The slides used in this section were obtained from the specimen database at the Department, of Pediatric Dentistry of the School of Dentistry of Ribeirão Preto, University of São Paulo (FORP–USP) and the material and methods were detailed in a previous study 6.
Briefly, after approval by the Animal Welfare Committee of the University of Washington, 4 beagle dogs (aged 12 to 18 months and average weight of 15 kg) were used. Thirty premolar roots (15 teeth) of the second and third upper premolars and of the second, third and fourth lower premolars were used. Each premolar with two roots was hemisected and each root was considered a specimen. Atraumatic root extraction was performed with number 150 forceps (Quinelato, Rio Claro, SP, Brazil) and the canals were instrumented and filled with gutta-percha cones (Dentsply-Herpo, Teresópolis, RJ, Brazil) and AH Plus cement (Dentsply-DeTrey, Konstanz, Germany) by the lateral condensation technique. The access cavities were restored with amalgam.
The groups were divided according to the period that the teeth were kept in an extraoral dry medium before replantation as follows: Group1: the roots were replanted after a 20 minutes extraoral time in a dry medium; Group 2: the roots were replanted after a 60 minutes extraoral time in a dry medium; Group 3: the roots were replanted after 90 minutes the extraoral time in a dry medium; Group 4 (negative control): healthy teeth, not extracted. The teeth were splinted with passive containment using a 0.4 mm nickel-titanium (Ni-Ti) wire and Z 250 composite (3M ESPE, St. Paul, MN, USA) for 10 days. The dogs were maintained on a canned food diet after the surgical procedure, and radiographic monitoring was performed monthly for 120 days. After 120 days, the dogs were killed by an overdose of 6% sodium pentobarbital solution administered intravenously.
Microscopic evaluation
Maxillary and mandibular blocks containing the roots and the surrounding alveolar bone were prepared and fixed in 10% formalin, decalcified in Formical solution (Decal Chemical Corporation, Congers, NY, USA) for 1 week followed by Immunocal solution (Decal Chemical Corporation, Tallman, NY, USA) for 2 months. Paraffin embedded sections were cut parallel to the long axis of the roots with a thickness of 5 µm at 90 µm intervals and stained with hematoxylin-eosin.
The frequency of inflammatory root resorption, replacement root resorption and periapical bone resorption in different groups was compared using the Fisher exact test (α = 0.05).
Determination of the presence of osteoclasts by histoenzymology analyses for TRAP
Deparaffinized tissue sections were incubated in a solution containing 8 mg of naphthol AS-MX di-sodium phosphate (Sigma-Aldrich) in 500 μL of NN-dimethylformamide followed by the addition of 50 mL of a 0.2 mol buffer solution/L sodium acetate (pH 5.0) containing 70 mg of Fast Red ITR (Sigma-Aldrich). Subsequently, the sodium tartrate substrate dihydrate (50 mmol/L) was added to the solution and incubated at 37°C for 12 h. Subsequently, the sections were washed in distilled water and counterstained with Fast Green. Quantitative analysis of the number of osteoclasts that were positive for the TRAP enzyme was performed taking into consideration the total number of osteoclasts per dental root. The groups were compared by means of one-way ANOVA followed by the Tukey's test (α = 0.05).
Immunohistochemistry
The slides were deparaffinized, hydrated in a decreasing series of ethanol, and kept in phosphate-buffered saline (PBS). Next, tissue sections were microwaved (7 x 12 seconds at 2-minute intervals) with sodium citrate buffer (pH = 6.0) for antigen retrieval. After temperature stabilization, the slides were washed with PBS (3x) for 5 minutes, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 40 minutes. Slides were further washed with PBS (3x) for 5 min and non-specific binding sites were blocked with 5% bovine serum albumin (Sigma-Aldrich) for 60 min. The tissues were then incubated with primary antibodies for RANK (sc-7626; Santa Cruz Biotechnology, Dallas, TX, USA), RANKL (sc-7628; Santa Cruz Biotechnology), OPG (sc-8468; Santa Cruz Biotechnology), alkaline phosphatase (ab54778; Abcam, Cambridge, MA, USA) and periostin (sc-67233; Santa Cruz Biotechnology Inc.) at 4 °C overnight. Next, the slides were washed and incubated with donkey anti-goat, goat anti-rabbit and rabbit anti-mouse biotinylated secondary antibody (Biocare Medical, Concord, CA, USA) for 1 h, washed in PBS, and incubated with streptavidin conjugated with horseradish peroxidase (HRP) for 20 min. The 3,3’-diaminobenzidine (DAB; Sigma-Aldrich) was used as the enzyme substrate for 5 min; the slides were washed with PBS, counterstained with haematoxylin for 15 s, washed with distilled water, dehydrated in increasing ethanol concentrations, and mounted in Entellan® (Merck, Darmstadt, Germany). Control slides in which the primary antibody was omitted were used to test the specificity of immunostaining. For this analysis, all slides were prepared in the same batch to obtain a standardized staining and were evaluated by an experienced blind examiner. Slides were scored according to the intensity of staining as (1) mild, (2) moderate, and (3) intense. The groups were compared using the Kruskal Wallis followed by the Dunn’s tests (α = 0.05).