Endogenous YAP1 activation drives immediate onset of cervical carcinoma in situ in mice.

Cervical cancer (CC) is usually initiated by infection with high-risk types of human papillomavirus (HPV). The HPV E6 and E7 proteins target p53 and RB, respectively, but other cellular targets likely exist. We generated uterus-specific MOB1A/B double KO (uMob1DKO) mice, which immediately developed cervical squamous cell carcinoma in situ. Mutant cervical epithelial cells showed YAP1-dependent hyperproliferation, altered self-renewal, impaired contact inhibition, and chromosomal instability. p53 activation was increased in uMob1DKO cells, and additional p53 loss in uMob1DKO mice accelerated tumor invasion. In human CC, strong YAP1 activation was observed from the precancerous stage. Human cells overexpressing HPV16 E6/E7 showed inactivation of not only p53 and RB but also PTPN14, boosting YAP1 activation. Estrogen, cigarette smoke condensate, and PI3K hyperactivation all increased YAP1 activity in human cervical epithelial cells, and PTPN14 depletion along with PI3K activation or estrogen treatment further enhanced YAP1. Thus, immediate CC onset may initiate when YAP1 activity exceeds an oncogenic threshold, making Hippo-YAP1 signaling a major CC driver.

UVA-activated riboflavin promotes collagen crosslinking to prevent root caries.

Root caries is an increasingly problem in aging societies with severe implications for the general health and wellbeing of large numbers of people. Strengthening type-I collagen, a major organic component of human dentin, has proved effective in preventing root caries. This study sought to determine whether exposure to riboflavin followed by UVA irradiation (RF/UVA) could promote additional collagen crosslinking, and thus improve the acid and enzymatic resistance of human dentin under simulated oral environments. If so, it could offer potential for treatment of the intractable problem of root caries. The greatest flexural strengths were found in dentin exposed to a 0.1% riboflavin solution for 1 minute followed by 1,600 mW/cm2 UVA irradiation for 10 minutes. Mineral loss and lesion depth were significantly lower in the RF/UVA group than in the control group. The microstructures of dentinal tubules and collagen networks after RF/UVA treatment retained their original forms after acidic and enzymatic degradation. In conclusion, RF/UVA treatment may be a new method for preventing root caries with promising prospects for clinical application.

Remineralization Potential of Mints Containing Bioactive Agents in Artificially Induced Root Caries.

This study investigated the remineralization effect of experimental mint formulations containing bioactive agents (xylitol; green tea extract, GT; and amorphous calcium phosphate, ACP) in the progression of artificially induced root caries. Root caries lesions were induced by demineralization solution (pH 4.6; 96 h; 37°C). The lesions were treated with mint A, mint B, mint C, xylitol, GT, ACP, or remineralization solution (RS; negative control). Specimens were pH-cycled through treatments (5×/day; 3 min) and 6 cycles of acidic (pH 5.0; 30 min) and neutral (pH 7.0; 10 min) buffers for 8 days. Bacterial collagenase (Clostridium histolyticum) was used overnight to simulate proteolytic challenge. Caries depth and porosity as well as mineral density were estimated using fluorescence microscopy (n = 15) and microcomputed tomography (n = 6). Analysis of variance (ANOVA, α = 0.05) showed no statistically significant difference in caries depth among all groups (p = 0.172). The highest fluorescence intensity decrease was observed for GT followed by mint C, with no significant difference between them (p = 0.868). There were significant differences among GT and mints A, B, and C when compared to RS (p < 0.001). No statistically significant differences in fluorescence intensity were observed among ACP, xylitol, and RS (p > 0.05). The mineral density of the lesions in GT, mints A, B, and C, and ACP was statistically similar (p > 0.05) and significantly higher than that in RS (p < 0.05). No significant difference was observed between xylitol and RS (p = 0.728). The experimental mints showed remineralization action on artificial root caries, and GT was found to be the main active ingredient in the investigated formulations.

Use of PIXE/PIGE for sequential Ca and F measurements in root carious model.

The progress of caries has conventionally been evaluated by checking changes in mineral density using transverse microradiography (TMR). Recent advances have seen development of a new measurement system, using in-air micro proton induced X-ray/gamma-ray emission (PIXE/PIGE). PIXE/PIGE enables analysis of distributions and concentrations of multiple mineral elements in a carious lesion. The aim of this study was to evaluate the effectiveness of PIXE/PIGE for investigating the development of root caries. In summary, we successfully established a multi-elemental sequential measuring method using in-air micro-PIXE/PIGE to identify the dynamic distributions and concentrations of Ca and F in human root dentin. The PIXE/PIGE potentially offers a useful advantageous technique for studying carious development by using as a combination with conventional techniques such as TMR and Micro-computed tomography (µCT).

