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After de- and subsequent remineralization at 0.03 and 1.0 ppm fluoride and either 0, 1, 10, 20 or 25 mM carbonate, artificial lesions in bovine enamel were demineralized again. The amount of secondary demineralization was determined after 15, 30 and 70 h of demineralization by means of quantitative microradiography. The results showed that fluoride incorporated during the remineralization period retards lesion formation during secondary demineralization. Carbonate incorporated during the remineralization period enhances secondary demineralization. An interaction between both ions was observed.

Some newly designed surface-active phosphates were investigated with respect to their capacity to inhibit caries lesion formation in vitro. On the labial or buccal surfaces of sound human third molars, windows were prepared for investigation. One half of each window was treated with the test compound at pH values of 7.5, 5.5, or 3.5. The other half served as a control. Both halves were demineralized and the generated caries lesions in both parts were compared by means of quantitative microradiography. Mono-, bis- and trisphosphates differed only marginally in their effect on the demineralization. The compounds diminished the demineralization down to 30% of that of untreated enamel. Per molecule at least one long alkyl chain (more than 12 C atoms) was required for achieving decreased demineralization, indicating the importance of a low surface tension. The results suggest that some of the tested compounds have promising properties for their use as anticaries agents, especially when applied in combination with fluoride.

The influence of fluoride, carbonate, and fluoride in combination with carbonate on the in vitro remineralization of bovine enamel was investigated with the use of a sandwich technique. After demineralization, enamel slices were subjected for 610 h to remineralizing solutions with 0.03 or 1.0 ppm fluoride. At each fluoride level, either 0, 1, 10, 20, or 25 mmol/L carbonate was tested. After 0, 22, 62, 126, 192, 329, and 610 h of remineralization, contact microradiographs were made by Cu Kα-radiation. At 0.03 ppm fluoride, carbonate had an inhibiting influence on remineralization. At 1.0 ppm fluoride, the inhibiting influence of carbonate changed into a stimulation of remineralization at 20 and 25 mmol/L carbonate. At 0, 1, and 10 mmol/L carbonate, fluoride had an inhibiting influence on remineralization. The differences in remineralization between the groups were explained by events concerning crystal growth, i. e., different types of minerals might have precipitated with differences in precipitation rates, and retardation of a precipitation step might have occurred under the various remineralization conditions. There was a mutual influence of fluoride and carbonate on the remineralization process. We conclude that the composition of the remineralizing solution with respect to fluoride and carbonate concentrations is important for the remineralization process.

To estimate permeability properties of bovine dental enamel, complex impedance measurements and radioisotope diffusion experiments were carried out before and after X-ray irradiation (single dose, 72 Gy) of enamel specimens. Neither impedance measurements nor diffusion experiments showed significant changes in permeability.

Subsurface lesions in bovine enamel slices were remineralized. The remineralization solutions contained either 0.03, 0.3, or 1.0 ppm fluoride at either pH 5.5 or 6.8. The amount of remineralization was determined after periods of up to 610 h, using quantitative microradiography. The results showed that after 126 h of remineralization in the presence of 0.03 ppm fluoride significantly (p less than 0.05) more remineralization occurred at pH 6.8 than at pH 5.5. At 0.3 and 1.0 ppm fluoride no significant differences between pH 5.5 and pH 6.8 were observed. An interaction between fluoride and pH was observed. The observed differences in the rates of remineralization are explained by the formation and subsequent transformation of the precursors octacalcium phosphate (pH 6.8) and brushite (pH 5.5) into (fluor)apatites.

Intact human enamel was demineralized in vitro to obtain artificial caries lesions. Part of the lesions was then treated with different surface-active compounds and remineralized with a remineralizing solution. The other part of the lesions was de- and remineralized in the same way, but was not treated with a surface-active compound. The results indicate that none of the surface-active compounds do affect the remineralization of the caries lesions. Possible applications of the tested compounds in combination with fluoride, for the prevention of dental caries, are discussed.

