2.1

Specimen preparation

Discs of commercially experimental PICN (PICN, n = 34), grade V titanium (Procera, Nobel Biocare, Göteborg, Sweden) (Ti, n = 34), Y-TZP (Procera, Nobel Biocare) (Zi, n = 34), lithium disilicate glass-ceramic (IPS e.max Press, Ivoclar Vivadent, Schaan, Liechtenstein) (eM, n = 34), and polytetrafluoroethylene (Sirris, Seraing, Belgium) (PTFE, negative control, n = 34), 13.5 mm in diameter, were produced. The experimental PICN (MaJEB sprl, Liège, Belgium) was made by infiltration of a pre-sintered glass-ceramic (Vita Mark II, Vita Zahnfabrik, Bad Säckingen, Germany) scaffold with a monomer (UDMA), and was polymerized under HP/HT (300 MPa, 180 °C) conditions. The PICN discs were cut out of a block and were rounded by polishing. Ti and Zi discs were manufactured by milling with the CAD-CAM Procera process (Nobel Biocare). eM samples were pressed into molds by following the manufacturer’s instructions.

All specimens were sequentially ground with 800-grit, 2400-grit and 4000-grit silicon carbide discs (Planopol 3 ^® , Struers, Ballerup, Denmark) to a 1 ± 0.02 mm thickness. They were ultrasonically cleaned in a detergent (Elmasonic One product, Singen, Germany and RBS T105 ^® 2%, Chemical Products, Brussels, Belgium) for 10 min, rinsed with phosphate-buffered saline solution (PBS, Lonza Group Ltd., Basel, Switzerland), rinsed 4 times with purified water (Milli-Q ^® system, Merck Millipore Corporation, Billerica, Massachusetts, United States) and then ultrasonically cleaned in ethanol 80%. Finally, discs were sterilized by UV exposure for 30 min per face.

2.2

Surface characterization

After cleaning and UV exposure, 4 disk samples per group were used for surface analyses using X-ray photoelectron spectroscopy, contact angle measurement, profilometry and scanning electron miscroscopy, respectively.

2.2.1

X-ray photoelectron spectroscopy

The elemental composition and distribution of the discs were studied using X-ray photoelectron spectroscopy (XPS). The XPS analyses were performed in a PHI-Quantum 2000 spectrometer (Physical Electronics, Chanhassen, Minnesota, USA) directly connected to the preparation chamber. The spectrometer is characterized by a monochromatized Al Kα primary X-ray beam and a photoelectron take-off angle of π /4 against the sample normal direction. Quantitative analysis was based on sensitivity coefficients suggested by the equipment manufacturer. Charge effects were compensated by electron and argon low energy beams.

2.2.2

Contact angle measurement

The sessile drop method was used for contact angle measurements with a dual-gradient density contact angle meter (Digidrop, DGD Fast/60, GBX, Bourg de Peage, France) coupled to Windrop software (GBX). The static contact angles were measured by the water-droplet method after deposition of 15 μL deionized water on the dry disk surfaces. Measurements were made at three different locations on each sample at 30 s, 60 s, 90 s and 120 s after application of each droplet at room temperature.

2.2.3

Profilometry

To determine surface roughness, images were acquired with a ContourGT-I 3D Optical Microscope (Bruker, Tucson, Arizona, USA) using the Vertical Scanning Interferometry mode (VSI). The vertical resolution in VSI mode is ∼2 nm independent of the objective. Five measurements were performed on each material at different locations. All images were acquired with the 50× objective (image size 0.13 × 0.10 mm ² , optical lateral resolution 0.5 μm). The tilt and cylinder terms were removed and no filtering was used.

2.2.4

Scanning electron microscopy

The morphological analysis of different surface structures was determined with an Environmental Scanning Electron Microscope with a Field Emission Gun (ESEM-FEG XL-30, FEI, Hillsboro, Oregon USA) used in high vacuum mode. Over-coated samples were observed using a detector of secondary electrons to determine the topography and a detector of backscattered electrons to acquire images in contrast of Z (elements atomic number).

