Combined effects of diets with reduced calcium and phosphate and increased fluoride intake on vertebral bone strength and histology in rats.

Ingested fluoride is incorporated into bone apatite and can affect the structural integrity of bone. Fluoride absorption in the gut and incorporation into bone is affected by the presence of other ions, including calcium. We hypothesized that a low calcium phosphate diet combined with high fluoride intake would have independent detrimental effects on vertebral bone strength. We measured the vertebral biomechanical properties and bone histology for rats given controlled diets with varied fluoride and calcium phosphate intakes for either 16 or 48 weeks. Diets were designed to produce mild calcium deficiency while keeping Ca:P ratio constant. Hence calcium deficiency was accompanied by a proportional phosphate deficiency. Calcium phosphate deficiency (25% of normal) significantly diminished vertebral strength only in younger rats. As the rats grew older, the effects of calcium phosphate deficiency were mitigated. Calcium phosphate deficiency reduced vertebral strength in young rats primarily by reducing the trabecular bone volume. The highest fluoride intake (50 mg/L) significantly diminished vertebral strength, but there was no effect of lower fluoride intakes on bone strength. In contrast to calcium phosphate deficiency, high fluoride intake had no effect on trabecular bone volume but instead increased the amount of unmineralized osteoid, particularly in older rats. This impairment of mineralization by fluoride appeared to be the primary cause of the diminished vertebral strength. The detrimental effects of fluoride on vertebral bone strength were not amplified by calcium phosphate deficiency.

In vitro fluoride dose-response study of sterilized enamel lesions.

Currently our intra-oral model uses enamel specimens that have been disinfected by soaking in buffered formalin (pH 6.8). However, because of increasing emphasis on infection control, it is important to identify a way to sterilize these specimens. The aim of this study was to determine if autoclaved, or gas sterilized, lesioned enamel responds to fluoride (F) in the same way alcohol-disinfected enamel lesions do. Seventy-two formalin-disinfected, human enamel specimens (3 mm) were lesioned in demineralizing solution for 96 h and were then divided into three groups. One group was autoclaved; one group was gas sterilized (ethylene oxide), and the remaining 24 specimens were further disinfected in 70% ethanol for 10 min. Specimens in each group were then treated 4 times/day for 4 weeks with 0, 250 or 1,100 ppm F dentifrice slurries in an in vitro cycling, remin/demin model. Following treatment, fluoride uptake was analyzed by microdrill biopsy, and lesion depth and mineral content changes (DeltaM) were determined by transverse microradiography. Data were analyzed by one-way ANOVA analysis. In all three groups of specimens there were significant (p<0.05) differences in fluoride uptake in response to different fluoride treatments. Autoclaved lesions failed to provide dose- response data with regard to changes in lesion mineral content. Because formalin and 70% alcohol are only disinfectants, and autoclaving altered the responsiveness of enamel lesions, results from this study suggest that, of the methods tested, gas sterilization is the preferred method for sterilizing enamel specimens that will be used in intra-oral studies.

An In vitro microbial-caries model used to study the efficacy of antibodies to Streptococcus mutans surface proteins in preventing dental caries.

The first step for a pathogenic bacterium to initiate infection is via attachment (i.e., through surface determinants) to a suitable receptor. An in vitro microbial artificial-mouth model was used to test the efficacy of polyclonal antibodies to Streptococcus mutans cell surface proteins (CsAb) and a cell surface 59-kDa protein (59Ab) in preventing S. mutans colonization and carious lesion formation. In study 1, groups of 12 human teeth specimens were inoculated with S. mutans, which were incubated with different concentrations of CsAb (A1 [positive control], sterile saline, no antibody; A2, 0.007 mg of antibody protein/ml; and A3, 0.7 mg of antibody protein/ml) for 1 h at 37 degrees C. The negative control group (B1) was not infected and was incubated with Trypticase soy broth (TSB) without dextrose supplemented with 5% sucrose (TSBS). In study 2, the same study design was used except that 59Ab was used instead of CsAb, normal rabbit serum was used in the positive control group (A1), and TSB supplemented with 1% glucose was used as the nutrient to control for sucrose-dependent colonization. All groups were exposed for 4 days to circulating cycles of TSBS and TSB (study 1 and study 2, respectively; 30 min each, three times per day) and a mineral washing solution (21 h per day). Prior to each nutrient cycle, 1 ml of the appropriate CsAb or 59Ab solution was administered to each group and allowed to mix for 30 min before cycling was resumed. Data obtained by confocal laser scanning microscopy demonstrated the presence of a significantly smaller (P < 0.05) lesion area and a smaller total lesion fluorescence in group A3 than in group A1 for both studies. In study 1, group A2 had significantly smaller values than A1 for lesion depth and area. There were no significant differences between groups A2 and A3 for lesion area or between groups A1 and A2 for total lesion fluorescence. In study 2, there were no significant differences among groups A1 and A2 for lesion depth or between groups A2 and A3 for all of the parameters studied. In both studies, there were no significant differences between S. mutans plaque CFU numbers among any of the groups. These studies demonstrated the efficacy of CsAb and 59Ab in reducing primary caries development in this model, although the underlying mechanism remains unclear.

