Influence of Consumption of Nitrate-rich Beetroot Juice on Lactate Production in Saliva and Oral Biofilm - A Clinical Trial.

PURPOSE: Diets rich in nitrates have the potential to prevent oral diseases such as caries or periodontitis. The reduced forms nitrite and nitric oxide have an antibacterial effect against cariogenic bacteria. The effect on bacterial acid production in saliva and oral biofilm is yet unknown. This study investigated the influence of consuming naturally nitrate-rich beetroot juice on bacterial lactate production in saliva and on the pH value of saliva and oral biofilm. MATERIALS AND METHODS: In addition to their usual diet, a study group of eight subjects consumed 50 ml of beetroot juice daily for a fortnight. After a two-week break, they rinsed with 0.2% chlorhexidine (CHX) for 14 days as a positive control. Bacterial lactate production was induced by rinsing with 50 ml apple juice and measured at different time points during the study. RESULTS: After two weeks of daily beetroot-juice consumption, an accumulation of nitrate and nitrite was measured in the saliva. No influence on the bacterial lactate production in saliva or the saliva and plaque pH was found. CONCLUSION: Commercially available beetroot juice showed no modulating effects on intraoral bacterial acid production, suggesting no caries-preventive properties under the tested conditions.

The abundance of lysozyme, lactoferrin and cystatin S in the enamel pellicle of children - Potential biomarkers for caries?

OBJECTIVE: In this study, the abundance of the protective salivary proteins lysozyme, lactoferrin, and cystatin S was quantified in the in situ formed pellicle of caries-free and caries-active children to determine whether they may be possible biomarkers for caries. DESIGN: Pellicle formation was performed in situ for 10 min on ceramic specimens from the oral cavity of children (5-8 years) with caries (n = 17) and without evidence of caries (n = 17). Additionally, unstimulated saliva was collected. Levels of lysozyme, lactoferrin, and cystatin S were measured in desorbed pellicle eluates and saliva using ELISA. RESULTS: No statistically significant differences were found in the occurrence of cystatin S and lysozyme in saliva and pellicle between caries-active and caries-free children. However, significantly higher amounts of lactoferrin were detected in the pellicle of caries-active children. CONCLUSION: The protective salivary protein lactoferrin may be a biomarker for caries susceptibility in children.

Associations of early childhood medication exposure and molar incisor hypomineralization (MIH): A routine data analysis.

OBJECTIVES: Molar incisor hypomineralization (MIH) has become a major oral health problem of widely unknown origin. Besides genetic predisposition, exposure to certain drugs in early childhood are suspected to be associated with MIH. Aim of this routine data analysis was to examine associations of MIH and exposure to medication as well as perinatal factors. METHODS: Individuals with MIH were identified in claims data using a validated predefined specific treatment pattern. The database was a comprehensive routine data set of a major national health insurance company (BARMER, Germany). Based on this treatment pattern a MIH group and an unaffected control group were formed for analysis. Various medical data including medical diagnoses and prescriptions were available. Associations were examined comparing results for a set of variables in both groups. Differences between the groups were tested for significance using T-tests (P<0.01). RESULTS: Between 2010 to 2019, a total of 298,502 children between 6 and 9 years of age were included in this analysis. 22,947 were assigned to the MIH group. For individuals in this group, significantly larger prescription quantities in the main ATC (Anatomical, Therapeutic, Chemical) groups J (antiinfectives for systemic use), R (respiratory system) and S (sensory organs) were found in the first 4 years of life compared to MIH unaffected individuals. With antibiotics, there were both significantly larger prescription quantities and significantly higher numbers of respective prescriptions in the first 4 years of life. The differences amounted up to about 10.62% in frequently used antibiotics to be found in ATC J01D (other beta-lactam antibacterials) for the number of prescriptions in the 4th year of life. No association was found for premature birth, mode of delivery or the use of antipyretic or anti-inflammatory medication. CONCLUSIONS: While perinatal factors do not seem to be associated with MIH development, early life exposure to antibiotics might play a role. CLINICAL SIGNIFICANCE STATEMENT: Although causal relations can still not be proven, a responsible use of the unquestionably beneficial antibiotics is encouraged from a clinical point of view.

Mucins 5b and 7 and secretory IgA in the oral acquired pellicle of children with caries and caries-free children.