Nerve Growth Factor Secretion From Pulp Fibroblasts is Modulated by Complement C5a Receptor and Implied in Neurite Outgrowth.

Given the importance of sensory innervation in tooth vitality, the identification of signals that control nerve regeneration and the cellular events they induce is essential. Previous studies demonstrated that the complement system, a major component of innate immunity and inflammation, is activated at the injured site of human carious teeth and plays an important role in dental-pulp regeneration via interaction of the active Complement C5a fragment with pulp progenitor cells. In this study, we further determined the role of the active fragment complement C5a receptor (C5aR) in dental nerve regeneration in regards to local secretion of nerve growth factor (NGF) upon carious injury. Using ELISA and AXIS co-culture systems, we demonstrate that C5aR is critically implicated in the modulation of NGF secretion by LTA-stimulated pulp fibroblasts. The NGF secretion by LTA-stimulated pulp fibroblasts, which is negatively regulated by C5aR activation, has a role in the control of the neurite outgrowth length in our axon regeneration analysis. Our data provide a scientific step forward that can guide development of future therapeutic tools for innovative and incipient interventions targeting the dentin-pulp regeneration process by linking the neurite outgrowth to human pulp fibroblast through complement system activation.

The proteome of the human parotid gland secretion in elderly with and without root caries.

OBJECTIVE: Saliva is important for oral health, including the prevention of dental decay. The purpose of the present work was to indicate whether the parotid gland has altered function in the elderly, particularly in terms of proteins secreted, and whether its functional status could be associated with the presence of root caries. MATERIAL AND METHODS: Ductal parotid gland secretion was obtained from 51 individuals: 21 elderly with carious roots (Patients), 20 elderly (Controls), and 10 adults (Young) without root caries. Pooled aliquots were analyzed by liquid chromatography/tandem mass spectrometry to yield lists of major proteins present in the three groups. RESULTS: Approximately 200 unique proteins were detected, of which 73 were identified repeatedly with high confidence and therefore included in the comparison. Some of the differences observed, when comparing Patients with respectively elderly Controls and the Young, resembled changes found in patients with Sjogren's syndrome, a condition associated with dental decay. Other changes involved proteins that are likely to impact on the oral microbiota, such as the absence of dermcidin and the presence of collagen in Patients. Cystatin S, a putative indicator of caries, was present at a higher level in Patients. CONCLUSIONS: Parotid function tends to change upon aging, with possible consequences as to caries activity. However, analyses of individual samples revealed considerable variations in protein patterns.

Micromorphological and micronanalytical characterization of stagnating and progressing root caries lesions.

Root caries is the predominant disease of the tooth tissues in the elderly population and differs in progression and micromorphology from coronal dentin caries. Therefore, different clinical concepts are needed for the treatment of these progressing and arrested lesions. It was the aim of this study to investigate the three-dimensional structure and volume of stagnating and progressing root caries lesions and to determine the mineral composition of the different lesion zones to achieve a better understanding of the natural history of root caries lesion formation. Of 21 extracted human teeth of patients between 42 and 77 years of age with stagnating and progressing root caries lesions serial sections were cut and investigated with polarized light and scanning electron microscopy. From the polarized light micrographs 3D reconstructions were made to determine the volumes of the lesion zones and their relations expressed in a demineralization index (DI). With increasing size of the demineralizing zone the DI increased indicating an increase in the size of the translucent zone. The 3D reconstructions showed distinct differences between stagnating and progressing root caries lesions. In the hypermineralized translucent dentin not all dentin tubules were obliterated by intratubular dentin and within the translucent dentin scattered dead tracts were found. Electron dispersive X-ray (EDX) analysis showed that the intertubular dentin of the translucent dentin was also demineralized to a certain extent. The results indicate distinct morphological differences between progressing and stagnating root caries lesions which may have consequences for treatment strategies.

Development of an in situ root caries model. A. In vitro investigations.