Fluoride intake from foods, beverages and dentifrice by young children in communities with negligibly and optimally fluoridated water: a pilot study.

UNLABELLED: While the level of fluoride intake that affords optimal cariostatic efficacy without causing dental fluorosis is not precisely known, it has been suggested that the threshold of fluoride exposure above which fluorosis may occur is between 0.05 and 0.07 mg/kg/day. OBJECTIVE: To monitor and compare fluoride intake from diet and dentifrice use (theoretical F: 0.10-0.11%) by three groups of 16- to 40-month-old children: two groups living in the negligibly water-fluoridated communities of San Juan, Puerto Rico, and Connersville, Indiana, and the third group residing in the optimally water-fluoridated region of Indianapolis, Indiana. METHODS: Fluoride intake from diet was monitored by the "duplicate plate" method, and fluoride ingested from dentifrice was determined by subtracting the amount of fluoride recovered after brushing from the amount originally placed on the child's toothbrush. RESULTS: The mean combined amount of fluoride ingested daily by children living in the negligibly fluoridated communities was not significantly different from that ingested by children in the fluoridated community. The major component of fluoride ingested by children in the negligibly fluoridated communities came from fluoridated dentifrice, and in the fluoridated area children ingested as much fluoride from toothpaste as they did from beverages. In San Juan mean daily fluoride intake was within the estimated range for safe fluoride exposure; however, in the "halo" community of Connersville and in Indianapolis, daily fluoride ingested by many of the children may have exceeded this level. CONCLUSION: Attention needs to be given, in negligibly water-fluoridated as well as in optimally water-fluoridated communities, to reducing the daily intake of fluoride by young children in order to avoid putting them at risk of developing dental fluorosis.

Intranasal immunization against dental caries with a Streptococcus mutans-enriched fimbrial preparation.

Streptococcus mutans has been identified as the major etiological agent of human dental caries. The first step in the initiation of infection by this pathogenic bacterium is its attachment (i.e., through bacterial surface proteins such as glucosyltransferases, P1, glucan-binding proteins, and fimbriae) to a suitable receptor. It is hypothesized that a mucosal vaccine against a combination of S. mutans surface proteins would protect against dental caries by inducing specific salivary immunoglobulin A (IgA) antibodies which may reduce bacterial pathogenesis and adhesion to the tooth surface by affecting several adhesins simultaneously. Conventional Sprague-Dawley rats, infected with S. mutans at 18 to 20 days of age, were intranasally immunized with a mixture of S. mutans surface proteins, enriched for fimbriae and conjugated with cholera toxin B subunit (CTB) plus free cholera toxin (CT) at 13, 15, 22, 29, and 36 days of age (group A). Control rats were either not immunized (group B) or immunized with adjuvant alone (CTB and CT [group C]). At the termination of the study (when rats were 46 days of age), immunized animals (group A) had significantly (P < 0.05) higher salivary IgA and serum IgG antibody responses to the mixture of surface proteins and to whole bacterial cells than did the other two groups (B and C). No significant differences were found in the average numbers of recovered S. mutans cells among groups. However, statistically fewer smooth-surface enamel lesions (buccal and lingual) were detected in the immunized group than in the two other groups. Therefore, a mixture of S. mutans surface proteins, enriched with fimbria components, appears to be a promising immunogen candidate for a mucosal vaccine against dental caries.

