2.1

Selection of materials and curing light

The examined composite materials were the RBC and OBLs with the largest purchasing volume in the public dental services in the Akershus Region of Norway). Akershus Region has 11.06% of the entire population in Norway and thus seemed representative for the entire nation ( Table 1 ). All of the composites were non-expired and of the same shade (A3).

All the composites were cured with the same LED curing light, LEDemetron II Light (1600 mW/cm2) (Kerr Corporation, Orange, CA, USA), selected on the basis of recommended light intensities for the different material manufacturers (Z250 > 400 mW/cm ² and 4U < 1000 mW/cm ² ). The OBL brand TD did not provide any recommendation on light intensity. The output of the curing unit was controlled by using the built-in radiometer.

2.2

Depth of cure

Depth of cure (DOC) was determined according to ISO 4049: 2009 (E) Dentistry − Polymer-based restorative materials (ISO 4049) . A stainless steel mold was used to prepare cylindrical test bodies with the same diameters (4 mm) and heights (6 mm). Six test bodies were made for every material, three from each batch. The cylindrical wells were filled with the composite paste, and all the materials were cured from the top side for 20 s. Curing depth (mm) was then calculated by dividing the measurement by two.

2.3

Flexural strength

Determination of three-point bending strength (flexural strength) was based on the standards put forth in ISO 4049 on a Zwicki (Zwick/Roell) with the testXpert (Zwick/Roell, Ulm, Germany) software. Five identical test bodies from each batch (n = 30) were prepared from stainless steel molds with the following dimensions and permissible deviations (25 ± 2)mm × (2.0 ± 0.1)mm × (2.0 ± 0.1)mm. The top and bottom surfaces were then polymerized with the use of six overlapping irradiations of 20 s on each side. The cured specimens, still embedded in the mold, were placed in a water bath (ISO 3696 grade 2 water [37 ± 1 °C)) for 15 min. The flexural strength (σ) of a material is defined as the maximum stress that a material can resist before failure when subjected to a bending load (Eq. (1) ) ; F is the maximum load (Newtons), L is the distance between supports, B is the width of the specimen, and H is the height of the specimen.

2.4

Degree of conversion

Attenuated total reflection Fourier transform infrared spectroscopy, ATR-FTIR (Spectrum 100, PerkinElmer Instruments, Waltham, MA, USA) was used to analyze the degree of conversion of monomers (percentage of reacted methacrylate) in every material on the top and bottom of the cured test specimens at the following time intervals post-cure: immediately following curing (T ₁ ); 0.5 h (T ₂ ); 1 h (T ₃ ); 4 h (T ₄ ); and 24 h (T ₅ ). A total of five specimens from each material batch (n = 30) were prepared in the previously described stainless steel metal molds. Samples were cured between two transparent polyester films to avoid oxygen inhibition of polymerization. The sampling was performed under following conditions: Mid-IR wavelength, 2 cm ⁻¹ resolution and 32 scans. The calculation of the degree of conversion (DC%) as previously described .

2.5

Polymerization shrinkage

Polymerization shrinkage was measured by utilizing a previously documented micro X-ray microcomputed tomography (μCT) methodology . μCT-scans were performed in dark conditions using desktop SkyScan 1172 (Bruker, Aartselaar, Belgium). Uncured samples (n = 3 for each composite type) with a mean size of 40.21 mm ² were mounted vertically in customized tubes. Scanning parameters were set to 17.77 μm pixel size, x-ray source with 100 kV and 100 mA and using 500 μm Al and 38 μm Cu filters. Samples were rotated 360° around their vertical axis with a rotational step of 0.7°. Next, the composites were cured for 60 s with the LEDemetron II Light to ensure optimal curing. This method allowed for scanning of 5 samples at a time. Shrinkage was calculated based on the differences in volume evaluated using calculation described by Sun & Lin-Gibson where shrinkage is S _μCT , volume of uncured composite is V1, and volume of cured composite is V1 (Eq. (2) ):.

