Microhardness Measurements on Tooth and Alveolar Bone in Rodent Oral Disease Models.

The mechanical property, microhardness, is evaluated in dental enamel, dentin, and bone in oral disease models, including dental fluorosis and periodontitis. Micro-CT (µCT) provides 3D imaging information (volume and mineral density) and scanning electron microscopy (SEM) produces microstructure images (enamel prism and bone lacuna-canalicular). Complementarily to structural analysis by µCT and SEM, microhardness is one of the informative parameters to evaluate how structural changes alter mechanical properties. Despite being a useful parameter, studies on microhardness of alveolar bone in oral diseases are limited. To date, divergent microhardness measurement methods have been reported. Since microhardness values vary depending on the sample preparation (polishing and flat surface) and indentation sites, diverse protocols can cause discrepancies among studies. Standardization of the microhardness protocol is essential for consistent and accurate evaluation in oral disease models. In the present study, we demonstrate a standardized protocol for microhardness analysis in tooth and alveolar bone. Specimens used are as follows: for the dental fluorosis model, incisors were collected from mice treated with/without fluoride-containing water for 6 weeks; for ligature-induced periodontal bone resorption (L-PBR) model, alveolar bones with periodontal bone resorption were collected from mice ligated on the maxillary 2nd molar. At 2 weeks after the ligation, the maxilla was collected. Vickers hardness was analyzed in these specimens according to the standardized protocol. The protocol provides detailed materials and methods for resin embedding, serial polishing, and indentation sites for incisors and alveolar. To the best of our knowledge, this is the first standardized microhardness protocol to evaluate the mechanical properties of tooth and alveolar bone in rodent oral disease models.

High contrast imaging of dental fluorosis in the short wavelength infrared.

Dental fluorosis is an increasing problem due to over exposure to fluoride from the environment. Fluorosis causes hypomineralization of the enamel during tooth development and mild fluorosis is visible as faint white lines on the tooth surface while the most severe fluorosis can result in pitted surfaces. It is difficult to quantify the severity of mild to moderate fluorosis and assessments are limited to subjective visual examinations. Dental fluorosis appears with very high contrast at short wavelength infrared (SWIR) wavelengths beyond 1400 nm and we hypothesize that these wavelengths may be better suited for detecting mild fluorosis and for estimating the severity on tooth surfaces. In this study, the contrast of fluorosis of varying severity on extracted human permanent teeth was measured at SWIR wavelengths ranging from 1300 to 2150 nm using an extended range of InGaAs camera and broadband light sources. The contrast was also measured in the visible range and with quantitative light-induced fluorescence (QLF) for comparison. The depth of hypomineralization and the integrated reflectivity were also measured with cross-polarization optical coherence tomography. The contrast of hypomineralization is significantly higher (P < 0.05) at 1460 and 1950 nm wavelengths than for the visible, fluorescence or other SWIR wavelengths from 1300 to 2150 nm. The highest correlation of the contrast with the depth of hypomineralization measured with cross-polarization-optical coherence tomography (CP-OCT) was at 1950 nm. This SWIR in vitro imaging study exploring wavelengths beyond 1400 nm has shown that hypomineralization on tooth surfaces can be viewed with extremely high contrast at SWIR wavelengths from 1460 to 2000 nm and that SWIR imaging has great potential for monitoring hypomineralization on tooth surfaces. New clinical methods are needed for the measurement of fluorosis that are valid, reliable, and feasible for surveillance at the community level. In addition, methods are needed for the quantitative assessment of fluorosis in vivo.

Ossification of posterior longitudinal ligament and fluorosis.

