism junctions were widened and the prism cores sometimes hypermineralized. Natural lesions sectioned in the prism long axis showed features previously seen with other techniques, e.g., cross-striations and striae of Retzius, but in much greater detail. Mineral enrichment at the prism periphery in the lesion body and a widening of the prism junction at the advancing fronts of lesions in permanent teeth were most obvious. Calculations showed that with an accelerating voltage of 30 kV, the images reflected mineral density up to 4 μm beneath the surface. BE microscopy produces a high-resolution image of mineral loss or gain in carious enamel, with relatively easy sample preparation.

The hypothesis that diffuse opacities in enamel result from a chronic, mild disturbance to ameloblast activities was tested using fluoride. Three sheep (HF) were dosed orally with 0.5, and 3 (LF) with 0.2 mg fluoride/kg body weight daily for 6 months. A control sheep (C) received no additional fluoride. The 7 sheep were killed at or close to the time of emergence of their permanent central incisors. One tooth from each sheep was sectioned longitudinally. The enamel related to the secretory (S) and maturation (M) phases of ameloblast activity at the start of fluoride dosing was determined from a tetracycline marker. The pattern of mineralization of the outer 150 μm of the cut labial enamel was assessed using microhardness testing. The SEM appearance of the acid-etched outer enamel was compared in S and M zones in 5 teeth. The enamel of the C tooth was translucent. Diffuse opacities, similar in appearance to human fluorosis, were present in all fluoride-treated teeth. Hardness values in the outer 70 μm of the enamel decreased as the fluoride dose increased and, in the HF teeth, were lower in the S zone than in the M zone. Fluoride given during the M phase induced a surface hypomineralization which increased in degree and depth when fluoride was also given during the S phase. The SEM appearance of M and S enamel was similar in 2 LF and 1 HF teeth but, in the other HF tooth, S enamel but not M enamel had a disordered prism structure and loosely-packed crystals in an abnormal organic matrix. Histological examination revealed that ameloblasts remained in only 4 of the 7 teeth and that their regression and the formation of the cementum adjacent only to the labial enamel were progressing abnormally.

Six subjects wore lower-arch intra-oral appliances supporting plaque-covered enamel units in the left and right buccal sulci. Units on both sides received identical cariogenic challenges by intermittent immersion in 0.28 M glucose. When one side was treated, in addition, with a mouthrinse designed to enrich plaque with Ca, P, and F, there was a 76% reduction in the softening and a 96% reduction in the porosity, created in enamel by the glucose exposures. F dentifrice extract, used similarly, caused a 67% reduction in enamel softening and a 93% reduction in porosity. When the two treatments were compared in the same experiment, the mouthrinse had a significantly greater effect in limiting enamel softening, but the porosity measurement technique was not sensitive enough to confirm this finding. Use of the mouthrinse caused variable deposition of fluorhydroxyapatite in plaque, and scanning electron microscopy examination of enamel showed small adherent hard deposits in some subjects. The polished enamel surface enabled backscattered electron imaging which revealed preferential dissolution of the core and tail regions of the prism. The results suggest that plaque mineral enrichment may be even more effective than F dentifrice in preventing dental caries.

As research progresses, laser energy moves closer to acceptable usefulness. Laser application to prepare dentin creates a more retentive surface for composite bonding.

In examining the microstructure of TEM specimens prepared from D+-implanted Cu for the presence of bubbles it was found that cuprous oxide (Cu2O) layers had formed over large areas of the specimen surfaces. The Cu was irradiated at normal incidence with 200 keV D+ ions at a temperature of 120 K to a dose of ~2 × 1021D+/m2. Ar+ ion milling at 330 K was used to erode irradiated surfaces to various depths prior to chemical back-thinning in a jet electropolishing bath. There was no evidence for the formation in the Cu of bubbles of either deuterium or argon, but dislocations at high density and planar defects were evident. Lattice fringes from {110}, {111} and {200} planes in Cu2O and moiré patterns formed by double diffraction in the Cu and overlaid Cu2O film were obvious features in bright-field micrographs. The moiré patterns include examples of magnified images of lattice defects.

Studies have shown synthetic calcium phosphates such as hydroxyapatite and beta tricalcium phosphate to be biocompatible in vivo. However, few studies have quantitated histological responses to the implants. The aim of this study was to develop a method for the quantitative assessment of tissue biocompatibility to ceramic materials in vivo and to use this method to compare noncarbonated and carbonated apatite implants. Synthetic sintered apatites of 0, 3, and 6% carbonate by weight were prepared and cut into implants 4 X 4 X 1 mm. These were placed 2 mm into the medial aspect of rat femurs. Following sacrifice at 4 weeks, the femurs were fixed in formalin, demineralized in formic acid, and embedded in glycol methacrylate. Sections were cut on an ultramicrotome set at 1.5 micron and stained with toluidine blue. A point counting technique using standard stereological grids and a low-power microscope was used to measure areas of new bone formation. The width of the connective tissue zone adjacent to muscle was measured using an image analyzer. All implants were well accepted by the host tissues judging from criteria of minimal inflammation and degree of fixation. Results showed an increase in new bone formed in the marrow cavity with increasing carbonate content. This may improve stability of the implant in the host bone, particularly during the initial healing period. A technique which should enable quantitative histological evaluation of different ceramic materials has been developed. The use of this method indicates that further studies are warranted to investigate carbonated apatite as an implant material.

Densely sintered synthetic hydroxyapatite (HA) is used as an implant material because of its excellent tissue biocompatibility. In order to maximize the biological potential of this calcium phosphate, we have investigated the incorporation of carbonate into HA to make a material which more closely resembles the mineral found in bones and teeth. The aim of the present study was to determine the conditions under which sintered carbonated apatites of specific carbonate content could be produced. The apatites were prepared by heating compressed pellets of precipitated carbonated apatite under a carbon dioxide/steam or nitrogen/steam atmosphere between 825 and 1050 degrees C. The products were analyzed chemically and the surfaces examined by x-ray diffraction, infrared spectroscopy, reflected light microscopy, and scanning electron microscopy. The results showed that carbonate loss during sintering could be reliably predicted, making it possible to produce materials with specific carbonate content, and with specific physical and chemical composition.