Indomethacin and celecoxib treatment
Sixty C57BL/6 6-week-old male mice (Mus musculus) weighting 20–22 g were acquired from the Central Animal Facility at University of São Paulo in Ribeirão Preto. All animals were kept in Animal Facility at the School of Dentistry of Ribeirão Preto-USP, housed in polypropylene cages with stainless steel lids, 15 × 20 cm (3 animals per cage), lined with autoclaved in a light temperature (22 ± 10%) and relative humidity (55 ± 10%), in a light, on a 12:12 h light–dark cycle, with a standard laboratory diet, free access to autoclaved water. The experiment had the approval by the Ethics Committee on Animal Use (CEUA), from the Ribeirão Preto Campus, University of São Paulo (USP) (#13.1.266.53.6). The animals were treated according to Brazilian Guideline for the Care and Use of animals in Teaching or Scientific Research Activities regulated by the national Council for the Control of Animal experimentation (Law 11.794/2008). Animal studies were conducted in compliance with ARRIVE guidelines36.
The animals were assigned into three groups: celecoxib (n = 20) or indomethacin (n = 20) treatment for a period of 28 days or received no medication (control group; n = 20). The non-selective COX-1 and COX-2 inhibitor Indomethacin (C19H16ClNO4; Cayman Chemical, Ann Arbor, MI, USA) solution was prepared in saline with 5% NaHCO3 and was given i.p. (5 mg/kg), daily throughout the experimental period. Selective COX-2 inhibitor Celecoxib (C17H14F3N3O2S; Pfizer Inc., La Jolla, CA, USA) was prepared in ethanol and saline, being provided by gavage (15 mg/kg), daily throughout the experimental period. Healthy incisor teeth from animals that did not receive medication were used as control. The animals were anesthetized, then euthanized in the CO2 chamber, and tissues containing bone and tooth were collected for further analysis.
Scanning electron microscopy–energy dispersive X-ray analysis
After the extraction of the lower incisors, the samples were dried, and then affixed to scanning electron microscopy (SEM) stubs, sputter-coated with carbon and examined with a JEOL-JSM-6610LV operating at 20 kV and 15–20 mm working distance. Quantitative element analysis was carried out with an Oxford Instruments INCA 300 EDX System (Abingdon, Oxfordshire, UK). The element content was calculated as the relative weight percentage of the total element content (100%). The count was conducted on the incisal and cervical areas of the lower incisors. The elements quantified were calcium (Ca), phosphorus (P), oxygen (O) and carbon (C). Statistical analyses of data were carried out using one-way ANOVA followed by Turkey test (α = 0.05).
Knoop microhardness test
Lower incisors were dried and the test was performed with a load of 10 gf for 5 s in a microhardness testing machine (Shimadzu–HMV-2, Kyoto, Japan) equipped with a Knoop diamond tip. The indentations were performed in three regions: the first was made in the tip of the teeth, the second one in the middle area of the enamel, and the third next to cervical region. Statistical analyses of data were carried out using one-way ANOVA followed by Turkey test (α = 0.05).
A high-resolution, desktop µCT system (Phoenix V tome xS240, GE, Boston, USA) was used to scan the samples. The samples were cleaned and dehydrated, and put in a plastic microtube. Scanning parameters were 70 kV and 200 µA, 0.1 mm Al/Cu filter and the voxel size was 5.4 μm. The projections were acquired over a full circle of rotation steps at 0.4° angle intervals, and each projection was composed of the average of 3 transmission images. The average time of scanning was around 2 h. The data from the tomography projection scans were reconstructed using the 3D Slicer Software37, and then analyzed using ImageJ (Wayne Rasband, National Institutes of Health, USA) software. Hydroxyapatite Ca5(PO4)3(OH) was used for standardization and calibration of measurements. The mean gray-value of the mineral grains were set to as the electron densities of 3.17 g/cm3. The volume, thickness and density of enamel was measured from the CT-scans central section of the incisors at the point just in front of the margin of the lower jaw bone.
The jaws were dissected and removed with surgical scissors. The blocks containing incisor teeth and bone were fixed in 10% buffered formalin for 24 h at room temperature and demineralized in 10% EDTA (Merck S.A. Chemical Industries, Rio de Janeiro, RJ) for approximately 21 days. After demineralization, the samples were submitted to routine histological processing, washed in running water for 24 h, dehydrated in increasing concentrations of alcohol, diaphanized in xylol, and embedded in paraffin. The blocks were sectioned transversally, in an axial direction, to obtain cuts with a thickness of 5 µm. Sections were then stained by hematoxylin and eosin (HE) for histological evaluation.
