* 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.
Participants must have a minimum of 2 natural teeth in each sextant (at least 12 teeth overall).
Individuals with clinically diagnosed gingivitis and/or early periodontitis (stage 1 and 2)
Must not have undergone periodontal maintenance therapy (PMPR) within the last 6 months.
Must not currently be wearing orthodontic appliances, due to their effect on oral microbiota and plaque composition.
Individuals under regular medication within one month of the study start date for any medical condition (such as hypertension and/or diabetes)
Pregnant or breastfeeding individuals.
Individuals requiring an interpreter or who are non-English speakers.
Individuals unable to provide written informed consent.
Parallel
Chlorhexidine (0.2%) mouthwash
Other interventionPMPR is a routine standard dental treatment for gingivitis and PD, and will not form part of the research intervention. The research intervention consists of using 0.2% chlorhexidine mouthwash (10 ml, rinsed for 1 minute, twice daily for 14 days).
Placebo mouthwash
Other interventionPMPR is a routine standard dental treatment for gingivitis and PD, and will not form part of the research intervention. The research intervention consists of a placebo mouthwash (10 ml, 1 min, twice daily for 14 days). The placebo mouthwash (control) is designed to resemble commercially available products but without the active ingredient. Use of this has been ethically approved (IRAS Project ID: 333173), validated and tested in the investigator's previous studies (Data to be published).
Proteomics - Salivary Protein Analysis
Purpose: To identify inflammatory and protective proteins, monitor changes post-PMPR in saliva. * Method ( Protocol from Imperial College, London): * Reduction \& Alkylation: A 10 μL aliquot of saliva will be thawed and mixed with 7 μL of ammonium bicarbonate buffer on ice. For reduction and alkylation, 5 μL each of TCEP and CAA will be added, maintaining a pH of 7-8 to ensure proper modification of disulfide bonds and cysteine residues. * SP4 Protocol: Following reduction and alkylation, the SP4 protocol will be implemented by adding 80 μL of LC/MS-grade acetonitrile to precipitate proteins; the mixture will be centrifuged to separate the supernatant, and the pellet will be washed three times with ethanol for thorough purification. * Digestion: The purified pellet will then be resuspended in stock trypsin by adding 20 μL 25 mM ammonium bicarbonate to lyophilised powdered trypsin (powdered sequencing grade modified trypsin) and incubate overnight at 37°C, and incubated overnight.
Salivary Flow-Rate
\- Unstimulated whole mouth saliva (uWMS) sample will be used to measure salivary flow rate. Salivary flow rate (mL/min) is calculated as: Salivary flow rate (mL/min) = (Weight of tube with saliva - Weight of empty tube) ÷ Collection time (min).
Salivary pH
* An unstimulated whole mouth saliva (uWMS) sample will be used to measure salivary pH. * Following saliva collection, salivary pH will be measured using a single-electrode digital pH meter (Lutron Electronic Enterprise Co., Ltd., Model PH-208, Taiwan).
Oral Microbial Composition
* Microbial composition of the saliva will be characterised by DNA extraction and 16S rRNA sequencing to observe oral microbiome shifts following PMPR.- * Purpose: To characterise oral microbiome shifts after PMPR. * Method/techniques: DNA extraction; 16S rRNA sequencing. * Bacterial genomic DNA will be extracted from WMS and AEP samples using standard methods or commercial kits. The extracted DNA will serve as the template for PCR amplification of 16S rRNA gene fragments (500-1,500 bp). - Prepared saliva and pellicle samples will be sent to the Oral Microbiome Research Laboratory at Temple University (USA) for DNA extraction and 16S rRNA sequencing, with a Material Transfer Agreement (MTA) in place. - Microbial DNA will be extracted from 1-3 mL of samples using Qiagen QIAamp or Vazyme VAMNE kits. DNA quantification will be performed using fluorometric methods (Qubit), and purity will be assessed by absorbance. Three sterile water blanks will be included as controls.
Acquired Enamel Pellicle (AEP) Protein Composition
\- Protein profile will be assessed in AEP. The purpose is to understand tooth-surface protective vs. pathogenic protein changes. Pellicle will be collected using filter strips; protein elution and analysis. Parameters Assessed: Structural and functional protein variations (such as- albumin, cystatins, mucins, PRPs). - Purpose: To detect and compare key structural protein changes across study time points. - Methods/techniques: SDS-PAGE and Western blot. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a discontinuous electrophoresis method commonly used to separate proteins with molecular weights between 5 and 250 kDa. SDS acts as a surfactant, masking the proteins' natural charges and giving them nearly identical charge-to-mass ratios. Under a constant electric field, proteins migrate toward the anode at speeds determined by their mass, allowing accurate size-based separation.Western blot is a molecular technique used to detect and quantify specific proteins
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Mahdi Mutahar
Lead sponsor
University of Portsmouth
Sponsor institution
Loughborough University
Collaborator
Temple University
Collaborator
Imperial College London
Collaborator
