The effect of 1,450 and 5,000 ppm sodium fluoride on polished dentin after citric acid erosion using change in step height.

PURPOSE: To measure step height change, using profilometry on dentin, after pre-treatment with sodium fluoride at 1,450 and 5,000 ppm and then erosion with citric acid. . METHODS: Dentin specimens (n= 150), sectioned from the coronal aspect of extracted human molars were randomly divided into three groups of 60 samples each and fully immersed in deionized water (control), or solutions of NaF with 1,450 ppm (F1450) or 5,000 ppm (F5000) for 3 minutes and then artificial saliva (not containing proteins) for 30 minutes. The samples were eroded for 10, 15, 20 or 25 minutes in 0.3% citric acid at pH 2.7. The mean step height change was calculated using confocal non-contact white light laser profilometry. . RESULTS: The mean (SD) step height for the control group at 25 minutes of acid exposure was 9.08 µm (± 0.74), for the F1450 fluoride group 8.74 µm (± 0.58) and for F5000 group 7.01 µm (± 0.56) µm, respectively. There were no statistically significant differences between the control group to the F1450 at any immersion times, whereas at F5000 there were statistically significant differences at all times (P< 0.0001). Within the limitations of this in vitro study, step height in dentin increased with time of exposure to citric acid and 5,000 ppm of sodium fluoride significantly reduced step height with artificial saliva. CLINICAL SIGNIFICANCE: 5,000 ppm NaF better protected dentin in an erosion model than concentrations commonly found in toothpastes.

Differences in the Natural Enamel Surface and Acquired Enamel Pellicle following Exposure to Citric or Hydrochloric Acid.

OBJECTIVES: The aim of this study was to investigate variations in the interaction between enamel, that is, the acquired enamel pellicle (AEP) and citric or hydrochloric acid. MATERIALS AND METHODS: A 24-h AEP was formed on natural enamel specimens (n = 40) from pooled whole mouth human saliva. Samples were randomly allocated to citric (0.3%, pH 3.2) or hydrochloric (HCl) acid (0.01 M, pH 2.38) exposure for 30 or 300 s. The total protein concentration (TPC), and phosphorous and calcium concentrations of the pellicle were determined before and after acid exposure, and again after re-immersion in saliva. Surface roughness and tandem scanning confocal microscopy imaging were used to assess enamel changes. RESULTS: After 300 s of citric acid exposure, the mean ± SD TPC reduced from 5.1 ± 1.1 to 3.5 ± 1.1 mg/mL (p < 0.05). In contrast, after 300 s of HCl exposure, the mean TPC did not reduce significantly from baseline (6.6 ± 1.1 to 5.7 ± 0.7 mg/mL) but was significantly reduced in the reformed pellicle to 4.9 ± 1.2 mg/mL (p < 0.001). This reduction occurred after significant release of calcium and phosphorous from the enamel surface (p < 0.001). Thirty seconds of exposure to either acid had no obvious effect on the AEP. The surface roughness of the enamel decreased after acid exposure but no differences between groups was observed. CONCLUSIONS: These findings indicate that citric acid interacted with proteins in the AEP upon contact, offering enamel protection. In contrast, HCl appeared to bypass the pellicle, and reduced protein was observed only after changes in the enamel chemical composition.

Terminology of Erosive Tooth Wear: Consensus Report of a Workshop Organized by the ORCA and the Cariology Research Group of the IADR.

Our understanding of erosive tooth wear and its contributing factors has evolved considerably over the last decades. New terms have been continuously introduced, which frequently describe the same aspects of this condition, whereas other terms are being used inappropriately. This has led to unnecessary confusion and miscommunication between patients, professionals, and researchers. A group of 15 experts, selected by the European Organization for Caries Research (ORCA) and the Cariology Research Group of the International Association for Dental Research (IADR), participated in a 2-day workshop to define the most commonly used terms in erosive tooth wear. A modified Delphi method was utilized to reach consensus. At least 80% agreement was achieved for all terms discussed and their definitions related to clinical conditions and processes, basic concepts, diagnosis, risk, and prevention and management of erosive tooth wear. Use of the terms agreed on will provide a better understanding of erosive tooth wear and intends to enable improved communication on this topic.

