Hygiene protocols for the treatment of denture-related stomatitis: local and systemic parameters analysis

Background The Denture-related stomatitis (DS) is a chronic multifactorial inflammation, strongly related to the presence of the biofilm. This oral condition has recently been correlated with deleterious cardiovascular alterations. The potential effect of hygiene protocols in the control of DS and Randomized clinical trials that address this oral condition with cardiovascular complications are necessary for clinical decision-making. Material/design A clinical trial, randomized, double-blind, of parallel groups, will be conducted in Brazil and the sample will be composed of 100 total edentulous individuals bi-maxillary, users of at least maxillary complete dentures, diagnosed with DS that will be allocated according to the different hygiene protocols: (1) brushing of the palate and immersion of the prosthesis in 0.25% sodium hypochlorite solution (positive control); (2) brushing of the palate and immersion of the prosthesis in 0.15% Triclosan solution; (3) brushing of the palate and immersion of the prostheses in lactose monohydrate; (4) brushing the palate with citric acid and immersing the prosthesis in lactose monohydrate. The response variables will be: heart rate variability and alteration of blood pressure (systemic level), remission of Denture-related Stomatitis, removal of biofilm, reduction of microbial load (CFU), mouth and prosthesis odor level, expression of MUC1, proinflammatory cytokines, C-reactive protein level - CRP, viscosity, pH and salivary flow (locally). Measurements will be performed in the Baseline and ten days after the interventions. The results obtained will be submitted to the pertinent statistical analysis with a level of significance of 0.05. Discussion This study will provide a guideline of clinical practice regarding the use of hygiene protocols in the treatment of oral diseases mediated by biofilm. Also, it may provide evidence of correlation of oral manifestation with cardiac risk.

vagal and sympathetic branches that reach the spleen, reflecting cholinergic and then noradrenergic signals, triggering splenic T-cells via adrenergic receptors. These memory T cells will secrete the vagal neurotransmitter -acetylcholine -responsible for the innate immune response, which binds to the alpha-7 nicotinic acetylcholine receptor (nAChR) in monocytes, resulting in the inhibition of the synthesis of inflammatory cytokines [26].
Scientific evidence of the correlation among the biofilm, DS, cardiovascular disease, and adequate denture-related stomatitis treatment, may contribute to the establishment of a protocol for the prevention and treatment of local inflammation; to be applied to primary care, which can significantly impact the costs of public health and quality of life of patients. To our knowledge, this is the first proposal of a clinical, controlled, randomized, and double-blind study that proposes to correlate the treatment of DS, through hygiene and brushing solutions, with analysis of local and systemic inflammatory responses and cardiovascular impairment.

Study hypothesis
The primary null hypothesis of the trial is that there is no difference between the protocols for the prevention and treatment of local and systemic inflammatory responses and cardiovascular risk.

Study setting
A randomized, controlled, double-blind, clinical trial with parallel groups named according to each hygiene protocol will be performed ( fig. 1). Figure 2 shows the study timeline, according to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) diagram. Additional file 1 presents the SPIRIT checklist.
The sample will be of convenience, composed of patients who will attend the discipline of Complete Denture, at the School of Dentistry of Ribeirão Preto -University of São Paulo (FORP / USP). A total of one hundred edentulous individuals will be invited, with at least the use of complete denture upper, diagnosed with denture-related stomatitis and biofilm in the total dentures.

Inclusion criteria
(1) Patients may be of both sexes; (2) must present good general health status; (3) totally edentulous, users of upper and lower conventional complete prostheses, or, necessarily, users of complete upper prosthesis (although edentulous mandibular) in good condition [28]; (4) prosthesis made from thermally polymerized acrylic resin and acrylic teeth; (5) presenting DS between types IB, II or III, according to the Newton Modified Classification [10]; and (6) their prostheses should present biofilm with a score equal to, or greater than 1, according to Additive Index Exclusion criteria.
Will be excluded: those patients who (1) present their prostheses with adaptation problems, with failures, repairs, or fractures; (2) allergy to any of the products studied; (3) Severe/serious illness that requires frequent hospitalization; (4) systemic conditions favorable to the development of Candida spp.; (5) use of antibiotics, anti-inflammatories, or antifungal agents in the last four weeks prior to the study; (6) other lesions on the oral mucosa; (7) practice of palatal mucous brushing already present during recruitment; (8) and replacement of prostheses in use during the experimental period.