The effect of fluoride treatment on bone mineral in rabbits.

Fluoride therapy has been used clinically for many years, but its use remains controversial and many basic questions remain unanswered. Accordingly, this study returns to an animal model to study the effects of high doses of fluoride on bone mineral in rabbits. Twelve rabbits, aged 3(1/2) months at the start of the study, received drinking water fluoridated at 100 ppm while their 12 control counterparts drank distilled water. All rabbits were sacrificed after 6 months. Fluoride was readily incorporated into femoral cortical bone (7473 +/- 966 ppm F versus 1228 +/- 57 ppm in controls; P < 0.00005). Fluoride therapy led to increased mineralization, as measured by density fractionation (P < 0.0005 for the distributions). The bone mineral itself was altered, with a significant increase in the width of crystals (66.2 +/- 2.0 A versus 61.2 +/- 0.9 A; P < 0.01). The microhardness of both cortical and cancellous bone in the femoral head of fluoride-treated rabbits was greater than that in the controls (P < 0.05). The phosphate, calcium, and carbonate contents in the bone was the same in both groups. Finally, fluoride administration did not affect the architecture or connectivity of cancellous bone in the femoral head. Previously published data [1] indicated that the mechanical properties of bone were adversely affected; this suggests that the effect of high doses of fluoride on the strength and stiffness of bone may be mediated by its effect on bone mineral.

Severe dental fluorosis in a Tanzanian population consuming water with negligible fluoride concentration.

OBJECTIVES: To identify risk factors for dental fluorosis that cannot be explained by drinking water fluoride concentration alone. METHODS: Two hundred eighty-four Tanzanian children ages 9 to 19 (mean 14.0+/-SD 1.69), who were lifetime residents at differing altitudes (Chanika, 100 m; Rundugai, 840 m; and Kibosho, 1,463 m; Sites 1, 2, and 3 respectively) were examined for dental fluorosis and caries. They were interviewed about their food habits, environmental characteristics and use of a fluoride-containing food tenderizer known locally as magadi. Meal, urine, water and magadi samples supplied by the participants were analyzed for fluoride content. Urine samples were also analyzed for creatinine concentration. Four magadi samples from Sites 1 and 3 were analyzed for complete element composition. RESULTS: Of the 13 water samples from Site 2, 10 contained > or =4 mg/L F, ranging from 1.26 to 12.36 mg/L with a mean+/-SD of 5.72+/-4.71 mg/L. Sites 1 and 3 had negligible water fluoride of 0.05+/-0.05 and 0.18+/-0.32 mg/L respectively. Mean TFI fluorosis scores (range 0-9) for Site 2 were high: 4.44+/-1.68. In Sites 1 and 3, which both had negligible water fluoride, fluorosis scores varied dramatically: Site 1 mean maximum TFI was 0.01+/-0.07 and Site 3 TFI was 4.39+/-1.52. Mean DMFS was 1.39+/-2.45, 0.15+/-0.73 and 0.19+/-0.61 at Sites 1, 2, and 3, respectively. There were no restorations present. Urinary fluoride values were 0.52+/-0.70, 4.34+/-7.62, and 1.43+/-1.80 mg/L F at Sites 1, 2, and 3, respectively. Mean urinary fluoride values at Site 3 were within the normal urinary fluoride reference value range in spite of pervasive severe pitting fluorosis. Meal and magadi analyses revealed widely varied fluoride concentrations. Concentrations ranged from 0.01 to 22.04 mg/L F for meals and from 189 to 83211 mg/L F for magadi. Complete element analysis revealed the presence of aluminum, iron, magnesium, manganese, strontium and titanium in four magadi samples. There were much higher concentrations of these elements in samples from Site 3, which was at the highest altitude and had severe enamel disturbances in spite of negligible water fluoride concentration. An analysis of covariance model supported the research hypothesis that the three communities differed significantly in mean fluorosis scores (P<0.0001). Controlling for urinary fluoride concentration and urinary fluoride:urinary creatinine ratio, location appeared to significantly affect fluorosis severity. Urinary fluoride:urinary creatinine ratio had a stronger correlation than urinary fluoride concentration with mean TFI fluorosis scores (r=0.43 vs r= 0.25). CONCLUSIONS: The severity of enamel disturbances at Site 3 (1463 m) was not consistent with the low fluoride concentration in drinking water, and was more severe than would be expected from the subjects' normal urinary fluoride values. Location, fluoride in magadi, other elements found in magadi, and malnutrition are variables which may be contributing to the severity of dental enamel disturbances occurring in Site 3. Altitude was a variable which differentiated the locations.