2.6

SEM-EDS

Morphology of fillers and distribution in the matrix were determined by scanning electron microscopy (SEM) in a Hitachi Analytical Table Top Microscope/Benchtop SEM TM3030 (Hitachi High-Technologies Europe, Berkshire, UK) operating at an accelerating voltage of 15 kV. For determination of the morphology of the filler uncured material was dissolved in acetone and chloroform, centrifuged and the particles dissolved in ethanol according to a washing technique by Beun et al. . The EtOH-filler suspension was smeared on a glass slide, desiccated at 37 °C for 6 h and gold sputtered before observation at ×3000 and ×5000 magnifications. Elemental analysis for the determination of elements in the filler particles was performed by the Bruker Quantax 70 energy dispersion X-ray spectrometer (EDS) attachment on the aforementioned apparatus on the inorganic fillers, but without any coating.

2.7

Hardness

Five square specimens of each material (n = 15) were cured in a steel mold (7 mm × 7 mm × 2 mm) between two strips of polyester to avoid oxygen inhibition of curing and to obtain a smooth surface. Ten indentations were made on both sides of the samples, at a load of 1.00 kg for 15 s using a Zwick/Roell ZVH30 (Zwick/Roell). The indentations were placed from one side to the other within the area directly under the curing tip.

2.8

Surface roughness parameters

Surface roughness parameters for the different materials were determined after three-body abrasion in a modified (Minimize, Buehler GmbH, Dusseldorf, Germany) toothbrush and slurry/reference toothpaste (ISO 11609: 2010 (E)) apparatus. Slurry/reference toothpaste was mixed from ISO Silica (SIDENT ^® , AT25747, EVONIK Industries, Hanau, Germany). Test bodies were polished with 4000 grit sandpaper surface parameters measured with 50× objective (Nikon, Japan) on a profilometer (Sensofar PLμ 2300, Terrassa, Spain) (n=9 per composite). The toothbrush bristle heads (Butler Gum 311, GUM, Chicago, IL, USA) were kept 24 h in ISO 3696 grade 2 water at 37 ± 1 °C prior to testing. After curing, the test bodies were stored at constant temperature (37 ± 1 °C) ISO 3696 grade 2 water before circular brushing (simulating Fone’s brushing technique) with 30,000 brush cycles commenced. After brushing, the test bodies dried at 37 °C for 24 h before surface parameters (surface roughness (Sa) and total peak height (St)) were measured and compared with measurements procured before abrasion.

2.9

Filler content

The total filler content (wt.%) of inorganic fillers was determined for each of the materials using the thermogravimetric analysis apparatus STA 449 F3 (Netzsch GmbH, Selb, Germany). The mass of a substance was monitored as a function of temperature or time as a sample specimen is subjected to a controlled temperature program . The composite sample was placed in an aluminum crucible (DSC/TG pan Al) and heated at a flow rate of 20 °C/min to 610 °C under nitrogen atmosphere.

2.10

Monomer elution (HPLC)

Residual monomer analysis was based on the guidelines for analysis of ISO 20795-1: 2013 (E) . The amount of residual monomer is presented as weight percentage of the organic matrix (resin). Cured material was stored in acetone for seven days prior to liquid chromatographic analysis with UV detection (UV wavelength 205 nm) in an Agilent 1100 HPLC (Agilent Technologies, Santa Clara, CA, USA). Chromatography was performed at ambient temperature using a Symmetry C18 column (150 mm × 150 mm, 5 μm particle size, 100 Å pore size) with an injection volume of 50 μl; flow rate: 1 ml/min; eluent A: acetonitrile in H ₂ O (50:50 v/v%); eluent B: acetonitrile. The following gradient was used: 0–5 min 100% eluent A, 5–10 min 20% eluent A, 10–20 min 20% eluent A, 20–22 min 100% eluent A. The external standards ( Table 2 ) were used for ten different concentrations from 0.1 μg/ml to 100 μg/ml with logarithmic increments. The standard curves were fitted with r ² > 0.99. The materials were tested for a selection of monomers based on composite composition given by the respective manufacturers ( Table 2 ).

