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Quantification of the degree of mineralization of bone in three dimensions using synchrotron radiation microtomography.

The availability of three-dimensional measuring techniques coupled to specific image processing methods opens new opportunities for the analysis of bone structure. In particular, synchrotron radiation microtomography may provide three-dimensional images with spatial resolution as high as one micrometer. Moreover, the use of a monoenergetic synchrotron beam, which avoids beam-hardening effects, allows quantitative measurements of the degree of mineralization in bone samples. Indeed, the reconstructed gray levels of tomographic images correspond directly to a map of the linear attenuation coefficient within the sample. Since the absorption depends on the amount of mineral content, we proposed a calibration method to evaluate the three-dimensional distribution of the degree of mineralization within the sample. First a theoretical linear relationship modeling the linear attenuation coefficient as a function of the hydroxyapatite concentrations was derived. Then, an experimental validation on phantoms confirmed both the accuracy of the image processing tools and the experimental setup used. Finally, the analysis of the degree of mineralization in four iliac crest bone biopsy samples was reported. Our method was compared to the reference microradiography technique, currently used for this quantification in two dimensions. The concentration values of the degree of mineralization were found with both techniques in the range 0.5-1.6 g of mineral per cubic centimeter of bone, both in cortical and in trabecular region. The mean difference between the two techniques was around 4.7%, and was slightly higher in trabecular region than in cortical bone.

Bone Density↗

Microvascular studies in human radiation bowel disease.

The microvasculature was investigated in the normal bowel (n = 43 patients) and in radiation bowel disease (n = 18 patients). Tissue samples obtained from postoperative colectomy specimens in which the intramural vessels had been perfused with barium sulphate suspension were examined. Microradiography was used to study vascular pattern which was abnormal in radiation bowel disease. A recently described radiograph fluorescence system was used to estimate barium concentration, and hence microvascular volume. The radiation group showed a highly significant reduction in barium concentration (p less than 0.001), when compared with the normal group. This reduction was diffuse in samples from 15 patients who had received combined intracavity and external radiotherapy, but localised in two patients who had received intracavity treatment only. It is concluded that microvascular compromise is an important factor in the natural history of radiation bowel disease.

Adult↗

Microvascular studies in non-specific inflammatory bowel disease.

The microvasculature was investigated in the normal bowel and in ulcerative colitis and Crohn's disease. Tissue samples from postoperative colectomy specimens in which the microvasculature had been perfused with barium sulphate suspension were examined. Microradiography was used to study intramural vascular pattern which was abnormal in both disease states. A recently described radiograph fluorescence system was used to estimate barium concentration and hence microvascular volume in tissue samples. Highly significant negative correlations were demonstrated between barium concentration and age in normal bowel (n = 44; r = -0.669; p less than 0.001) and in segmental Crohn's disease (n = 11; r = -0.698; p = 0.017). Barium concentration was significantly reduced (p less than 0.05) in segmental Crohn's disease (n = 11) but remained normal in diffuse Crohn's disease of the colon (n = 6) and ulcerative colitis (n = 7). It is postulated that ischaemia may be a factor in the pathogenesis of segmental Crohn's disease in older patients.

Adolescent↗

The pattern of intramural veins of the left ventricle of the human heart.

A detailed injection, microradiographic, and histological necropsy study of 18 hearts showed that the pattern of myocardial veins differed from the arterial pattern. In "normal" hearts, large drainage veins began in the subendocardial zone and coursed fairly directly towards the epicardium, maintaining a comparatively even calibre throughout. Smaller, but similar, drainage veins also begain in the middle of the myocardial wall. In the outer myocardium small groups of these vessels converged to form a single vein which then entered, almost at right angles, the pericardial veins. In general, small veins within the myocardium entered directly into the large drainage veins without any extensive intermediate sized venous network. In "abnormal" hearts with left ventricular hypertrophy the myocardial veins appeared basically normal but were more widely separated and consequently overall less dense on microradiography. Interruption of main drainage veins and loss of normal pattern were seen in areas of myocardial fibrosis associated with coronary artery disease. In a heart with severe generalized coronary artery atheroma the normal pattern in the inner half of the wall was replaced by a "plexus" of small veins in which the majority of vessels coursed circumferentially. The possible significance of both the normal and abnormal vein pattern is discussed.