OBJECTIVE: Ossification of posterior longitudinal ligament (OPLL) is a progressive disease that causes spinal canal compromise and serious neurological sequelae in advanced cases. The incidence of OPLL in the Asiatic population is 2%-3%, but the incidence is more in the background of fluorosis. Our aim was to study the association of OPLL with fluorosis by comparing urine fluoride levels and to study the types of OPLL. MATERIALS AND METHODS: Thirty consecutive patients with OPLL, observed on cervical skiagram, and confirmed by the computed tomography (CT) of the cervical spine, underwent a 24-h urine fluoride level assessment by the ion-selective electrode method. Due consent of all the patients was obtained and the data was collected. Thirty patients with a normal cervical radiograph were taken as a control group and their 24-h urine fluoride levels were compared with the test group. The 24-h urine fluoride level above 1.6 mg/L was taken as the diagnostic parameter of fluorosis. Imaging analysis of the study group focused on the subtype of OPLL, the mass occupying ratio, the sagittal cervical angle, the signs of dural penetration, and the spinal levels involved. Urinary fluoride levels were correlated with the presence of OPLL and different types of OPLL. RESULTS: Of the 30 patients with OPLL, 25 were males and 5 were females. The most common presentation was myelopathy. Continuous type of OPLL was seen in 11 (36.6%), segmental in 8 (26.6%), focal in 5 (16.6%), and mixed variant in 6 (20%) patients. 24-h urinary fluoride levels ranged from 0.26 mg/L to 12.2 mg/L. 18 (60%) of the patients in the study group were found to have urinary fluoride levels above 1.6 mg/L and only 1 patient (4%) of the control group had the urine fluoride level >1.6 mg/L. This difference was statistically significant. Patients with continuous and mixed types of OPLL had a higher mean urine fluoride level than those with a segmental and focal type of OPLL. The continuous variant of OPLL had a statistically significant occupancy ratio when compared to the other three variants, and the high mass occupancy ratio of the OPLL was directly associated with the presence of dural penetration. CONCLUSION: Fluorosis is associated with a higher incidence of OPLL. Higher urinary fluoride levels correlate with the severe forms of OPLL.

Further opportunities for digital imaging in dental epidemiology.

Dental epidemiological research permits accurate tracking of the prevalence and distribution of oral disease across population groups, enabling planning and evaluation of public health interventions and healthcare service provision. This first section of this paper aimed to review traditional assessment methods in dental epidemiology and to consider the methodological and logistical benefits provided by digital imaging, both generally and specifically in relation to an established dual-camera system. The remainder of this paper describes the results of a semi-structured examination of an image archive from previous research utilising a dual-camera system, exploring whether the diagnostic yield of the images might be increased. Common oral conditions are presented alongside suggestions of the diagnostically useful data displayed in example images. Possible scoring mechanisms are discussed with consideration of the limitations that might be encountered for each condition. The retrospective examination suggests further data is obtainable from images acquired using the dual-camera system, however, consideration should be given to how best to validate this clinically. Additionally, other imaging modalities are discussed whilst taking into account the potential limitations of the dual-camera system.

Automatic detection and classification of dental fluorosis in vivo using white light and fluorescence imaging.

OBJECTIVES: To assess a novel method of automatic fluorosis detection and classification from white light and fluorescent images. METHODS: Dental images from 1,729 children living in two fluoridated and two non-fluoridated UK cities were utilised. A novel detection and classification algorithm was applied to each image and TF scores were obtained using thresholding criteria. These were compared to clinical reference standard images. Comparisons between reference and automated assessments were undertaken to record correct and incorrect classifications and the ability of the system to separate the fluoridated and non-fluoridated populations. RESULTS: The automated system performed well and was able to differentiate the two populations (P < 0.0001) to the same degree as the reference standard. When using the highest score from the clinical assessment the agreement between automated and clinical assessments was 0.56 (Kappa SE = 0.0160, p < 0.0001). CONCLUSIONS: Assessment of dental fluorosis is typically undertaken by clinical examiners in epidemiological studies. The training and calibration of such examiners is complex and time consuming and the assessments are subject to bias - frequently because of the examiner's awareness of the water fluoridation status of subjects. The use of remote scoring using photographs has been advocated but still requires trained examiners. This study has shown that image-processing methodologies applied to white light and fluorescent images could automatically score fluorosis and statistically separate fluoridated and non-fluoridated areas. The system requires further refinement to manage confounding factors such as the presence of non-fluoride opacities and tooth stain.