Remineralization experiments of bovine enamel with subsurface lesions were carried out with the use of a sandwich technique. The remineralizing solutions contained no or 2 ppm fluoride added to the solution. The amount of remineralization was determined after remineralization periods up to 324 by means of quantitative microradiography. After 84 h the group with fluoride showed significantly (p less than 0.05) less remineralization than the nonfluoride group. This inhibition of fluoride on the remineralization may be explained by the inhibiting effect that fluoride at certain concentrations has on the crystal growth of apatites.

Literature data on equilibria between solid calcium phosphates having the apatite structure and aqueous solutions containing Na+ and CO3 2- ions were reexamined in view of the present data about the complexation of ions in such systems. Recalculations showed that all solutions were undersaturated or just saturated with respect to either brushite or octocalciumphosphate or more or less finely dispersed calcite and supersaturated with respect to pure hydroxylapatite. Sections through the quasi-sextary phase diagram were constructed which are representative for the system under simulated in vivo conditions.

Third molars just erupting and premolars (before eruption or half a year or 3.5 years after eruption) were extracted and sawn vertically through the buccolingual plane. Tracings of the Ca, P, Na and Mg contents of the outer 100-µm layer of the enamel were made with an electron microprobe. Care was taken to limit the diameter of the excited volume to about 1 µm and to suppress the evaporation of Na. On the unerupted and freshly erupted surfaces the Na and Mg content varied from 50 to 80% of the content at 100 µm depth. However, half a year or 3.5 years after eruption the Na and Mg contents were negligibly small at the tooth surface and they remained smaller than in unerupted or freshly erupted teeth up to a depth of 10–30 µm. These data suggest that posteruptive maturation occurs in the mineral of the outer layer of tooth enamel whereby it builds up a higher resistance to carious breakdown. This interpretation is corroborated by clinical findings reported in the literature. The consequences of wear and abrasion on these phenomena are not known yet. The above-mentioned depth of 10–30 µm agrees with the penetration depth of pH changes estimated to result from the consumption of foods and drinks with varying pH.

Point count analyses were made with the electron microprobe for Ca, Na and Mg in a vertical cross-section through the enamel of a freshly erupted human pre-molar in the approximal area. A low excitation energy was chosen to limit the size of the excited volume and to suppress the evaporation of Na. The Na content showed variations with a wavelength of 6 ± 1 µm, which equals the distance of cross-striations. The Mg content showed variations with a wavelength of 25 ± 4 µm which equals the distance of striae of Retzius, whereby these striae probably contain a higher Mg content. On the assumption that higher Na and/or Mg contents represent mineral with a higher solubility, the observed variations in mineral composition help to explain the fine structure of carious lesions as observed frequently by microscopy and microradiography.

Tritiated proline was administered to domestic cats during the development of their permanent dentition. The amount of residual radioactivity was determined autoradiographically at various times after cavity preparation in mature molars and premolars. Two wk after cavity preparation, the amount of radiolabel beneath the axial cavity wall decreased to about 50% of that present in corresponding contralateral cavity-free teeth. The observed decreases are ascribed to an enhancement of the protein metabolism in dentin. At 45 wk, 80-90% of the radiolabel has disappeared. This paper discusses the role of an eventual effect of microleakage on the latter results.

The adherence of 4 strains of oral bacteria to hydroxyapatite and to chemically modified hydroxyapatite was investigated in vitro. It was found that successive treatment of hydroxyapatite beads with potassium phytate and hexadecylamine lead to complete inhibition of the adherence of three Gram-positive strains, Streptococcus mutans, Streptococcus sanguis and Actinomyces viscosus, but not of the Gram-negative Fusobacterium strain. Coating of the hydroxyapatite surface with saliva prior to treatment with phytate and amine did not significantly change bacterial adherence. When phytate and amine-treated hydroxyapatite was post-treated with saliva, the adherence of S. mutans and A. viscosus, but not of S. sanguis, was still completely blocked.