Adult↗

Postoperative radionuclide evaluation of osteoid osteomas.

Five cases of clinically suspected osteoid osteomas were studied by preoperative injection of technetium-99m methylene disphosphonate, intraoperative localization with a radiation-sensitive scintillation probe, and postoperative examination of the entire tissue specimen (including the presumed nidus and surrounding bone). Microradiography and light microscopy were also used. In addition, a new autoradiography technique was introduced in which the excised surgical specimen was placed on undeveloped x-ray film for pathologic localization, diagnosis of the lesions, and a study of the relative intensity of radioactive uptake in the nidus vs. surrounding bone. Autoradiography revealed that the nidus showed the greatest concentration of radioactivity, followed by the surrounding bone. The authors conclude that 99mTc can be used clinically in localizing osteoid osteomas and that preoperative and intraoperative scanning can assist in conservative surgical excision, e.g., minimal extirpation of bone in delicate areas such as the spine. For small lesions, autoradiography assists the pathologist in identifying an osteoid osteoma.

Adolescent↗

CT of the middiaphyseal femur: cortical bone mineral density and relation to porosity.

PURPOSE: To determine whether computed tomography (CT) can be used to quantify age- and site-related changes in cortical bone mineral density (cBMD) at the middiaphyseal femur and whether cBMD differences are related to intracortical porosity. MATERIALS AND METHODS: Cortical bone specimens from 163 femurs were studied with CT and microradiography. Femurs were from 77 males and 86 females in a white anthropologic collection covering a broad age spectrum. In each sample, the cBMD was measured in the entire cortical width and in periosteal, midcortical, and endosteal subregions of interest. Age- and site-related changes in cBMD were tested for significance by using a two-way analysis of variance for both sexes. By using linear regression, cBMD was compared with porosity in the entire cortical width and in each subregion. RESULTS: There were significant age-related differences in cBMD (P <.001 in females, P =.008 in males). In addition, cBMD values were significantly different between the three cortical subregions (P <.001 for both sexes), decreasing from the periosteum to the midcortex to the endosteum. The cBMD values were closely related to porosity, and porosity contributed to 71.6% of the variance in cBMD in the overall population. CONCLUSION: CT is effective in the measurement of age- and site-related changes in cBMD. Decreases in cBMD are closely correlated with increased cortical porosity.

Adolescent↗

Inhibition of dentin demineralization by fluoride in vitro.

Compared with the knowledge accumulated on enamel-fluoride interactions, relatively little data is available regarding fluoride effects on dentin. This applies to both laboratory and clinical studies into the efficacy of fluoride schemes for the prevention of root surface caries. This study aimed to determine the effects of fluoride and pH on the demineralization of dentin, such as to provide information necessary to develop preventive programmes. Bovine dentin blocks were subjected to undersaturated calcium- and phosphate-containing solutions in the pH range 4.0-6.0 with fluoride added at concentrations between 0.5 and 10 ppm. Non-fluoride solutions served as controls. Mineral loss was assessed chemically and by transversal microradiography. Comparisons were made with similar studies on enamel demineralization. The results showed that demineralization of dentin depends on both pH and fluoride concentration in the demineralizing solution. Inhibition of demineralization that could be relevant from a clinical point of view was found at fluoride values 5-10 times the corresponding values for enamel. Also rapid depletion of fluoride from the solutions was observed, indicating the high uptake capacity of dentin for fluoride. Lesion depth depended on pH of the solution while the fluoride levels were associated with the surface layer, both in mineral content and depth. For dentin we propose a demineralization mechanism where acid penetrates rapidly into the tissue, presumably through the tubules, after which the released calcium and phosphate is partly trapped by the inward diffusing fluoride. This leads to the formation of a surface layer, which may even be hypermineralized compared to sound dentin.

Animals↗

Effect of toothpicks with and without fluoride on De- and remineralization of enamel and dentine in situ.