Digital epidemiology - Calibrating readers to score dental images remotely.

BACKGROUND: There is growing interest to use digital photographs in dental epidemiology. However, the reporting of procedures and metric-based performance outcomes from training to promote data quality prior to actual scoring of digital images has not been optimal. METHODS: A training study was undertaken to assess training methodology and to select a group of scorers to assess images for dental fluorosis captured during the 2013-2014 National Health and Nutrition Examination Survey (NHANES). Ten examiners and 2 reference examiners assessed dental fluorosis using the Deans Index (DI) and the Thylstrup-Fejerskov (TF) Index. Trainees were evaluated using 128 digital images of upper anterior central incisors at three different periods and with approximately 40 participants during two other periods. Scoring of all digital images was done using a secured, web-based system. RESULTS: When assessing for nominal fluorosis (apparent vs. non-apparent), the unweighted Kappa for DI ranged from 0.68 to 0.77 and when using an ordinal scale, the linear-weighted kappa for DI ranged from 0.43 to 0.69 during the final evaluation. When assessing for nominal fluorosis using TF, the unweighted Kappa ranged from 0.67 to 0.89 and when using an ordinal scale, the linear-weighted kappa for TF ranged from 0.61 to 0.77 during the final evaluation. No examiner improvement was observed when a clinical assessment feature was added during training to assess dental fluorosis using TF, results using DI was less clear. CONCLUSION: Providing examiners theoretical material and scoring criteria prior to training may be minimally sufficient to calibrate examiners to score digital photographs. There may be some benefit in providing an in-person training to discuss criteria and review previously scored images. Previous experience as a clinical examiner seems to provide a slight advantage at scoring photographs for DI, but minimizing the number of scorers does improve inter-examiner concordance for both DI and TF.

Mineralization-defects are comparable in fluorotic impacted human teeth and fluorotic mouse incisors.

OBJECTIVE: Fluoride excess of 0.05-0.07mgF/kgbw/day in water or food additives like salt is the principal cause of endemic dental fluorosis. How fluoride causes these defects is not clear yet. Recent studies in rodents suggest that development of enamel fluorosis is associated with insufficient neutralization of protons released during the formation of hypermineralized lines. DESIGN: Here we examined whether hypermineralization could also be assessed by MicroCT in developing molar enamel of humans exposed to fluoride. RESULT: Micro-CT analysis of hypomineralized enamel from human fluorotic molars graded by the Thylstrup-Fejerskov (TF) Index as III-IV showed weak hypermineralized lines and hypermineralized patches not seen in TF-I/II grade enamel. The mesio-distal sides of these molar teeth were significantly smaller (∼18%, p=0.02) than in TF-I/II teeth. CONCLUSION: The patterns of changes observed in human fluorotic teeth were similar to those in fluorotic rodent incisors. The data are consistent with the hypothesis that also in developing human teeth fluoride-stimulated local acidification of enamel could be a mechanism for developing fluorotic enamel.

Validation of ICMR index for identification of dental fluorosis in epidemiological studies.