2.3

Cell study

2.3.1

Cell culture and seeding

All manipulations were done under sterile conditions (L2 laminar flow hood). The cells were cultured under the condition of 5% CO ₂ and 37 °C (Binder incubator, Tuttlingen, Germany). Primary cultures of human gingival fibroblasts were established using the explant technique. The gingival tissue samples were obtained from healthy adult patients undergoing dental surgery at the oral and maxillofacial surgery department at the University Hospital of Liège. For tissue harvest, informed consent was obtained from the patients and the protocol was approved by the Ethics Committee of the University Hospital of Liège, Belgium (October 8, 2013). Explants were immediately stored at 4 °C in a transport medium composed of Dulbecco’s Modified Eagle Medium (DMEM high glucose, GlutaMAX™ Supplement, pyruvate, Gibco, Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum (FBS, Gibco), 500 μg/ml gentamicin (Gibco), 200 IU/ml penicillin-200 μg/ml streptomycin (Lonza, Verviers, Belgium) and 25 μg/ml fungizone (Gibco). Explants were minced into small fragments (1 mm ³ ) using a blade and were distributed on a Petri dish. 5 ml of complete medium composed of DMEM (DMEM high glucose, GlutaMAX™ Supplement, pyruvate, Gibco) supplemented with 10% FBS (Gibco), 50 μg/ml ascorbic acid (Sigma Aldrich, St Louis, USA) and 100 IU/ml penicillin-100 μg/ml streptomycin (Lonza), were gently added, taking care to not detach the fragments. The cultures were kept for 2–4 weeks in the incubator until near confluence. Cells were washed with phosphate-buffered saline (PBS) solution without calcium and magnesium (DPBS, Lonza) and detached using 0.05% trypsin-EDTA solution (Gibco). Cells were expanded in T75 flasks by passing a 1:3 split. Stocks from different patients were stored in liquid nitrogen (complete medium—dimethylsulfoxide 10%). All experiments were conducted at the 3rd to 6th passage.

Cell seeding was performed following a method described previously , using specific cylindrical inserts, which ensure an efficient seal around the discs (Insert-Based System for Rigid biomaterial, IBS-R). These cylindrical inserts (2 mm thick, 15.5 mm high and 15.5 mm in diameter) were machined from medical-grade PTFE (Fluteck P2000, Sirris, Liège, Belgium) (Hardinge Model HLV-H Precision, Hardinge Inc., Elmira, NY, United States). In the thickness of the bottom of the cylinder, a 1 mm high and 1 mm thick bevel was milled in order to clip it to a disk sample with a light friction ( Fig. 3 ). 15 “disk-insert” systems were assembled for SEM analysis: one for each material (PICN, Ti, Zi, eM, PTFE), the test being triplicated with cells coming from 3 different patients (biological triplicate); and 135 for cell viability assay and immunofluorescence staining: 3 (technical triplicate) for each material and for each incubation time (24 h, 48 h and 72 h), in biological triplicate. The systems were distributed in 24-well plates cultures. Cells were seeded at a concentration of 5 × 10 ³ cells in 1 ml of culture medium. After the different incubation times, the PTFE inserts were removed from the discs.

Drawing (a) and picture (b) of the PTFE insert from Cytotechnology, a new method using insert-based systems (IBS) to improve cell behavior study on flexible and rigid biomaterials, 2016, Grenade C, Moniotte N, Rompen E, Vanheusden A, Mainjot A, De Pauw-Gillet M-C, . With permission of Springer.

2.3.2

Cell viability assay

To evaluate the living cell population on the different materials, a viability test was then performed directly on discs at 24 h, 48 h and 72 h. Cellular viability of 100% was attributed to the HGF grown in control polystyrene (PS) wells without discs. Cellular viability was quantified by a colorimetric assay using 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (inner salt MTS, Promega, Madison, USA). MTS solutions were prepared according to the manufacturer’s instructions. Discs were rinsed with 1 ml of DMEM/F-12 (Gibco) and 1 ml of fresh DMEM/F-12 with MTS solution (10%) was applied. The plates were incubated at 37 °C in the absence of light for 45 min. The plates were then shaken for 15 s. 200 μl of supernatant was removed from each well and placed in 96 well microplates. The absorbance of supernatant aliquots was read at 492 nm using the Powerwave X microplate spectrophotometer (Biotek instrument Inc., Winooski, VT, USA) and the viability was calculated and normalized from the absorbance of control samples taken as 100% (PS).