The aim of this investigation was to study the effect of the interproximal use of fluoride (F)-impregnated and non-impregnated birch toothpicks on the degree of de- and remineralization of enamel and dentine in situ. Ten volunteers with complete dentures in the upper jaw participated. Each subject had four specimens: (1) sound enamel, (2) demineralized enamel, (3) sound dentine and (4) demineralized dentine; placed pairwise at two approximal sites (15/16 and 25/26) of the maxillary prosthesis. The study involved three test periods (A, B and C), each lasting 4 weeks. In A, the subjects used F toothpicks (impregnated in 4% NaF) and, in B, nonimpregnated toothpicks 3 times daily. During period C, no toothpicks were used. Dentifrice or other F-containing products were not allowed during the 4-week periods. Transversal microradiography was used to determine lesion depth (ld) and mineral loss (DeltaZ). The results revealed that all the sound samples lost mineral during the three experimental periods; DeltaZ for both enamel and dentine was less for A and B compared with C (p<0.01) and less for A compared with B and C for dentine (p<0.05, p<0.01). The demineralized samples also lost mineral, apart from dentine, during periods A and B, i.e. when F-impregnated and non-impregnated toothpicks were used; ld for enamel and DeltaZ for dentine were less for A compared with C (p<0.05). Four weeks' use of toothpicks, especially F-impregnated toothpicks, thus reduces the demineralization of enamel and dentine at approximal sites in situ.

Aged↗

Efficacy of sterilisation methods and their effect on enamel demineralisation.

The aim of this project was to determine the effectiveness of sterilisation methods for dental enamel for use in intra-oral cariogenicity tests, and their possible effect on the degree of demineralisation of enamel. Bovine incisors were cut vertically into five portions and each assigned to one of five groups. Group 1 was used as a control while the other four groups were subjected, respectively, to gamma irradiation ( congruent with 25kGy), steam autoclaving (121 degrees C for 15 min), sodium hypochlorite (12% w/v for 24h) and povidone-iodine (7.5% w/v for 24h). Total viable counts of microorganisms remaining following sterilisation of the specimens were performed following incubation of the specimens for 24h at 37 degrees C. Caries-like lesions were produced in each specimen using an acidic buffer solution (pH4.5). Sections were cut from each specimen, ground to 80-microgram thickness, and microradiographed. Mineral loss and lesion depth were quantified using transverse microradiography. Statistical analysis was by ANOVA. Dunnett's and Tukey's tests. Microbial growth (Staphylococcus aureus and bacilli) was observed only in control specimens in both brain heart infusion broth and on blood agar plates. The sterilisation methods affected the enamel surface as follows: gamma irradiation (cream discolouration), NaOCl (bleaching), and povidone-iodine (white spot-like lesion). Compared with the control, there was no significant difference in mineral loss and lesion depth with any of the groups, but the numerical values of mineral loss and lesion depth in groups can be ranked as follows: gamma irradiation <povidone-iodine <control <autoclave <NaOCl. In conclusion, the four sterilisation methods were all effective to sterilise enamel, but gamma irradiation proved the most acceptable method for enamel to be used in cariogenicity tests having the least adverse effect.

Acids↗

Fluoride in enamel lining pits and fissures of the occlusal groove-fossa system in human molar teeth.

The fluoride (F) distribution in enamel lining the occlusal fissures of human molars and premolars is difficult to investigate by normal microsampling techniques, yet this information is of importance as fissures are particularly susceptible to caries. We have used the proton probe to map the distribution of F and Ca in sections of 17 molar teeth collected from Danish and New Zealand populations. The caries status of the sectioned fissure was determined by microradiography or visually after drying. The probe scans were graphed as density images, surface plots and topographical plots. Sound enamel bordering grooves and fissures showed a high-F surface layer, in 1 sample approximately 60 microm wide near the fissure bottom but gradually widening to double this width near the fissure opening, and up to 200 microm wide in grooves, while underlying enamel had a low and almost constant F level. In this respect fissure and groove enamel resembled smooth surface enamel. The F concentration fluctuated along the surface layer, reaching maximum values ranging from 1,800 to 4,200 ppm in 5 non-carious fissures. Incipient caries in fissure enamel usually but not always resulted in an increase in F in the outer layer, the F maximum values in 5 such samples ranging from 1,900 to 7,200 ppm. F maximum values in outer enamel were higher in 7 samples showing advanced caries, 2, 700-10,000 ppm. The lesion subsurface body usually showed normal F values, as did carious dentine in the advanced lesions. The variable characteristics of the outer layer in sound fissure enamel are likely to be the result of a developmental process rather than environmental influences. The fact that we normally failed to find increased F concentrations in subsurface carious fissure enamel or in underlying carious dentine suggests that F does not diffuse into the deep part of fissure lesions, and probably has minimal effect on slowing the progress of such lesions, a suggestion in accord with clinical findings on the F effect on fissure caries.

