Formulation composition
Our study used EGCG derived from green tea and available on 5 µm solids provided in the solid-state (E41430; Sigma–Aldrich; St Louis, MO, USA). We prepared the EGCG-based paste for endodontic use as an intracanal dressing as previously reported [16] at a 1 mg/mL concentration using polyethylene glycol 400 (PEG 400, Galena Química e Farmacêutica Ltda., Campinas, SP, Brazil) as a vehicle and zinc oxide (2 g) (SS White Artigos Dentários Ltda., Rio de Janeiro, RJ, Brazil) as a radio-opacity agent. We used calcium hydroxide paste (Calen®, S.S. White Dental Articles Ltda.) as a control because it is currently the most common medication used during endodontic treatment. Calen paste is comprised of 2.5 g calcium hydroxide and 0.05 g colophony, with 0.5 g zinc oxide as a radio-opacity agent and 2 mL polyethylene glycol 400 (PEG 400) as a vehicle.
Animals
All animal procedures in this study were performed while conforming to protocols reviewed and approved by the Animal Care Committee of the University of São Paulo (Protocol #11.1.1405.53.8). The studies also followed standards recommended by the International Organization for Standardization (ISO) no 7405/2008 (dogs), with the exception of the recommended experimental periods (28 and 90 days), aiming to restrict the number of animals to the minimum necessary to obtain conclusive results, in accordance with the current ARRIVE (Animal Research: Reporting of In Vivo Experiments).
Laboratory Animal Care Facility at the Medical School of Ribeirão Preto, Brazil, gave in three beagle dogs of both genders at 12 months of age and 33 pounds. For the experiment, we selected the 2nd and 3rd upper premolars and the 2nd, 3rd and 4th lower premolars, totaling 35 teeth (70 roots). In cases of gingivitis and/or gingival calculus, the animals received prophylaxis, scraping, straightening and dental polishing, followed by the application of 0.12% chlorhexidine digluconate (Periogard - Colgate - Palmolive - Indústria Ltda. - Brazil).
Operative procedures
The same operator performed all procedures. He preanesthetized and subsequently anesthetized the animals intravenously with zolazepam. For induction of periapical lesions after crown access, the operator removed the pulp tissue and left the root canals exposed to the oral cavity for 7 days to allow microbial contamination [22]. Afterward, he sealed the cavities with zinc oxide-eugenol cement (IRM®, Dentsply Industria e Comércio LTDA - Petrópolis - Brazil). Before the operative procedures, the operator took standardized periapical radiographs until the development of periapical radiolucencies, which occurred after 45 days.
After this period, the teeth were isolated with a rubber dam and disinfection of the operative field was made with 2% chlorhexidine gluconate. The working length was determined to be 1 mm short of the radiographic apex and confirmed by xRay periapical radiography. Apical delta was perforated by using #20 to #25 K-files at the length of the tooth, thus creating a standardized apical opening. The root canals were instrumented by ProTaper Universal rotary system (DentsplyMaillefer, Ballaigues, Switzerland) in the sequence recommended by the manufacturer (S1 0.18/.02, S2 0.20/.04, F1 0.20/.07, F2 0.25/.08, F3 0.30/.09, F4 0.40/.06 and F5 0.50/.05) using the XSmart™ endodontic micromotor (Dentsply Maillefer Instruments; Ballaigues, Switzerland) under irrigation with 3.6 mL 2.5% NaOCl at each file change. The ethylene-diamine-tetra-acetic acid (EDTA) solution was used as a penultimate wash for 3 minutes under agitation with a k file followed by final rinse with NaOCl solution. Five groups were formed according to the following experimental conditions (Table 1):
Table 1
Groups evaluated and the distribution of the number of teeth and roots.
Clinical conditions and treatments | Number of teeth (roots) |
Healthy tooth | 5 teeth (10 roots) |
Tooth with untreated periapical lesion | 5 teeth (10 roots) |
Tooth treated endodontically in a single session | 5 teeth (10 roots) |
Tooth treated endodontically in two sessions: paste based on EGCG | 10 teeth (20 roots) |
Tooth treated endodontically in two sessions: paste based on calcium hydroxide | 10 teeth (20 roots) |
Healthy group (healthy and nontreated teeth): MMP-2 and MMP-9 expression in healthy tissues was characterized.
Untreated group (teeth with untreated periapical lesions): MMP-2 and MMP-9 expression in periapical lesions experimentally induced and nontreated endodontically was characterized.
One session group (teeth with induced periapical lesions submitted to endodontic treatment performed in a single session): the root canal filling was finished in the same session after chemomechanical preparation.
EGCG group (teeth with induced periapical lesions submitted to endodontic treatment performed in two sessions with EGCG-based intracanal dressing): root canal filling was performed 14 days after the application of endodontic medication.