Quantitative Swept-Source Optical Coherence Tomography of Early Enamel Erosion in vivo.

Swept-source optical coherence tomography (SS-OCT) shows potential for the in vivo quantitative evaluation of micro-structural enamel surface phenomena occurring during early erosive demineralization. This randomized controlled single-blind cross-over clinical study aimed to evaluate the use of SS-OCT for detecting optical changes in the enamel of 30 healthy volunteers subjected to orange juice rinsing (erosive challenge) in comparison to mineral water rinsing (control), according to wiped and non-wiped enamel surface states. Participants were randomly allocated to 60 min of orange juice rinsing (pH 3.8) followed by 60 min of water rinsing (pH 6.7) and vice versa, with a 2-week wash-out period. In addition, the labial surfaces of the right or left maxillary incisors were wiped prior to SS-OCT imaging. An automated ImageJ algorithm was designed to analyse the back-scattered OCT signal intensity (D) after orange juice rinsing compared to after water rinsing. D was quantified as the OCT signal scattering from the 33 µm sub-surface enamel, normalised by the total OCT signal intensity entering the enamel. The back-scattered OCT signal intensity increased by 3.1% (95% CI 1.1-5.1%) in the wiped incisors and by 3.5% (95% CI 1.5-5.5%) in the unwiped incisors (p < 0.0001). Wiping reduced the back-scattered OCT signal intensity by 1.7% (95% CI -3.2 to -0.3%; p = 0.02) in comparison to the unwiped enamel surfaces for both rinsing solutions (p = 0.2). SS-OCT detected OCT signal changes in the superficial sub-surface enamel of maxillary central incisor teeth of healthy volunteers after orange juice rinsing.

In vitro culture of previously uncultured oral bacterial phylotypes.

Around a third of oral bacteria cannot be grown using conventional bacteriological culture media. Community profiling targeting 16S rRNA and shotgun metagenomics methods have proved valuable in revealing the complexity of the oral bacterial community. Studies investigating the role of oral bacteria in health and disease require phenotypic characterizations that are possible only with live cultures. The aim of this study was to develop novel culture media and use an in vitro biofilm model to culture previously uncultured oral bacteria. Subgingival plaque samples collected from subjects with periodontitis were cultured on complex mucin-containing agar plates supplemented with proteose peptone (PPA), beef extract (BEA), or Gelysate (GA) as well as on fastidious anaerobe agar plus 5% horse blood (FAA). In vitro biofilms inoculated with the subgingival plaque samples and proteose peptone broth (PPB) as the growth medium were established using the Calgary biofilm device. Specific PCR primers were designed and validated for the previously uncultivated oral taxa Bacteroidetes bacteria HOT 365 and HOT 281, Lachnospiraceae bacteria HOT 100 and HOT 500, and Clostridiales bacterium HOT 093. All agar media were able to support the growth of 10 reference strains of oral bacteria. One previously uncultivated phylotype, Actinomyces sp. HOT 525, was cultivated on FAA. Of 93 previously uncultivated phylotypes found in the inocula, 26 were detected in in vitro-cultivated biofilms. Lachnospiraceae bacterium HOT 500 was successfully cultured from biofilm material harvested from PPA plates in coculture with Parvimonas micra or Veillonella dispar/parvula after colony hybridization-directed enrichment. The establishment of in vitro biofilms from oral inocula enables the cultivation of previously uncultured oral bacteria and provides source material for isolation in coculture.

Effect of Model Variables on in vitro Erosion.

