Explanation for the choice of comparators {6b}
The use of a drill, which is the conventional and most commonly used method, will be compared with the use of Papacarie™ with or without the Bixa orellana extract, and with or without the irradiation for aPDT. This study will show which of these treatments will be more efficient with regard to microbiological, radiographic, and clinical aspects. The models, types, and details of all instruments have been detailed upon the first appearance in the text.
Intervention description {11a}
Group 1 – Caries removal with a low-speed drill (control group):
1.
Initial periapical radiography (Children's Insight IP-01 Periapical – Carestream, Rochester, Nova York, EUA);
2.
Prophylaxis with a toothbrush and fluoride toothpaste (Colgate® Children Dr. Dentuço, Nova York, EUA) and fluoride toothpaste (Herjos Prophylactic Paste, Vigodent, Rio de Janeiro, Brazil);
3.
Relative isolation with cotton roll (Dental Roller Cotton – Cremer, Santa Catarina, Brazil) and aspirator (Disposable Dental Sucker – SSPlus, Paraná, Brazil);
4.
Microbiological sampling with ear curette (Meyhoefer auricular n° 2 curette (ABC Instrumentos Cirúrgicos, São Paulo, Brazil) for standardization of volume of carious tissue;
5.
Removal of carious dentin with carbide burs (Carbide Drill No. 6 Spherical CA – Angelus, Paraná, Brazil) and manual instruments (Double Dentin Digger n° 17/18 - Millennium – Golgran, São Paulo, Brazil);
6.
Second microbiological sampling with ear curette (Meyhoefer auricular n° 2 curette (ABC Instrumentos Cirúrgicos, São Paulo, Brazil) for standardization of volume of carious tissue;
7.
Clinical inspection of texture of the remaining dentin with an exploratory probe (Eighth Exploration Probe – Golgran, São Paulo, Brazil), to check the absence of soft tissue;
8.
Restoration with glass ionomer cement (Ketac Molar EasyMix – 3M ESPE); and
9.
Clinical inspection for signs of infiltration and radiographic follow-up immediately, 1 week, and 1, 3, 6, and 12 months after treatment.
Group 2 – Partial removal of carious tissue with the administration of Papacarie™:
1.
Initial periapical radiography;
2.
Relative isolation with a cotton roll and aspirator;
3.
Microbiological sample with otoscope curette to standardize the volume of carious tissue;
4.
Application of Papacarie™ (Fórmula e Ação, São Paulo, SP, Brazil) for 5 min, removal of carious tissue around lateral walls of the cavity with a noncutting curette and no removal of carious tissue on pulp floor (partial removal) [
10];
5.
Washing of the cavity with water and drying with absorbent paper;
6.
Second microbiological sampling;
7.
Clinical inspection of texture of the remaining dentin with an exploratory probe, to check the absence of soft tissue;
8.
Restoration with glass ionomer cement (Ketac Molar EasyMIx 3M ESPE); and
9.
Clinical inspection for signs of infiltration and radiographic follow-up immediately, 1 week, and 1, 3, 6, and 12 months after treatment.
Group 3 – Partial removal of carious tissue with the administration of Papacarie™ and application of
Bixa orellana extract (20% - seed extract diluted in mineral oil):
1.
Initial periapical radiography;
2.
Relative isolation with a cotton roll and aspirator;
3.
Microbiological sample with otoscope curette to standardize the volume of carious tissue;
4.
Application of Papacarie™ with
Bixa orellana extract (20%) (Fórmula e Ação, São Paulo, SP, Brazil) for 5 min, removal of carious tissue around lateral walls of the cavity with a noncutting curette and no removal of carious tissue on pulp floor (partial removal) [
10];
5.
Washing of the cavity with water and drying with absorbent paper;
6.
Second microbiological sample of remaining dentin with curette;
7.
Clinical inspection of texture of the remaining dentin with an exploratory probe, to check the absence of soft tissue;
8.
Restoration with glass ionomer cement (Ketac Molar EasyMIx 3M ESPE); and
9.
Clinical inspection for signs of infiltration and radiographic follow-up immediately, 1 week, and 1, 3, 6, and 12 months after treatment.
Group 4 – Partial removal of carious tissue with the administration of Papacarie™, application of
Bixa orellana extract (20%) and LED (aPDT):
1.
Initial periapical radiography;
2.
Relative isolation with a cotton roll and aspirator;
3.
Microbiological sample with otoscope curette to standardize the volume of carious tissue;
4.
Application of Papacarie™ with
Bixa orellana extract (20%) for 5 min and the light-emitting diode (LED) light curing device (Valo Cordless Ultradent®, South Jordan, UT, USA) an office appliance, with a coupled radiometer, and a spectrum of 440–480 nm will be used. Both the volunteer to be treated and the professional will be using specific eye protection glasses. The active end of the LED will be coated with clear disposable plastic (PVC), thus avoiding cross-contamination. Removal of carious tissue around lateral walls of the cavity with a noncutting curette and no removal of carious tissue on pulp floor (partial removal) [
10‐
12];
5.