Artificial laminated carious lesions were made on the buccal surface of human premolars. The volume percentage of mineral was determined as a function of depth by microradiography. Using the electron microprobe, the signals for Ca, Na, Mg, P and Cl were recorded as a function of depth both through the lesions and through the adjacent sound enamel. In the demineralized parts of the lesions there was a preferential loss of Na and a preferential retention of Cl. In the surface layers and the laminations the Na/Ca and Cl/Ca ratios were practically the same as in the adjacent sound enamel. This indicates that these surface layers and laminations were not formed by gross dissolution of the original mineral followed by gross precipitation of another, less soluble calcium phosphate, but that they remained there during the continuous demineralization due to the fact that the microcrystals became resistant to dissolution, perhaps due to protection by a thin layer of precipitated fluorapatite or fluoridated hydroxyapatite. In a previous study the same was found for surface layers and laminations in natural laminated carious lesions. This indicates that during continuous in vitro demineralization tooth enamel mineral behaves similarly as during intermittent in vivo demineralization.

The permeability of dental enamel can be estimated by means of diffusion measurements with radio-isotopes. Because of the low intrinsic permeability of enamel membranes, determination via this route may take two weeks. A much faster electrochemical method is presented, in which the real and imaginary part of the membrane impedance are measured at discrete intervals in the frequency-range 1 Hz-1 MHz. Using the principles of network-analysis, a phenomenological interpretation of these electrochemical data in terms of a 6-parameter model is given. The model contains R0, the intrinsic membrane resistance, R∞, the residual membrane resistance and the four parameters, necessary to describe the postulated enamel diffusion impedance, which bears strong resemblance to the Warburg impedance. Starting from this model, optimal estimates (with standard-errors) of a membrane's R0 and vmax (the log of the frequency where the imaginary part of the membrane impedance is maximal) were derived. These parameters were predictors of the permeability of enamel.

The effect of orthophosphate, pyrophosphate, trimetaphosphate, hexametaphosphate, phytate and 1,1-hydroxyethyldiphosphonate on the ion-selective properties of bovine tooth enamel was investigated using electrochemical methods. All phosphates increased the ion-selectivity of enamel, an effect that increased with increasing number of phosphate groups. The effects of phytate and the diphosphonate were more pronounced than those of the corresponding phosphates.

In this study complex impedance measurements and radioisotope diffusion experiments are used to estimate physical parameters for enamel such as pore-diameter, membrane-porosity and membrane-charge. The assumptions implied in deriving a model and in estimating the membrane-parameters and ionic radii were Verified by comparing the experimental observations with values calculated with the proposed model. A good congruence was found between the measured and calculated values. The membrane-porosity appeared to be the main determinant for the experimentally observed wide ranges in permeability and conductance. The dependence of conductance on the electrolyte-concentration (2 to 100 mmol/1) and ionic species (RbCl, KCl, KNO3 and Mg(NO3)2) are shown to be explained by the model in a satisfactory way.

Six lesions were made on the buccal surfaces of premolars. The volume percentage of mineral was determined as a function of depth by microradiography. Using the electron microprobe, the signals for Ca, Na, Mg, P and Cl were recorded as a function of depth both through the lesions and through the adjacent sound enamel. In the demineralized parts, there was a preferential loss of Na and a preferential retention of chloride. In the surface layers, the Na: Ca and Cl: Ca ratios were almost the same as in the adjacent sound enamel, indicating that the surface layers were not formed by gross dissolution of the original mineral followed by gross reprecipitation of another, less-soluble calcium phosphate, but remained probably because their microcrystals were protected by a thin layer of precipitated fluorapatite or fluoridated hydroxyapatite. The same had been found for surface layers in natural caries.

Aliphatic amines with chain lengths from C 2 to C 18 were investigated for their effect on the diffusion of [ 3 H]-sorbitol, [ 14 C]-glycerol, 36 Cl − and 86 Rb + . Before amine application, enamel was pretreated with a phytate solution. C 12 C 18 amines reduced the rate of diffusion to values of about 20–30 per cent of the values found before treatment. Amines with chain lengths below C 12 were less effective. The presence of an unsaturated carbon-carbon bond (C 18:1 ) seems to promote the adsorbance of the amine to enamel.