BACKGROUND & OBJECTIVES: The Indian Council of Medical Research (ICMR) formulated a Task Force on dental fluorosis and recommended the subgroup to develop a simplified index for identification and grading of dental fluorosis to be used by the health workers. This study was conducted to pre-test the 'ICMR Index for Dental Fluorosis' in the field to check its reliability and reproducibility. METHODS: A total of 600 photographs were taken, 150 in each grade of fluorosis by screening 14-17 yr school children from eight schools of Hisar (Haryana) and South west Delhi. Eighty photographs were finalized (20 in each grade) before calibration to be used for training of field workers. Calibration exercise was conducted involving the five member survey team on 100 diagnosed cases of dental fluorosis. The members again screened 74 children with dental fluorosis in the field to categorize in to different grades of fluorosis for assessment of inter-examiner reliability. RESULTS: The ICMR criteria showed more difference in agreement in very mild and mild categories during calibration. The inter-examiner reliability (κ) ranged from 0.59-1. The criteria was further modified and inter- examiner reliability (κ) found to be 0.83-0.98 which was almost perfect agreement. INTERPRETATION & CONCLUSIONS: The tool developed by the ICMR to assess dental fluorosis can be used in a field set up by non-dental personnel reliably with high degree of reproducibility.

Grading and quantification of dental fluorosis in zebrafish larva.

OBJECTIVE: The prevalence and severity of dental fluorosis in primary teeth are different from permanent teeth. Previous animal models of dental fluorosis mainly focus on juvenile rats, mice and zebrafish. Our experiment aims to set a dental fluorosis model using zebrafish larva and explore the characteristics of the first generation teeth by fluoride treatment. MATERIALS AND METHODS: After the zebrafish eggs were laid, they were exposed to excess fluoride (19ppm, 38ppm and 76ppm) for five days. The morphological characteristics of first generation teeth were examined by H&E staining, whole-mount alizarin red and alcian blue staining, and scanning electron microscope (SEM) technique. RESULTS: With whole-mount alizarin red and alcian blue staining, the tooth cusps presented red in normal control. 19ppm and 38ppmm fluoride resulted in extensive red staining from tooth cusps to the lower 1/3 of teeth. 76ppm fluoride caused malformed teeth with uneven red staining. H&E staining showed that excess fluoride caused cystic-like changes in 38ppm and 76ppm groups. SEM revealed the dose dependent pathological changes in zebrafish enameloid with fluoride treatment. Based on SEM findings, we set 0-4 dental fluorosis index (DFI) score to label the severity of dental fluorosis. CONCLUSIONS: Excess fluoride presented a dose dependent fluorosis changes in the teeth of zebrafish larva. The DFI scores in our experiment reflect dose dependent fluorosis changes in a good way and will benefit the future research of dental fluorosis.

Developmental and Post-Eruptive Defects in Molar Enamel of Free-Ranging Eastern Grey Kangaroos (Macropus giganteus) Exposed to High Environmental Levels of Fluoride.

Dental fluorosis has recently been diagnosed in wild marsupials inhabiting a high-fluoride area in Victoria, Australia. Information on the histopathology of fluorotic marsupial enamel has thus far not been available. This study analyzed the developmental and post-eruptive defects in fluorotic molar enamel of eastern grey kangaroos (Macropus giganteus) from the same high-fluoride area using light microscopy and backscattered electron imaging in the scanning electron microscope. The fluorotic enamel exhibited a brownish to blackish discolouration due to post-eruptive infiltration of stains from the oral cavity and was less resistant to wear than normally mineralized enamel of kangaroos from low-fluoride areas. Developmental defects of enamel included enamel hypoplasia and a pronounced hypomineralization of the outer (sub-surface) enamel underneath a thin rim of well-mineralized surface enamel. While the hypoplastic defects denote a disturbance of ameloblast function during the secretory stage of amelogenesis, the hypomineralization is attributed to an impairment of enamel maturation. In addition to hypoplastic defects, the fluorotic molars also exhibited numerous post-eruptive enamel defects due to the flaking-off of portions of the outer, hypomineralized enamel layer during mastication. The macroscopic and histopathological lesions in fluorotic enamel of M. giganteus match those previously described for placental mammals. It is therefore concluded that there exist no principal differences in the pathogenic mechanisms of dental fluorosis between marsupial and placental mammals. The regular occurrence of hypomineralized, opaque outer enamel in the teeth of M. giganteus and other macropodids must be considered in the differential diagnosis of dental fluorosis in these species.