2.11

Biocompatibilty and cytotoxicological analyzes

The biocompatibility and cytotoxicological analyzes were based on ISO 10993-5: 2009 (E) and ISO 7405: 2008 (E) . The cytotoxicity and metabolic activity were assessed in cell cultures of A549 cells (a human epithelial, lung carcinoma cell line), human gingival fibroblasts (HGF), and on primary human osteoblasts after 24 h and 48 h incubation in conditioned medium. Twelve identical struts for each composite type were prepared according to the method described for flexural testing. Two struts made up one sample and six replicates were prepared for each composite type (n = 18). After curing the samples were rinsed with deionized water. The composite struts (200 mm ² ) was incubated in 6.25 ml of cell culture medium, corresponding to a ratio of 6.25 ml/day per tooth (following a major work’s recommendation ).

The culture growth medium for the A549 cells contained low glucose DMEM GlutaMAX cell culture medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Biosera, Boussens, France), 100 U/ml penicillin and 100 μg/ml streptomycin (Biowest, Nuaille, France). The A549 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS, PAA Laboratories), 100 U/ml penicillin and 100 μg/ml streptomycin (Biowest, Nuaille, France). Cells were subcultured 1:4 before reaching confluence using PBS and trypsin/EDTA .

HGF were obtained from Provitro GmbH (Berlin, Germany). Cells were cultured at standard conditions of 37 °C and 5% CO ₂ , and maintained in low glucose DMEM GlutaMAX cell culture medium (Life Technologies,) supplemented with 10% foetal bovine serum (Biosera, Boussens, France), 100 U/ml penicillin and 100 μg/ml streptomycin (Biowest, Nuaille, France). Cells were subcultured1:4 before reaching confluence using PBS and trypsin/EDTA .

Primary human osteoblast (NHO) (Lonza, Walkersville, MD, USA) were cultured in osteoblast basal media (OBM; Lonza) supplemented with 10% fetal bovine serum, 0.1% GA-1000 and 0.1% ascorbic acid.

The composite samples were stored for 24 h at 37 °C and 5% CO ₂ . The resulting extracts were subsequently transferred to sterile microcentrifuge tubes: “24 h extracts”; and stored at 4 °C. Immediately after taking the first “24 h extracts”, the composites were transferred to fresh 6.25 ml of cell culture media to see if any additional monomer eluted from the composites after another 24 h would still have a cytotoxic potential. All groups were placed for 24 h at 37 °C and 5% CO ₂ . The resulting extract was subsequently transferred to sterile microcentrifuge tubes: “48 h extracts”; and stored at 4 °C. All extracts were pre-warmed at 37 °C for 12 h prior to cytotoxicity testing. To test the effect of the liquid extracts of the different dental composites on cell toxicity, 2 × 10 ⁴ cells were seeded in each well (48-well plate) and cultured with growth medium for 72 h. After this period, growth media was changed and replaced with the liquid extracts of dental composites (n = 6) for 24 h. In addition, untreated cells with culture media (low control, n = 6) and cells cultured with culture media supplemented with Triton X-100 1% (high control, n = 6) were used as assay controls according to manufacturer’s instruction (Roche Diagnostics, Mannheim, Germany).

Lactate dehydrogenase (LDH) activity in the culture media after 24 h incubation with the exudates was used as an index of cell death. LDH activity was determined spectrophotometrically after 30 min incubation at 25 °C of 100 μl of culture and 100 μl of the reaction mixture by measuring the oxidation of NADH at 490 nm in the presence of pyruvate, according to the manufacturer’s kit instructions (Roche Diagnostics). Results are presented relative to the LDH activity in low control and high control (set to be 100% cell death) :

For cell morphology visualization of the HGF and A549 cells, cells cultured for 24 h with the different extracts were fixed for 15 min with 4% formaldehyde in PBS at room temperature. Representative phase-contrast images of cells were taken at 10× of magnification, and compared to untreated cells at the same time point.