Slices of bovine enamel and intact human enamel were treated with (a) a two-step treatment consisting of phytate followed by hexadecyl amine, or (b) 2-O-stearoylglycerol-1,3-diphosphate (Glydip), or (c) phosphatidic acid. The effect of these agents on the rate of caries lesion formation and on the permeability of dental enamel was quantitatively determined. Glydip had the most pronounced effect on lesion formation and showed a strong inhibiting effect on the rate of demineralization, combined with a reduction of the permeability. The results indicate that at least a part of the effect of Glydip on the demineralization was caused by a reduction of the rate of dissolution of the enamel mineral. The reduction in permeability may have had an additional effect.

The rate of dissolution of whole human enamel was studied by carrying out artificial caries experiments in buffers of known pH and known degree of undersaturation with respect to hydroxyapatite and by

Diffusion coefficients of 86 Rb + , 36 Cl − , [ 3 H]-sorbitol and [ 14 C]-glycerol through enamel were determined both in the absence and the presence of urea or t -butanol which are known for their structure-breaking and structure-promoting properties respectively in aqueous solutions. Their effect on the diffusion coefficients could be explained solely on the basis of the respective shifts in the relative viscosities of the aqueous solutions, indicating that the water of the transport phase is probably free water, in accord with explanation for the extra increase of diffusion coefficients with temperature (Burke and Moreno, Archs oral Biol. 17 , 433–437 (1975)) by the hypothesis that the amount of free water increases with temperature at the expense of bound water.

To investigate the permeability of the dental enamel of erupted and unerupted human pre-molars and its variation with depth, we determined the resistivity of successive 100-μm-thick layers. The electrical resistance of halved tooth crowns at zero Hertz, which according to Scholberg et al. (1982, 1984) is inversely related to the permeability, was measured before and after removal of successive enamel layers from the outer surface toward the dentino-enamel junction (D.E.J.). The resistivity of the successive enamel layers increased from the D.E.J. toward the outer surface in approximately the same way in erupted as well as in unerupted pre-molars, except within the outermost layer of 100 to 200 μm thickness. The resistivity of this layer in erupted pre-molars was considerably higher than that in unerupted pre-molars, which may be due to post-eruptive mineralization.

Intact roots of human molars, which had not been exposed to the oral environment, were exposed to buffer solutions of pH 5.5, which contained, besides calcium and phosphate, various concentrations offluoride. Densitometric measurements on contact-microradiograms of transverse sections of these roots produced data which were used to calculate the rate of demineralization ( Vdem). Vdem appeared to be reduced with increasing fluoride concentration and with decreasing pIOHA (i.e., the negative logarithm of the ionic product of hydroxyapatite) of the buffer solution. In addition, it was observed that, after some days of exposure, remineralization started to occur within the surface layer at some μm from the outer surface and that the region within which remineralization took place was extended inward with increasing time of exposure. These data suggest that fluoride is effective in the prevention of root surface caries. The mechanism of the interaction of fluoride with the root mineral is discussed.

Extensive ‘white spot’ and ‘brown/white spot’ lesions with intact surfaces in 11 molars were investigated by microradiography and electron microprobe analysis for Fe, Ca, Na and Cl. At the surface of the brown spots some Fe was found. Within the lesions a preferential dissolution of Na and a slight enrichment of Cl was found. This suggests that Na and Cl are sited in different parts of the mineral and the Na-containing part appears to be more soluble. The Na and Cl contents of a lamination approach those of adjacent sound enamel. This indicates that laminations do not result from major reprecipitation of a less soluble calcium phosphate within the formed lesion, but that the mineral is protected from local dissolution early in the process. A possible mechanism is discussed.

