Remineralization of artificial enamel lesions by theobromine.

OBJECTIVE: This study investigated the remineralization potential of theobromine in comparison to a standard NaF dentifrice. METHODS: Three tooth blocks were produced from each of 30 teeth. Caries-like lesion was created on each block using acidified gel. A smaller block was cut from each block for baseline scanning electron microscopy imaging and electron-dispersive spectroscopy (EDS) analysis for surface Ca level. A tooth slice was cut from each lesion-bearing block for transverse microradiography (TMR) quantification of baseline mineral loss (Δz) and lesion depth (LD). Then baseline surface microhardness (SMH) of each lesion was measured. The three blocks from each tooth were assigned to three remineralizing agents: (1) artificial saliva; (2) artificial saliva with theobromine (0.0011 mol/l), and (3) NaF toothpaste slurry (0.0789 mol/l F). Remineralization was conducted using a pH cycling model with storage in artificial saliva. After a 28-day cycle, samples were analyzed using EDS, TMR, and SMH. Intragroup comparison of pre- and posttest data was performed using t tests (p < 0.05). Intergroup comparisons were performed by post hoc multistep comparisons (Tukey). RESULTS: SMH indicated significant (p < 0.01) remineralization only with theobromine (38 ± 32%) and toothpaste (29 ± 16%). With TMR (Δz/lD), theobromine and toothpaste exhibited significantly (p < 0.01) higher mineral gain relative to artificial saliva. With SMH and TMR, remineralization produced by theobromine and toothpaste was not significantly different. With EDS, calcium deposition was significant in all groups, but not significantly different among the groups (theobromine 13 ± 8%, toothpaste 10 ± 5%, and artificial saliva 6 ± 8%). CONCLUSION: The present study demonstrated that theobromine in an apatite-forming medium can enhance the remineralization potential of the medium.

Management of sleep-time masticatory muscle activity using stabilisation splints affects psychological stress.

To treat sleep bruxism (SB), symptomatic therapy using stabilisation splints (SS) is frequently used. However, their effects on psychological stress and sleep quality have not yet been examined fully. The objective of this study was to clarify the effects of SS use on psychological stress and sleep quality. The subjects (11 men, 12 women) were healthy volunteers. A crossover design was used. Sleep measurements were performed for three consecutive days or longer without (baseline) or with an SS or palatal splint (PS), and data for the final day were evaluated. We measured masseter muscle activity during sleep using portable electromyography to evaluate SB. Furthermore, to compare psychological stress before and after sleep, assessments were made based on STAI-JYZ and the measurement of salivary chromogranin A. To compare each parameter among the three groups (baseline, SS and PS), Friedman's and Dunn's tests were used. From the results of the baseline measurements, eight subjects were identified as high group and 15 as low group. Among the high group, a marked decrease in the number of bruxism events per hour and an increase in the difference in the total STAI Y-1 scores were observed in the SS group compared with those at baseline (P < 0·05). No significant difference was observed in sleep stages. SS use may be effective in reducing the number of SB events, while it may increase psychological stress levels, and SS use did not apparently influence sleep stages.

Observer performance in diagnosing osteoporosis by dental panoramic radiographs: results from the osteoporosis screening project in dentistry (OSPD).