Calcium↗

Effect of tooth-bound fluoride on enamel demineralization/ remineralization in vitro.

The effect of tooth-bound fluoride (F) on enamel caries formation was investigated under the condition that loosely bound F was essentially absent. Eighteen thin enamel sections, prepared from the lingual or buccal surfaces of extracted human molars, were embedded in acrylic resin with the enamel surfaces exposed. The sections were placed in a pH 7 remineralizing solution (RS; 1.2 mmol/l Ca, 0.72 mmol/l P, 30 mmol/l KCl, 50 mmol/l HEPES) for 5 days, and were randomly divided into 3 groups: (1) control group that received no treatment, (2) acidulated phosphate fluoride (APF) group that received 5 cycles of a 4 min treatment with APF gel followed by immersion in the RS for 2 days (RS changed daily) and (3) dicalcium phosphate dihydrate (DCPD) - APF group that received 5 cycles of a 4-min pH 2.1 DCPD-forming solution followed by 4 min APF gel and then placed in the RS for 2 days. After the treatment cycles, the sections were washed in a constant composition F titration system to remove loosely bound F. An in vitro model, which consisted of cycles of de- (6 h) and remineralization (18 h) each day for 5 days, was used to produce caries-like lesions in the specimens. The DeltaZ (mineral loss) values, measured by quantitative microradiography, of the lesions formed in the three groups were (mean +/- standard deviation; n = 6) 91.2+/-12.3 microm for the control group, 41.3+/-10.1 microm for the APF group and 21.2+/-4.8 microm for the DCPD-APF group. The same system produced lesions in untreated shark enamel with a mean DeltaZ of 4.4+/-0.3 microm (n = 12). One-way fixed-effects ANOVA indicated that mineral loss was significantly different among the different groups (p<0.05). The results showed that enamel resistance to lesion formation increased with increasing tooth-bound F content. Shark enamel was much more resistant to demineralization than human enamel.

Acidulated Phosphate Fluoride↗

Comparison of QLF and DIAGNOdent for quantification of smooth surface caries.

The aim was to compare the detection and quantification of smooth surface caries by KaVo DIAGNOdent and quantitative laser/light-induced fluorescence (QLF) using a laser and a lamp as two different light sources. On 40 premolar teeth extracted from adolescents, 71 non-cavitated approximal surfaces were assessed. For QLF, both mean and maximum fluorescence losses were registered. To provide a gold standard for verification, the teeth were then sectioned and lesion depth, stratified on a five-point scale, was determined by histopathology and microradiography. The correlation between the gold standard and the two methods was assessed by Spearman's rank correlation coefficient. The sensitivity and specificity of the methods for detection of smooth surface caries at D(3) level were also assessed. For lesion depth, correlation with the gold standard was similar for QLF and DIAGNOdent: about 0.85. With respect to dentinal caries detection, sensitivity for DIAGNOdent was 0.75 and specificity 0.96, with a cut-off point of 9. The corresponding values for QLF were 0.94 and 1, with a cut-off value of 20% of fluorescence loss. Spearman's rank correlation coefficients for enamel mineral loss and QLF and DIAGNOdent, respectively, were 0.76 and 0.67. It was concluded that for quantification of smooth surface caries, the methods are of equal merit, but for scientific purposes, QLF offers the advantage of closer correlation with changes in mineral content.

Adolescent↗

Effectiveness of two fluoridation measures on erosion progression in human enamel and dentine in vitro.