Chronic fluoride exposure does not cause detrimental, extraskeletal effects in nutritionally deficient rats.

On the basis of observations that endemic fluorosis occurs more often in malnourished populations, a series of studies tested the hypothesis that deficient dietary intake of calcium, protein or energy affects fluoride metabolism so that the margin of safe fluoride exposure may be reduced. The objective of the investigation was to determine whether changes in fluoride metabolism in nutritionally deficient rats resulted in manifestation of any extraskeletal toxic fluoride effects not observed in healthy animals. This investigation included two studies, one that monitored the effect of calcium deficiency on the effects of chronic fluoride exposure, and a second study that observed fluoride effects in rats that were deficient either in protein or in energy and total nutrient intake. Control and experimental rats received drinking water containing 0, 0.26 (5), 0.79 (15) or 2.63 (50) mmol fluoride/L (mg/L) for 16 or 48 wk. Control rats were fed optimal diets and experimental rats were fed diets deficient in calcium (Study 1) or protein (Study 2). An additional group of experimental rats (Study 2) was provided with a restricted amount of diet; thus these rats were deficient in energy and total nutrient intake. The intake, excretion and retention of fluoride were monitored; after the rats were killed, tissue fluoride levels and biochemical markers of tissue function were analyzed. Bone marrow cells were harvested from some of the rats, after 48 wk of treatment, for determining the frequency of sister chromatid exchange, a marker of genetic damage. Although there were significant differences among fluoride treatment groups in fluoride excretion and retention that resulted in significantly greater fluoride levels in tissues of the experimental rats, we were unable to detect any harmful, extraskeletal biochemical, physiologic or genetic effects of fluoride in the nutritionally deficient rats.

Measurement of enamel remineralization using microradiography and confocal microscopy. A correlational study.

Tooth minerals are lost and regained constantly in a normal, human oral environment. Different methods have been developed to measure this gain and loss in enamel minerals; however, these methods deal with different problems, such as being time consuming or involving the use of X-rays. The aim of this study was to determine if remineralization measured in a thin enamel section (TS) by transversal microradiography (MR) can be reliably monitored by measuring lesion parameters (area, total and average dye fluorescence) on the same TS or on half a tooth (HT) with confocal laser scanning microscopy (CLSM). Thirty-six human enamel specimens were demineralized for 96h, and then half of each specimen was covered with an acid-resistant nail varnish. Specimens were divided into three groups (12/group) and subjected for 20 days to a cyclic remineralization regimen with consisted daily of a 4-hour acid challenge, four 1-min treatment periods with 0, 250 or 1,100 ppm F dentifrice slurries (1:2; dentifrice:water) and 20 h in pooled, human saliva, at room temperature. Specimens were cut and analyzed by MR, then stained with a fluorescent dye (0.1mM rhodamine B) for 1 h and analyzed using CLSM. Both MR and CLSM detected significantly greater remineralization (p<0.05) in the specimens treated with the fluoride-containing dentifrices than in the specimens treated with 0 ppm F. Significant differences were detected between specimens treated witht the fluoride-containing dentifrices by MR and CLSM (HT area and total fluorescence). Statistically significant (p<0.05) Pearson correlation coefficients were calculated between the MR and CLSM data: difference in MR mineral content (DeltaM) versus HT lesion area = 0.71; DeltaM versus HT total fluorescence = 0.70; DeltaM versus HT average fluorescence = 0.61; DeltaM versus TS lesion area = 0.88; DeltaM versus TS total fluorescence = 0.63, and DeltaM versus TS average fluorescence = 0.40. It is concluded that confocal microscopy in either TS or HT may provide valid surrogates (area and total fluorescence) for MR measurements in enamel remineralization studies.