Previously we showed that phospholipase C treatment and hexane extraction of cattle rhodopsin in rod outer segment membrane preparations removed 95% of the phospholipids originally present in these preparations, reducing thereby the phosphatidyl ethanolamine and phosphatidyl serine contents from 30 and 9 to 0.2 and 1.6 mole per mole rhodopsin, respectively, while leaving the pigment completely intact. Now it is shown that, upon incubation of this rhodopsin preparation with phospholipase A 2 from Crotalus adamanteus , most of the remaining phosphatidyl serine was hydrolyzed to lysophosphatidyl serine and fatty acid. Repeated extraction of this preparation with aqueous bovine serum albumin solution completely removes the lyso compound. After this procedure 64% of the rhodopsin was still spectrally intact. After removal of some “free” retinaldehyde, originating from decomposed rhodopsin, by means of endogenous retinol dehydrogenase activity and added NADPH, the resulting rhodopsin had the same absorption spectrum, molar absorbance value, and photolytic properties as native rhodopsin. Quantitative analysis of the phospholipids present in the final product yielded values of 0.10 mole per mole rhodopsin for each of the two amino group containing phospholipids phosphatidyl ethanolamine and phosphatidyl serine. These findings prove that the “chromophoric” retinaldehyde cannot be linked to either phospholipid in native cattle rhodopsin.

Tritiated proline was administered as protein marker to domestic cats during the development of their permanent dentition (age 11-18 wk). At 30, 43, 56, and 75 wk, the amount of tritium in the mature dentin was determined by quantitative autoradiography. At 43, 56, and 75 wk the residual amount of label was 92, 92, and 77%, respectively, of that at 30 wk. The metabolism of dentinal collagen (with possibly a recycling of proline) and the metabolism of non-collagenous proline-containing proteins are suggested as factors that may contribute to the decrease of tritium label.

A radiochemical method was developed to determine quantitatively and simultaneously the transport of up to four different compounds through dental enamel. The compounds chosen were [3H]-sorbitol, [14C]-glycerol, 36Cl− and 86Rb+. Effective diffusion coefficients of these compounds determined at 4 °C were considerably different for different specimens of enamel. Thus values for [3H]-sorbitol varied from 0.04 × 10−8 to 2.5 × 10−8cm2s−1. Most enamel membranes showed an ion selective behaviour by which the cations were more mobile than the anions. A molecular sieve effect was observed for glycerol and sorbitol.

In this study a mathematical model for the caries process is presented. It includes diffusion of ions, dissolution of mineral and complexation of ions. The conclusions from experiments with the model

The effect of fluoride and monofluorophosphate on the ion selective properties of bovine tooth enamel was investigated using electrochemical methods. The effect of monofluorophosphate was fundamentally different from that of fluoride. The anti-cariogenic properties of fluoride or monofluorophosphate cannot be deduced from their effect on the ion selectivity of enamel.

Crowns and roots of human molars, the roots from which had not been exposed to the oral environment, were exposed for 0, 3.5, 7, and 14 days to buffer solutions which were undersaturated or supersaturated with respect to hydroxyapatite. Densitometric measurements on contact-microradiograms of transverse sections of the crowns and of the cervical parts of the roots yielded plots of the mineral content as a function of the distance to the outer surface. From these plots, the rate of demineralization was calculated. It was found that the mineral of the roots dissolved even in buffer solutions which were supersaturated with respect to hydroxyapatite. Comparison of the results obtained from the crowns with those from the roots showed that the root hard tissues were more vulnerable to demineralization than was the dental enamel.

The effect of hexadecylamine on the permeability of bovine dental enamel was investigated using a radiochemical diffusion technique. Treatment with an aqueous solution of hexadecylamine reduced the permeability of the enamel by about 20 per cent. Pre-treatment of the enamel with an aqueous solution of phytate followed by hexadecylamine yielded a reduction in permeability of approximately 70 per cent. The observed effects were similar for ionic (86Rb+ and 36Cl-) and non-ionic [( 3H]-sorbitol and [14C]-glycerol) compounds. The strong permeability-reducing effect of the combined phytate-amine treatment may also be anticariogenic.

The fixed charge of enamel and the ratio of the diffusion coefficients of cations to anions of some common electrolytes within enamel were calculated from steady state electromotive forces developed in concentration cells across enamel caps of whole human teeth, sections of human enamel or synthetic hydroxyapatite membranes. The electrolytes were KCl, KF, NaCl, CaCl 2 , KH 2 PO 4 and K 2 HPO 4 . The results indicate that enamel behaves like an ion exchange membrane of which the fixed charge depends on the electrolytic environment and the sequence of exposure to various electrolytes.