Planned Interventions
All patients will receive verbal and written instructions according to the hygiene protocols and they should do to brushing with soft toothbrush and water during 2 minutes the palate region, once a day (Toothbrush CS 5460C Adulto Ultra Macia, Curaprox, Curaden Swiss do Brasil Imp. Exp. LTDA, São Caetano do Sul, São Paulo, Brasil). To immerse their dentures in the specific product, once a day, according to the time proposed by the manufacturer, as well as to perform the brushing of the prosthesis for 2 minutes with a specific brush (Prosthesis Brush BDC150/152/153, Curaprox, Curaden Swiss do Brasil Imp. Exp. LTDA, São Caetano do Sul, São Paulo, Brasil) and neutral soap, 3 times a day. In addition, all patients will be instructed to remove the prosthesis during the night period and leave them in a container with clean water; and at morning, rinsing the prosthesis under running water, before inserting them into the oral cavity. Neutral soap and the solutions will be available to participants in identical dosing vials and sufficient quantity for continuous use for ten days; for greater control and monitoring of the hygiene protocol. Citric acid in the form of an effervescent tablet will be removed from the package and placed in neutral packaging. If the patient has a complaint about the products under investigation or adverse events, theys may interrupt the treatment at any time, and notifying us of what has happened. To verify if the patient is performing the protocol, there will be a consultation five days after the beginning of the treatment, for clarification of doubts and follow-up. The patients will be instructed to avoid the consumption of beverages or foods that alter the metabolism, such as coffee, soda, alcohol, chocolate, as well as physical exercises, 24 hours before the tests.

Randomization, allocation, and blinding
The study will be double-blind, and for this, each researcher (R) will have an assignment during the experiment: Participants will be distributed into groups taking into account a random numerical sequence (on a 1:1:1:1 ratio), generated by a computer; the numbers of identification of the patients will be placed into an envelope by a blinded researcher, not involved in the clinical steps, which also will perform step the preparation of the products (R1). Another researcher will be responsible for opening the envelope at the moment of the delivery of the appropriate product to the patients, according to the hygiene protocols, and will perform the examination of patients and the collection of samples (R2). The R3 will perform the distribution of the protocols and the orientation of the participant, and finally R4: perform statistical analysis of data (blind). The researchers involved in the clinical steps (R2 and R3) and patients will not be blinded because of the nature of the intervention. Patients' allocated interventions will not be revealed until the statistical analysis is completed.

Primary: Denture-related stomatitis remission (DS)
To evaluate the effect of the hygiene protocols on DS remission, the participants will be examined under the Baseline condition and also ten days after starting their specific of the use of the protocol. To quantify the inflammation, standardized photographs of the palate will be obtained (Digital Camera, Canon EOS, Canon EF 100 mm / 2: 8 Macro Lens and Canon ML3 Circular Flash), with the focus centered on the median raphe region. The images will be transferred to the computer and two blinded, previously trained researcher, will assign scores according to the classification of Kabawat et al. [10].

Systemic evaluation
The indirect measurement of the patients' blood pressure will be performed by the oscillatory sphygmomanometer method using an automated device (HEM7130, Omron Healthcare Brasil, São Paulo, SP); at which, 2 to 3 measurements with a 5-minute interval will be performed, recording the systolic (maximum) and diastolic (minimum) blood pressure values. The technique for obtaining and classifying the individuals will follow the categorization proposed by the American Heart Association, (2019).
Patients will be referred for continuous electrocardiographic monitoring with the Einthoven's II lead, combined with the monitoring of the respiratory rate with an elastic strap holding a stretch sensor around the thorax. The electrocardiogram and the respiratory sensor signal shall be filtered (100 Hz to 0.5 kHz), amplified (BioAmp ADInstruments, Bella Vista Australia), digitalized (PowerLab 2/20 ADInstruments Bella Vista Australia) and sampled (1000 Hz), continuously, using an IBM / PC. The files with the electrocardiogram recordings will be processed using a computer program (ECG Module for LabChart, ADInstruments, Bella Vista, Australia), which identifies the QRS complex of the electrocardiogram and calculates the duration of successive intervals between R waves (RR interval or cardiac interval). This processing will allow the generation of time series, beat-to-beat, from the cardiac interval values.
From these recordings, it will be performed the analysis of the cardiac interval variability (spectral analysis). The series with RR interval values will be re-sampled at 3 Hz by cubic interpolation, to regularize the interval between beats. The series with interpolated RR interval values will be divided into segments with 512 values each, with a 50% overlap.
The stationarity of each segment will be examined visually, and those with artifacts, or transients, will be excluded. Each segment will have its spectrum calculated by the Fast The Sampling Entropy (SampEn) will be calculated from the IC series with the help of the JBioS software. The n practical terms, SampEn quantifies the (logarithmic) probability that near-size patterns will continue to + 1. In other words, of the size patterns that are similar, SampEn indicates which percentage of these will remain similar for + 1, that is, when an extra point is considered. High probability of the patterns continuing close indicates regularity, yielding low values of entropy. Cardiac variability and blood pressure will be recorded in the control period and after the treatment of DS through hygiene protocols. Thus, each will be the control of himself.