Irradiation of dental tissue for 1 min on a single point;
6.
Washing of the cavity with water and drying with absorbent paper;
7.
Second microbiological sample of remaining dentin with curette;
8.
Clinical inspection of texture of the remaining dentin with an exploratory probe, to check the absence of soft tissue;
9.
Restoration with glass ionomer cement (Ketac Molar EasyMIx 3M ESPE); and
10.
Clinical inspection for signs of infiltration and radiographic follow-up immediately, 1 week, and 1, 3, 6, and 12 months after treatment.
Relevant concomitant care permitted or prohibited during the trial {11d}
Children involved in the study may present caries involving enamel, deficient restorations, small carious lesions on dentin with no access for manual scalers, hidden caries, sign or symptom of pulp involvement, clinical impossibility of restoration, evidence of pulp involvement, or carious lesion extending beyond 2/3 of dentin in other teeth, which are not involved in the study. In these cases, the researchers will provide the necessary care for these other conditions.
Provisions for post-trial care {30}
No major harms are expected, but possible intercorrences, such as tooth pain, will be monitored and recorded. Any additional assistance participants may need, will be provided.
Outcomes {12}
For the treatment of caries lesions to be considered successful, it should be able to remove infected tissue and microorganisms in a sufficient amount, so that, in the control session, the teeth that were once infected do not show any signs of remaining infected tissue beneath the restorative material. Consequently, the microbiological evaluation had been chosen as the primary outcome. However, factors such as the radiological exam and the clinical aspects are also important for the control section, to show the presence or lack of success. Therefore, the microbiological, radiographic, and clinical evaluations should be considered as primary outcomes.
Microbiological evaluation
This is one of the primary outcomes of the study. A sample of caries-affected dentin will be taken from each selected tooth before the removal of the carious tissue. The samples will be standardized with the use of a Meyhoefer auricular curette n° 2 (ABC Instrumentos Cirúrgicos, São Paulo, Brazil), because it has a hole in its tip, and placed into test tubes containing 3.8 ml of the transport medium (Phosphate Buffered Saline (PBS) - Thermo Fisher Scientific, Massachusetts, EUA). The dental tissue will be dispersed in the transport tube (Microtubo Tipo Eppendorf Cap. 1.5 ml – GlobalPastic, São Paulo, Brazil) containing glass pearls through agitation at maximum speed in a vortex device (Agitador Para Tubos Vortex – Kasvi, Paraná, Brazil) for 30 s to homogenize the biological material. The biofilm will be diluted in series on the order of 101 to 106 in peptone water and inoculated in culture media in Petri dishes (Placa de Petri Descartável Estéril - PRO-LAB MATERIAIS PARA LABORATORIOS LTDA, São Paulo, Brazil). Aliquots of dilutions 104, 105, and 106 will be sewn on the surface of Brucella agar (Difco Laboratories, Detroit, Michigan) containing defibrinated sheep blood (50ml/L), hemin (5 mg/ml), and menadione (10mg/ml) for the determination of the total number of viable microorganisms (VM). Aliquots of dilutions 103 and 104 will be sewn on Mitis Salivarius agar (Difco Laboratories, Detroit, Michigan) for the determination of the total number of streptococcus (S). Aliquots of dilutions 101 and 102 will be sewn on Mitis Salivarius agar with the addition of debacitracin for the determination of the population of streptococcus of the mutans group (SM).
Aliquots (100 ml) from each dilution will be sewn onto the surface of agar and spread with the aid of a Drigalski spatula. Undiluted aliquots and aliquots from dilution 10
2 (100 ml) will be pour-plated on Rogosa SL agar (Difco Laboratories, Detroit, Michigan) for the determination of lactobacilli (LB). The Brucella agar dishes will be incubated in an anaerobic chamber (PLAS by LABS, Lansing, MI) at 37°C for 7 days. The Mitis Salivarius agar and Mitis Salivarius Bacitracin dishes will be incubated in a 10% CO
2 atmosphere (CO
2 greenhouse, Shel Lab, mod. 2123, Oregon) at 37°C for 48 h. The dishes containing Rogosa agar will be incubated in a 10% CO
2 atmosphere at 37°C for 72 h. After incubation, the characteristic colonies in each dish will be counted with the aid of a stereomicroscope at a magnification of 10 times in dilutions with 30 to 300 colonies per dish. All procedures will be performed in duplicate, and the mean of the counts will be calculated. The results will be expressed in colony-forming units (CFU) of SM and LB as well as in proportion of streptococcus (% S/VM), SM group (% SM/VM and lactobacilli (% LB/VM) in relation to the total of viable microorganisms (VM). For SM, the proportion in relation to the total of streptococci (% SM/S) will also be calculated. Immediately after the removal of the carious tissue, samples of the remaining dentin will be taken with a Meyhoefer auricular n° 2 curette and the procedures will be repeated [
14].
