The Effect of Nitrate Supplement on the Oral Microbiome and Saliva in Dental Erosion

Dental erosion happens when teeth lose some of their parts because of acid from the things people eat and/or drink or even from their stomach. It can make their teeth hurt and become sensitive. The mouth has a protective shield called salivary pellicle that helps, and there are some special bacteria called Nitrate-reducing bacteria (NRB) that can be good for the mouth and heart. Eating some fruits and vegetables or drinking beetroot can make these bacteria thrive, which might help the heart and blood pressure. This study wants to see how drinking beetroot juice affects these good bacteria in your mouth for people with healthy teeth and those with erosion. This study aims to find out if drinking beetroot juice can change the saliva and bacteria in the mouth for the better, in people with both healthy teeth and those with dental erosion.

The proposed study is part of a PhD educational project and will be a double blind, randomised design, crossover trial for the effect of nitrate supplement on healthy control and people with dental erosion (PwDE). This study aims to explore the effect of nitrate supplement on oral microbiome in WMS and SP and its impact on blood pressure and arterial stiffness in healthy controls and PwDE.

Upon consent being granted, several samples and measurements will be collected from the participant during this research study spanning over 3 visits.

The proposed study is part of a PhD educational project and will be a double blind, randomised design, crossover trial for the effect of nitrate supplements on healthy control and PwDE. This study aims to explore the effect of nitrate supplements on oral microbiome recovery in whole mouth saliva (WMS) and salivary pellicle (SP) and its impact on blood pressure and arterial stiffness in PwDE and healthy controls. The reason this is coming to the HRA is because we do not have an HTA license at the University of Portsmouth.

This study will assess various biomarkers, including salivary proteins and oral microbiome, vascular function, nitrate-reducing activity of oral bacteria, and oxidative stress markers, to understand the impact of chlorhexidine mouthwash on oral and vascular health in PwDE and healthy individuals. The effect of CHX on the structure and composition of the oral microbiome in healthy and PwDE will be analysed. Furthermore, this study will analyse micro and macrovascular endothelial function changes in both groups (healthy and PwDE groups), linking changes to oral microbial communities and cardiovascular function.

Two groups of participants are required for this study \[healthy (n=10) and PwDE (n=10) groups\] from volunteer participants from the university of Portsmouth (UoP) who have no signs of or reported medical problems (detailed protocol of recruitment attached). Based on power calculation, 12 individuals will be recruited for each group (healthy and PwDE groups) to account for a 16% drop-out rate.

Participants will be approached to volunteer for the study by the chief investigator and PhD researcher through word of mouth, emails via departmental staff lists, University of Portsmouth Dental Academy (UPDA) updates newsletter, UPDA department meetings and presentations about the study during the UPDA's journal club events. A participant information sheet (PIS) and consent form (see attached forms) will be distributed either via email or by handing out hard copies to those wishing to partake in the research. Participants who have returned their completed consent form will be invited to come to UPDA for further assessment of suitability (visit 1).

The participants who has agreed to take part in this research will be invited to attend the University of Portsmouth Dental Academy, Beatty Building (UPDA) for three consecutive visits. They will be invited to attend the UPDA's dental clinics for their first visit for clinical screening and baseline sampling then for their consecutive sampling visits (2 visits) to attend Spinnaker Building, School of Sport, Health \& Exercise Science, University of Portsmouth (SbUoP) for further sampling.

Visit 1 (30 minutes-1 hour): This visit will include clinical screening, consent and baseline samples taken and will take place at UPDA dental clinics. The mouths of participants will be screened for suitability to fit in either of the groups (healthy or dental erosion groups) depending on the presence or absence of dental erosion in their teeth. A scoring system called BEWE will be used which is a simple, reproducible and convenient scoring system that can be used for recording clinical findings of dental erosion. If BEWE score is greater than 8 per extant cumulative score and one score 3 in at least one sextant then this means the participant's mouth shows evidence of dental erosion. If a participant is found to be suitable based upon inclusion/exclusion criteria (see attached detailed protocol), they will be invited to attend further appointments.

A medical history, dental history and two further questionnaires (Diet Questionnaire and Reflux Disease Questionnaire) will be completed with participants to ensure that they are suitable for this research study.

After the oral assessment and completing all forms and questionnaires, participants will be assigned to two groups of participants \[healthy (n=10) and dental erosion (n=10) groups\]. Within each group, the ten participants will be randomly and blindly assigned to further two subgroups by an independent researcher to receive one of the treatments \[Beetroot Juice (BJ) (n=5) and placebo (flavoured water with discolouring dyes) (n=5)\] as can be seen in the attached protocol. In the same visit, they will then be invited to come to the science laboratory (Beatty Building, UPDA) for several sample collections. As described in the protocol and participant information sheet, these will include measuring participants' blood pressure and brachial artery endothelium-dependent function. Next, venous blood will be collected via venepuncture from the forearm in 4 mL aliquots. After that, saliva and salivary pellicle from teeth and tongue will be collected.

