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

Upon consent being granted, several samples and measurements will be collected from the participant during this research study spanning over 15 visits. The proposed study will be a double blind, randomised design, crossover trial for the effect of CHX mouthwash on healthy control and PwDE. Two groups of participants are required for this study [healthy (n=12) and PwDE (n=12) 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, 14 individuals will be recruited for each group (healthy and PwDE groups) to account for a 14% 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 event. 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 fifteen 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 (14 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 BEWE will be used to screen as whether participants have erosion or not. It 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 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=12) and dental erosion (n=12) groups]. Within each group, the twelve participants will be randomly and blindly assigned to further two subgroups by an independent researcher to receive one of the mouthwash treatments [[placebo CHX group (n=6) and antibacterial CHX mouthwash group (n=6)] 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 10 mL CHX placebo mouthwash (ultrapure mint-flavoured water) or another 14 tubes containing 10 ml antibacterial mouthwash containing 0.2% CHX (Corsodyl Mint, GlaxoSmithKline, UK) depending on the randomisation process. Both versions of the mouthwash will be identical in taste, appearance, colour, texture, and smell using plain, identical bottles for both solutions. They will be instructed to administer each product in a reverse order which will thoroughly be explained by a member of our research team during this visit (visit 1). Specifically, participants will be instructed to rinse their mouth with the selected mouthwash for 1 minute, twice a day for 7 days taking the final tube the morning before their next visit (visit2). For study standardisation, participants will also be given a standardised toothpaste to use throughout the duration of the study.

Visit 2 - 8 (30-40 minutes per visit):

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 administered 2 hours prior to their scheduled appointment time. Upon arrival, blood pressure of the brachial artery (forearm) will be measured following 30 minutes of seated rest in a quiet room using an automated sphygmomanometer. A total of five successive measurements will be taken, with 1-minute rest between readings, with the mean of the final three measurements to be recorded. The mean of the systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP) measurements will also be recorded. Endothelial function test and brachial artery endothelium-dependent function will be measured as described below in the analysis section. Next, venous blood will be collected via venepuncture from the forearm in 4 mL aliquots as described in the analysis section. Finally, WMS, AEP and tongue SP samples will then be collected as described in the detailed sample collection section below.

All participants will then be invited to come to the (SbUoP) for further six visits in which same samples and measurements will be collected.

Upon completion of final sample collections (visit 8), each participant from each group [healthy control (n=6) and PwDE(n=6)] will receive the reverse order of the product they had received in visit 1 (cross over) to be used after a washout period of 12-weeks. Each participant will either receive 14 tubes containing 10 mL CHX placebo mouthwash (ultrapure mint-flavoured water) or another 14 tubes containing 10 ml antibacterial mouthwash containing 0.2% CHX (Corsodyl Mint, GlaxoSmithKline, UK) in the reverse order (crossover design) depending on what they have had before the washout period (visit1) as seen in figure1.

Washout and Reverse treatment:

After visit 8, participants will be instructed to carry out a washout period of 12-weeks before starting the 7-day reverse order of the given product administration as above. Specifically, the 6 participants from each group [healthy (n=12) and PwDE (n=12)] who had administered placebo CHX mouthwash before visit 2 will be given same amount of antibacterial mouthwash containing 0.2% CHX (Corsodyl Mint, GlaxoSmithKline, UK) to be administered in the same conditions as explained above (1 min, twice daily for 7 days) with the final tube taken the morning before their next visit (visit 9). The other 6 individuals will be instructed to administer the placebo CHX in the reverse order (crossover) of their 5 counterparts during visit 2 as shown in figure 1, twice daily, with the final tube to be taken the morning before visit 9.

Visit 9 - 15 (30-40 minutes per visit):

Visit 9 will take place after the 12-week washout period followed by seven days of administering the reverse product as seen in figure 1. Participants will be invited to return to the (SbUoP), between 9AM-12PM, with the final dose being taken 2 hours prior to their scheduled appointment time (visit 9). Upon arrival, same sample and measurements will be repeated as visits (2-8). Upon completion of final sample collections (visit 15), this will be the end of volunteer participation.