Mice (6 months old) were anesthetized using ketamine HCl (62.5 mg/kg) and xylazine (12.5 mg/kg) in sterile PBS by intraperitoneal injection and then mounted on a jaw retraction board. For tooth injury without pulp exposure, the occlusal surfaces of the right maxillary and right mandibular first molars were injured by cuspal grinding (50% depth) using a No. 2 round bur drill and air-dried. For acute tooth injury with pulp exposure, the occlusal surfaces of the right maxillary first molars were injured until pulp was exposed, using a No. 2 round bur drill with water spray, followed by an endodontic size K6-file (Flexofile, Maillefer, Dentsply-Sirona) for final exposure, air-dried, and sealed with glass ionomer cement (Fuji II, GC company, Chicago, IL, USA), as previously described [33 (link)]. The right injured maxillae were used for histology, and the right injured mandibles were used for micro-computed tomography (CT) or gene expression analysis. The left side of the maxillae was used as a control (non-injured site) for histology or the analysis of gene expression. For tooth injury without pulp exposure, mouse heads were collected 4 weeks following tooth injury. For acute tooth injury with pulp exposure, 3- and 6-month-old mouse heads were collected after 6 or 24 h of contamination in oral fluids (6 mice per genotype for each age and contamination time point).
Flexofile
Manufactured by Dentsply
Sourced in Switzerland, United States
Flexofile is a dental laboratory instrument used for shaping and preparing root canals during endodontic procedures. It features a flexible, file-like design to navigate and conform to the complex anatomy of the root canal system.
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7 protocols using flexofile
1
Dental Injury Model in Mice
2
Standardized Root Canal Treatment Protocol
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Root canal treatment (RCT) was performed on teeth in groups 3 to 6. Access cavity preparation was achieved using a diamond bur (FG 8514; Intensiv, Grancia, Switzerland). Root canals were negotiated with size 10 K-file (Flexofile; Dentsply Maillefer, Ballaigues, Switzerland), and the working length was established at 1 mm short of the apical foramen. Canals were instrumented to the working length, enlarging the apex to F3, using ProTaper Universal (Dentsply Maillefer). During endodontic treatment, deionized water irrigation was intermittently deposited using side-vented 30-G needles. The canals were then dried with paper points and filled with 30/0.06 taper gutta-percha (Conform Fit; Dentsply Maillefer) and an epoxy resin-based sealer AH Plus (Dentsply Maillefer) using single-cone obturation technique. The gutta-percha was removed to the depth of 1 mm below CEJ. When the endodontic treatment was completed, all the samples were examined using a stereomicroscope at 15× magnification; no cracks were found among the surface. For teeth in group 6, the access cavity and cervical defects were filled with temporary filling material Ceivitron (Triune Med Tec, Cambridgeshire, UK) for a week before restoration to allow the endodontic sealer to set.
3
Endodontic Treatment Using K3 System
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Initially, the entrance orifice of the canal and the cervical and middle thirds were prepared using Largo No. 2 (Dentsply Maillefer, Rio de Janeiro, RJ, Brazil) and No. 4, 3, and 2 Gates Glidden drills (Dentsply-Maillefer, Ballaigues, Switzerland), respectively, according to the crown-down technique. The working length was determined by inserting a size 15 Flexo-File (Dentsply-Maillefer) up to the apical foramen, and subtracting 1 mm from this measurement.
The K3 system (SybronEndo, Scafati, Italy) was used for instrumentation, with a file of size 55/0.04 as the final instrument. An amount of 2.5 mL of 2.5% NaOCl (Formula e Ação, São Paulo, SP, Brazil) was used at change of each file, using disposable syringes and 30 G NaviTip needles (Ultradent, South Jordan, UT, USA) calibrated at 1 mm short of the working length. The root canals were dried with absorbent paper cones (Tanari, São Paulo, SP, Brazil), and filled using the lateral condensation technique and Fillapex sealer (Angelus, Londrina, PR, Brazil). Next, the specimens were stored at 37°C and 100% humidity for 7 days to allow the sealer to set.
The K3 system (SybronEndo, Scafati, Italy) was used for instrumentation, with a file of size 55/0.04 as the final instrument. An amount of 2.5 mL of 2.5% NaOCl (Formula e Ação, São Paulo, SP, Brazil) was used at change of each file, using disposable syringes and 30 G NaviTip needles (Ultradent, South Jordan, UT, USA) calibrated at 1 mm short of the working length. The root canals were dried with absorbent paper cones (Tanari, São Paulo, SP, Brazil), and filled using the lateral condensation technique and Fillapex sealer (Angelus, Londrina, PR, Brazil). Next, the specimens were stored at 37°C and 100% humidity for 7 days to allow the sealer to set.
4
Endodontic Root Canal Preparation and Obturation
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All procedures were performed by a single operator.