Phenotypic variation of fluoride responses between inbred strains of mice.

Excessive systemic exposure to fluoride (F) can lead to disturbances in bone homeostasis and dental enamel development. We have previously shown strain-specific responses to F in the development of dental fluorosis (DF) and in bone formation/mineralization. The current study was undertaken to further investigate F responsive variations in bone metabolism and to determine possible relationships with DF susceptibility. Seven-week-old male mice from FVB/NJ, C57BL/6J, C3H/HeJ, A/J, 129S1/SvImJ, AKR/J, DBA/2J, and BALB/cByJ inbred strains were exposed to NaF (0 or 50 ppm as F(-)) in drinking water for 60 days. Sera were collected for F, Ca, Mg, PO(4), iPTH, sRANKL, and ALP levels. Bone marrow cells were subjected to ex vivo cell culture for osteoclast potential and CFU colony assays (CFU-fibroblast, CFU-osteoblast, CFU-erythrocyte/granulocyte/macrophage/megakaryocyte, CFU-granulocyte/macrophage, CFU-macrophage, and CFU-granulocyte). Femurs and vertebrae were subjected to micro-CT analyses, biomechanical testing, and F, Mg, and Ca content assays. DF was evaluated using quantitative fluorescence and clinical criteria. Strain-specific responses to F were observed for DF, serum studies, ex vivo cell culture studies, and bone quality. Among the strains, there were no patterns or significant correlations between DF severity and the actions of F on bone homeostasis (serum studies, ex vivo assays, or bone quality parameters). The genetic background continues to play a role in the actions of F on tooth enamel development and bone homeostasis. F exposure led to variable phenotypic responses between strains involving dental enamel development and bone metabolism.

Child skeletal fluorosis from indoor burning of coal in southwestern China.

OBJECTIVES: We assess the prevalence and pathogenic stage of skeletal fluorosis among children and adolescents residing in a severe coal-burning endemic fluorosis area of southwest China. METHODS: We used a cross-sectional design. A total of 1,616 students aged between 7 and 16 years in Zhijin County, Guizhou, China in late 2004 were selected via a cluster sampling of all 9-year compulsory education schools to complete the study questionnaire. Any student lived in a household that burned coal, used an open-burning stove, or baked foodstuffs over a coal stove was deemed high-risk for skeletal fluorosis. About 23% (370) of students (188 boys, 182 girls) were identified as high-risk and further examined by X-ray. RESULTS: One-third of the 370 high-risk participants were diagnosed with skeletal fluorosis. Overall prevalence of child skeletal fluorosis due to indoor burning of coal was 7.5%. Children aged 12-16 years were significantly more likely to be diagnosed with skeletal fluorosis than children aged 7-11 years (OR = 1.84, 95% CI: 1.17-2.90; P = .0082). Four types of skeletal fluorosis were identified: constrictive (60.7%), raritas (15.6%), mixed (16.4%), and soft (7.4%). Most diagnosed cases (91%) were mild or moderate in severity. In addition, about 97% of 370 high-risk children were identified with dental fluorosis. Dental fluorosis was highly correlated with skeletal fluorosis in this study. CONCLUSIONS: Skeletal fluorosis among children may contribute to poor health and reduced productivity when they reach adulthood. Further efforts to reduce fluoride exposure among children in southwestern of China where coal is burned indoors are desperately needed.

Fluoride's effect on human dentin ultrasound velocity (elastic modulus) and tubule size.