OBJECTIVE: The objective of this study was to determine whether differences in the abundance of mucins 5b and 7 as well as secretory IgA exist in the oral acquired pellicle between children with active caries and caries-free children. DESIGN: Pellicle formation was performed for 10 min in-situ on ceramic slabs in the oral cavity of children (5-7 years of age) with caries (n = 15) and without signs of caries (n = 13). Furthermore, unstimulated saliva was collected. Concentrations of Muc5b, Muc7 and sIgA were measured in desorbed pellicle eluates and in saliva. RESULTS: Significantly larger concentrations of Muc5b, Muc7 and sIgA were detected in the pellicle obtained from children with caries compared to caries-free children. However, in the salivary samples concentrations of mucins Muc5b and Muc7 as well as sIgA did not differ significantly between the two groups. CONCLUSIONS: All three pellicle components Muc5b, Muc7 as well as sIgA could be identified as potential biomarkers for early childhood caries with high sensitivity and specificity. This could contribute to a better understanding of the different caries susceptibility in children.

Targeted metabolomics of pellicle and saliva in children with different caries activity.

Pellicle is the initial proteinaceous layer that is formed almost instantaneously on all solid surfaces in the oral cavity. It is of essential relevance for any interactions and metabolism on the tooth surface. Up to now, there is no information on the metabolome of this structure. Accordingly, the present study aims to characterise the metabolomic profile of in-situ pellicle in children with different caries activity for the first time in comparison to saliva. Small molecules such as carbohydrates, amino acids, organic acids, and fatty acids, putatively involved in the formation of caries were quantified using mass spectrometry (MS)-based techniques, such as (stable isotope dilution analysis)-ultra-performance liquid chromatography-tandem MS and gas chromatography/electron ionisation-MS. Pellicle and corresponding saliva samples were collected from caries-active, caries-free and caries-rehabilitated 4- to 6-year-old children. The most abundant analytes in pellicle were acetic acid (1.2-10.5 nmol/cm2), propionic acid (0.1-8.5 nmol/cm2), glycine (0.7-3.5 nmol/cm2), serine (0.08-2.3 nmol/cm2), galactose (galactose + mannose; 0.035-0.078 nmol/cm2), lactose (0.002-0.086 nmol/cm2), glucose (0.018-0.953 nmol/cm2), palmitic acid (0.26-2.03 nmol/cm2), and stearic acid (0.34-1.81 nmol/cm2). Significant differences depending on caries activity were detected neither in saliva nor in the corresponding pellicle samples.

Activity and distribution pattern of enzymes in the in-situ pellicle of children.

OBJECTIVE: This study investigated, for the first time, pellicle enzymes with respect to their activity, distribution and fluorescence pattern in children with different caries experience. DESIGN: In-situ pellicles were collected from 41 children (aged 4-6 years) with different caries status; 17 of them were caries-free (dmf = 0), 12 had dental restorations but no current caries (dmf ≥ 2) and 12 had at least two carious lesions (dmf ≥ 2). Bovine enamel samples were fixed on individual upper jaw braces for pellicle formation. After 30 min of intraoral exposure, the pellicle and saliva samples were analysed for the activities of amylase, lysozyme, peroxidase and glucosyltransferase (GTF). The distribution of these enzymes, including GTF-isoforms B, C and D, and the pellicle ultrastructure were examined by gold-immunolabelling and transmission electron microscopy (TEM). Furthermore, interactions between pellicle enzymes and adherent bacteria were visualised using combined fluorescence and immunofluorescence labelling. RESULTS: There were no significant differences in the pellicle enzyme activities between the study groups. TEM analysis revealed the absence of GTF C and D in the pellicle of caries-active children. Amylase, peroxidase and GTF-isoforms showed a random distribution within the pellicle layer; lysozyme was found in the form of clusters. A similar ultrastructural pattern was observed for all subjects. Fluorescence labelling technique enabled visualisation of all enzymes, except for GTF B. CONCLUSION: Pellicle enzyme activities and ultrastructure are not associated with children's caries status. Further investigation is needed to assess the influence of individual GTF-isoforms on caries susceptibility in children.

Initial microbial colonization of enamel in children with different levels of caries activity: An in situ study.

