Biocompatibility, bioactivity, porosity and sealer/dentin interface of ready-to-use bioceramic sealers using a dentin tube model

Approval by the research ethics committee

The following methods were performed in accordance with the Declaration of Helsinki and this study was approved by the University Research Ethics Committee (protocol number: 12647319.8.0000.5416). The University Research Ethics Committee waived informed consent because all teeth used in this study were obtained from the University Human Tooth Bank. In addition, the Animal Experimentation Ethics Committee approved this protocol at the University (#04/2019). The experimental and analytical methods were conducted in accordance with the ARRIVE 2.0 (Animal Research: Reporting of In Vivo Experiments) guidelines. All animal experiments followed all relevant guidelines and regulations.

Preparation of dentin tubes

This study used single-rooted human teeth to produce dentin tubes. All tubes were subjected to the standardization process, and samples that did not fit the template were rejected. Dentin tubes were sectioned using a precision cutting machine (ISOMET; Buehler, Lake Bluff, IL, USA) and prepared using Gates-Glidden No. 5 burs (Dentsply Sirona, Charlotte, NC, USA). Samples of 5 mm length, 1.3 mm internal diameter, and 1.5 mm wall thickness were obtained. Measurements were confirmed using a digimatic caliper (Mitutoyo Corporation, São Paulo, SP, Brazil) and an iwanson caliper (Golgran Millennium, São Caetano do Sul, SP, Brazil). Dentin tubes were subjected to a protocol to remove the “sludge layer”, using 17% EDTA, 1% sodium hypochlorite and distilled water, and were then sterilized in an autoclave (Cristófoli Equipamentos de Biossegurança, Campo Mourão, PR, Brazil) using a test tube containing 100 ml of distilled water, wrapped in surgical grade paper (15 minutes, 1700 W, 127 °C), without a drying cycle. After autoclaving, the tubes were stored in an oven (37 °C, 95% humidity) and kept hydrated with distilled water until filling with sealants.

Analysis of biological properties

Experience design

Thirty-two adult male Holtzman rats (Rattus norvegicus albinus) weighing between 270 and 300 g were used and housed in polyethylene cages, and maintained under a 12:12 light-dark cycle at controlled temperature (23 ± 2 °C) and humidity (55 ± 10%), with food and water provided ad libitum. The animals were divided into five groups (n = 6 tubes per group): dentin tubes were filled with endodontic sealants (Table 1) and empty tubes were used as controls (control group – CG). Sample size was calculated using G*Power 3.1.7 software (Heinrich-Heine Universität, Düsseldorf, Germany). The calculation was based on an alpha of 0.05 and a beta power of 0.99 for all variables. Previous studies were used to determine the specific effect size of capsule thickness, 1.06¹⁹; number of inflammatory cells, 3.61¹⁹; cells immunolabeled with interleukin-6, 1.17²⁹; and osteocalcin-immunolabeled cells, 2.04⁴⁴Six samples per group/period were indicated as the ideal size required to observe significant differences.

Intraperitoneal anesthesia with xylazine hydrochloride (4 mg kg-1 body weight; União Química, São Paulo, SP, Brazil) and ketamine hydrochloride (80 mg kg-1 body weight, Virbac do Brasil, São Paulo, SP, Brazil) was applied to the animals. A 2-cm cranio-caudal incision and tissue disvulsion were performed in the dorsal region. Four tubes were inserted per animal corresponding to the different experimental groups^3.4. Suture was performed with 4/0 silk thread (ETHICON, São José dos Campos, SP, Brazil). After 7, 15, 30, and 60 days after implantation, animals were euthanized with an overdose of anesthesia and the implanted tubes with adjacent tissues were extracted. Dentin tubes were isolated from surrounding tissues and processed for micro-CT analysis to assess porosity and dentin-sealer interface. Surrounding tissues were used for biocompatibility and bioactivity assessments.

Histological procedures

Adjacent tissues were removed and immersed in 4% formaldehyde solution, buffered with 0.1 M sodium phosphate and pH 7.2 for 72 hours. After fixation, specimens were dehydrated, diaphanized, immersed in liquid paraffin (60 °C) for 4 hours, and embedded in paraffin. Longitudinal sections with a thickness of 6 µm were obtained. Unserial sections were stained with hematoxylin-eosin (H&E) to estimate the number of inflammatory cells and capsule thickness. Additional unserial sections were mounted on 4% silane-treated slides (Sigma-Aldrich, Saint Louis, MO, USA) and underwent immunohistochemistry to detect osteocalcin (OCN) and interleukin-6 (IL-6).

Numerical density of inflammatory cells

Inflammatory cell counts were obtained from three HE-stained sections (with a minimum distance of 100 µm between sections) captured under a magnification of × 695 (each field with 0.09 mm²). The number of inflammatory cells was calculated using an image analysis program (Image-Pro Express 6.0 program; Olympus Corporation, Tokyo, Japan) and divided by the total area (number of inflammatory cells per mm²)^3,4,11,28.

Thickness of capsules

Three images of H&E-stained nonserial sections per specimen were captured at 65× magnification using a camera (DP-71; Olympus Corporation, Tokyo, Japan) attached to a light microscope (BX-51; Olympus Corporation, Tokyo, Japan). Capsule measurements were performed using image analysis software (Image-Pro Express 6.0 program, Olympus Corporation, Tokyo, Japan) according to previous studies^3,4,24,28,29.