The aim of this study was to investigate the effects of tooth type (molar/premolar), tooth surface (buccal/lingual), smear layer or no smear layer, storage of specimens in deionised water, mode and speed of agitation, and rinsing method between cycles on mean step height loss and Knoop microhardness (KHN) change. Polished human enamel specimens embedded in acrylic resin were prepared from sound permanent molar and premolar tooth surfaces. A 0.3% (pH 3.2) solution of citric acid was used to erode the specimens, in a cycling procedure, consisting of 10 min immersion followed by rinsing in deionised water for 30 s with a spray bottle, for 5 cycles. The specimens were analysed with a non-contact white light profilometer and KHN. Molar teeth (148.99±24.49 KHN) and buccal surfaces (155.62±30.35 KHN) produced significantly less microhardness change compared to premolar (186.40±20.74 KHN) and lingual surfaces (179.76±23.21 KHN; p<0001). The effect of storage and rinsing showed little difference in mean step height loss (<1 µm) and microhardness change. With no smear layer a significantly lower mean step height loss and microhardness change (p<0.001) was observed. Agitation was performed with Orbital, Gyro and See-Saw rockers at 30, 40, 60, and 70 rpm. The mean step height loss was largest for See-Saw at 70 rpm (11.73±0.91 µm) and lowest for Orbital at 30 rpm (2.76±1.12 µm). A statistical difference was found between all types of agitation and speeds (p<0.001). In conclusion, this study has shown that the variables investigated here have a significant impact on the measurable outcome, highlighting the importance of accurate and detailed method sections.

Oral health and elite sport performance.

While the research base is limited, studies have consistently reported poor oral health in elite athletes since the first report from the 1968 Olympic Games. The finding is consistent both across selected samples attending dental clinics at major competitions and more representative sampling of teams and has led to calls from the International Olympic Committee for more accurate data on oral health. Poor oral health is an important issue directly as it can cause pain, negative effects on appearance and psychosocial effects on confidence and quality of life and may have long-term consequences for treatment burden. Self-reported evidence also suggests an impact on training and performance of athletes. There are many potential challenges to the oral health of athletes including nutritional, oral dehydration, exercise-induced immune suppression, lack of awareness, negative health behaviours and lack of prioritisation. However, in theory, oral diseases are preventable by simple interventions with good evidence of efficacy. The consensus statement aims to raise awareness of the issues of oral health in elite sport and recommends strategies for prevention and health promotion in addition to future research strategies.

The future of faculty recruitment: Inspiring students into an academic career.

The dilemma surrounding faculty shortages within dental education continues to present significant challenges for the dental profession. There remains a tremendous need to create an effective and sustainable pathway for the recruitment of faculty into dental academia, with an emphasis on the establishment of a more diverse and representative faculty composition. This perspective paper proposes a blueprint to nurture and inspire dental students into academia.

Validation of a Cumulative Hypersensitivity Index (CHI) for dentine hypersensitivity severity.

AIM: This study developed the Schiff Index into a Cumulative Hypersensitivity Index (CHI) to measure dentine hypersensitivity (DH) severity per subject. It validates this score by investigating if it compares to one comprising a percentage score from all tooth surfaces and as a highest score per subject. MATERIALS AND METHODS: Overall, 350 subjects were recruited from hospital and general practice in south-east England. Buccal, occlusal and oral Schiff Index scores were collected and percentages calculated based on scores 1, 1 and above, 2 and above and 3. CHI scores and highest Schiff Index scores were also recorded per subject. Spearman correlation coefficients (p-values) assessed the relationship between CHI scores, Schiff Index percentages and Schiff Index highest per subject. RESULTS: In subjects with a Schiff Index score of 1 or more on at least one tooth surface (indicating DH was present), correlation of the CHI score to the Schiff Index percentage score was 0.982 (p < 0.001). Correlation of the sextant score to the highest Schiff Index score per subject was 0.963, (p < 0.001). DH was more likely on tooth surfaces with gingival recession (p < 0.001) and without bleeding on probing (p < 0.01). CONCLUSIONS: This study has validated a novel CHI score to indicate DH severity per subject and may help guide clinical management.

Single-centre, single-blinded, randomised, parallel group, feasibility study protocol investigating if mandibular advancement device treatment for obstructive sleep apnoea can reduce nocturnal gastro-oesophageal reflux (MAD-Reflux trial).