PURPOSE: To investigate patterns of overnight in situ microbial colonization of enamel in children. METHODS: Overall, 29 children (aged 5-9 years) participated in the study. Nine were caries-free with no decayed, missing, or filled teeth (DMFT), 11 were caries-rehabilitated (DMFT ≥ 2, no active carious lesions), and nine were caries-active (DMFT ≥ 2, at least two carious lesions). Bovine enamel samples were fixed on individual upper jaw splints stored overnight in situ. 4',6-diamidino-2-phenylindole (DAPI) combined with Concanavalin A staining was applied for fluorescence microscopic visualization of total adherent bacteria and glucans. Fluorescence in situ hybridization (FISH) was used for distinction of eubacteria, streptococci, and Candida albicans. Salivary samples were investigated for Streptococcus mutans (S. mutans) by using CRT bacteria test and yeasts with Calcofluor white (CFW) staining. RESULTS: With all fluorescence methods, bacteria but not Candida albicans were detected on enamel samples. No statistically significant differences were observed in distribution patterns of the adherent bacteria between the groups. CFW staining indicated fungal structures in saliva samples of all participants. Based on CRT test results, the lowest amount of S. mutans were observed in caries-free children. Thus, initial microbial colonization patterns of enamel in children are not influenced by caries activity. CLINICAL SIGNIFICANCE: Caries activity in children may influence the process of initial bioadhesion and thus distribution patterns of bacterial attachment to the enamel surface. Investigation of in situ biofilm formation might provide valuable insights regarding the varying caries susceptibility in children.

Effect of Tannic Acid on the Protective Properties of the in situ Formed Pellicle.

OBJECTIVES: In the present in situ/ex vivo study the impact of tannic acid on the erosion-protective properties of the enamel pellicle was tested. Additionally, the antiadherent and antibacterial effects of tannic acid were evaluated. METHODS: The pellicle was formed in situ on bovine enamel samples fixed on individual splints worn by 6 subjects. Following 1 min of pellicle formation the volunteers rinsed for 10 min with tannic acid. After further oral exposure for 19 min, 109 min, and 8 h overnight, respectively, slabs were incubated in HCl ex vivo (pH 2.0, 2.3, 3.0) over 120 s. Subsequently, kinetics of calcium and phosphate release were measured photometrically. Samples after a 1-min fluoride mouth rinse as well as enamel samples with and without a 30-min in situ pellicle served as controls. Antiadherent effects were evaluated after a 1-min rinse with tannic acid and oral exposure of the slabs overnight. DAPI (4',6-diamidino-2-phenylindole) combined with concanavalin A staining and live/dead staining was used for fluorescence microscopic visualization and quantification of adherent bacteria and glucans. Modification of the pellicle's ultrastructure by tannic acid was evaluated by transmission electron microscopy (TEM). RESULTS: Tannic acid significantly improved the erosion-protective properties of the pellicle in a pH-dependent manner. Bacterial adherence and glucan formation on enamel were significantly reduced after rinses with tannic acid as investigated by fluorescence microscopy. TEM imaging indicated that rinsing with tannic acid yielded a sustainable modification of the pellicle; it was distinctly more electron dense. CONCLUSION: Tannic acid offers an effective and sustainable approach for the prevention of caries and erosion.

Effect of Inula viscosa on the pellicle's protective properties and initial bioadhesion in-situ.

OBJECTIVES: The present in situ study investigated the effect of Inula viscosa tea on the pellicle's acid protective properties and on initial oral biofilm formation. DESIGN: Biofilm formation was performed on bovine enamel slabs on individual maxillary splints. Following 1min of pellicle formation, eight subjects rinsed for 10min with Inula viscosa tea and the splints remained for 8h intraorally. Samples carried after 1-min rinsing with CHX (0.2%) or without rinse served as controls. BacLight™ staining, 4',6-diamidino-2-phenylindole (DAPI)-staining and fluorescence in situ hybridization (FISH) were used for fluorescence microscopic detection of adherent bacteria. For investigation of acid protective properties, three subjects rinsed for 10min with Inula viscosa tea after 1min pellicle formation and kept the splints intraorally for further 19min. Physiological 30-min pellicles and native enamel samples served as controls. After HCl incubation of the samples ex-vivo over 120s (pH 2.0, 2.3, 3.0) calcium- and phosphate release were quantified photometrically. Potential influences on the pellicle's ultrastructure by Inula viscosa tea were evaluated by transmission electron microscopy (TEM). RESULTS: Application of Inula viscosa tea yielded a significant reduction of adherent bacteria on all enamel samples as detected by fluorescence microscopy. For calcium- and phosphate release no significant effect was recorded. TEM investigation indicated a modification of the pellicle's ultrastructure, but no enhanced protection against erosive noxae. CONCLUSION: Rinsing with Inula viscosa tea influences the bacterial colonization on enamel in situ over 8h but has no impact on acid protective properties of the pellicle.