Radiographic evaluation
This is one of the primary outcomes of the study. The radiographic examinations will be carried out in the following manner: (1) periapical radiographs will be used as a diagnostic aid; (2) after the incomplete removal of the demineralized dentine and the temporary filling of cavity, a bitewing radiograph it will be taken to allow the analysis of the radiolucent zone (RZ), that is, the amount of demineralized dentine left; (3) immediately, 1 week, and 1, 3, 6 and 12 months, periapical and bitewing radiographs will be taken to analyze the integrity of the periapical area and the eventual changes in the RZ.
To obtain geometric standardization of films, bitewing film holders will be used. The images obtained before and after the treatment will be digitized by a ScanJet 6100 scanner Hewllet-Packard (OR, USA). The images will be stored in maximum-quality JPG format. The Imagelab software (version 2.3, SoftiumSistemas de Informatica, São Paulo, Brazil) will be used to analyze the images (equalize and subtract). Change in the density measurement of the RZ during treatment will be evaluated blind through digital subtraction of radiographic images in relation to the initial exams in the experimental periods (immediately, 1 week, and 1, 3, 6, and 12 months).
The radiographic subtraction process will consist of comparing two images. By definition, a digital subtraction image from a site where no change in density has occurred would show a complete cancelation of all anatomical structures, eliminating the constant structures present in both. Any change in the structures indicates a change in density. This method requires standardized radiographs regarding the X-ray projection, exposure time, film, and development. Nonetheless, the procedures will be standardized; radiographs will be performed at different moments, which causes them to have different densities. To correct for any changes in density, the gray-level histograms of the two images will be compared and adjusted using a nonparametric contrast correction algorithm. The image radiograph equalization will be done in the following way: the radiograph taken immediately after the treatment and the one taken after each period of treatment (immediately, 1 week, and 1, 3, 6, and 12 months) will be placed beside one another on the computer screen. The radiograph that shows the best distribution of gray tonalities on the histogram will be chosen as a model, and the other image will be equalized according to the model.
The subtraction will be carried out the following way: the radiograph taken immediately after the treatment and the images obtained after each of the experimental periods will be overlapped. The anatomic details of the two images will be matched by rotation or moving the images horizontally or vertically. The radiolucent zone beneath the restoration and two control areas (CA1 and CA2) will be selected. The control areas will be dentinal areas (mesial and distal) close to the RZ. The difference in radiographic density between the two radiographs will be determined for each selected area using a gray tonalities scale. There are 256 individual shades of gray in an 8-bit pixel going from 0 (black) to 255 (white). This is the pixel’s grayscale resolution. An average gray level value of 128 (the middle of the digitizer gray level range set by the software) would show up at each pixel. Areas with gray levels <128 in the subtraction image would indicate a loss in density and gray levels >128 would indicate an increase in density [
15].
Evaluation of time required for the procedure (secondary outcome)
The time required for each procedure will be measured using a digital stopwatch (Kenko, Hong Kong) in minutes and seconds from the onset of treatment until the complete removal of the carious tissue. The time will be recorded on a specific chart for analysis. The need or non-need for anesthesia will also be recorded.
Evaluation of the need for local anesthesia during intervention and degree of pain/discomfort of children during the procedure (secondary outcome)
All interventions will be initiated without the prior administration of local anesthesia. The children will be told that anesthesia could be administered at any time during the intervention. A face scale with different expressions will be used to evaluate the need for local anesthesia and the child will be asked to point to the expression that most corresponds to his/her degree of pain/discomfort.
Interpretation of face scale:
Clinical evaluation
This is one of the primary outcomes of the study. The clinical evaluation will be performed by a researcher blinded to the different treatment groups. The criteria used for the evaluation will be the retention of the restorative material in the cavity and the occurrence of secondary caries. The evaluation scores will be based on the criteria below. Digital photographs of the restorations will also be taken and serve to complement the clinical and radiographic findings. The visual demonstration will contribute to any necessary clarifications and facilitate the discussion and documentation of the cases. Thus, digital photographs will be taken of all teeth in the different groups before and after the interventions. We believe participants will be present for the follow-ups due to concerns and other possible treatments in the clinic.
-
0 = present; no defects;
-
1 = present; small marginal defects measuring less than 0.5 mm in depth; no need for repair;
-
2 = present; small marginal defects measuring 0.5 mm to 1mm in depth; need for repair;
-
3 = present; large marginal defects measuring 1 or more mm in depth; need for repair;
-
4 = absent; restoration nearly or completely lost; need for treatment;
-
5 = absent; additional treatment having been performed for some reason;
-
6 = tooth absent for any reason;
-
7 = present; surface wear measuring less than 0.5 mm in depth; no need for replacement;
-
8 = present; surface wear greater than 0.5 mm in depth; need for replacement; and
-
9 = impossible to diagnose.