Slides were deparaffinized, hydrated in a decreasing ethanol series, and kept in phosphate-buffered saline (PBS). Next, tissue sections were microwaved (7 × 12 s at 2-min intervals) with sodium citrate buffer (pH = 6.0) for antigen retrieval. After temperature stabilization, the slides were washed with PBS (3×) for 5 min, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 40 min. Slides were further washed with PBS (3×) 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 antibody for COX-2 (sc-1746, Santa Cruz Biotechnology, Santa Cruz, USA), DSP H-300 (sc-33587, Santa Cruz Biotechnology), MMP-20 (sc-26926, Santa Cruz Biotechnology) and Runx2 (ab-23981, Abcam, USA) at 4 °C overnight. Next, slides were washed and incubated with mouse anti-goat (sc-2491, Santa Cruz Biotechnology) and mouse anti-rabbit (sc-2489, Santa Cruz Biotechnology) biotinylated secondary antibody for 1 h, washed in PBS, and incubated with streptavidin conjugated with horseradish peroxidase (HRP) for 20 min. 3,3′-diaminobenzidine (DAB, Sigma-Aldrich) was used as the enzyme substrate for 5 min; the slides were washed with PBS, counterstained with hematoxylin for 15 s, washed with distilled water, dehydrated in increasing ethanol concentrations, and mounted in Entellan® (Merck, Darmstadt, Germany). Negative control slides, in which the primary antibody was omitted, were used to test the specificity of immunostaining. For quantification of intensity of the immunostaining in the ameloblast layer, the Software Image J (National Institutes of Health, Bethesda, MD, USA) and the image deconvolution plugin (Color Deconvolution) were used. DAB vector was applied and then the selected channel and threshold were manually adjusted, at 10× magnification38. Analysis was carried out by blinded examiners to the sample group assignment at the cervical third of the root, using in 1 section per slide, 1 tooth per animal, from 10 different mice. All immunohistochemistry staining for the same antibody was done in the same batch, with a rigid control of time and temperature that we described in the manuscript. Data obtained were analyzed using one-way ANOVA followed by Turkey test (α = 0.05).
In situ zymography
Five μm-thick tissue sections were immersed in sodium borohydride (1 mg/ml) followed by incubation with a fluorescein isothiocyanate (FITC)-bound gelatin substrate (DQ™ Gelatin, Molecular Probes, Eugene, OR) dissolved in agarose (0.1 mg/ml) for 2 h at 37 °C in a humidified light-protected chamber. Nuclei were counterstained by adding 4′-6-Diamidino-2-phenylindole (DAPI; 0.5 μg/ml) to the incubation medium. Control slides were preincubated in 20 mM ethylene diamine tetraacetic acid (EDTA, Sigma, St Louis, MO) for 1 h, and then EDTA was added to the incubation medium. Quantification of gelatinolytic activity in the sections was assessed by counting the number of spots of fluorescence in the ameloblast layer (10× magnification) and expressed as number of spots of enzymatic activity per mm2.
To investigate the translocation of runt-related transcription factor 2 and its role in the synthesis of biomineralization proteins and as an indicator of cell differentiation, indirect immunofluorescence assays were conducted.
The slides were prepared as described above. Next, the slides were washed with PBS (3×) for 5 min and 1 mg/mL sodium borohydride solution (3x) (Dinâmica Química Contemporânea Ltda., Diadema, SP, Brazil) for 15 min. Non-specific binding sites were blocked with 5% bovine serum albumin (Sigma-Aldrich) for 60 min. Immunolabeling was done using primary antibody for runt-related transcription factor 2 (ab-23981, rabbit polyclonal, Abcam) at 4 °C overnight. The following day, the slides were washed in PBS (3×) and incubated with secondary antibody (mouse anti-rabbit IgG conjugated with fluorescein) for 1 h in a dark chamber. The slides were further washed with PBS (3×) and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (0.5 µg/ml) (Santa Cruz Biotechnology Inc., Dallas, TX, USA) for 5 min. Slides were mounted with ProLong Gold Antifade (Molecular Probes Inc., Eugene, USA). The amount of positively stained cells was determined in the ameloblast layer and then the percentage of cells that presented Runx-2 translocated to the nucleus was calculated. First, the regions were photographed by fluorescence microscopy at 20× magnification using fluorescein isothiocyanate (FITC) and DAPI filters. Next, the images were analyzed using Image J software (U.S. National Institutes of Health, Bethesda, MD, USA) to determine the number of positively stained cells by and selecting the “analyze particles” tool: pixel size was set as the average size of previously measured cell nuclei. Total cell count and stained cell count were expressed as a percentage and staining was compared across groups using one-way ANOVA followed by Tukey’s test (α = 0.05).