The aim of the present study was to evaluate the effect of fluoride on the progression of erosive demineralisation in human enamel and dentine using a cyclic de- and remineralisation model in vitro. The mineral content expressed in micrometres was determined daily by longitudinal microradiography (LMR) and presented as cumulative mineral loss over 5 days. For erosive demineralisation, all samples were immersed in 0.05 M citric acid (pH 2.3) for 6x10 min/day and stored in a remineralisation solution. Fluoridation measures were performed as follows: group 1: control, no fluoridation; group 2: toothpaste fluoridation 3x5 min/daily (NaF, 0.15% F-); group 3: toothpaste fluoridation as group 2 and additionally application of a fluoride mouthrinse (Olaflur/SnF2, 0.025%F-) 3x5 min/daily and on days 1 and 3 gel fluoridation (Olaflur/NaF; 1.25% F-) for 1x5 min. After the first experimental day, no significant differences were found between the groups. However, after 5 days the erosive mineral loss values for enamel were 147.5+/-18.7 microm in the control group, 128.1+/-15.0 microm in group 2 (p< or =0.05) and 116.1+/-12.4 microm in group 3 (p< or =0.001). In dentine, the respective values were 136.7+/-16.4, 111.8+/-26.9 (p< or =0.001) and 60.3+/-17.8 (p< or =0.001). The intensive fluoridation significantly reduced erosion progression in enamel but had a more pronounced effect on dentine. The results suggest that subjects with erosive lesions should use an intensive fluoridation measure.

Analysis of Variance↗

Comparative study to quantify demineralized enamel in deciduous and permanent teeth using laser- and light-induced fluorescence techniques.

Caries is a disease that affects both deciduous and permanent dentitions. Caries progresses more rapidly in deciduous enamel than in permanent enamel. Therefore, new caries diagnostic methods need to be tested on the deciduous teeth as well. Quantitative laser-induced fluorescence (QLF I) as well as the quantitative light-induced fluorescence (QLF II) seem promising for the quantification of mineral loss from dental caries but have only been tested on the permanent dentition. The objective of this study was to determine and compare the ability of QLF I and QLF II to quantify mineral loss from carious lesions in both deciduous and permanent teeth. Thirty sound deciduous and 30 sound permanent teeth were cleaned and divided into three groups each containing 10 deciduous and 10 permanent teeth. Windows on the buccal or labial enamel surfaces were demineralized for 48, 72, or 96 h. Images of demineralized enamel were captured using QLF I and QLF II. The images were analyzed to determine the mean change in fluorescence radiance (Delta F, %). The teeth were then sectioned for assessment of lesion depth (microm) and integrated mineral loss (IML, vol% x microm) using transverse microradiography (TMR), as the 'gold standard' for lesion analysis. The results indicated a good correlation for Delta F between QLF I and QLF II in both deciduous (r = 0.96) and permanent teeth (r = 0.98). There was a good correlation between Delta F and TMR (lesion depth and IML) in deciduous teeth (r = 0.76 and 0.84 with QLF I, r = 0.81 and 0.88 with QLF II). In permanent teeth, the correlation between Delta F and TMR (lesion depth and IML) was lower than in deciduous teeth (r = 0.07 and 0.53 with QLF I, r = 0.15 and 0.62 with QLF II). From these results it can be concluded that either QLF method is capable of quantifying mineral loss in early carious lesions in deciduous teeth. Moreover, under the conditions of this study, the use of either QLF method to quantify mineral loss in early carious lesions in deciduous teeth is slightly more accurate than in permanent teeth.

Analysis of Variance↗

Fluoride and urea chewing gums in an intra-oral experimental caries model.

The aim of the present investigation was to evaluate the effect of sugar-free chewing gums containing fluoride (F) and urea in an intra-oral experimental caries model. Placebo chewing gums (without any active ingredient) and no gum served as controls. Fifteen subjects participated in a cross-over, single-blind study. Demineralised enamel and dentine blocks were embedded in circular plastic discs and bonded to the buccal surfaces of the lower canines and first premolars. The discs were removed and analysed using transversal microradiography after each of the six 4-week periods during which the subjects used either test or placebo products or no product. The results revealed that frequent use of sugar-free chewing gum is sufficient to inhibit further demineralisation of previously demineralised enamel and dentine specimens in the oral cavity. Comparing F, urea and placebo gums, the data showed that there was little or no difference between the products, except for an inhibitory effect on the chewing side of the dentition after using F chewing gums.

Adult↗

Remineralisation of enamel lesions with daily applications of a high-concentration fluoride gel and a fluoridated toothpaste: an in situ study.