Mandibular cortical erosion detected on dental panoramic radiographs (DPRs) may be useful for identifying women with osteoporosis, but little is known about the variation in diagnostic efficacy of observers worldwide. The purpose of this study was to measure the accuracy in identifying women at risk for osteoporosis in a worldwide group of observers using DPRs. We constructed a website that included background information about osteoporosis screening and instructions regarding the interpretation of mandibular cortical erosion. DPRs of 100 Japanese postmenopausal women aged 50 years or older who had completed skeletal bone mineral measurements by dual energy X-ray absorptiometry were digitized at 300 dpi. These were displayed on the website and used for the evaluation of diagnostic efficacy. Sixty observers aged 25 to 66 years recruited from 16 countries participated in this study. These observers classified cortical erosion into one of three groups (none, mild to moderate, and severe) on the website via the Internet, twice with an approximately 2-week interval. The diagnostic efficacy of the Osteoporosis Self-Assessment Tool (OST), a simple clinical decision rule based on age and weight, was also calculated and compared with that of cortical erosion. The overall mean sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the 60 observers in identifying women with osteoporosis by cortical erosion on DPRs were 82.5, 46.2, 46.7, and 84.0%, respectively. Those same values by the OST index were 82.9, 43.1, 43.9, and 82.4%, respectively. The intra-observer agreement in classifying cortical erosion on DPRs was sufficient (weighted kappa values>0.6) in 36 (60%) observers. This was significantly increased in observers who specialized in oral radiology (P<0.05). In the 36 observers with sufficient intra-observer agreement, the overall mean sensitivity, specificity, PPV, and NPV in identifying women with osteoporosis by any cortical erosion were 83.5, 48.7, 48.3, and 85.7%, respectively. The mean PPV and NPV were significantly higher in the 36 observers with sufficient intra-observer agreement than in the 24 observers with insufficient intra-observer agreement. Our results reconfirm the efficacy of cortical erosion findings in identifying postmenopausal women at risk for osteoporosis, among observers with sufficient intra-observer agreement. Information gathered from radiographic examination is at least as useful as that gathered from the OST index.

Functional differences in the acinar cells of the murine major salivary glands.

In humans, approximately 90% of saliva is secreted by the 3 major salivary glands: the parotid (PG), the submandibular (SMG), and the sublingual glands (SLG). Even though it is known that all 3 major salivary glands secrete saliva by a Cl(-)-dependent mechanism, salivary secretion rates differ greatly among these glands. The goal of this study was to gain insight into the properties of the ion-transporting pathways in acinar cells that might account for the differences among the major salivary glands. Pilocarpine-induced saliva was simultaneously collected in vivo from the 3 major salivary glands of mice. When normalized by gland weight, the amount of saliva secreted by the PG was more than 2-fold larger than that obtained from the SMG and SLG. At the cellular level, carbachol induced an increase in the intracellular [Ca(2+)] that was more than 2-fold larger in PG and SMG than in SLG acinar cells. Carbachol-stimulated Cl(-) efflux and the protein levels of the Ca(2+)-activated Cl(-) channel TMEM16A, the major apical Cl(-) efflux pathway in salivary acinar cells, were significantly greater in PG compared with SMG and SLG. In addition, we evaluated the transporter activity of the Na(+)-K(+)-2Cl(-) cotransporters (NKCC1) and anion exchangers (AE), the 2 primary basolateral Cl(-) uptake mechanisms in acinar cells. The SMG NKCC1 activity was about twice that of the PG and more than 12-fold greater than that of the SLG. AE activity was similar in PG and SLG, and both PG and SLG AE activity was about 2-fold larger than that of SMG. In summary, the salivation kinetics of the 3 major glands are distinct, and these differences can be explained by the unique functional properties of each gland related to Cl(-) movement, including the transporter activities of the Cl(-) uptake and efflux pathways, and intracellular Ca(2+) mobilization.

Bone Aging by Advanced Glycation End Products: A Multiscale Mechanical Analysis.

