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.
Eligibility criteria.
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.
The parallel groups according to different hygiene protocols are:
G1 – brushing the palate with a soft brush and prosthesis immersion in 0.25% sodium hypochlorite solution (control group);
G2 - brushing the palate with a soft brush and prosthesis immersion in 0.15% triclosan solution;
G3- brushing the palate with a soft brush and prosthesis immersion in citric acid (Nitradine, Bonyf AG, Liechtenstein, Swiss); and
G4- prosthesis immersion and brushing of the palate with citric acid and soft brush (Nitradine, Bonyf AG, Liechtenstein, Swiss).
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.
Study outcomes
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].
Secondary
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 Fourier Transform (FFT), after Hanning windowing. The RR range spectra will be integrated into low (LF: 0.04 to 0.15 Hz) and high frequency (HF: 0.15 to 0.50 Hz) bands. The relative power (normalized units) of the RR interval spectra, in each frequency band, as well as the ratio of the LF and HF (LF / HF) powers thereof, will be determined.
Symbolic Analysis searches for patterns of changes between successive cardiac interval value, classify these changes and quantifies their occurrence. Sequences of 3 symbols will then be analyzed and classified into four different families, according to the number of variations found. The frequency of occurrence of each pattern will be analyzed, and indicated, as 0V%, 1V%, and, 2V%. The frequency of variations of type 0V (sympathetic) and 2V (vagal) are of interest as indicators of cardiocirculatory autonomic modulation.
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 𝑚 will continue to 𝑚 + 1. In other words, of the size patterns 𝑚 that are similar, SampEn indicates which percentage of these will remain similar for 𝑚 + 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 Staphylococcus spp. (Mannitol Salt Agar, Kasvi Imp. e Dist. de Prod. para Laboratórios Ltda, Curitiba, Brasil), Gram-negative bacterias (MacConkey Agar, Himedia Laboratórios PVI Ltd, Mumbai, Índia), Candida spp. (CHROMagar™ Candida, Becton Dickinson, Paris, França) and Streptococcus mutans (SB20 Modified Agar with Casitone, Himedia Laboratories PVI Ltd, Mumbai, Índia), and incubated in a microbiological stove (De Leo Equipamentos Laboratoriais, Porto Alegre, RS, Brasil) at 37 ° C for 48 hours. The cultivation of S. mutans will be in an environment of microaerophilic in jar of anaerobiosis (Permution, Curitiba, PR, Brasil). After the incubation period, the CFU count will be performed to quantify the microbial load.
The Breath Alert ™ portable device (Tanita Corporation®-Japan), used according to the manufacturer's instructions, will measure the odor of the cavity with and without the denture. The odor level will be given using by score, presented in the apparatus, with values that can vary from 1 to 4. Thus, the classification of the odor is classified according to the scores [30] 1) Odorless, normal; 2) Mild, normal odor; 3) Moderate, bad breath - perceptible; 4) Strong odor, noticeable. The evaluation will be performed with the 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 kits (Enzyme Linked Immunosorbent Assay – ELISA) Multiplex Human Cytokine Magnetic Bead Kit (Millipore, United States), according to the manufacturer's instructions [32]. The determination of C-reactive protein in saliva will be performed using CRP ELISA kits (Salimetrics Europe Ltda.) [33]. Both ELISA methodologies will be performed in duplicates on two standard 96-well microplates, according to the protocol provided by the respective suppliers.
Characterization of the sample
Socio-demographic characteristics of the participants of the study will be collected during the first consultation through medical and dental history reports. Information will be collected such as edentulism time, age of prostheses in use, drug profile, hygiene habits (use of oral antiseptics or chemical hygiene of prostheses, frequency of hygiene of the prostheses), continuous nocturnal use, and smoking.
The Quality of Life associated with Oral Health will be evaluated by applying the Oral Health Impact Profile questionnaire, specific for edentated patients (OHIP-EDENT), validated for the Brazilian population [34]. The questionnaire presents 19 questions for four domains: "complaints related to chewing", "psychological discomfort and incapacity", "social incapacity", and "pain and mouth discomfort". Participants will be asked to answer the questions according to their feelings according to one of the following: 'never', 'sometimes', or 'almost always'.
Patient satisfaction will be assessed by the frequency of specific symptoms, such as local pain, burning sensation, bad breath, and buccal dryness. The responses will be collected based on a 100 mm visual analog scale (VAS), which will provide parameters for assessing heterogeneity of the eligible sample in the Baseline. They will also be asked to give an open-label response to other sensory side effects [10, 29].
During clinical examination, the conditions of the prostheses in use, such as stability and retention according to Anastassiadou et al. [28], as well as biofilm deposits and visible debris, will be observed [11]. Data on the shape and resilience of the edentulous maxillary ridge will also be collected. The data on the quality of life - related to oral health, quality of the prostheses and general satisfaction will be collected for the characterization of the sample in the baseline [29].
Sample size estimation
Sample size for the quantitative outcomes was determined based on the primary outcome of this study (Denture-related stomatitis remission). According to previous trial [11], considered the standard deviations 2.19 (1-saline group) and 1.79 (sodium hypochlorite group 2), a 95% confidence interval (bilateral), and a detectable difference of at least 2 logs. Based on a power of 80%, this study clinical requires at least 21 participants. An additional 20% will be added to the planned sample to compensate for possible dropouts, thus resulting in a total of 25 participants.
Statistical analysis
The data collected for the groups, prior to adherence to the hygiene protocols, such as age, gender, and grade of schooling, will be compared to ascertain the initial similarity. The effect of groups on primary and secondary outcomes will be assessed. When applicable (ex. OHIP-EDENT), pre-treatment values will be applied as a co-variable in the statistical model. The significance level of the tests will be 0.05. Noncompliant participants will be followed up, such as those requiring interruption of one of the hygiene protocols, and to evaluate the significance of protocol deviations. In other words, the statistical analysis will consider the participants according to the treatment received (per protocol), as well as according to the planned treatment (intention-to-treat - ITT); and the results will be confronted.
The data will be analyzed for normal distribution and homogeneity; once met, parametric tests will be performed. In the analysis of the quantitative data, it will be performed repeated measures ANOVA. For the categorical variables (questionnaire) it will be applied the Friedman test to compare the different times, and the Kruskal-Wallis test to compare the groups. A correlation test will be performed between the quality of life indices and the quantitative variables (Pearson correlation coefficient). If the quantitative data do not present normal distribution or homoscedasticity, the non-parametric Friedman and 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.