2.3.3

Immunofluorescence staining

After the MTS assay, the discs were rinsed with PBS and the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 20 min. An immuno-staining was then performed and the number of cells, their coverage on discs and their morphology were determined from microscopic fluorescent images. Cells were permeabilized with 0.5% Triton X-100 (Sigma) at 4 °C for 20 min. Blocking was performed with 1% BSA (Sigma) in PBS at 37 °C for 1 h. Actin was stained by incubating in 0.05% Tween-20 (Sigma) in PBS solution with 1:40 dilution of Alexa fluor 488-labeled phalloidin (Life Technologies, Carlsbad, California, United States) at 37 °C for 1 h. A nuclear stain dye DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) (D8417-1MG, Sigma)/PBS 1:5000 was then added and allowed to set in at room temperature for 10 min. The stained sample surface was imaged with an IX81 optical inverted microscope equipped with an UPlanFL objective at 10× magnification and with an XCite-iris IX fluorescence unit and a C-BUN-F-XC50 charge-coupled-device camera (Olympus Optical Co., Ltd). An image analysis software, “CellSens” (Olympus), was used to quantify the number of cells and their coverage on discs. 350 pictures (×10 magnification) per sample were performed to allow the analysis of the entire disk surface. Cell spreading was calculated by dividing the covered surface by the number of cells.

2.3.4

Cellular morphology

A qualitative analysis of cellular morphology on discs was conducted with the fluorescent actin staining at the tree time points and by classical SEM methods at 48 h using glutaraldehyde (2.5%) in a sodium cacodylate buffer 0.1 M, pH 7.3, desiccated to critical point and shadowed with platinum. The SEM used was a Jeol JSM-840A microscope (Jeol, Tokyo, Japan) operated at 22 kV.

2.3.5

Statistical analysis

For each variable, the data were treated by a variance analysis with respect to fixed effects (materials, time and interactions between them) and random effects. Materials were compared two by two by multiple comparisons of Scheffe. To highlight the part of the variation due to the subject and due to the disk specimen, the variance components (VC) were calculated by time and material. The level of significance was set at 5% ( p < 0.05). The calculations were carried out by means of the software SAS version 9.4 (SAS Institute, Cary, NC, the USA).

2.1

Specimen preparation

Discs of commercially experimental PICN (PICN, n = 34), grade V titanium (Procera, Nobel Biocare, Göteborg, Sweden) (Ti, n = 34), Y-TZP (Procera, Nobel Biocare) (Zi, n = 34), lithium disilicate glass-ceramic (IPS e.max Press, Ivoclar Vivadent, Schaan, Liechtenstein) (eM, n = 34), and polytetrafluoroethylene (Sirris, Seraing, Belgium) (PTFE, negative control, n = 34), 13.5 mm in diameter, were produced. The experimental PICN (MaJEB sprl, Liège, Belgium) was made by infiltration of a pre-sintered glass-ceramic (Vita Mark II, Vita Zahnfabrik, Bad Säckingen, Germany) scaffold with a monomer (UDMA), and was polymerized under HP/HT (300 MPa, 180 °C) conditions. The PICN discs were cut out of a block and were rounded by polishing. Ti and Zi discs were manufactured by milling with the CAD-CAM Procera process (Nobel Biocare). eM samples were pressed into molds by following the manufacturer’s instructions.

All specimens were sequentially ground with 800-grit, 2400-grit and 4000-grit silicon carbide discs (Planopol 3 ^® , Struers, Ballerup, Denmark) to a 1 ± 0.02 mm thickness. They were ultrasonically cleaned in a detergent (Elmasonic One product, Singen, Germany and RBS T105 ^® 2%, Chemical Products, Brussels, Belgium) for 10 min, rinsed with phosphate-buffered saline solution (PBS, Lonza Group Ltd., Basel, Switzerland), rinsed 4 times with purified water (Milli-Q ^® system, Merck Millipore Corporation, Billerica, Massachusetts, United States) and then ultrasonically cleaned in ethanol 80%. Finally, discs were sterilized by UV exposure for 30 min per face.

2.2

Surface characterization

After cleaning and UV exposure, 4 disk samples per group were used for surface analyses using X-ray photoelectron spectroscopy, contact angle measurement, profilometry and scanning electron miscroscopy, respectively.

2.2.1

X-ray photoelectron spectroscopy

The elemental composition and distribution of the discs were studied using X-ray photoelectron spectroscopy (XPS). The XPS analyses were performed in a PHI-Quantum 2000 spectrometer (Physical Electronics, Chanhassen, Minnesota, USA) directly connected to the preparation chamber. The spectrometer is characterized by a monochromatized Al Kα primary X-ray beam and a photoelectron take-off angle of π /4 against the sample normal direction. Quantitative analysis was based on sensitivity coefficients suggested by the equipment manufacturer. Charge effects were compensated by electron and argon low energy beams.

