Biological and chemical properties of five mineral oxides and mineral trioxide aggregates repairing high plasticity: an in vitro study

Antibiofilm activity

Five different inocula of P. gingivalis (ATCC 33277), P.endodontalis (ATCC 35406), P.micra (ATCC 23195), F.nucleatum (ATCC 25586) and P. intermediate (ATCC 33563) were prepared, standardized in saline solution (NaCl 0.9%) (Eurofarma, São Paulo, SP, Brazil) and at 1 × 108 CFU/mL in a spectrophotometer (V-5000 visible spectrophotometer, Shanghai Metash Instruments, China). Then, 100 μl of corresponding inoculum was added to each well of 96-well microplates. The plates were incubated (37°C) with shaking (75 rpm) for 90 min. Then the supernatant was discarded, 100 µL of Brucella broth (Himedia, Mumbai, India) was added and the plates were incubated (37°C) for 7 days under anaerobic conditions; the culture medium was replaced every 48 h. After the formation of the biofilms, the materials were brought into contact with the biofilms to be evaluated for 24 h, and the plates were incubated (37° C.) under anaerobic conditions. Subsequently, the biofilm measurement test (MTT) was performed. Two independent experiments were performed with 5 replicates each, totaling n=10 for each experimental group.

The experimental groups were: (I) saline solution (negative control group) (Eurofarma, São Paulo, SP, Brazil); (II) 2.5% sodium hypochlorite (NaOCl) (Biodynamics, Ibiporã, PR, Brazil) (positive control group); (III) 5MO (Golden Yatti LLC, Muscat, Oman); (IV) MTA Repair HP (Angelus, Londrina, PR, Brazil); and (V) MTA (Angelus, Londrina, PR, Brazil). All CSCs were spatulated according to the proportions specified in the manufacturer’s instructions (3 × powder: 1 × liquid) and applied immediately fresh. After 24 hours of contact with the biofilms, the CSCs were analyzed by adding 100 µL of MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA). The plates were incubated in the dark for 1 hour in an anaerobic chamber at 37°C. Then, the MTT solution was removed, 100 µL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO., USA) was added, the plates were again incubated at 37°C for 10 min and placed on a constant shaker for 10 min. Finally, the plates were read in a microplate reader at 570 nm (BIO-TEK Instruments, Highland Park, Winooski, VT, USA). The optical densities (OD) obtained were converted into percentage of cell viability of the microorganisms using the following formula:

$$% {text{Viability}} = left( {{text{OD of treated group}} times 100} right)/{text{mean OD of control group}}).$$

Cytotoxicity analysis

Cultures of mouse macrophages (RAW 264.7) (Rio de Janeiro Cell Bank – APABCAM, RJ, Brazil) and osteoblasts (MG-63) (Rio de Janeiro Cell Bank – APABCAM, RJ, Brazil) were used. Cells were stored in cell culture flasks (TPP, Zollstrasse, Switzerland) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (LGC Biotechnology, Cotia, SP, Brazil), supplemented with 10% fetal bovine serum ( FBS) (Invitrogen, Grand Island, NY, USA), incubated at 37°C, atmospheric humidity and 5% CO2. The culture medium was changed every 48 h, and the cells were transferred to another cell flask when a cellular subconfluent state of the cells was observed. The cells were transferred to a Falcon tube where they were centrifuged for 5 min at 2000 rpm. The number of viable cells was quantified by the Trypan blue exclusion test (0.4%, Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured in 96-well microplates. A 200 µl aliquot of DMEM was added and supplemented with 10% FBS containing 2 × 104 viable cells, after which the plates were incubated (37°C, 5% CO2) for 24 h to promote cell adhesion. Then the supernatant was discarded and the cells were washed with PBS. Control wells containing only cells with culture medium were used. The incubation period was 5 min and 24 h, and there were 12 wells per group (n=12).

CSCs were manipulated in a 24-well plate following the manufacturer’s instructions. Additional group of Ca(OH)2 (Biodynamic Chemicals and Pharmaceuticals, Ibiporã, PR, Brazil) was tested. It was handled using a powder/liquid ratio of 1:1. The materials were mixed and each well containing the respective cement was filled with 2 mL of culture medium (DMEM) supplemented with 10% FBS, after which the plates were incubated at 37°C for 24 h with 5% of CO2.

Then, 100 µL per well of the conditioned DMEM from each experimental group was applied to the cells. The NaOCl group was used as a control. Culture viability was determined by analyzing mitochondrial activity of viable cells using MTT reduction in formazan, where 100 µL of MTT solution was added per well, and the plates were incubated (37°C, 5 % CO2) for 1 h, protected from light. This solution was then discarded and 100 µL of DMSO was added per well to expose the formazan crystals produced after viable cells took up the MTT salt. After the wells were incubated for 10 min and shaken for the same time, their absorbance was read in a microplate reader with a wavelength of 570 nm. The OD obtained was converted into percentage of cell viability according to the following formula:

$$% ,{text{Viability}} = left( {{text{OD of Treated Group}} times 100} right)/{text{Average OD of Control Group}}.$$

Morphological analysis and chemical composition of groups analyzed using energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD)

These analyzes were carried out on CSC discs manipulated according to the manufacturer’s instructions, compacted between two glass plates in silicone molds (5 mm × 2 mm) until the cements had completely set. Discs were placed in Petri dishes and stored at 100% humidity for 3 days at 37°C, after which they were either (a) maintained at 100% humidity at 37°C (control), or (b) immersed in distilled water. for 4 h followed by air drying for 12 h.

All heels have been covered with carbon to promote electrical conductivity. The samples were visualized by SEM (MIRA3-TESCA, Brno-Kohoutovice, Czech Republic). Images of the different components of the material microstructure were captured at different magnifications up to 100 k× in electron backscatter mode.

The chemical composition of the discs was determined by EDX using SEM. Phase analysis was performed on samples from each experimental group, using Cu Kα radiation XRD and a double crystal monochromator, in an automated powder diffractometer. The samples were presented in powder form, in a monocrystalline sample holder, which was used to avoid unwanted diffraction peaks. Phase identification was performed using search matching software available in the International Center for Diffraction Data (ICDD) database.

Radiopacity analysis

The radiopacity of the CSCs was assessed using a graduated aluminum scale of increasing thickness (1 to 8 mm). The CSC disks were compacted into silicone rings 5 ​​mm in diameter and 2 mm in height. The CSC discs and aluminum scale were taped onto a charge-coupled device (CCD) sensor of a cephalometric device, to ensure standardization of the distance between the image receptor and the X-ray source, and to obtain a digital image. The radiographic device used was an ORTHOPHOS XG 5 (Dentsply Sirona, York, PA, USA) operating with acquisition parameters of 60 kVp, 10 mA and an exposure time of 9.1 s. The image was produced from a single acquisition and subjected to pixel intensity analysis in an image analysis program (ImageJ 1.53e, Wayne Rasband and Contributors, National Institute of Health, USA).

The regions of interest corresponding to the images of the cements and the steps of the aluminum ladder were determined with the selection tool, and were analyzed individually and progressively to obtain the histogram of the distribution of the pixel values ​​(mean , standard deviation, maximum and minimum values). A comparative analysis was then carried out between the average values ​​of the cements and those corresponding to the rungs of the aluminum stepwedge scale, in order to determine the closest equivalence between the density of the materials studied and the thickness of the aluminum (in mm).

statistical analyzes

All data were subjected to a normality test, then analyzed with the Kruskal Wallis test, supplemented by Dunn’s test, considering a significance level set at α ≤ 0.05, using GraphPad Prism 6 (La Jolla , CA, USA).