OBJECTIVES: The paper describes preliminary in vitro investigations, the objectives of which were to examine the influence of certain experimental parameters on artificial carious lesion formation in root hard tissues, and their remineralisation. These experiments formed part of a wider study that aimed to develop an in situ model of root caries, based on the existing coronal caries model used in Liverpool. The present studies examined the effects (a) of the anatomical origin of the dentine, the presence or absence of cementum, the exposure time and the type of demineralising system, on lesion development, and (b) of baseline lesion size on the extent and location of mineral re-precipitation. METHODS: Mineral content parameters in plano-parallel sections taken from dentine lesions were determined by computer-controlled transverse microradiography. RESULTS: The importance of the anatomical origin of the dentine on lesion formation was investigated by comparing in vitro lesion formation in premolar and molar dentine, and in dentine from apical, middle and coronal thirds of the root: no difference was observed between these sites. Lesions formed more rapidly in acid buffer solutions than in acid gel systems, and were more reliably produced when cementum was removed. The effect of baseline lesion size on subsequent in vitro remineralisation demonstrated that a small baseline mineral content was associated with a larger percentage mineral gain. The location of mineral deposition throughout the lesion was also influenced by baseline mineral content parameters. CONCLUSIONS: The results form a basis for the further development of an in situ dentinal caries model, providing data to suggest that manipulation of parameters involved in the preparation of artificial carious lesions has a significant effect on the behaviour of the lesion, particularly the phenomenon of remineralisation. Further work is needed to investigate the behaviour of the model in situ.

Development of an in situ root caries model B. In situ investigations.

OBJECTIVES: The aims of this pilot study were to assess the suitability of an in situ model for root caries studies, and to investigate the effect of sub-surface lesion mineral content and ultrastructural organisation on remineralisation. METHODS: Subjects were selected by criteria generally used for coronal caries models. Plaque pH responses and salivary flow rates were determined by standard methods. The chosen substrate was human dentine with artificially induced caries-like demineralisation and sterilised by gamma-irradiation (4100 Gy). Two levels of baseline demineralisation were induced by exposure to an acid buffer for 2 or 5 days. Mineral content was measured by computer-controlled quantitative microradiography. RESULTS: Remineralisation in situ was greater in smaller lesions with lower surface mineral content than in larger lesions in which the surface mineral content was high. CONCLUSIONS: The model is suitable for further development for investigating dentinal caries. More work is needed to validate the model by means of a fluoride dose-response study.

Effect of saliva composition on experimental root caries.

The aim of this study was to determine the effect of saliva composition on caries lesion development independently of the flow rate of unstimulated whole saliva (UWS) and other caries-related variables such as lesion progression time, oral hygiene level, and fluoride exposure. We hypothesized that this could be done by developing experimental root caries under carefully controlled conditions in situ in test subjects with UWS flow rates within a narrow window of normalcy. Fifteen female and 5 male subjects (66 +/- 6 years) were selected for the study according to their UWS flow rates between 0.2 and 0.4 ml/min. All subjects developed experimental root caries lesions during a 62-day period in which UWS as well as stimulated whole saliva (SWS) were repeatedly collected and analysed for flow rate, pH, buffer capacity, inorganic, and organic composition. Caries lesion development was determined by quantitative microradiography. The mean UWS flow rate was 0.30 +/- 0.05 ml/min. Significant negative correlations were obtained between UWS total phosphate concentration and mineral loss (DeltaZ; r(s) = -0.72, p < 0.001) and UWS total protein concentration and DeltaZ (r(s) = -0.70, p < 0.01). SWS and its constituents had only limited or no effect on DeltaZ. Qualitative UWS protein analysis (SDS-PAGE) revealed that subjects with low DeltaZ values had broader and more stained amylase bands than subjects with high DeltaZ values. These findings were confirmed quantitatively by HPLC. We conclude that, within a group of subjects with normal UWS flow rates, the UWS composition was more important for caries lesion development than the SWS composition. Furthermore, high UWS concentrations of phosphate, protein, and amylase were caries-protective.

Influence of the mineral content and morphological pattern of artificial root caries lesion on composite resin bond strength.

Dentine substrates with different mineral contents and morphological patterns were created by submitting root slabs to the following treatments: (A) immersion in artificial saliva during the experimental period (control), (B) demineralization for 32 h to induce caries lesion (demineralized group), and (C) demineralization for 32 h followed by remineralization for 8 d (remineralized group). The slabs were longitudinally sectioned, the mineral content was determined by cross-sectional microhardness, and the bond strength of an adhesive system/composite resin was assessed using a microtensile bond strength test. The dentine morphology after the treatments as well as the failure pattern of the debonded specimens was examined by scanning electron microscopy. Statistically significant differences were found in mineral content. Morphological analysis showed marked differences between the patterns of demineralized and remineralized substrates. The bond strength mean value of the control A did not differ from the group B, but was statistically higher than the group C. Since no linear relationship was found between dentine mineral content and bond strength values, it could be suggested that the morphological pattern may be more relevant than the mineral content to explain the bond strength of composite resin to dentine.

Penetration of an antibacterial dentine-bonding system into demineralized human root dentine in vitro.