2.2.2

Contact angle measurement

The sessile drop method was used for contact angle measurements with a dual-gradient density contact angle meter (Digidrop, DGD Fast/60, GBX, Bourg de Peage, France) coupled to Windrop software (GBX). The static contact angles were measured by the water-droplet method after deposition of 15 μL deionized water on the dry disk surfaces. Measurements were made at three different locations on each sample at 30 s, 60 s, 90 s and 120 s after application of each droplet at room temperature.

2.2.3

Profilometry

To determine surface roughness, images were acquired with a ContourGT-I 3D Optical Microscope (Bruker, Tucson, Arizona, USA) using the Vertical Scanning Interferometry mode (VSI). The vertical resolution in VSI mode is ∼2 nm independent of the objective. Five measurements were performed on each material at different locations. All images were acquired with the 50× objective (image size 0.13 × 0.10 mm ² , optical lateral resolution 0.5 μm). The tilt and cylinder terms were removed and no filtering was used.

2.2.4

Scanning electron microscopy

The morphological analysis of different surface structures was determined with an Environmental Scanning Electron Microscope with a Field Emission Gun (ESEM-FEG XL-30, FEI, Hillsboro, Oregon USA) used in high vacuum mode. Over-coated samples were observed using a detector of secondary electrons to determine the topography and a detector of backscattered electrons to acquire images in contrast of Z (elements atomic number).

2.3

Cell study

2.3.1

Cell culture and seeding

All manipulations were done under sterile conditions (L2 laminar flow hood). The cells were cultured under the condition of 5% CO ₂ and 37 °C (Binder incubator, Tuttlingen, Germany). Primary cultures of human gingival fibroblasts were established using the explant technique. The gingival tissue samples were obtained from healthy adult patients undergoing dental surgery at the oral and maxillofacial surgery department at the University Hospital of Liège. For tissue harvest, informed consent was obtained from the patients and the protocol was approved by the Ethics Committee of the University Hospital of Liège, Belgium (October 8, 2013). Explants were immediately stored at 4 °C in a transport medium composed of Dulbecco’s Modified Eagle Medium (DMEM high glucose, GlutaMAX™ Supplement, pyruvate, Gibco, Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum (FBS, Gibco), 500 μg/ml gentamicin (Gibco), 200 IU/ml penicillin-200 μg/ml streptomycin (Lonza, Verviers, Belgium) and 25 μg/ml fungizone (Gibco). Explants were minced into small fragments (1 mm ³ ) using a blade and were distributed on a Petri dish. 5 ml of complete medium composed of DMEM (DMEM high glucose, GlutaMAX™ Supplement, pyruvate, Gibco) supplemented with 10% FBS (Gibco), 50 μg/ml ascorbic acid (Sigma Aldrich, St Louis, USA) and 100 IU/ml penicillin-100 μg/ml streptomycin (Lonza), were gently added, taking care to not detach the fragments. The cultures were kept for 2–4 weeks in the incubator until near confluence. Cells were washed with phosphate-buffered saline (PBS) solution without calcium and magnesium (DPBS, Lonza) and detached using 0.05% trypsin-EDTA solution (Gibco). Cells were expanded in T75 flasks by passing a 1:3 split. Stocks from different patients were stored in liquid nitrogen (complete medium—dimethylsulfoxide 10%). All experiments were conducted at the 3rd to 6th passage.

Cell seeding was performed following a method described previously , using specific cylindrical inserts, which ensure an efficient seal around the discs (Insert-Based System for Rigid biomaterial, IBS-R). These cylindrical inserts (2 mm thick, 15.5 mm high and 15.5 mm in diameter) were machined from medical-grade PTFE (Fluteck P2000, Sirris, Liège, Belgium) (Hardinge Model HLV-H Precision, Hardinge Inc., Elmira, NY, United States). In the thickness of the bottom of the cylinder, a 1 mm high and 1 mm thick bevel was milled in order to clip it to a disk sample with a light friction ( Fig. 3 ). 15 “disk-insert” systems were assembled for SEM analysis: one for each material (PICN, Ti, Zi, eM, PTFE), the test being triplicated with cells coming from 3 different patients (biological triplicate); and 135 for cell viability assay and immunofluorescence staining: 3 (technical triplicate) for each material and for each incubation time (24 h, 48 h and 72 h), in biological triplicate. The systems were distributed in 24-well plates cultures. Cells were seeded at a concentration of 5 × 10 ³ cells in 1 ml of culture medium. After the different incubation times, the PTFE inserts were removed from the discs.

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