Fluoride treatment increased serum IGF-1, bone turnover, and bone mass, but not bone strength, in rabbits.

We hypothesized that fluoride partly acts by changing the levels of circulating calcium-regulating hormones and skeletal growth factors. The effects of oral fluoride on 24 female, Dutch-Belted, young adult rabbits were studied. The rabbits were divided into two study groups, one control and the other receiving about 16 mg fluoride/rabbit/day in their drinking water. After 6 months of fluoride dosing, all rabbits were euthanized and bone and blood samples were taken for analyses. Fluoride treatment increased serum and bone fluoride levels by over an order of magnitude (P < 0.001), but did not affect body weight or the following serum biochemical variables: urea, creatinine, phosphorus, total protein, albumin, bilirubin, SGOT, or total alkaline phosphatase. No skeletal fluorosis or osteomalacia was observed histologically, nor did fluoride affect serum PTH or Vitamin D metabolites (P > 0.4). BAP was increased 37% (P < 0.05) by fluoride; serum TRAP was increased 42% (P < 0.05); serum IGF-1 was increased 40% (P < 0.05). Fluoride increased the vertebral BV/TV by 35% (P < 0.05) and tibial ash weight by 10% (P < 0.05). However, the increases in bone mass and bone formation were not reflected in improved bone strength. Fluoride decreased bone strength by about 19% in the L5 vertebra (P < 0.01) and 25% in the femoral neck (P < 0. 05). X-ray diffraction showed altered mineral crystal thickness in fluoride-treated bones (P < 0.001), and there was a negative association between crystal width and fracture stress of the femur (P < 0.02). In conclusion, fluoride's effects on bone mass and bone turnover were not mediated by PTH. IGF-1 was increased by fluoride and was associated with increased bone turnover, but was not correlated with bone formation markers. High-dose fluoride treatment did not improve, but decreased, bone strength in rabbits, even in the absence of impaired mineralization.

An in situ interproximal model for studying the effect of fluoride on enamel.

This crossover study determined the ability of an interproximal, intra-oral model to demonstrate a fluoride dose response to 0-, 250- and 1,100-ppm fluoride (sodium fluoride) dentifrices with respect to fluoride uptake into, and remineralization of, incipient subsurface enamel lesions. Following a 1 week 'lead in' period during which 30 panelists were randomly assigned to use one of the products, two enamel specimens with artificial carious lesions were mounted into a specially designed functional partial denture worn by each panelist. Panelists continued to brush three times daily with their test dentifrice for 4 weeks, after which the specimens were removed and analyzed for fluoride uptake and remineralization. The procedure was repeated until each panelist had followed all three treatment regimens. Fluoride analyses were performed using a microdrill biopsy technique, and mineral content changes were determined by transverse microradiography. Fluoride uptake data were significantly different (p < 0.01) for all three products with the effect of 1,100 ppm F > 250 ppm F > placebo. The 1,100 ppm F dentifrice also effected significantly greater remineralization (p < 0.01) than did the 250-ppm-F or placebo dentifrices. Relative efficacy of the three fluoride dentifrices tested in this study was similar to that established in a clinical trial, and, therefore, supports the use of this model for in situ studies of the effects of fluoride-containing products on enamel lesions.

Lack of effect of long-term fluoride ingestion on blood chemistry and frequency of sister chromatid exchange in human lymphocytes.

Two studies were conducted to assess the potential for adverse physiologic and genotoxic effects of long-term fluoride ingestion in adults residing in three communities with varying water fluoride levels (0.2 ppm, 1.0 ppm, and 4.0 ppm). All were long-time (> or = 30 years) residents of their respective communities. Plasma and urine samples were collected for fluoride analyses. Additional plasma was collected to determine blood chemistry, and plasma lymphocytes were examined to determine the frequency of sister chromatid exchange. Significant differences in urine (P = 0.001) and plasma (P = 0.0001) fluoride levels were found in the three communities. Seven of the blood parameters were statistically different among the communities, although all were within the defined normal range of the pathology laboratory. Sister chromatid exchange frequency was statistically higher in the 4.0 ppm fluoride community as compared to the other communities. Because of the higher SCE frequency in the 4.0 ppm fluoride community, a second study was performed to determine if the increased frequency was potentially related to the fluoride level of the communal water supply. Of the 58 adults recruited; 30 had used city water and 28 had used well water (< or = 0.3 ppm fluoride) as their principal water source for 30 years. Data analyses showed that the sister chromatid exchange frequency did not differ between the groups, indicating that the increased sister chromatid exchange frequency was not related to the fluoride level of the communal water. The investigation provided evidence that the long-term ingestion of water containing 4.0 ppm fluoride did not have any clinically important physiologic or genotoxic effects in healthy adults.