The quality and quantity of mandibular bone are essential prerequisites for osseointegrated implants. Only the Hounsfield unit on preoperative computed tomography is currently used as a clinical index. Nevertheless, a considerable mismatch occurs between bone quality and the Hounsfield unit. Loss of bone toughness during aging has been accepted based on empirical evidence, but this concept is unlikely evidence based at the level of mechanical properties. Nonenzymatic bone matrix cross-links associated with advanced glycation end products predominate as a consequence of aging. Thus, loss of tissue integrity could diminish the bone toughening mechanism. Here, we demonstrate an impaired bone toughening mechanism caused by mimicking aging in rabbits on a methionine-rich diet, which enabled an enhanced nonenzymatically cross-linked bone matrix. A 3-point bending test revealed a greater reduction in femoral fracture resistance in rabbits on a methionine-rich diet, despite higher maximum and normalized breaking forces (287.3 N and 88.1%, respectively), than in rabbits on a normal diet (262.2 N and 79.7%, respectively). In situ nanoindentation on mandibular cortical bone obtained from rabbits on a methionine-rich diet did not enable strain rate-dependent stiffening and consequent large-dimensional recovery during rapid loading following constant displacement after a rapid-load indentation test as compared with those in rabbits on a normal diet. Such nanoscale structure-function relationships dictate resistance to cracking propagation at the material level and allow for the overall bone toughening mechanism to operate under large external stressors. The strain-dependent stiffening was likely associated with strain-energy transfer to the superior cross-linked bone matrix network of the normal diet, while the reduction in the enzymatically cross-linked matrix in bone samples from rabbits on a methionine-rich diet likely diminished the intrinsic bone toughening mechanism. The present study also provides a precise protocol for evaluating bone mechanical properties at the material level based on observations from a series of nanoindentation experiments.

In vitro collagen synthesis of tooth germs from newborn rats with protein-energy malnutrition.

Mineralization and matrix formation are closely related processes in the growth and development of both incisor and molar tooth germs. Addition of ascorbic acid does not affect in vitro collagen synthesis of tooth germs from protein-energy malnourished rats.

The effect of protein-energy malnutrition on the growth of tooth germs in newborn rats.

The effect of protein-energy malnutrition on the growth of molar and incisor tooth germs was studied. Significant differences were found between control and malnourished groups in the weights and calcium contents of both tooth germs. The data point to the importance of the effect of nutrient deficiency on the critical growth period of an oral structure.

The effect of calcitonin in vitro on tooth germs in protein-energy malnourished rats.

The effect of calcitonin on the development of incisor and molar tooth germs was studied in normal and nutritionally deprived newborn rats. Calcitonin affected the calcium uptake of nutritionally deprived molar tooth germs in the early postnatal period; this interaction was not seen in the later postnatal period, however.

Detection of vascular disease risk in women by panoramic radiography.

Low bone mineral density and rapid bone loss of the skeleton are associated with mortality risk from vascular diseases in post-menopausal women. Panoramic radiographic measurements are considered as indicators of skeletal bone mineral density or bone turnover. We hypothesize that such measurements may be associated with vascular disease risk in post-menopausal women. Associations of mandibular cortical shape and width on panoramic radiographs with skeletal bone mineral density and risk factors related to vascular diseases were investigated in 87 post-menopausal women. Cortical shape was associated with skeletal bone mineral density, low-density lipoprotein cholesterol, apolipoprotein B, resting heart rate, and endothelial dysfunction. Cortical width was associated with skeletal bone mineral density, low-density lipoprotein cholesterol, and apolipoprotein A1. Dentists may be able to refer women with increased risk of vascular diseases, as well as low bone mineral density, to medical professionals for further examination by panoramic findings.

Alteration of femoral structure in later life by chronically feeding caffeine during rapid growing period in newborn female rats.

The effects of caffeine intake in early life on bone structure later in life were studied in rats. At day 9 of gestation, dams were divided into 2 groups. Group 1 (control) received a 20% protein diet; group 2 received the 20% protein diet supplemented with caffeine (2 mg/100 g body weight). After birth pups were continuously fed their respective diets until day 93, when the diet of group 2 was replaced with a noncaffeine 20% protein diet. On day 388 animals from both groups were weighed, killed, and femora and mandibles were removed. Calcium, phosphorus, magnesium, zinc, hydroxyproline, and hexosamine concentrations were measured. Radiographs of some femora were taken and paraffin cross sections were made at the midshaft of others. Femora in the caffeine group were wider, periosteal bone area/total bone area was greater, the cross sectional area of femoral bone was smaller, and there were fewer osteocytes/bone area than in controls. Calcium, phosphorus, zinc, and hydroxyproline concentrations in the caffeine group were less in both bones of the caffeine group. These results indicate that if animals are exposed to caffeine during the rapidly growing period, changes occur in femoral bone which are similar to those that occur with aging.