Calcium hydroxide group (teeth with induced periapical lesions submitted to endodontic treatment performed in two sessions with calcium hydroxide-based intracanal dressing): The root canal filling was applied 14 days after the application of endodontic medication.
In the EGCG and calcium hydroxide groups, the operator applied an intracanal dressing with each material 1 mm beyond the working length to promote a very slow extrusion of the medication into the periradicular tissues, which was radiographically assessed. This procedure was performed with the aid of an ML threaded syringe (S.S. White Artigos Dentários Ltda.; Rio de Janeiro, Brazil) and a long needle 27G (Septoject XL; Septodont, France). Sealing was achieved with glass-ionomer-based cement for 14 days. At the end of this period, the operator removed intracanal dressing by irrigation and performed root filling.
To carry out the root filling, we used AH Plus sealer (De Trey; Dentsply, Konstanz, Germany) and gutta-percha cones by lateral condensation with a final radiographic confirmation. Then, the operator restored all teeth with a base of glass–ionomer cement and silver amalgam.
Histotechnique processing
After 120 days of the first section of endodontic treatment, all animals were euthanized. Tooth presenting extruded filling was excluded from study. The maxilla and mandible were removed, dissected, sectioned, fixed, washed and subjected to decalcification. Subsequently, the pieces were neutralized, washed, dehydrated in alcohol, cleared in xylol and embedded in paraffin. Serial longitudinal sections 5µm-thick were cut in mesiodistal orientation. For histopathological analysis, the sections were initially stained with hematoxylin and eosin (H&E), and evaluated by conventional light microscopy (Leica DMR, Leica Microsystem Wetzlar Gmbh; Wetzlar, Germany).
Microscopic analysis was performed by two examiners, with the Kappa (K) concordance test at k = 0.81 without prior knowledge of the group to which the analyzed specimen belongs. They evaluated the integrity of the extracellular matrix and the presence and intensity of the inflammatory infiltrate on slides stained with H&E under conventional and fluorescent light microscopy by means of quantitative analysis. An assessment of the degree of tissue disorganization was carried out to characterize the repair stage through the reinsertion and neoformation of the fibers of the periodontal ligament attached to the apical third. We classify the degree of collagen fiber disruption and disorganization as (1) absent, (2) minimal, (3) moderate or (4) severe to indicate the level of apical periodontal ligament destruction6. We use this same criterion to assess the integrity of the adjacent cementum and alveolar bone.
Immunofluorescence processing
In order to evaluate the expression of matrix metalloproteinases and the distribution of these enzymes in tissues (pulpal, apical and periapical region), immunofluorescence assays were performed for MMP-2 and MMP-9.
The slides were deparaffinized, hydrated in a decreasing series of alcohols, and washed under running water. The antigenic recovery with Proteinase K 1: 500 (Invitrogen, Carlsbad, USA) performed for 10 minutes. The slides were washed in phosphate buffered saline (PBS) for 5 minutes (3 times). After that, sodium borohydride 1mg / mL (Dinâmica Química Contemporânea LTDA, Indaiatuba, Brazil) was applied for 15 minutes (3 ×). Washed again in PBS for 5 minutes (3 ×) and the nonspecific binding sites blocked with 1% bovine serum albumin (Sigma, St Louis, USA) for 60 minutes. The tissues were incubated with the primary antibodies in 1:50 concentration for MMP-2 (5 g / mL; 53630, Santa Cruz Biotechnology, Dallas, USA) and MMP-9 (5 g / mL; 21736, Santa Cruz Biotechnology) at 4 ºC overnight. Then, the slides were removed from the refrigerator and placed at room temperature for 1 hour. After that, they were washed in PBS for 5 minutes (3 times). The slides were then incubated with biotinylated secondary anti-mouse FITC antibodies at a concentration of 1: 200 for 1 hour (Rabbit anti-mouse IgG-FITC sc-358916, Santa Cruz Biotechnology), washed in PBS for 5 minutes (3 times), and finally, the coverslips were placed using UltraCruz® Aqueous Mounting Medium with DAPI (24941, Santa Cruz Biotechnology, Dallas, USA. Control slides were used to test the specificity of the immunostaining in which the primary antibody was omitted and the slides were incubated in phosphate buffered saline (PBS).
The microscopic analysis was performed by two examiners with the Kappa (K) concordance test at k = 0.83, without prior knowledge of the group to which the analyzed specimen belongs.
Statistical analysis
The quantitative histopathological results (tissue disorganization, inflammatory infiltrate and MMP-2 and MMP-9 9 expression) are ordinal, independent variables with three different categories and sizes for the experimental and control groups. Thus, for each variable, the Kruskall-Wallis nonparametric test was used, followed by Dunn's post-test for multiple comparisons. The statistical software used was SPSS version 25 (IBM, Chicago, USA) with a significance level of 5% (p < 0.05).