High fluoride intakes cause osteomalacia and diminished bone strength in rats with renal deficiency.

Renal insufficiency is known to increase plasma fluoride levels, which may increase the risk of fluorosis and osteomalacia. The purpose of this study was to determine the effects of fluoride on skeletal fragility and mineralization in renal-deficient animals. We evaluated the skeleton of rats with surgically induced renal deficiency (4/5 nephrectomy) that were chronically exposed to fluoridated water at concentrations of 0, 5, 15, and 50 ppm for a period of 6 months. The chosen fluoride doses caused plasma fluoride levels equivalent to those in humans consuming fluoridated water levels of 0, 1, 3, and 10 ppm, respectively. Animals with renal deficiency drank about 60% more water and excreted 85% more urine than control animals. Glomerular filtration rate (GFR) was decreased 68% and plasma BUN was increased fourfold in rats with renal deficiency. Plasma fluoride was strongly correlated with 1/GFR and was greatly increased by renal deficiency in all animals consuming fluoridated water. There was a strong positive, nonlinear relationship between plasma fluoride and bone fluoride levels, suggesting nonlinear binding characteristics of fluoride to bone. The amount of unmineralized osteoid in the vertebral bone was related to the plasma fluoride levels. Vertebral osteoid volume was increased over 20-fold in animals with renal deficiency that received 15 or 50 ppm fluoride, suggesting osteomalacia. Should osteomalacia be defined as a tenfold increase in osteoid volume, there appeared to be a threshold plasma fluoride level of about 20 micromol/L, above which osteomalacia was observed consistently. This plasma fluoride level was not achieved in control rats regardless of fluoride intake, nor was it achieved in renal-deficient rats receiving 0 or 5 ppm fluoride. A fluoride concentration of 50 ppm reduced femoral bone strength by 11% in control rats and by 31% in renal-deficient rats. Vertebral strength also was decreased significantly in renal-deficient rats given 50 ppm fluoride. In conclusion, fluoridated water in concentrations equivalent to 3 and 10 ppm in humans, caused osteomalacia and reduced bone strength in rats with surgically-induced renal deficiency.

Absence of detrimental effects of fluoride exposure in diabetic rats.

This study is part of a comprehensive programme to investigate fluoride toxicity and the hypothesis that fluoride ingested by "medically compromised' animals will result in altered physiological function. Its objectives were to monitor fluoride retention, tissue fluoride concentrations and genetic variables in diabetic and control rats chronically exposed to fluoride, and to determine whether or not adverse effects occurred. Male, Zucker fatty diabetic rats and Zucker age-matched lean controls were fed a low-fluoride diet ( < 1.2 parts/10(6) F-) ad libitum and received 0, 5, 15 or 50 parts/10(6) fluoride in their drinking water for 3 or 6 months. Fluoride metabolic balance was determined for 4 days before the end of each study phase. Plasma and urine were analysed for biochemical markers of tissue function, and plasma, urine, faeces and tissues were analysed for fluoride. Bone marrow cells from animals killed after 6 months of treatment were examined for frequency of sister chromatid exchange, a marker of genetic damage. The diabetic rats consumed, excreted and retained significantly greater amounts of fluoride than the controls (p < 0.05). There were dose-related increases in fluoride excretion, retention and tissue concentrations in both classes of animals, which were significantly greater in the diabetic rats. In spite of greater amounts of fluoride in the tissues of diabetic animals, there was no evidence, under these experimental conditions, that any of the fluoride exposures tested caused measurable adverse effects on the physiological, biochemical or genetic variables that were monitored.