Differential effects of collagen in oral tissues and skin by protein-energy malnourished newborn rats and lactating dams.

Effects of protein-energy malnutrition were studied in newborn rats and their dams. Upon delivery, dams received 6%, 12% or 20% protein diets. At Day 15 pups received 14C(U)-proline. The posterior tongue, hard palatal mucosa, soft palatal mucosa and skin were analyzed for collagen and counts incorporated (collagen synthesis, that is, the rate of 14C-proline converted to 14C-hydroxy-proline into gingival collagen). These regions of the dams were also removed to study collagen content. Although soft palatal mucosal collagen of newborns in the 6% and 12% protein groups was decreased, that of skin in the 6% protein group was increased. No such differences were observed in tongue and hard palatal mucosa. Counts incorporated was decreased in the tongue, soft palatal mucosa and skin, but not in hard palatal mucosa. Collagen contents of tongue and hard palatal mucosa and skin of the dams showed no differences, whereas that of soft palatal mucosa in the 6% protein groups was increased. Effects of protein-energy malnutrition on oral tissues and skin were therefore different between newborns and lactating dams. Furthermore, one part of oral mucosa is affected differently from other parts of mucosa and in both, degree of malnutrition has different influences.

Effect of liquid protein diet on collagen metabolism of the gingiva of young rats.

The effects of liquid protein diet (LPD) on gingival collagen metabolism were evaluated in young rats. Experimental animals received a LPD, while pair-fed and ad libitum groups received a 20% casein diet. Prior to sacrifice at days 23, 35 and 48 post-partum, animals from each group were injected with C14-proline. Gingival tissues from the palate were excised and analyzed for collagen content and collagen synthesis. Results of body-weight changes at day 48 showed a 20% decrease in the LPD group, while pair-fed and ad libitum groups increased 47% and 267%, respectively. Collagen content per gram of gingiva was not significantly different among the three groups. Gingival collagen synthesis, however, did show significant differences among the three groups. It was concluded that while LPD effects on the overall body condition were profound, LPD exerts minimal effect on gingival collagen metabolism in young rats.

Combined effects of caffeine and prostaglandin E2 on the proliferation of osteoblast-like cells (UMR106-01).

BACKGROUND: The general public widely consumes caffeine (1,3,7-trimethylxanthine), which is contained in various foods, beverages and over-the-counter medications. We have shown previously that caffeine intake could affect bone metabolism in vivo. METHODS: Because prostaglandin E2 (PGE2) is shown to be elevated in the periodontally diseased site, the possible interaction between caffeine and PGE2 was investigated in the present study using UMR106-01 rat osteoblast-like cells in vitro. RESULTS: Although neither 0.1 mM caffeine nor 0.1 microg/ml of PGE2 alone showed any inhibitory effects on cell proliferation, the combination of caffeine and PGE2 showed significant inhibition. However, in order to have inhibitory effects, both caffeine and PGE2 had to be present at least 72 or 96 hours in the medium. Addition of the endogenous PGE2 synthesis inhibitor, indomethacin, showed no effects on cell proliferation. Neither cAMP-inducing agent IBMX (0.01 mM and 0.1 mM) nor forskolin (0.001 mM) inhibited cell proliferation, but combined with PGE2 these agents strongly inhibited proliferation as was observed with the combination of caffeine and PGE2, suggesting possibly that the increase of intracellular cAMP concentration plays an important role in the inhibitory effects of cell proliferation. CONCLUSIONS: The present data for the first time demonstrate the possible implication of routine caffeine intake in the acceleration of pathological conditions of periodontitis. Thus, we propose that chronic caffeine intake is one of the possible risk factors in the advancement of pathology in the periodontitis patient. Further research in this area is warranted.

Effects of maternal caffeine with zinc intake during gestation and lactation on bone development in newborn rats.