INTRODUCTION: Just under half of patients with obstructive sleep apnoea (OSA) also have gastro-oesophageal reflux disease (GORD). These conditions appear to be inter-related and continual positive airway pressure (CPAP) therapy, the gold standard treatment for OSA to prevent airway collapse, has been shown to reduce GORD. As the impact of mandibular advancement devices, a second-line therapy for OSA, on GORD has yet to be investigated, a feasibility study is needed prior to a definitive trial. METHODS: This will be a single-centre, single-blinded, tertiary-care based, interdisciplinary, parallel randomised controlled study. Potential OSA participants presenting to the sleep department will be pre-screened for GORD using validated questionnaires, consented and invited to receive simultaneous home sleep and oesophageal pH monitoring. Those with confirmed OSA and GORD (n=44) will be randomly allocated to receive either CPAP (n=22) or MAD therapy (n=22). Following successful titration and 3 weeks customisation period, participants will repeat the simultaneous sleep and oesophageal pH monitoring while wearing the device. The number of patients screened for recruitment, drop-out rates, patient feedback of the study protocol, costs of interventions and clinical information to inform a definitive study design will be investigated. ETHICS AND DISSEMINATION: Health Research Authority approval has been obtained from the Nottingham 2 Research Ethics Committee, ref:22/EM/0157 and the trial has been registered on ISRCTN (https://doi.org/10.1186/ISRCTN16013232). Definitive findings about the feasibility of doing 24 hour pH oesophageal monitoring while doing a home sleep study will be disseminated via clinical and research networks facilitating valuable insights into the simultaneous management of both conditions. TRIAL REGISTRATION NUMBER: ISRCTN Reg No: 16013232.

First impressions count.

UNLABELLED: The art and craft of recording intra-oral anatomy successfully with dental impressions relies on the interaction of three critical factors--the 'golden triangle of impression-taking': an appreciation of the anatomical features to be recorded, the material used to take the impression and the clinical handling/operative technique applied. This paper aims to discuss the three factors and their inter-relationships, detailing clinical tips for successful, reproducible and consistent outcomes. CLINICAL RELEVANCE: Obtaining accurate dental impressions is the key to success in a wide range of clinical restorative procedures. This paper offers clinical advice to practitioners to plan and then take predictable, good quality impressions for their restorative cases.

Functional changes in the oral microbiome after use of fluoride and arginine containing dentifrices: a metagenomic and metatranscriptomic study.

BACKGROUND: Tooth decay is one of the most prevalent diseases worldwide, and efficient tooth brushing with a fluoride-containing dentifrice is considered fundamental to caries prevention. Fluoride-containing dentifrices have been extensively studied in relation to enamel resistance to demineralization. Arginine (Arg) has also been proposed as a promising prebiotic to promote pH buffering through ammonia production. Here, we present the first metagenomic (DNA sequencing of the whole microbial community) and metatranscriptomic (RNAseq of the same community) analyses of human dental plaque to evaluate the effect of brushing with fluoride (Fl) and a Fl+Arg containing dentifrices on oral microbial composition and activity. Fifty-three patients were enrolled in a longitudinal clinical intervention study with two arms, including 26 caries-active and 27 caries-free adults. After a minimum 1-week washout period, dental plaque samples were collected at this post-washout baseline, 3 months after the use of a 1450-ppm fluoride dentifrice, and after 6 months of using a 1450-ppm fluoride with 1.5% arginine dentifrice. RESULTS: There was a shift in both the composition and activity of the plaque microbiome after 3 months of brushing with the fluoride-containing toothpaste compared to the samples collected at the 1-week post-washout period, both for caries-active and caries-free sites. Although several caries-associated bacteria were reduced, there was also an increase in several health- and periodontitis-associated bacteria. Over 400 genes changed proportion in the metagenome, and between 180 and 300 genes changed their expression level depending on whether caries-free or caries-active sites were analyzed. The metagenome and metatranscriptome also changed after the subjects brushed with the Fl+Arg dentifrice. There was a further decrease of both caries- and periodontitis-associated organisms. In both caries-free and caries-active sites, a decrease of genes from the arginine biosynthesis pathway was also observed, in addition to an increase in the expression of genes associated with the arginine deiminase pathway, which catabolizes arginine into ammonia, thereby buffering acidic pH. Bacterial richness and diversity were not affected by either of the two treatments in the two arms of the study. CONCLUSIONS: Our data demonstrate that long-term use of both assayed dentifrices changes the bacterial composition and functional profiles of human dental plaque towards a healthier microbial community, both in caries-free and caries-active sites. This observation was especially apparent for the Fl+Arg dentifrice. Thus, we conclude that the preventive benefits of tooth brushing go beyond the physical removal of dental plaque and that the active ingredients formulated within dentifrices have a positive effect not only on enamel chemistry but also on the metabolism of oral microbial populations. Video Abstract.