Measurement of enamel demineralization using microradiography and confocal microscopy. A correlation study.

Substantial amounts of tooth minerals are lost during dental caries formation. Transversal microradiography, a well-accepted method used to quantify mineral loss, is a time-consuming technique which requires a thin enamel section (100 microns) and involves the use of x-rays. In an attempt to solve these difficulties, a procedure has been developed in which a human tooth specimen with demineralized enamel is cut in half (HT), stained with a fluorescent dye (rhodamine B) and analyzed using a laser scanning confocal microscope. A series of three studies was conducted to correlate measurements of enamel demineralization obtained from enamel thin (100 microns) sections (TS) using transversal microradiography with three parameters (area of the lesion; total and average dye fluorescence intensities) measured on the same TS or on a thicker section (HT) of the same specimen by laser scanning confocal microscopy. Results showed that a 0.1 mM rhodamine B solution provided the most adequate imaging conditions for confocal microscopy. Pearson's correlation coefficients, calculated between microradiography and confocal microscopy data obtained using a 0.1 mM rhodamine B solution, were: delta Z vs. HT lesion area = 0.95; delta Z vs. HT total fluorescence = 0.80; delta Z vs. HT average fluorescence = 0.74; delta Z vs. TS lesion area = 0.95; delta Z vs. TS total fluorescence = 0.74; delta Z vs. TS average fluorescence = 0.55. All these correlations coefficients were statistically significant (p < 0.01). It is concluded that in enamel demineralization studies statistically significant correlations exist between parameters measured using transversal microradiography and parameters quantified using confocal microscopy.

An in vitro microbial model for studying secondary caries formation.

Secondary caries is a major reason for the replacement of restorations. Because it is hypothesized that the development of secondary caries is closely associated with pathogenic oral bacteria, an in vitro microbial model has been developed to produce secondary carious lesions. A mixture of overnight cultures of Streptococcus mutans and Lactobacillus casei in dextrose-free trypticase soy broth, supplemented with 5% sucrose (TSBS), at 37 degrees C was used in this model as the inoculum for the experimental groups. Uninoculated control groups were incubated with medium only. Groups of human tooth specimens restored using composite, together with their respective controls, were exposed for 7 or 12 days to circulating cycles of TSBS (30 min each, 3 times per day) and a mineral wash solution (for a total of 22.5 h per day), at 37 degrees C. The pH of the experimental groups dropped to 4.l-4.5 during the test periods. The pH of the control groups remained at 6.8-7.0. The inoculated bacteria remained viable throughout the study. No contamination of experimental or control samples occurred. Laser scanning confocal microscopy demonstrated the development of incipient surface and wall lesions in all the specimens of experimental groups in as few as 7 days. Reproducibility of the model was confirmed in a second investigation. Therefore, it was concluded that this model can be used for studying the microbial etiology and prevention of secondary caries.

Fluoride reduces bone strength in older rats.

In response to recent concerns about the effect of water fluoridation on hip fracture rates, we studied the influence of fluoride intake on bone strength. Four groups of rats were fed a low-fluoride diet ad libitum and received 0, 5, 15, or 50 ppm of fluoride in their drinking water. Animals were euthanized after 3, 6, 12, or 18 months of treatment. Mechanical strength of the right femur was measured by three-point bending. Fluoride content for the left femur was measured, and static histomorphometric measurements were made on a lumbar vertebra. Femoral failure load was not significantly decreased in rats treated for 3 and 6 months, but was decreased as much as 23% in rats treated 12 and 18 months at 50 ppm fluoride. Extrapolation from regression equations predicted that older rats lose 36% of femoral bone strength when bone fluoride content is increased from 0 to 10,000 ppm, while younger rats will lose only 15%. Thus, the decreased strength appeared to be due to the combined effects of fluoride intake and age on bone tissue and was not associated with a decrease in bone density or mineralization defects. There were only small effects of fluoride on bone histomorphometry. Fluoride intake at high levels had no negative effects on bone mineralization. Fluoride intake was associated with slight increases in trabecular bone volume and trabecular thickness, but these effects could not be demonstrated consistently. The mechanism by which large amounts of fluoride affect bone strength more severely in older animals is unknown.

