PMPR and Chlorhexidine on Periodontal Disease and Vascular Function

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.

• 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).

• 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).

• 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.

• 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

• Purpose: To evaluate the oral microbiota's nitrate-reducing capacity.
• To evaluate bacterial role in nitric oxide pathways.
• Method: Nitrate rinse; incubation; centrifugation; nitrite quantification.

To assess nitrate-reducing activity in WMS samples, a stock solution will be prepared by dissolving 1011 mg potassium nitrate in 1 L of ultrapure water. Aliquots of 10 mL will be stored in 15 mL Falcon tubes at -20°C until use.

• For the procedure, one aliquot will be thawed and used as a rinse solution. Participants will rinse their mouths with 10 mL of the solution for 5 minutes under timed supervision. The expectorated rinse will be collected in a 50 mL Falcon tube and transferred into microcentrifuge tubes.
• Samples will be centrifuged at 10,000 rpm for 10 minutes. The supernatant will be collected, transferred into a new tube, and stored at -20°C for later analysis of nitrite concentration.

• Brachial artery FMD will be used to assess endothelial function following a 5-minute ischemic stimulus induced by forearm cuff inflation. Measurements will be performed in the supine position on the right arm, with the cuff placed distal to the olecranon process.
• A 12-MHz linear array ultrasound probe will be used to image the brachial artery while simultaneously recording B-mode images and Doppler blood velocity traces. Depth, focus, and gain settings will remain consistent, and the transducer location will be documented for reproducibility.
• After a 60-second baseline, the cuff will be inflated to 220 mmHg for 5 minutes. Ultrasound recordings will continue during inflation and for 3 minutes post-deflation. All scans will be performed by the same researcher for each participant.
• Brachial artery diameter, blood flow, and shear rate will be analysed using automated edge-detection and wall-tracking software to minimise investigator bias.

• Iontophoresis will be used to assess microvascular endothelial function through transdermal drug delivery with a low-intensity electric current. Participants will acclimatize for 30 minutes in a room maintained at 23°C before receiving acetylcholine (ACh, 1%) and sodium nitroprusside (SNP, 0.01%) on the volar forearm.
• The skin will be cleaned with water for injection, and two perspex rings will be placed as anode and cathode, connected to the iontophoresis controller. Each chamber will contain 0.5 mL of drug solution. The stimulation protocol will include four pulses at 25 μA, followed by single pulses at 50, 100, 150, and 200 μA, each lasting 20 seconds with 120-second intervals.
• Skin blood flow will be measured using Laser Doppler probes connected to a perfusion monitor, with data recorded via PowerLab and LabChart software. Cutaneous vascular conductance (CVC) will be calculated as skin flux/MAP.

• Cardiac output (CO) will be measured noninvasively using the Physio Flow PF-05 Lab1 device with thoracic electrode placement. Two electrodes placed at the sternal manubrium and lower thorax will monitor EKG for heart rate, while four electrodes at the neck base and xiphoid process will measure impedance signals.
• Skin will be prepared by shaving and cleaning to optimise signal quality. For participants with pacemakers, neck electrodes will be positioned opposite the device. Calibration will be performed by acquiring stable signals over 30 heartbeats with simultaneous blood pressure measurement.
• The system will provide heart rate, stroke volume, CO, and other hemodynamic parameters, with data reviewed and stored for analysis.

• Pulse Wave Analysis (PWA) will be performed to assess central aortic hemodynamic parameters. After a 5-minute rest, brachial pressure waveforms will be recorded and used to derive aortic waveforms via a transfer function. Output will include pulse pressure (PP), augmentation pressure (AP), and augmentation index (AI75). Results will be displayed graphically and compared with population norms using the SphygmoCor XCEL system.

• Pulse Wave Velocity (PWV) will be measured as an index of arterial stiffness. Participants will rest in the supine position for 5 minutes before measurement. A femoral cuff will be placed on the thigh, and carotid pressure will be recorded with a tonometer to calculate transit time and derive PWV in m/s. Results will be compared with population data.

• Ozone-based chemiluminescence, a biochemical test, will be used to measure nitric oxide (NO) metabolites, primarily nitrite (NO₂-), in saliva samples. In this method, NO reacts with ozone to produce an excited form of nitrogen dioxide, which is detected as a chemiluminescent signal.
• Saliva samples will be analysed for nitrite concentrations using a Sievers nitric oxide analyser (Sievers NOA 280i).

• Ozone-based chemiluminescence, a biochemical test, will be used to measure nitric oxide (NO) metabolites, such as nitrate (NO₃-), in saliva samples. In this method, NO reacts with ozone to produce an excited form of nitrogen dioxide, which is detected as a chemiluminescent signal.
• Saliva samples will be analysed for nitrate concentrations using a Sievers nitric oxide analyser (Sievers NOA 280i).