On day 9 of gestation, pregnant dams were randomly divided into 3 groups. Dams of group 1 were fed a 20% protein diet as a control. Dams of group 2 were fed a 20% protein diet supplemented with caffeine. Dams of group 3 were fed a 20% protein diet supplemented with caffeine and zinc. The amount of caffeine added to the maternal diet was 2 mg/100 g body weight; the amount of zinc was 0.6 g/kg of diet. At birth, pups were mixed within each group, and 8 randomly selected pups from each group were assigned to each dam of the respective group and were continuously fed the same diet. On day 15, the pups were killed and cranial bones, mandibles and femurs removed. The bones were measured, and the mineral content of the mandibles and femurs was determined. Although there were no differences in the dimensions of the cranial bones among the groups, the measurements and mineral content of the mandibles and femurs were consistently affected by the caffeine in the diet. On the other hand, supplementation of the caffeine-added diets with zinc led to greatly improved bone development, reaching values up to or beyond control levels. Thus zinc supplementation of a caffeine diet given to the dams during gestation and lactation can favourably influence the otherwise impaired bone development of their offspring.

Effects of maternal caffeine intake on the growth of rat tooth germs in protein-energy malnourished neonates.

Fourteen rat dams with 8 pups each were fed either a 6, 12 or 20 per cent protein diet upon birth. Another group of 12 dams with the same number of pups was pair-fed either a 6, 12 or 20 per cent protein diet supplemented with caffeine (2 mg/100 g body weight). At day 15, randomly-selected pups were injected with [14C]-proline to determine collagen synthesis of the incisor and molar tooth germs. Another group of pups was used to determine calcium content of these tooth germs. Body weight, incisor weight and total calcium contents of tooth germs of pups from dams fed with 6 per cent protein diet were greater in the caffeine-supplemented group, whereas in the 20 per cent protein diet with caffeine group, these parameters were lower. The molar weights of the 12 per cent protein diet with caffeine animals were greater than the 12 per cent group without caffeine. The total hydroxyproline content of the incisor tooth germs from animals in the 12 per cent protein diet with caffeine was greater than is the non-caffeine group. However, total hydroxyproline of the molar tooth germs in the 20 per cent protein groups with caffeine was less than in the non-caffeine group. The rate of collagen synthesis of the incisor and molar tooth germs showed no difference in the presence or absence of caffeine in the 6, 12 and 20 per cent protein groups. Incisor and molar tooth germs are thus affected differently by the interaction of protein and caffeine, possibly due to differences in the pattern of tooth development.

Effects of orally-administered caffeine on the growth of tooth germs in protein-energy malnourished newborn rats.

Lactating dams with 8 suckling pups were fed either a 6 or 20 per cent protein diet from the birth of the pups to day 15. Pups were divided into caffeine and non-caffeine groups and, every other day between days 3 and 13, were either intubated with caffeine (1 mg/100 g body wt) dissolved in 0.1 ml physiological saline solution, or 0.1 ml saline solution as a control. At day 15, the weight of incisors and molar germs, hydroxyproline content, collagen synthesis and calcium contents were determined. Nutritional factors exercised consistent effects on all the parameters, but effects attributable to caffeine intubation were minor. There was lower molar weight in the 20 per cent protein group with caffeine, and collagen synthesis of incisor and molar germs in the caffeine and non-caffeine animals in the 6 and 20 per cent group were different. These parameters showed a significant interaction between nutrition and caffeine. Thus the direct administration of a small amount of caffeine to newborns causes only slight effects on tooth-germ development under some nutritional conditions.

Biochemical and physical alterations of bones in newborn rats due to excess methionine administered either by gastric intubation or by maternal milk.

In one series of experiments, methionine dissolved in physiological saline solution was gastric-intubated into newborn rats every other day from 3 to 13 days after birth. In a second series, diets supplemented with methionine were fed daily to lactating dams from one day after birth of the pups. Pups were killed 15 days after birth and the mandible and long bones removed. The weight and 45Ca uptake of long bone of the groups receiving 0.1 g/100 g body weight of methionine were increased compared with either the group receiving no methionine supplement or that given 0.2 g/100 g body weight. The bone effects of high levels of methionine in the maternal diet were more severe than after intragastric administration. Dietary methionine supplementation during the early growth period is necessary for the normal development of bones in newborn rats, but excess intragastric methionine impaired long-bone development and altered collagen synthesis in long bones and mandibles. High maternal methionine impaired mandibles and long bones, and led to decreased linear dimensions.