Pulpotomy for the Management of Irreversible Pulpitis in Mature Teeth (PIP): a feasibility study.

BACKGROUND: Progression of dental caries can result in irreversible pulpal damage. Partial irreversible pulpitis is the initial stage of this damage, confined to the coronal pulp whilst the radicular pulp shows little or no sign of infection. Preserving the pulp with sustained vitality and developing minimally invasive biologically based therapies are key themes within contemporary clinical practice. However, root canal treatment involving complete removal of the pulp is often the only option (other than extraction) given to patients with irreversible pulpitis, with substantial NHS and patient incurred costs. The European Society of Endodontology's (ESE 2019) recent consensus statement recommends full pulpotomy, where the inflamed coronal pulp is removed with the goal of keeping the radicular pulp vital, as a more minimally invasive technique, potentially avoiding complex root canal treatment. Although this technique may be provided in secondary care, it has not been routinely implemented or evaluated in UK General Dental Practice. METHOD: This feasibility study aims to identify and assess in a primary care setting the training needs of general dental practitioners and clinical fidelity of the full pulpotomy intervention, estimate likely eligible patient pool and develop recruitment materials ahead of the main randomised controlled trial comparing the clinical and cost-effectiveness of full pulpotomy compared to root canal treatment in pre/molar teeth of adults 16 years and older showing signs indicative of irreversible pulpitis. The feasibility study will recruit and train 10 primary care dentists in the full pulpotomy technique. Dentists will recruit and provide full pulpotomy to 40 participants (four per practice) with indications of partial irreversible pulpitis. DISCUSSION: The Pulpotomy for the Management of Irreversible Pulpitis in Mature Teeth (PIP) study will address the lack of high-quality evidence in the treatment of irreversible pulpitis, to aid dental practitioners, patients and policymakers in their decision-making. The PIP feasibility study will inform the main study on the practicality of providing both training and provision of the full pulpotomy technique in general dental practice. TRIAL REGISTRATION: ISRCTN Registry, ISRCTN17973604 . Registered on 28 January 2021. Protocol version Protocol version: 1; date: 03.02.2021.

Effect of rinsing with ethanol-containing mouthrinses on the production of salivary acetaldehyde.

It has been suggested that the use of alcohol-containing mouthrinses could lead to the presence of acetaldehyde in saliva. In this cross-over study, salivary acetaldehyde levels and microbial profiles were determined before and after rinsing with ethanol-containing mouthrinses with essential oils (EO) and cetyl pyridinium chloride (CPC) as the active ingredients, and with 21.6% ethanol and water controls. After rinsing with all ethanol-containing rinses, acetaldehyde was detected in saliva after 30 s but declined to low levels after 5 min. The highest peak levels were seen with the ethanol control (median = 82.9 µM at 2 min) and were significantly higher than those seen at the same time after rinsing with the EO rinse (43.1 µM). There was no correlation between microbial counts or plaque scores and acetaldehyde levels, although dividing the subjects on the basis of a peak acetaldehyde salivary concentration of > 90.8 µM after the ethanol rinse revealed that the high responders were highly significantly more likely to harbour salivary yeasts than were the low responders. Rinsing with ethanol-containing mouthrinses causes a rapid, but transient, increase in salivary acetaldehyde levels.

Comparison of metrology created by profilometry and digital microscopy on polished dentine in an erosion/abrasion model.