Despite fluoride (F) use in caries prevention, not much is known about its effects on tooth quality. This study evaluated the effect of tooth F concentration ([F]) on selected dentin structural and mechanical properties. Third molars (n = 136) from Toronto, which has 1 part per million (p.p.m.) water [F], Montreal (0.2 p.p.m. water [F]), and Fortaleza (Brazil) (0.7 p.p.m. water [F]), were analyzed for [F], dental fluorosis (DF) severity, ultrasound velocity, and dentin tubule size and density. The enamel [F] was found to vary between 32 and 940 p.p.m., the dentin [F] was found to vary between 110 and 860 p.p.m., while the DF severity varied between TF0 and TF4. The enamel [F] showed no correlation with dentin [F], DF severity, ultrasound velocity, dentin tubule size or density. The dentin [F] correlated with DF severity, dentin tubule size, and ultrasound velocity. DF severity showed a correlation with dentin [F] and ultrasound velocity. It was concluded that dentin [F] is an indicator of dentin structural properties (dentin tubule size and ultrasound velocity), while DF severity is an indicator of dentin mechanical properties (ultrasound velocity).

Brick tea fluoride as a main source of adult fluorosis.

An epidemiological survey was conducted in Naqu County, Tibet in September 2001 to investigate the manifestations of fluorosis in adults caused by the habitual consumption of brick tea. Profiles were obtained for the total daily fluoride intake, environmental fluoride levels and average urinary fluoride concentration, and a physical examination and a skeletal radiographic study were conducted. One hundred and eleven 30-78-year-old adults were enrolled. It was found that the fluoride level of water sources in Naqu County was 0.10+/-0.03 mg/l; no evidence of fluoride air pollution was found, but the brick tea water processed foods--zamba and buttered tea--had fluoride contents of 4.52+/-0.74 mg/kg and 3.21+/-0.65 mg/l, respectively. The adult daily fluoride intake reached 12 mg, of which 99% originated from the brick tea-containing foods. The positive rate of clinical symptoms by physical examination was 89%; furthermore, 42 of the 111 subjects were diagnosed by X-ray. The positive examination rate was 83%. Although the osteosclerosis-type skeletal fluorosis (overall increased bone matrix density) affected 74%, arthropathy and arthritis affected a significant number of the patients, resulting in functional disability. The results suggest that this brick tea-type fluorosis had even more severe adverse effects on human health compared with both the water-type and coal combustion-type fluorosis that occurred in other areas of China.

Skeletal and dental fluorosis: two case reports.

Two case reports from a high fluoride (10 ppm) rural community. They presented with severe degrees of dental fluorosis, hyper-sensitivity of teeth and skeletal fluorosis all arising from the ingestion of high amount of fluoride in water over a long period of time. Both cases had deformities of the upper and lower limbs. However, the deformities were more pronounced in the lower limbs than in the upper limbs, resulting in knock knee. Radiological finding showed osteosclerosis of the axial bones while the appendicular bones exhibited osteoporosis. There was marked change of bone structure observed as osteomalacia, and course trabecular bone pattern. Osteoporosis was also associated with cortical thinning. Periosteal bone apposition was observed in the bones: and genu valgum of the limbs. Biochemical tests revealed normal values for serum calcium and inorganic phosphate. However, the serum alkaline phosphatase was elevated. This may be an indication of a pathological condition where there are possible compensatory mechanisms to maintain normal levels of serum calcium and inorganic phosphate. One case which had undergone corrective surgical intervention of the lower limbs four years earlier, had continued to live in the same environment using drinking water with 10 ppmF after corrective surgery, and showed no improvement.

[Deciduous teeth with fluorosis from a morphological and microanalytical point of view]. / Zeby mleczne z fluoroza w obrazie morfologicznym i mikroanalitycznym.

Deciduous teeth with enamel fluorosis obtained from a 7 year-old child and normal deciduous teeth were studied by light microscopy and X-ray microanalysis. In the fluorotic teeth, altered architecture of enamel prisms and marked damage of enamel were observed. X-ray microanalysis revealed disturbances in the mineral metabolism of the fluorotic tooth tissues: as compared with normal teeth, an increase in calcium, phosphorus and magnesium content as well as lowered calcium/phosphorus ratio were found in the enamel prisms, interprismatic substance, dentinal tubules and intertubular dentin.