For the rate of demineralization of enamel during caries, three physicochemical processes are of importance: the diffusion into and out of the enamel, the dissolution of mineral and the complexation of ions. The interpretation of diffusion experiments in conjunction with artificial caries experiments and computer simulations is described. In the caries process, relevant diffusion coefficients of small ions and molecules are of the order of magnitude 10(-9)--10(-7) cm2 s-1.

Slices of enamel were demineralized in weak acid solutions at pH 5. The solutions were analysed for Ca, P, Na and Mg. A substantial increase of the Ca P ratio in the solution after about 6 h of demineralization was ascribed to brushite formation. The ratios of liberated Ca Na , P Na , Ca Mg and P Mg were always lower than the correspondent ratios in sound enamel. It was concluded that precipitation of brushite, and a preferential dissolution of Na and Mg compounds from the enamel both play a role in the dissolution-precipitation reactions in dental enamel during acid attack.

P ermanent filling materials have some deficiencies such as dehydration of dentin by the withdrawal of water during the hardening process, penetration of pulpirritating components of the filling material into dentin, and microleakage around the filling due to lack of adaptation. To protect the pulp against these irritations, an applied intermediary base material that has good sealing properties should be used. Intermediary base materials can be divided into three groups: varnishes, liners (varnish with additives such as calcium hydroxide or zinc oxide), and cements. In vitro the amount of most varnishes applied as a continuous layer has slight permeability.‘” However, in vivo experiments suggest that these varnishes are not always as effective as pulp protectors as might be expected from the results of the in vitro experiments.‘-” This discrepancy may occur because in most in vitro experiments the investigated materials are applied on materials different from dentin. For testing the sealing capacity of varnishes, the kind of supporting material is important. A supporting material with bad wetting properties may lead to discontinuities in the obtained layers.* This investigation studied the sealing properties of some intermediary base materials applied in vitro on dentin in a way that corresponds as much as possible with the in vivo situation in dental practice.

Tritiated proline was administered to domestic cats during the development of their permanent premolars. The metabolic activity of collagen in the mature premolar dentin was determined by quantitating the amount of tritiated hydroxyproline in the dentin as a function of time. It has been demonstrated that the metabolic activity of the dentinal collagen was extremely low and remained within the experimental error for a period of 45 weeks.

A radiochemical method was used to determine quantitatively the effect of RbCl, CaCl 2 and MgCl 2 on the transport of [ 3 H]-sorbitol, [ 14 C]-glycerol, 45 Ca 2+ , 36 Cl − and 86 Rb + through dental enamel. The findings indicate that the transport of 45 Ca 2+ is influenced strongly by surface exchange reactions. The influence of Ca 2+ and Mg 2+ on the transport of 86 Rb + and 36 Cl − indicates that, at a concentration of ~10mmol/l, these ions alter the fixed charge of enamel from negative to less negative, or positive values. Previously adsorbed 45 Ca 2+ ions are desorbed at higher Ca 2+ and Mg 2+ concentrations. Rb + ions at 10 mmol/l have no such effect. The results indicate that Ca 2+ and Mg 2+ can be adsorbed reversibly on the crystal surfaces or in their Stern layers in the pores of dental enamel.

Whole roots of molars that had never been exposed to the mouth were exposed to calcium and phosphate-containing buffer solutions with discrete values of pIOHA [i.e. -log(a10Ca2+ X a6PO4(3-) X a2OH-)] at various pH. From densitometric measurements on contact-microradiographs of transverse sections of the roots the rate of demineralization (Vdem) was calculated. Vdem changed non-linearly as a function of pIOHA and became zero at a pIOHA-value of 105.3 +/- 0.4; this is substantially lower than the corresponding value of 118 +/- 1 for enamel. Thus root mineral is more soluble than enamel mineral. The critical pH for root caries appears to be about 6.7, provided that the plaque fluid follows about the same pH-pIOHA- pathway as acidified saliva.

AN accurate value for the molar absorbance of the visual pigment rhodopsin is important in quantitative studies on the biochemistry of vision. A recent controversy regarding this value1,2 prompted the present study.