OBJECTIVE: Digital microscopy offers the ability to scan surfaces to produce 3D reconstructions, allowing step height measurements with high accuracy. The aims of this study were to compare the step heights from the gold standard, non-contact profilometry, to digital microscopy in an erosion/abrasion model. METHODS: Dentine specimens (n = 60) were immersed in deionised water, 1450 ppm and 5000 ppm fluoride as sodium fluoride for 3 min, eroded for a total of 25 min in a cycled protocol in 0.3% citric acid (pH 2.7) and abraded with 120 and 240 brushing strokes. Samples were scanned by a non-contacting profilometer with a 0.1 µm vertical resolution and then the same samples imaged with a digital microscope and the step heights compared. Data were analysed in GraphPad Prism 7.00. Data were normally distributed and a 3 way ANOVA with post hoc analysis used to assess for differences between groups. Agreement between the measurement method was assessed using IntraClass Correlations and Bland Altmans plots. RESULTS: The mean step heights from the profilometry and the digital microscope on the same samples were not statistically significant different. The magnitude of the differences was less than 0.5 µm. The results of the ANOVA demonstrated that the individual factors fluoride concentration and number of strokes were significant (P<0.05), however, the method of analysis was not (p = 0.74). ICC's between the two methods of analysis were excellent (0.996, p<0.001) with no proportional bias. CONCLUSIONS: This study reports that step height on dentine from a digital microscopy and non-contact profilometry were not significantly different. The digital microscope, although slower, allows visual inspection of the samples as well as measurement. SIGNIFICANCE: Digital microscope's offer the ability to scan, 2D or 3D images and perform meteorological analysis of samples. In this investigation both showed that 5000 ppm fluoride prevents erosive tooth wear in vitro.

The use of the Modified Gingival Margin Plaque Index (MGMPI) method to investigate the inhibitory effect of various toothpastes on dental plaque formation.

OBJECTIVE: Colgate Total (CTT) is the only FDA-approved toothpaste for antiplaque and antigingivitis benefits. The objective of this study was to compare the impact of Colgate Total Pharma (CTP), a new variant of Colgate Total, with Colgate Regular Toothpaste (CRT) on plaque formation over a 24-hour period following a single use of the dentifrice. METHODS: CTP and CRT were the two test products. CRT was used for a washout product as well. Fifteen male/female subjects who met the inclusion/exclusion criteria were included into this single-blind (preliminary phase) and double-blind (randomized phase) crossover study. Ethical approval and written informed consent were obtained. Preliminary phase: After a one-week washout with CRT, subjects brushed in the dental clinic with CRT before a one-minute use of a test dentifrice. A baseline Modified Gingival Margin Plaque Index (MGMPI) score was calculated. Subjects refrained from oral hygiene for 24 hours, and returned to the clinic for their 24-hour MGMPI score. Subjects entered the second washout phase to repeat as per the crossover design. The above procedures were conducted three times by three independent examiners. Randomized phase: Subjects were randomized to the groups according to a computer-generated randomization schedule. The procedure was carried out as in the preliminary phase, except the washout period between the two products was at least one week and the products (CTP or CRT) were used in a randomized double-blind manner. Plaque scores were recorded as above. RESULTS: CTP provided a significant (p = 0.01) antiplaque effect versus CRT. The results are consistent with previously reported data for CTT. All three examiners demonstrated a strong correlation for this clinical study utilizing the MGMPI methodology. CONCLUSION: This clinical investigation examined the efficacy of a new variant of a commercial dentifrice, historically shown to provide antiplaque and antigingivitis efficacy. It is important to confirm the continued efficacy of new products to consumers and to the profession. Additionally, this clinical trial demonstrated the usefulness of the clinical methodology with respect to consistency in results by three independent clinical examiners. Because this methodology is often employed to document antiplaque benefits of new and existing technologies, it is important to periodically evaluate and confirm its reliability and reproducibility.

Proteins from whole mouth saliva mediate greater protection against severe erosive tooth wear than proteins from parotid saliva using an in vitro model.