The inhibition of enamel demineralisation and the enhancement of remineralisation are positively but not linearly related to the concentration of fluoride, especially when high fluoride concentrations are used. The aim of this in situ experiment was to determine the maximum amount of enamel remineralisation that can be achieved with daily applications of very high concentrations of fluoride. For this purpose we compared the efficacy of a daily application of fluoridated topical gel (12500 ppm F, partly as NaF, Olafluor and Dectafluor, pH 4.5) in combination with a fluoridated toothpaste (1450 ppm F as NaF), with fluoridated toothpaste alone. Participants (n = 26, with partial dentures) were fitted with a demineralised enamel specimen (mean mineral loss of 1674 vol%.micro m) and were instructed to use one of the two fluoride treatments. After 4 weeks of treatment, the specimens were retrieved, a section was cut and analysed with microradiography. The remainder of each of the specimens was used for analysis of the 'loosely bound' and 'bound' fluoride. Fluoride was measured with gas-liquid chromatography. After 4 weeks in the mouth, the original lesion was reduced in size by 54% in the toothpaste + gel group (n = 14) and by 44% in the toothpaste-only group (n = 12), but the difference between the groups was not statistically significant. The mineral content profiles showed remineralisation of the lesions throughout the depth of the lesion. The enhancement of remineralisation by the high amounts of fluoride was most pronounced in the surface layer. For both the 'loosely bound' and 'bound' fluoride, a statistically significant increase in fluoride concentration could be found in the toothpaste + gel group. In the 4-week in situ period the use of high amounts of fluoride resulted in a maximum remineralisation rate. This is illustrated by an increase in remineralisation and higher fluoride concentrations in the toothpaste + gel group compared to the toothpaste-only group.

Dental Enamel↗

Influence of enamel thickness on quantification of mineral loss in enamel using laser-induced fluorescence.

The objective of this study was to evaluate the effect of enamel thickness on the quantification of white-spot lesions with laser fluorescence. One hundred and twenty 3-mm-diameter human ground and polished enamel specimens were used. Specimens were illuminated by a 488-nm argon laser, images were acquired through a 540-nm high-pass filter and stored on the Quantitative Light-Induced Fluorescence (QLF) program. Specimens were divided into two groups and demineralized for 48 or 96 h and QLF images were again acquired. Fluorescence radiance (FR) of both sound (FR(s)) and demineralized (FR(d)) enamel was determined using the QLF program. Change in fluorescence radiance (Delta FR, %) was determined as follows: Delta FR = FR(d)/FR(s) x 100. One thin section was obtained for transverse microradiography analysis. Enamel and dentin thickness of the thin sections were measured. There was a good correlation between QLF parameters and enamel thickness. It can be concluded that the FR observed for similar lesions depends on the actual enamel thickness.

Dental Enamel↗

Influence of olive oil emulsions on dentin demineralization in vitro.

The effect of two different concentrations of olive oil emulsions on development of artificial caries-like dentin lesions under severe demineralizing conditions was investigated. Bovine dentin samples (n = 180) were ground flat, polished, divided into four groups, and subjected to three demineralization cycles per day. Samples were stored in one of the following solutions for 5 min prior to demineralization in a buffer solution (pH 5): Group 1: 50% oil emulsion (olive oil and distilled water); group 2: 5% oil emulsion; group 3: distilled water; and group 4: 1,500 ppm sodium fluoride. Daily up to 9 days, lesion depth (ld) and mineral loss (deltaZ) were determined by means of microradiography and analyzed by ANOVA and Tukey's studentized range test (p < or = 0.05). Lesion depth increased with time for all groups. Mineral loss increased in groups 1-3. A small but significant decrease in mineral loss was observed following treatment with lipid emulsions as compared to treatment with distilled water, but fluoride treatment was considerably more effective. Mean mineral loss (means +/- SD in vol% x microm) averaged over the study period was 4,368 +/- 1,599, 4,536 +/- 1,823, 4,849 +/- 1,798, and 789 +/- 177 for group 1, 2, 3, and 4, respectively. Ratio (deltaZ/ld) remained constant around 30 vol% for groups 1-3, but decreased for group 4. In conclusion, externally provided lipids have the potential to reduce dentin demineralization in vitro.

Analysis of Variance↗