PMPR and Chlorhexidine on Periodontal Disease and Vascular Function

* Gum inflammation is called gingivitis. Gum disease, known as periodontal disease, is a long-term inflammation of the gums and bone around teeth, leading to tooth loss. Both gingivitis and periodontal disease are also linked to other health problems, including heart disease. Additionally, salivary proteins play a role in maintaining oral health. For example, a protective layer called the salivary pellicle (SP) forms on teeth after tooth brushing and helps defend against harmful bacteria. * Nitrate-reducing bacteria (NRB) are also present in saliva and play a role in oral and cardiovascular health by converting nitrate to nitric oxide (NO). This compound supports blood vessel health and overall cardiovascular function. Therefore, NRB's abundance is linked to lower blood pressure and improved cardiovascular health. * Standard treatments for gum disease include deep cleaning (also called professional mechanical plaque removal, or PMPR), and sometimes chlorhexidine (CHX) mouthwash is used with PMPR to help reduce mouth bacteria. However, while CHX is effective against plaque (a sticky film of bacteria on the tooth surface), it may reduce the activity of "nitrate-reducing" bacteria (NRB), which are important for producing nitric oxide (NO). * This study investigates the impact of deep cleaning (PMPR) with and without Chlorhexidine mouthwash on salivary and SP proteins, as well as blood vessel function, in people who have gum disease. By examining these links, the investigators aim to gain a deeper understanding of how oral care practices may impact overall health, particularly heart health.

* \*\*Introduction\*\* * Periodontal disease (PD) is a chronic inflammatory condition affecting the tissues supporting teeth, including gums, bone, and ligaments, and is associated with a 19% increased risk of cardiovascular disease. It starts with gum inflammation, known as gingivitis. Both chronic gingivitis and PD have multiple causes including poor oral hygiene, smoking, diabetes, genetics, stress, age, and certain medications, and PD contributes significantly to healthcare costs ($54 billion direct, $25 billion indirect annually). * The key approach for managing PD is non-surgical, typically involving Professional Mechanical Plaque Removal (PMPR), with or without adjunct antiseptic mouthwashes such as 0.2% chlorhexidine (CHX). While CHX is effective in reducing dental plaque, concerns exist regarding its impact on beneficial oral nitrate-reducing bacteria (NRB), which play a critical role in systemic nitric oxide (NO) pathways and cardiovascular homeostasis. Altering the oral microbiome may influence vascular function, including blood pressure and endothelial health. * This study investigates the effects of PMPR, performed as standard periodontal therapy, and adjunctive CHX mouthwash on salivary and acquired enamel pellicle (AEP) proteins, oral microbiome, NRB activity, and vascular function in individuals with PD. * \*\*Study Design Overview\*\* * A total of 30 adult participants with clinically confirmed gingivitis and/or periodontal disease will be enrolled and followed at four assessment points over a three-month period. * Recruitment Process and Practicalities Under the supervision of the academic supervisor, potential research participants will be recruited via the following steps: * Research participants recruited from SDHCP dental clinics: * Step 1: Providing Study Information to Potential Participants Clinicians at the University of Portsmouth (SDHCP), Dr. Anish Patel, Prof. Chris Louca, and Dr. Bhavin Dedhia, will inform patients about the study during their routine clinical appointments. Study materials, including the Participant Information Sheet and Invitation Letter, will be provided at least 24 hours before the first appointment, allowing individuals time to consider participation. * Step 2: Participant Follow-Up Participants who wish to volunteer for the study will then be contacted by the research team. The PhD researcher will explain the study in more detail, answer any questions, and confirm their willingness to participate. This process ensures that participants have at least 24 hours to consider their involvement before their appointment. The consent form will be signed by the individuals in person, before their oral screening, on their first visit. * Eligibility Screening: Consented participants will undergo a routine clinical examination by the clinicians using the standard British Society of Periodontology (BSP) 2017 classification, focusing on individuals with BPE codes 1 to 4 for gingivitis and subsequent diagnosis of periodontitis (stages 1 and 2). * Research participants recruited from community centres: \-- Step 1: Providing Study Information to Potential Participants: The PhD researcher will engage with the public by visiting community centres in Portsmouth and delivering informational talks about the study. Study materials, including the Participant Information Sheet and Invitation Letter, will be provided to allow potential participants at least 24 hours before their first appointment. * Step 2: Participant Follow-Up Interested