• Plasma nitrite (NO₂-) levels will be quantified using ozone-based chemiluminescence. In this method, nitric oxide reacts with ozone to generate excited nitrogen dioxide, producing a measurable chemiluminescent signal. Plasma samples will be centrifuged and stored at -80 °C until analysis with a Sievers Nitric Oxide Analyser (NOA 280i)

• Plasma nitrate (NO₃-) levels will be quantified using ozone-based chemiluminescence. In this method, nitric oxide reacts with ozone to generate excited nitrogen dioxide, producing a measurable chemiluminescent signal. Plasma samples will be centrifuged and stored at -80 °C until analysis with a Sievers Nitric Oxide Analyser (NOA 280i)

• Blood pressure in the brachial artery will be measured after 30 minutes of seated rest in a quiet room, using an automated sphygmomanometer. Five consecutive readings will be taken, with a 1-minute rest between each measurement.
• The average of three readings will be recorded, including the mean values for SBP, DBP, and MAP.

• Enzyme-Linked Immunosorbent Assay (ELISA) Duoset kits will be used to quantify blood biomarkers, including IL-6, IL-10, and TNFα, and to assess systemic inflammation and its association with periodontal disease and vascular function. The kits use a sandwich ELISA approach, with a capture antibody pre-coated onto a microplate, followed by sample application.
• This method allows precise measurement of cytokines and acute-phase proteins from participants' blood samples. High specificity and sensitivity enable detection of both pro-inflammatory (IL-6, TNFα) and anti-inflammatory (IL-10) markers. Results will provide insights into the inflammatory response and its modulation before and after PMPR treatment.

To evaluate and assess the efficacy of Periodontal Mechanical Plaque Removal (PMPR) combined with 0.2% CHX mouthwash on Bleeding on Probing (BoP)

• BOP will be recorded at six sites per tooth as: 0 (minimum) = no bleeding, 1 (maximum) = bleeding on probing.
• Scores will be expressed as the percentage of bleeding sites per participant, with a minimum to maximum range (0-100%).
• Lower scores indicate a better periodontal outcome; a reduction reflects improvement following PMPR ± CHX mouthwash.
• This outcome will assess the short-term clinical efficacy of CHX mouthwash adjunctive to PMPR in individuals with periodontal disease.

To evaluate and assess the efficacy of Periodontal Mechanical Plaque Removal (PMPR) combined with 0.2% CHX mouthwash on Probing Pocket Depth (PPD)

• PPD will be measured in millimetres at six sites per tooth and categorised as: 0 (minimum) = ≤3 mm, 1 = 4-5 mm, 2 (maximum)= ≥6 mm. Lower scores indicate a better periodontal outcome. - Efficacy will be assessed as mean change in PPD (mm) and as percentage of pocket closure, defined as sites with baseline PPD ≥4 mm reducing to ≤3 mm at follow-up.
• This outcome will assess the short-term clinical efficacy of CHX mouthwash adjunctive to PMPR in individuals with periodontal disease.

To evaluate and assess the efficacy of Periodontal Mechanical Plaque Removal (PMPR) combined with 0.2% CHX mouthwash on the Clinical Attachment Level (CAL)

• Clinical Attachment Level (CAL) will be calculated by combining probing pocket depth and gingival recession measurements.
• CAL will be measured in millimetres at six sites per tooth and categorised as: 0 (minimum) = 0-2 mm, 1 = 3-4 mm, 2 (maximum) = ≥5 mm.
• The mean change in CAL (mm) will then be compared between the two groups (PMPR + CHX vs. PMPR + placebo).
• Lower scores indicate a better periodontal outcome; a reduction reflects attachment gain following use of PMPR ± CHX mouthwash.
• This index will be used to assess the short-term clinical efficacy of CHX mouthwash, adjunctive to PMPR, in individuals with periodontal disease.

To evaluate and assess the efficacy of Periodontal Mechanical Plaque Removal (PMPR) combined with 0.2% CHX mouthwash on Plaque index (PI).

• A score of 0,1,2,3,4,5 will be used to record the PI on the buccal and lingual surfaces of the teeth (except third molars) using the Turesky-modified scoring criteria.
• Scoring system:
• 0 (minimum)= No plaque; 1 = flecks at cervical margin; 2 = thin band ≤1 mm; 3 = band >1 mm covering <⅓ surface; 4 = plaque covering ≥⅓-<⅔; 5 (maximum)= plaque covering ≥⅔ of the surface.
• Scores will be averaged across all surfaces to obtain a mean PI score from minimum to maximum, with a range of 0-5.
• Lower scores indicate better oral hygiene; a reduction reflects improvement following PMPR ± CHX mouthwash.
• This index will be used to assess the short-term clinical efficacy of CHX mouthwash, adjunctive to PMPR, in individuals with periodontal disease.

To evaluate and assess the efficacy of Periodontal Mechanical Plaque Removal (PMPR) combined with 0.2% CHX mouthwash on Löe & Silness Gingival Index (GI).

• GI will be assessed at four sites per tooth (mesial, distal, buccal, lingual) and scored (on a minimum 0 to a maximum 3) with: 0 = normal, 1 = mild inflammation, 2 = moderate inflammation, 3 = severe inflammation. Scores will be averaged across all sites to give a mean GI score (range 0-3). Lower scores indicate better gingival health; a reduction reflects improvement following PMPR ± 0.2% CHX mouthwash.
• Gingivitis severity classification:
• Mild gingivitis: 0.1-1.0; Moderate gingivitis: 1.1-2.0; Severe: 2.1-3.0
• This index will be used to assess the short-term clinical efficacy of CHX mouthwash on GI, adjunctive to PMPR, in individuals with periodontal disease.