In this study, the penetration of three proprietary dentine-bonding agents (Prime & Bond 2.1, Single Bond, Liner Bond 2) and experimental dentine-bonding systems incorporating an antibacterial monomer, 12-methacryloyloxydodecylpyridinium bromide (MDPB), into artificial root caries lesions was evaluated, and the bactericidal activity of each material against Streptococcus mutans or Lactobacillus casei impregnated into demineralized dentine blocks was assessed. All of the commercial dentine-bonding agents were capable of penetrating into the artificial carious lesions to more than 150 microm. The depth of penetration of the experimental systems, which were based on Liner Bond 2, was not significantly different from that of their parent product. Liner Bond 2 primer exhibited the greatest bactericidal effects among the three proprietary dentine-bonding agents tested. Bactericidal activities of experimental primers containing MDPB were greater than those of any other products, and the application of 4% MDPB-containing primer resulted in complete killing of bacteria in demineralized dentine. The results indicate that the penetration of dentine-bonding agents into extensively demineralized root dentine is possible in vitro, and the experimental dentine-bonding systems containing the antibacterial monomer MDPB are capable of killing bacteria within demineralized dentine. This could be of benefit when managing root caries lesions.

Studies of dental root surface caries. 2: The role of cementum in root surface caries.

Artificial caries lesions were produced in roots of teeth using an acetate buffer system, when the layer of cementum was either normal in thickness, excessively thickened by hypercementosis, or had been removed completely. The rates of lesion progression were measured in each case using polarized light microscopy to measure lesion depth. Analysis of calcium (Ca) and phosphorus (P) loss during the demineralizing process was carried out. The removal of cementum was found to significantly increase the initial rate of penetration of the lesion into the root, although this rate progressively reduced to a level consistent with that found in normal roots after seven days of demineralization. The overall depth remained consistently greater than that observed in normal roots, or when lesions were produced entirely within hyperplastic cementum. Chemical analysis also showed removal of cementum resulted in an initial doubling of the Ca and P lost from the root surface. Prior direct exposure of segments of normal roots to the oral environment was found not to significantly alter the rate of artificial lesion progression, in comparison with that in the originally protected segment of the root surface. It was concluded that an intact cementum layer has the intrinsic ability to protect the underlying dentine of exposed tooth roots against acidic demineralization and that prior exposure to the oral environment does not significantly alter this ability.

Studies of dental root surface caries. 1: Comparison of natural and artificial root caries lesions.

Qualitative polarized light microscopy and cross-sectional microhardness testing profiles were used to compare artificial root caries lesions with those naturally present in extracted human teeth. The artificial lesions were produced by immersion of roots with exposed windows in chemical buffers of acetate and lactate with no added enzymes or bacteria. The three buffer systems used produced lesions with the range of histological characteristics of natural lesions. These included a 'body' or 'surface' zone of the lesion when viewed in water, and a 'frontal' zone of reverse birefringence when viewed in quinoline. The 'frontal' zone was noted to first develop after three days of immersion in acetate buffer. Advanced natural lesions exhibited a wide, diffuse, advancing front, which was similar to that produced by 28-60 day lesions in acetate. Microhardness profiles of natural lesions could all be matched by one or more of the artificial lesions. The demineralizing process was seen to reduce mineral content across the lesion from 45 per cent in normal dentine to between 15 and 25 per cent.

[The dynamics of pH, free calcium and total proteins in root plaque fluid and their relationships to the cariogenic potential].

OBJECTIVE: To enhance knowledge of root caries by analysis of the dynamics of dental plaque fluids on root surface and their relationships with the caries history. METHODS: The pH, free calcium and total proteins of dental plaque fluids on sound root surfaces were analyzed before and after the sucrose challenge. Subjects were either root caries-free (RCF) or caries-positive (RCP). RESULTS: After a sucrose challenge, the pH of plaque fluids dropped significantly and the concentration of free calcium increased significantly. There were no statistical differences in pH (RCF group 6.24 +/- 0.74, RCP group 5.96 +/- 0.70) and free calcium [RCF group (0.60 +/- 0.46) mmol/L, RCP group (0.89 +/- 0.54) mmol/L] between RCF and RCP group before sucrose challenge. Following sucrose exposure, pH (5.14 +/- 0.19) and free calcium [(1.73 +/- 0.74) mmol/L] in RCP group were significantly lower than RCP group [(pH 5.28 +/- 0.16), free calcium (2.73 +/- 1.25) mmol/L]. No interrelationship has been found between the amount of total proteins and free calcium. Components (contents, change trend) in root plaque were strongly related to that in coronal plaque. CONCLUSIONS: The process of root caries may be consistent to coronal caries regarding to the acid production of bacteria after a sucrose challenge. Plaque compositions show a stronger cariogenic potential in RCP group.