individuals will be contacted by the research team to review the study participant information sheet (PIS), address any questions, and confirm their willingness to participate. Eligible participants will then be invited to the University of Portsmouth SDHCP dental clinics to receive routine dental treatment, which will include participation in the research study. * After screening and completion of questionnaires and forms, participants will be matched based on factors that may affect salivary proteins at baseline, including age (30-75 years), smoking status, BPE gingival score, periodontal disease classification, clinical severity of caries, dental erosion, and presence of diabetes. * The study will be composed of two sequential phases: * (i) Observational phase: Measure changes in salivary and Acquired Enamel Pellicle (AEP) or salivary pellicle (SP) protein composition and vascular function before and after routine PMPR. * (ii) Randomised, double-blind, placebo-controlled interventional phase: Participants will receive either CHX mouthwash or placebo mouthwash for two weeks following PMPR to evaluate combined or isolated effects on oral and vascular parameters. * Unstimulated whole mouth saliva (WMS) and AEP samples will be collected at baseline (Day 0), Day 1, Day 14, and Day 90. Proteomic and protein quantification techniques will map changes in both host and bacterial proteins. DNA-based microbiome analysis (16S rRNA sequencing) will examine shifts in bacterial composition, with particular attention to NRB and periodontal pathogens. * Vascular Function Assessments will include: * (i) Flow-Mediated Dilation (FMD): Large blood vessel endothelial function (ultrasound) * (ii) Pulse Wave Analysis (PWA) and Pulse Wave Velocity (PWV): Arterial stiffness * (iii) Iontophoresis: Small blood vessel endothelial function * (iv) Cardiac Output: The volume of blood pumped by the heart into the systemic circulation per minute, will be measured using PhysioFlow * (v) Blood Pressure: Including Mean Arterial Pressure (MAP) * (vi) Blood samples: Inflammatory plasma biomarkers (IL-6, IL-10, TNF-alpha) * \*\*Power and Sample Size Calculations\*\* * (i) Observational phase: N = 6, paired t-test, effect size 0.87, 80% power, α \< 0.05 * (ii) Interventional phase: Based on salivary biomarker MMP-8. ANOVA repeated measures with 90% power, α \< 0.05, effect size f = 0.87, requiring 12 participants per group. Accounting for 14% dropout, n = 15 per group, total N = 30. * Standardised procedures, examiner calibration, identical toothpaste and mouthwash containers, and allocation concealment will minimise bias and variability. * Participant Materials Key study documents, such as the participant information sheet and consent form, were shared with the PPI group for review. They provided explicit feedback on the clarity and accessibility of these materials, resulting in revisions to ensure the language is easy to understand and free of jargon. This has made the study more approachable for potential participants. * \*\*Storage of Data\*\* \-- No identifiable personal data will be published. All data will be linked-anonymised. Results published in journals or data repositories will contain only anonymised data. * \*\*Analysis of Data\*\* * i) Saliva Proteomics Analysis - Samples generated at School of Dental, Health and Care Professions (SDHCP), University of Portsmouth; proteomics analysis at Imperial College London. Saliva samples will be analysed for changes in salivary proteins, using Perseus software (Perseus\_v2.1.3.0.zip), run on .NET Desktop Runtime. * ii) Saliva Oral Microbiome Analysis - Samples generated at SDHCP, analysed at Temple University, USA, using Next-generation sequencing methods. * iii) Acquired Enamel Pellicle Proteins - Analysed using SDS-PAGE and Western blotting. * iv) Nitrate-Reducing Bacteria Activity - Generated at SDHCP, analysed at Loughborough University. * v) Vascular Function Data - Generated and analysed at School of Psychology, Sport and Health Sciences (SPSHS), University of Portsmouth. * \*\*Clinical Significance\*\* --This study will reveal how PMPR and adjunctive CHX mouthwash modulate the oral proteome and microbiome, and how these changes relate to vascular function. Findings will clarify the systemic effects of commonly used oral antiseptics, particularly on NRB and vascular function. Results will inform best practices for periodontal therapy, shaping future oral and systemic health guidelines. * \*\*Dissemination\*\* The findings from this study will be disseminated through multiple channels: * (i) Peer-reviewed journals: Results will be submitted to high-impact dental, oral health, and cardiovascular journals. * (ii) Scientific conferences: Findings will be presented at national and international conferences in periodontology, dentistry, and vascular research. * (iii) Public engagement: Lay summaries will be made available to participants and the public via the University of Portsmouth website and social media platforms. * (iv) Data sharing: De-identified datasets and analytical methods may be shared with other researchers upon reasonable request, following ethical approval and data protection guidelines.