2.12

Statistics

Statistical analyzes were performed using the statistical software SigmaPlot 13.0 (Systat Software, San Jose, USA). All tests were performed at a confidence level of 95% and post hoc retrospective power analyzes were performed to find the statistical power of the tests (alpha = 0.050). Normality (Shapiro-Wilk [p-value to reject 0.050]) and equal variance tests (Brown-Forsythe [p-value to reject 0.050]) were performed prior to further statistical testing of the combined batch values. When the datasets were found normally distributed, statistical comparison of the different groups was performed using one-way analysis of variance (ANOVA) test followed by post hoc tests for pairwise comparisons performed using Student–Newman–Keuls test. The datasets that failed normality or equal variance test were analyzed using non-parametric Kruskal–Wallis one-way ANOVA with multiple comparisons performed using Tukey test, or Dunn’s method in case of differences in the group sizes. Statistical significance was considered at a probability p < 0.05. Comparison of the means was performed using Student t -test after testing for normality. Mann-Whitney Rank Sum Test was used for failed normality for Student’s t -test. Statistical significance was considered at p < 0.050.

A correlation study was performed on all tested parameters with a bivariate regression analysis, Spearman two-tailed, using the computer software Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA) version 22.0 for Windows. The results were interpreted as follows: no correlation if |r| < 0.3, correlation if 0.3 < |r| < 0.5, and strong correlation if 0.5 < |r| < 1. A negative r indicated a negative correlation while a positive r indicated a positive correlation. .

2.1

Selection of materials and curing light

The examined composite materials were the RBC and OBLs with the largest purchasing volume in the public dental services in the Akershus Region of Norway). Akershus Region has 11.06% of the entire population in Norway and thus seemed representative for the entire nation ( Table 1 ). All of the composites were non-expired and of the same shade (A3).

All the composites were cured with the same LED curing light, LEDemetron II Light (1600 mW/cm2) (Kerr Corporation, Orange, CA, USA), selected on the basis of recommended light intensities for the different material manufacturers (Z250 > 400 mW/cm ² and 4U < 1000 mW/cm ² ). The OBL brand TD did not provide any recommendation on light intensity. The output of the curing unit was controlled by using the built-in radiometer.

2.2

Depth of cure

Depth of cure (DOC) was determined according to ISO 4049: 2009 (E) Dentistry − Polymer-based restorative materials (ISO 4049) . A stainless steel mold was used to prepare cylindrical test bodies with the same diameters (4 mm) and heights (6 mm). Six test bodies were made for every material, three from each batch. The cylindrical wells were filled with the composite paste, and all the materials were cured from the top side for 20 s. Curing depth (mm) was then calculated by dividing the measurement by two.

2.3

Flexural strength

Determination of three-point bending strength (flexural strength) was based on the standards put forth in ISO 4049 on a Zwicki (Zwick/Roell) with the testXpert (Zwick/Roell, Ulm, Germany) software. Five identical test bodies from each batch (n = 30) were prepared from stainless steel molds with the following dimensions and permissible deviations (25 ± 2)mm × (2.0 ± 0.1)mm × (2.0 ± 0.1)mm. The top and bottom surfaces were then polymerized with the use of six overlapping irradiations of 20 s on each side. The cured specimens, still embedded in the mold, were placed in a water bath (ISO 3696 grade 2 water [37 ± 1 °C)) for 15 min. The flexural strength (σ) of a material is defined as the maximum stress that a material can resist before failure when subjected to a bending load (Eq. (1) ) ; F is the maximum load (Newtons), L is the distance between supports, B is the width of the specimen, and H is the height of the specimen.

Stay updated, free dental videos. Join our Telegram channel

Join