OBJECTIVES: to investigate how the composition of the acquired enamel pellicle (AEP) affected a laboratory model of erosive tooth wear (ETW) on human enamel by comparing whole mouth saliva (WMS) to parotid saliva (PS). METHODS: 60 enamel specimens were prepared from extracted human teeth and were randomly assigned to 4 experimental groups: WMS (n = 20), PS (n = 20), artificial saliva (AS, n = 10) and deionised water (DW, n = 10). Following incubation, a subset of WMS (n = 5) and PS (n = 5) groups were used to collect the AEP before the erosive challenge. The rest of the blocks, had their AEP collected after five cycles of acid, wash and saliva and were then assessed for mean step height changes using a non-contacting profilometer (n = 10 each). AEP samples were collected from the enamel specimens by rubbing with filter papers soaked in sodium dodecyl sulfate. Total protein in AEP was quantified using BCA assay, individual protein components of AEP were separated and analysed using SDS-PAGE and western blot for [mucin 5b, albumin, carbonic anhydrase VI (CA VI), statherin]. Specific antibody binding was quantified using purified protein standards of known concentration. Samples of AEP were also analysed by LC/MS/MS sequencing. RESULTS: WMS group had significantly (p < 0.0001) less acid-induced erosion (step height [4.16 (0.9) µm]) than PS group [6.41 (0.3) µm]. The amount of total protein, mucin 5b and albumin were more dominant in WMS pellicles than PS (p < 0.0001) whereas CA VI and statherin were dominant in PS pellicles (p < 0.0001). CONCLUSION: The composition of the acquired enamel pellicle influences the degree of protection from acid attack, possibly by altering the mechanism of protection. The in-vitro model used in this study was severe enough to cause tissue loss as opposed to just softening of the surface structure. AEP from WMS was more protective than that of PS, and its likely mechanisms of protection seem to be as a physical barrier rather than stabilising the crystal structure. SIGNIFICANCE: The protective salivary proteins against in-vitro erosion models differ from in-vivo studies. Therfore, it can be recommended that in-vitro laboratory models of ETW need to be assessed carefully to represent the clinical environment more closely.

Evidence for Proline Utilization by Oral Bacterial Biofilms Grown in Saliva.

Within the mouth bacteria are starved of saccharides as their main nutrient source between meals and it is unclear what drives their metabolism. Previously oral in vitro biofilms grown in saliva have shown proteolytic degradation of salivary proteins and increased extracellular proline. Although arginine and glucose have been shown before to have an effect on oral biofilm growth and activity, there is limited evidence for proline. Nuclear magnetic resonance (NMR) spectroscopy was used to identify extracellular metabolites produced by bacteria in oral biofilms grown on hydroxyapatite discs. Biofilms were inoculated with stimulated whole mouth saliva and then grown for 7 days using sterilized stimulated whole mouth saliva supplemented with proline, arginine or glucose as a growth-medium. Overall proline had a beneficial effect on biofilm growth-with significantly fewer dead bacteria present by biomass and surface area of the biofilms (p < 0.05). Where arginine and glucose significantly increased and decreased pH, respectively, the pH of proline supplemented biofilms remained neutral at pH 7.3-7.5. SDS-polyacrylamide gel electrophoresis of the spent saliva from proline and arginine supplemented biofilms showed inhibition of salivary protein degradation of immature biofilms. NMR analysis of the spent saliva revealed that proline supplemented biofilms were metabolically similar to unsupplemented biofilms, but these biofilms actively metabolized proline to 5-aminopentanoate, butyrate and propionate, and actively utilized glycine. This study shows that in a nutrient limited environment, proline has a beneficial effect on in vitro oral biofilms grown from a saliva inoculum.

Mixed aerobic-anaerobic incubation conditions induce proteolytic activity from in vitro salivary biofilms.

Oral biofilms have not been studied using both metabolome and protein profiling concurrently. Bacteria produce proteases that lead to degradation of functional salivary proteins. The novel protocol described here allows for complete characterisation of in vitro oral biofilms, including proteolytic, metabolic, and microbiome analysis. Biofilms were grown on hydroxyapatite discs from whole mouth saliva, using sterilised saliva as a growth-medium, in different growth environments. Salivary protein degradation was assessed from spent saliva growth-medium using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and metabolic activity by nuclear magnetic resonance (NMR). Discs were assessed for depth and coverage of biofilms by confocal laser scanning microscopy (CLSM), and biofilms were collected at the end of the experiment for 16S rRNA gene sequence analysis. There was a significant difference in biofilm viability, salivary protein degradation, and metabolites identified between biofilms grown aerobically and biofilms exposed to an anaerobic environment. Bacterial 16S rRNA gene sequencing showed the predominant genus in the 7-day aerobic biofilms was Streptococcus, in aerobic-anaerobic and anaerobic 7-day biofilms Porphyromonas, and in aerobic-anaerobic and anaerobic 13-day biofilms Fusobacterium. This data suggests new growth requirements and capabilities for analysing salivary biofilms in vitro, which can be used to benefit future research into oral bacterial biofilms.