Effect of aging on animal response to chronic fluoride exposure.

This study was conducted to test the hypothesis that physiological changes which occur during aging increase the biological impact of fluoride and reduce the threshold of safe fluoride exposure. Four groups of rats were fed a low-fluoride diet (< 1.2 ppm) ad libitum and received 0, 5, 15, or 50 ppm fluoride in their drinking water. Animals were killed after three, six, 12, or 18 months of treatment. Blood and urine were monitored for biochemical markers of tissue function, and plasma, urine, feces, and representative tissues were analyzed for fluoride. In addition, bone marrow cells from animals killed after 18 months of treatment were examined for frequency of sister chromatid exchange (SCE), a marker of genetic damage. Study results indicated that, within treatment groups, fluoride intake, excretion, and retention did not change significantly between three and 18 months. Fluoride concentration in soft tissues did not change with treatment duration in the fluoride-treated animals. Mineralized tissue fluoride concentration and the total fluoride in the carcasses increased continually as the animals aged. In spite of significant, dose-related differences in tissue fluoride levels which occurred in all age groups in this study, there were no indications that increased fluoride in the tissues caused any adverse physiological or genotoxic effects. None of the monitored clinical "wellness" markers of tissue integrity and function was altered by fluoride in a clinically significant manner. Therefore, there was no evidence from this study that aging reduces the threshold of safe chronic fluoride exposure.

Degradation of insoluble bovine collagen and human dentine collagen pretreated in vitro with lactic acid, pH 4.0 and 5.5.

The purpose of the study was to test the hypothesis that both insoluble pure type I collagen from bovine Achilles tendon and dentine collagen in root dentine powder from human teeth required acid pretreatment for subsequent degradation by trypsin, a non-specific protease. Pure type I collagen or dentine powder was treated with lactic acid, at pH 4 or 5.5, or distilled, deionized water (pH 7) as a negative control. After incubation at 37 degrees C for 24 h, extracts of pure type I collagen solutions were analysed for soluble collagen with the hydroxyproline assay. Extracts of dentine powder solution were analysed for Ca2+, total protein, final pH, and hydroxyproline. Residual, undegraded pellets were washed and then treated with trypsin or collagenase. After 24 h of incubation, the soluble fractions from the enzyme-treated pure type I collagen and dentine powder solutions were analysed for hydroxyproline. Results showed that almost no pure type I collagen was degraded during acid pretreatment. Trypsin degraded significantly more pure type I collagen in the pH 4-treated group than in the other groups. Collagenase degraded about 70% of the pure type I collagen irrespective of acid pretreatment. While acid pretreatment at pH 4 did not degrade dentine collagen, data from Ca2+ analyses and collagen breakdown by trypsin suggested that pretreatment at pH 4 demineralized and denatured dentine collagen so that the collagen could be subsequently degraded by enzymes. After pretreatment at pH 4, about 27 and 57% of the dentine collagen was degraded by trypsin and collagenase, respectively, in contrast to minimal degradation of non-acid-treated dentine collagen by the same enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

[A comparative study on a human model of the in situ absorption by enamel of fluorine obtained from the NaF and Na2PO3F contained in tooth pastes]. / Sravnitel'noe izuchenie na chelovecheskoi modeli in situ pogloshcheniia émal'iu ftora, poluchennogo iz NaF i Na2PO3F, soderzhashchikhsia v zubnykh pastakh.

A denture model was used in situ in cross experiment to assess fluorine absorption by impaired enamel from toothpastes containing NaF and Na2PO3F. Six toothpastes were used in experiment: two contained NaF, three Na2PO3F, and one contained no fluorine. Twenty-five volunteers participated in experiment, who were fitted with dentures with sites of impaired enamel located close to each other. Each participant in the experiment was to accidentally clean his or her teeth with one of the pastes three times daily for two weeks. Then the enamel specimens were removed and analysed for fluorine content. The experiment lasted till each volunteer spent all the six pastes offered. The results indicated a much higher efficacy of NaF-containing pastes as regards fluorine absorption by impaired enamel than the ones containing Na2PO3F; the presence of decalcinated phosphate did not enhance the activity of Na2PO3F.