The effects of retinoic acid on the growth of mandible and long bone in fetal rats.

Timed-pregnant rats were injected with a total of 90 microgram of retinoic acid from day 13 until delivery. Other pregnant groups were given either a total of 390 microgram of retinol from day 5 or a total of 270 microgram of retinol from day 13 until delivery. Although the weight gains and food intake of the dams during gestation showed no difference between groups, the long bones were slightly heavier in pups born from retinoic acid-injected dams. Total collagen content in both mandibles and long bones did not show differences between retinoic acid- and retinol-injected groups, but collagen synthesis in both bones was greatly increased in the retinoic acid-injected groups. Calcium content of the mandible in the retinol-injected group from day 13 of gestation was increased, whereas 45Ca uptake of long bone of this group was increased. Retinoic acid, when injected in physiological doses during gestation, may have some effects on the organic phase of bone, long bones being slightly more sensitive to retinoic acid than the mandibles.

Physical examination of caffeine's effects on the enamel surface of first molar in new-born rats.

Samples of the first molars of offspring whose dams were fed a diet supplemented with caffeine were examined by scanning electron microscopy, X-ray diffractometry and electron microprobe analysis. Scanning microscopy of the enamel surface of the caffeine group revealed a consistently rougher surface than in the non-caffeine controls, both before and after acid exposure. X-ray diffraction analysis of the pulverized whole tooth in the caffeine group showed broader diffraction peaks for the lattice plane reflections (202) and (300), indicating smaller crystallites. Pure enamel samples of the caffeine group examined with a Gandolfi X-ray camera also revealed more diffuse diffraction lines than in the non-caffeine controls, further indicating smaller crystallites in the enamel. The calcium and phosphorus contents of the acid-exposed samples in both control and caffeine groups were lower than the non-acid exposed control and caffeine groups by electron microprobe analysis. After exposure to acid, the calcium and phosphorus contents of the outer surface of the enamel in the caffeine group were greatly reduced as compared to that of the non-caffeine controls. Thus various methods consistently indicate that caffeine ingestion during early growth affects the enamel surface of the first molars, resulting in impaired mineralization. Caffeine intake may therefore have a negative effect on amelogenesis and possibly increases susceptibility to dental caries.

The effect of prenatal caffeine exposure on the enamel surface of the first molars of newborn rats.

Timed-pregnant rats were randomly divided into three groups at day 7 of gestation. A caffeine tablet was implanted subcutaneously in the experimental group and a placebo tablet in the control group. The third group was used as surrogate dams. At birth, eight randomly selected pups born either to the experimental or control dams were placed with surrogate dams that had produced pups on the same day; these were then used in either the experimental or the control group, and so the surrogate dams raised pups that came from either the experimental or control group. At day 22 after birth, the pups were killed, and their first and second maxillary and mandibular molars were removed. They were then placed in a specially designed chamber and exposed to an acid solution. The outflowing acid solution was collected every 20 min and up to 80 min. Then the calcium, phosphorus, and magnesium contents of each fraction were measured. The enamel surfaces of untreated and acid-treated first molars of the caffeine and control groups were examined by scanning electron microscopy. Untreated teeth were powdered and separated into enamel and dentine. Pure enamel samples were examined with a Gandolfi X-ray powder camera to measure the crystallite size. At 20-, 40- and 60-min intervals, calcium and phosphorus contents of the first molars of the caffeine group were significantly higher than those of the control. The enamel surface of the untreated and acid-treated first molars of the caffeine group had significantly different scanning microscopic appearances from those of the non-caffeine untreated and acid-treated control groups.(ABSTRACT TRUNCATED AT 250 WORDS)