Paper points

Fifty (50) single-rooted teeth were selected. Roots were sectioned at cementum-enamel junction, with a diamond disc (Dhpro, Paranaguá, PR, Brazil) under water-cooling. The working length (WL) was visually established, with a #10 hand instrument (Dentsply-Maillefer, Ballaigues, Jura-Nord Vaudois, Switzerland) that was inserted into the canal until its tip reached the apical foramen. The WL was determined 1 mm shorter to the apex. The root canals were prepared using the standardized K-files series (Dentsply-Maillefer, Ballaigues, Jura-Nord Vaudois, Switzerland), from a #10 K-file to a #60 K-file in the entire WL, to facilitate the LPS introduction and collection. At each change of instrument, the canals were flushed with 2 mL of NaOCl 1% (Biodinamica, Ibiporã, PR, Brazil). After preparation, canals were filled with EDTA 17% (Biodinamica, Ibiporã, PR, Brazil) for 3 minutes and irrigated with 5 mL of sterile saline solution. Canals were dried with paper points (Dentsply, Rio de Janeiro, RJ, Brazil). Each sample was fixed in epoxy resin (Loctite, São Paulo, SP, Brazil) in a well, in 12-well cell culture plates (Kasvi, Curitiba, PR, Brazil).

One specialist in endodontics performed all the endodontic procedures. A 10 K-file was introduced into the canal until it was visible at the apical foramen and the length measured. The working length (WL) was calculated by subtracting 0.5 mm from this length. Root canals were prepared with the ProTaper Universal system (Dentsply Maillefer, Ballaigues, Switzerland), using a crown-down technique, according to the manufacturer’s instructions, up to the F4 instrument, which has an ISO 040 tip size, a 6% apical third taper, and then a progressively decreasing taper in the body. A size 10 K-file was inserted after each file to maintain apical patency. Canals were irrigated between each instrument with 0.5 mL of freshly prepared NaOCl 2.5%, and after instrumentation, with 10 mL EDTA 17% followed by 3 mL of 5.25% NaOCl for 3 minutes using a 30 G Navi Tip needle (Ultradent, South Jordan, UT, USA) and introduced 3 mm shorter than the WL (17 (link)). After all the procedures, each canal was irrigated with 10 mL of distilled water then dried using paper points (Dentsply Maillefer).

Teeth were sectioned apically 12 mm below and coronally 2 mm above the cementoenamel junction using a diamond disk. Remnant pulpal soft tissue was removed using a size 30 Hedström file (Dentsply Maillefer, Ballaigues, Switzerland) [Figure 1]. Working length was determined radiographically using size 20 K-files (Dentsply Maillefer, Ballaigues, Switzerland) and was kept 2 mm more than normal to over-instrument the canals simulating immature open apices. Rotary instrumentation was carried out using ProTaper rotary instruments (Dentsply Maillefer, Ballaigues, Switzerland) to standardize to master apical size 30 corresponding to F-3. Next Peeso reamers (Dentsply Maillefer, Ballaigues, Switzerland) were used sequentially from #1 to #5 to obtain larger canal space and foramen. The canals were irrigated with 2 mL 2.5% sodium hypochlorite (NaOCl) (Sigma-Aldrich, St. Louis, MO) between the instrument changes. A final flush was applied using 5 mL 17% ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, St. Louis, MO) for 1 min and 5 mL 2.5% NaOCl for 1 min and then dried using paper points (Dentsply Maillefer, Ballaigues, Switzerland)

Forty bovine central incisor teeth were selected and sectioned
horizontally to standardize the root lengths at 17
mm. The working length was established at 16 mm. The root
canals were instrumented with using hand instrumentation
technique up to the #80 K- file, and they were prepared with
#1-6 Peeso reamer drills. At each instrument change, 2 mL of
1.5% sodium hypochlorite (NaOCl) was used for irrigation.
After finishing the instrumentation protocol, 20 mL of 1.5%
NaOCl (5 min), 5 mL of distilled water, and 20 mL of 17% ethylenediaminetetraacetic
(EDTA) (5 min) were applied to the
root canals. Then, the root canals were dried using paper
points (Dentsply Maillefer, Ballaigues, Switzerland). Equal
portions of metronidazole (Eczacibasi, Istanbul, Turkey), ciprofloxacin
(Biofarma, Istanbul, Turkey), and minocycline (Ratiopharm,
Ulm, Germany) were used for the TAP preparation.
The medicament was prepared by mixing the powder with
distilled water (with a powder to liquid ratio of 1 mg/1 mL)and it was introduced into the root canals with a Lentulo spiral
at 900 rpm until the medicament extrusion was visible at
the apical foramen. The apical portions of the canals were
sealed with a flowable composite resin, and the access cavities
were temporarily sealed (Cavit G; 3M ESPE, Seefeld, Germany).
The teeth were stored at 37 °C and 100% humidity.

The post space preparation of all roots was initiated after 7 days to allow the sealer to set. Post spaces were prepared to a depth of 10 mm with a special preparation drill (No. 3 FRC Postec Plus drill; Ivoclar Vivadent, Schaan, Liechtenstein) which had a 1.0-mm diameter apically and a 0.02 taper. After post space preparation, the canals were irrigated using sterile water and dried with paper points (Dentsply Maillefer). The presence of any residual gutta-percha on the walls of the post space was evaluated by radiographic imaging.