A coronal access cavity was prepared using diamonds burs 1013 and 3080 (KG Sorensen, São Paulo, Brazil). The root canals were negotiated using a size of 10 K-files (Dentsply Sirona, Ballaigues, Switzerland), and the working length (WL) was established at 1 mm from the major apical foramen. Root canal preparation was performed by step-back technique to the size 35 Flexofile (Dentsply Sirona, Ballaigues, Switzerland). Irrigation was accomplished using 2 mL of 2.5% sodium hypochlorite (NaOCl) at each instrument change (Miyako do Brasil Industria e Comercio Ltda., São Paulo, Brazil).
Eighty specimens were dried with paper points (Dentsply Ind. Com. Pirassununga, São Paulo, Brazil). For the obturation (lateral condensation technique), size 35 gutta-percha cones and accessory cones (Dentsply Indústria e Comércio Ltda., Pirassununga, Brazil), with AH Plus sealer (Dentsply Sirona GmbH, Konstanz, Germany) were used. Zinc oxide/zinc sulfate cement (Citodur®; DoriDent, Wien, Austria) was placed over the filling material as a temporary restorative material. Negative control was composed of empty root canals just after preparation (n = 20), and POS of canals irrigated with EDTA 17% (Biodinâmica, Ibiporã, Brazil) for 3 minutes and filled with CH paste just after preparation. These specimens were not obturated, retreated, or submitted to CBCT.
A coronal access cavity was prepared using diamonds burs 1013 and 3080 (KG Sorensen, São Paulo, Brazil). The root canals were negotiated using a size of 10 K-files (Dentsply Sirona, Ballaigues, Switzerland), and the working length (WL) was established at 1 mm from the major apical foramen. Root canal preparation was performed by step-back technique to the size 35 Flexofile (Dentsply Sirona, Ballaigues, Switzerland). Irrigation was accomplished using 2 mL of 2.5% sodium hypochlorite (NaOCl) at each instrument change (Miyako do Brasil Industria e Comercio Ltda., São Paulo, Brazil).
Eighty specimens were dried with paper points (Dentsply Ind. Com. Pirassununga, São Paulo, Brazil). For the obturation (lateral condensation technique), size 35 gutta-percha cones and accessory cones (Dentsply Indústria e Comércio Ltda., Pirassununga, Brazil), with AH Plus sealer (Dentsply Sirona GmbH, Konstanz, Germany) were used. Zinc oxide/zinc sulfate cement (Citodur®; DoriDent, Wien, Austria) was placed over the filling material as a temporary restorative material. Negative control was composed of empty root canals just after preparation (n = 20), and POS of canals irrigated with EDTA 17% (Biodinâmica, Ibiporã, Brazil) for 3 minutes and filled with CH paste just after preparation. These specimens were not obturated, retreated, or submitted to CBCT.
5
Microbiological Sampling and Tooth Restoration
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Microbiological samples were extracted from tooth canals before irrigation. Three different absorbent paper points were sterilized and used for different canal sizes. These points were inserted in canals for 30 seconds after the canals were accessible. An intraoral periapical radiograph was used preoperatively to determine the workable length of the tooth canals. Some sterile saline solution was used to irrigate the canals, if they were dry before the points were inserted. The paper points used for the roots were placed in a tube containing thioglycolate. Conventional teeth treatment followed sample collection. FlexoFiles (Dentsply, Switzerland) instruments were used with 0.5 mL of the identified irrigant after filing. The canal was wetted and dried one last time after instrumentation and before sealing the tooth. A postirrigation sample was obtained from the canals using another set of three sterilized paper points. Lastly, iodoform paste (Vitapex) was inserted in the canal. An intraoral periapical radiograph was obtained after the operation. Stainless steel crowns (3M) were employed for after-treatment restoration.
6
Endodontic Procedure Protocol
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Teeth isolation was performed using sterilized cotton rolls, and high-speed evacuation was used to control saliva. The teeth were cleaned with 0.12% chlorhexidine gluconate (Listermix; Sigma Pharmaceutical Industries, Quesna, Egypt). After gaining occlusal access to the pulp chambers of the involved teeth, the pulps were extirpated and the root canals were cleaned and shaped using Flex-o-Files (Dentsply Tulsa Dental, Tulsa, OK, USA). The canals were filled with warm vertical compaction of gutta-percha and AH Plus root canal sealer (Dentsply Tulsa Dental). The coronal access cavities in all teeth were then restored with amalgam (Ivoclar Vivadent, Amherst, NY, USA) [17 (link)].
7
Mandibular Incisor Microstructural Analysis
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Twenty‐two mandibular incisors extracted for causes not related to this study were collected (IRB protocol 00010445) and stored in 0.1% Thymol. Teeth were evaluated by microcomputed tomography for suitability for the study and discarded if resorptions, root cracks, open apices, previous root canal therapy, presence of multiple canals, long oval canals (aspect ratio >4) or existing indirect restorations were present. Conventional endodontic accesses cavities were prepared and #10 FlexoFiles (Dentsply) were introduced to confirm patency and to record specimen's canal lengths. Then, specimens were autoclaved and stored in distilled water. Two additional samples were used for electron scanning microscope analysis.
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