1H-NMR analysis of microbial-derived organic acids in primary root carious lesions and saliva.

In addition to lowered pH values, the molecular profile and concentrations of microbial-derived organic acids in carious dentin are important demineralization parameters involved in the induction, development and progression of dental caries. High-resolution proton ((1)H) NMR spectroscopy was employed to examine the organic acid status of primary root carious lesions. (1)H-NMR analysis of post-neutralized perchloric acid extracts of active carious lesions revealed that at an operating frequency of 600 MHz, the (1)H-NMR-detectable organic acid composition of carious dentin samples (mean molecular percentage content +/- standard error; the mean molecular percentage content is defined here as the mean of the concentration of each (1)H-NMR-visible organic acid/anion expressed as a percentage of total (1)H-NMR-detectable organic acid/anion level in each sample) was acetate 51 +/- 2%, formate 37 +/- 2%, lactate 5 +/- 1%, propionate 3 +/- 0.8%, pyruvate 2.4 +/- 0.3%, n-butyrate 1.2 +/- 0.2%; succinate 0.1 +/- 0.1%; iso-butyrate, n- and iso-valerate, and n- and iso-caproate (total) <0.2%. Further components detectable included alanine, glycine, choline, phosphorylcholine, trimethylamine oxide, methanol, glycolate and assorted saccharides. In view of their high dissociation constants (K(a)), our results demonstrate that formic and pyruvic acids (K(a) = 1.77 x 10(-4) and 3.20 x 10(-3) mol/dm(3), respectively) contribute substantially to the decreased pH values associated with active caries lesions (cf. lactate K(a) = 1.40 x 10(-4) mol/dm(3)), and hence the pathogenesis of primary root caries.

Fluoride distribution in sound and carious root tissues of human teeth.

Proton probe analysis has been used to provide for the first time quantitative F concentration data in carious root tissues from subjects consuming water containing 1 ppm F. In small lesions at the neck of the tooth with minimal tissue loss the F concentration was significantly higher at the outer lesion edge than at the outer edge of adjacent sound root tissue. In one sample with high F values the lesion edge had 19,000 ppm F and the adjacent sound root surface 5,400 ppm F microg F/g apatite). In large lesions with extensive cavitation F was again concentrated in the outer edge of the lesion and was significantly higher (1,800-4,100 ppm) than in adjacent sound inner dentine (190-290 ppm). Fluoride concentrations varied markedly along the outer edge of both normal and carious root tissues. Fluoride increase at the lesion edge is not an effect of tissue shrinkage but probably a result of remineralisation events during caries. This additional F may be expected to increase tissue resistance to further acid attacks.

A computer-assisted videodensitometric method to visualize mineral distributions in in vitro and in vivo formed root caries lesions.

A computer-assisted videodensitometry (CAV) method is presented for the microradiographical mineral determination and mineral visualization of in vitro and in vivo formed dentin lesions. The method employs as essential steps (1) image digitizing by a CCD camera, (2) conversion of gray values to equivalent aluminium thicknesses and (3) conversion of aluminium thickness to vol% of mineral. Procedure and accuracy are described. The CAV method is illustrated for dentin lesions formed in vitro as well as for clinical root caries lesions. The CAV method is especially advantageous for clinical lesions because it can be employed for very complex mineral distributions and irregular lesion outlines. In given areas of interest, the mineral distribution (in vol%) can be plotted in 3D form in pseudo-colors. Conventional microradiographical profiles can be obtained directly from the CAV images. For in vitro lesions, the mineral distribution parameters, lesion depth, mineral loss, and mineral acquisition values can be generated from CAV images with a reproducibility of better than 7%. The CAV method is a valuable tool in quantitative mineral studies of clinical lesions in extracted teeth as well as in in situ lesion investigations.

Penetration of varnishes into demineralized root dentine in vitro.

In this paper the penetration of three different varnishes employed in caries prevention (Duraphat, Fluor Protector and Cervitec) into demineralized dentine is quantified using confocal laser scanning microscopy. The results show that the varnish penetration into lesions about 85 microns in depth if for Cervitec about 35 microns and considerably less for Duraphat and Fluor Protector. The penetration is into the dentinal tubules and is influenced by dentinal tubule direction. The drying procedure--pretreatment of the dentine--influences the penetration, though sizeably only for Cervitec applications. This paper shows that varnish penetration into the tissue and presumably 'sealing' tubules completely or partly is valuable with respect to root caries prevention and hypersensitivity.