Local parameters
To verify the biofilm removal of the upper prosthesis, the technique described will be performed in according to Badaró et al. [11], that from the biofilm evidence, the prostheses will be photographed in standardized positions. The areas of biofilm and the surface of the prosthesis will be calculated using software and will be applied in a formula to identify the amount of total area of the biofilm, before and after treatment.
The microbial load of the prostheses and the palate will be evaluated. The biofilm collection of these sites will be followed according to the protocol recommended by Kabawat et al.
[10] and de Souza et al. [29]. Serial dilutions will then be obtained which will be seeded in Petri dishes, with culture medium specific for the growth of participant without the prostheses in position, and then with the prostheses seated in the oral cavity. Thus the odor related to the prosthesis will be calculated based on the difference between the odor of the cavity with the prosthesis and odor of the cavity without the prosthesis.
Saliva samples will be collected to evaluate salivary parameters. The non-stimulated total saliva will be collected for 10 min by the method of spitting, which will be subjected to viscosity analysis and pH measurement. The calibration of pH will be performed in a pHmetro (PHTEK, Curitiba, Paraná, Brazil) after calibration of the equipment. The kinematic viscosity analysis of saliva will be measured using glass viscometer, and the liquid viscosity coefficient will be calculated according to Shekhar et al. [31]. The total stimulated saliva will be collected for 5 min using the habitual chewing of 1g of gum base [19], from which the calculation of saliva volume will be obtained to evaluate the salivary flow. Subsequently, saliva samples will be centrifuged at 10,000 xg, for 15 min, at 4°C, to remove cellular debris. Aliquots of supernatant will be stored at -80°C for the analyses.
The precipitates will be evaluated by ELISA [22] in identification and quantification of MUC1 expression. The absorbance at 405 nm will be measured after 30-45 min in an ELISA reader.
As a control, wells without saliva will be used. The assay for saliva will be performed in triplicate, and the results will be presented as the mean difference between optical density (OD) readings in experimental and control wells. Salivary concentrations of cytokines (IL-6 and TNF-α) will be measured using enzyme-linked immunosorbent assay Kruskal-Wallis tests will be applied for comparison between times and groups, respectively.
A flowchart of the participants will be prepared for a detailed explanation of the characteristics of the sample and the quantification of quitters and missing participants.
This part will provide the number of individuals examined, and reasons for exclusion, as well as the recruited, treatment-allocated participants who complete the trial and analyze it at the end. The flowchart will provide the reasons for any deviation from the protocol.

Data management, monitoring, and auditing
A data monitoring committee composed of an independent researcher will check collected data regularly. This researcher shall have no relationship with the trial sponsors.
Moreover, the Institutional Board at Sao Paulo University may conduct an independent audit at any time.

Ethical Considerations and Dissemination
This study protocol was approved by the Research Ethics Committee of the School of Dentistry of Ribeirão Preto (CAAE 93712418.1.0000.5419), and Registered on November 9, 2018, on the ReBec platform RBR-4hhwjb, and will be reported in compliance with the CONSORT statement.
Detailed information will be given to each potential participant of eligibility, before the initial examinations, while we will obtain free and informed consent as a prerequisite for recruitment. The informed consent will be obtained from all study participants. As a consent clause, we will grant the individuals the right to withdraw from the study at any time. All documents relating to the participants, such as terms of consent and clinical data, will be kept in a locked cabinet to guarantee their confidentiality.
Electronic data handled by the researchers will contain numerical codes in place of the names. Any changes to the protocol will be conducted after the opinion of the Research Ethics Committee and development agencies. Also, the authors will disclose the results of this proposal, regardless of the findings. The results of the RCT will be disseminated in a peer-reviewed journal.

Discussion
This RCT will provide a guideline of clinical practice regarding the use of hygiene protocols in the treatment of oral diseases mediated by biofilm (DS). Also, it may provide evidence of correlation of oral manifestation with cardiac risk.
The DS has been recently associated with systemic implications (variations in blood pressure and endothelial dysfunction) that precede the development of serious cardiovascular disorders, such as atherosclerosis and hypertension, which are changes in general health with high mortality / morbidity rates [16][17][18]. However, the mechanisms that correlate oral inflammation and cardiovascular effects are not yet fully described, but one of the most important hypotheses is the pre-activation of the immune system [17].
The information from this study will also improve clinical decision-making and potentially protect edentate patients from harm caused by ineffective treatment, as well as, the incorporation of hygiene protocols for oral tissues and prostheses can achieve favorable results associated with low costs and minimal adverse effects, besides possibly avoid the involvement of opportunistic diseases, which may lead to an decreased risk of cardiac diseases. The resulting will be published information will provide evidence for the development of clinical recommendations for DS, which will not only be used for publication in indexed journals but also for public health information services.
Thus, we predict a favorable impact on public health, while the results will provide the basis for future investigations of the characterization of possible changes in HRV, correlated or not, with changes in blood pressure, salivary pro-inflammatory markers and salivary parameters with DS. Given the vast prevalence of denture stomatitis in this elderly population, such knowledge may be crucial for effective control as well as for the detection of cardiovascular disease risk.

Trial status
First version (01)