Then each participant from each subgroup will either receive 14 tubes containing 70 ml each of BJ containing nitrate supplements (n=5) or 14 tubes containing the placebo (flavoured water with discolouring dyes) (n=5) depending on the randomisation process. Both versions of the beetroot juice will be identical in taste and appearance. Participants will be blinded to the active nitrate supplement of interest (BJ). Each group will be instructed to intake each product in a reverse order as shown in (figure 1) which will thoroughly be explained by a member of our research team during this visit (visit 1). Specifically, 5 individuals from each group \[healthy (n=10) and PwDE (n=10) groups\] will be instructed to intake 70 mL each of BJ containing nitrate supplements twice daily for 7 days with the final tube to be taken the morning before their next visit (visit 2) as shown in figure 1. The other 5 individuals will be instructed to intake 70 mL placebo (flavoured water with discolouring dyes) twice daily for 7 days with the final tube to be taken the morning before visit 2. For standardisation, each participant will also be given the same toothpaste to use throughout the duration of the study.

Visit 2 (30-40 minutes per visit) AND Washout and Reverse treatment:

Seven days after visit 1, participants will be invited to return to the (SbUoP) for visit 2, between 9AM-12PM, with the final dose being taken 2 hours prior to their scheduled appointment time. Upon arrival, same samples will be collected and same measurements will also be recorded as that in baseline (blood pressure, brachial artery endothelium-dependent function, venous blood, saliva and salivary pellicles).

After visit 2, each group of participants will be instructed to undertake a four week wash out period before swapping to using the other nitrate supplement (twice a day) for seven more days. The study will end with a final sampling visit on the seventh day of the other nitrate supplement (visit 3) as shown in figure 1.

Visit 3 (30-40 minutes per visit):

Seven days after switching to the alternative nitrate treatment, participants will be asked to take it twice a day and continue this treatment for an additional seven days (visit 3). Then, on the seventh day of this new nitrate treatment (visit 3), participants will be invited for a final sampling visit with final dose taken 2 hours before the oral assessment, as indicated in Figure 1. Upon arrival, same sample and measurements will be repeated as visits 2. Upon completion of final sample collections (visit 3), this will be the end of volunteer participation.

Eligibility Criteria:
Inclusion Criteria:

Eligibility Criteria A) Healthy volunteers Group

Inclusion Criteria:

1. Be aged 18 to 75 years inclusive
2. Have a minimum of 20 natural uncrowned teeth (excluding 3rd molars) present
3. Give written informed consent
4. Be in good general health and oral health (without active caries lesions, gingivitis, periodontitis.
5. No diseases of the soft or hard tissues of the oral cavity
6. No other oral condition affecting the composition of the oral fluids and presented normal salivary flow (stimulated salivary flow \>1 mL/min, unstimulated salivary flow \> 0.25 mL/min; salivary pH \> 6)
7. Show no evidence of dental erosion or/and active dental caries (with BEWE score ≤8 per extant cumulative score per patient and no clinically visible decayed teeth) B) Dental erosion group Inclusion Criteria

1. Same as above of healthy volunteers (1-6). 2. Show evidence of dental erosion (with BEWE score \> 8 per extant cumulative score per patient and one score 3 in a sextant. BEWE is a simple, reproducible and convenient scoring system that can be used with the diagnostic criteria for recording clinical findings and examining dental hard tissues other than for caries, guiding the management of erosive tooth wear.

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Exclusion Criteria:

1. Smokers, using mouthwash or tongue scrapes.
2. Consumption of beetroot juice in normal diet (This will be determined from responses on the diet form).
3. Diagnosed with alcoholism (or drinking more than four times a week, or consuming at least 40 g in males and 20 g in females per day)
4. Presenting conditions associated with vomiting (e.g. anorexia or bulimia), bruxism and Gastro-oesophageal reflux disease (GORD)
5. Occupations associated with increased risk of erosion, such as swimmers and battery, charging and galvanizing workers.
6. Wearing certain types of dental work on specific teeth (e.g., orthodontic appliances, extensive dental bridges, or recent oral surgery
7. Use of antibiotics three (3) months prior to or during this study
8. Use of any over the counter medications other than analgesics or have a known medical condition or currently taking any medication
9. Pregnant or breastfeeding

8. Participation in another clinical study in the month preceding this study 9. Medical condition which requires pre-medication prior to dental visits/procedures.

10. Participants who have eaten/had anything to drink up to two hours before providing a sample (Two hours prior to the trial, participants will be instructed to eat a self-selected light meal and drink 500 ml of water. No other food or fluid will be allowed