Immunohistochemical detection of IL-6 and osteocalcin (OCN)

Deparaffinized sections were immersed in 0.001 M sodium citrate buffer pH 6.0 and heated at 98 °C in a microwave for 30 min for IL-6 detection and 10 min for OCN detection. After cooling, the slides were washed with 0.05 M Tris-HCl buffer pH 7.4 and endogenous peroxidase was inactivated by treatment with 5% aqueous hydrogen peroxide solution for 20 min. Then, the sections were washed and incubated with 2% bovine serum albumin (BSA; Sigma-Aldrich, St Louis, MO, USA) for 30 min. Sections were then incubated overnight in a humid chamber at 4 °C with a mouse anti-IL-6 primary antibody (Abcam, Cambridge, UK, England, Ab code 9324) diluted 1:100 or a rabbit anti-osteocalcin primary antibody diluted 1:500 (code SAB1306277; Sigma-Aldrich, St Louis, MO, USA). After washing, sections were incubated with a biotinylated anti-mouse IgG secondary antibody (LSAB, Dako., Carpinteria, CA, USA) at room temperature. The chromogen 3,3′-diaminobenzidine (ImmPACTTM DAB; Vector, Burlingame, CA, USA) revealed peroxidase activity, and sections were then counterstained with Carazzi hematoxylin. Sections were incubated with non-immune serum instead of anti-IL-6 and anti-OCN antibody as negative control.

The number of IL-6 and OCN immunostaining cells was estimated in all samples. In each sample, a standardized field was captured at 695× magnification (0.09 mm²) using a digital camera (DP-71, Olympus Corporation, Tokyo, Japan) attached to the optical microscope (BX-51, Olympus Corporation). In these images, the number of immunolabeled cells (in brown/yellow color) per mm² has been calculated^3,4,28,29.

Von Kossa reaction and analysis under polarized light

The von Kossa method was used for the detection of calcium deposits in capsules. The sections were deparaffinized, hydrated, and immersed in 5% silver nitrate solution for 1 hour under sunlight. Then, the sections were rinsed with distilled water for 3 minutes, immersed in 5% sodium hyposulfite solution for 5 minutes. After washing with distilled water for 5 minutes, the sections were stained with picrosirius red for 1 hour, dehydrated, and mounted in resin medium.^3.45.

Evaluation of birefringent structures in capsules was performed using unstained sections viewed under polarized light (BX51; Olympus Corporation, Tokyo, Japan)^3,4,28,29.

Analysis of physicochemical properties

After the implantation periods of 7, 15, 30, and 60 days, dentin tubes filled with SP, BIOC, TF, and AHP (n = 6 per group) were extracted from the subcutaneous tissue of rats. Sample size was calculated using G*Power 3.1.7 software (Heinrich-Heine Universität, Düsseldorf, Germany). One-way ANOVA was used with an alpha error of 0.05 and a beta power of 0.99 for all variables. A previous study¹⁹ was used to calculate the specific effect size for each variable: 2.21 for porosity and 1.11 for dentin-material interface. In order to observe a significant difference between the experimental groups and the CG, six samples per group/period were indicated as the ideal number required. Dentin tubes were preserved for 24 hours with gauze moistened in distilled water and stored in an oven (37 °C, 95% humidity). For comparison, samples (n = 6) were prepared using new dentin tubes filled with freshly applied sealants (reference), which were preserved for 48 hours with gauze moistened in distilled water and stored in an oven (37 °C, 95% humidity) for a complete set¹⁹.

Samples were evaluated by microtomography (micro-CT, SkyScan 1176; Bruker-microCT, Kontich, Belgium), with the following parameters: current of 313 µA, 80 kV, pixel size of 9 µm and 360° rotation with a Cu + Al filter. Image reconstruction was performed using the NRecon program (V1.6.4.7; Bruker-MicroCT, Kontich, Belgium) with correction parameters for beam hardening, smoothing and annular artifacts defined for each material. Image analysis was performed using the CTAn software (V1.11.8; Bruker-MicroCT, Kontich, Belgium).

Porosity

The porosity of the grouting materials was measured in cubic millimeters and percentages. The porosity values ​​for each grouting material and for each time period were compared to the baseline. CTAn and CTVol software (V2.0; Bruker-MicroCT, Kontich, Belgium) were used to create 3D models of the filled cavities^19.21.

Dentin-material interface

The method of evaluating the differences in void percentages at the interface between the dentinal surface of the root canal walls and the sealers was based on previous studies^6.19. The 3D distribution of interface voids in a predefined volume of interest (VOI), including the canal wall dentin and sealer, was calculated for each group and compared to the baseline. Voids from a size of 9 μm in the VOI were detected using the gray level threshold. 3D models of the voids were then created using CTAn software.

statistical analysis

All data were statistically analyzed using GraphPad Prism 9 software (Jandel Scientific, Sausalito, CA, USA). Data passed the Kolmogorov-Smirnov normality test. Biological property data were analyzed using two-way ANOVA followed by Tukey’s test, while porosity and interface analysis data were subjected to one-way ANOVA with Tukey’s test. A significance level of P ≤ 0.05 was accepted.