2.1

Specimen preparation

Discs of experimental PICN (PICN, n = 40) and commercial 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 = 40), 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 scaffold (Vita Mark II, Vita Zahnfabrik, Bad Säckingen, Germany) 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, the discs were sterilized by UV exposure for 30 min per face.

2.2

Surface characterization

The surface of all samples was characterized in a previous study by X-ray photoelectron spectroscopy, contact angle measurement, profilometry and scanning electron microscopy .

2.3

HGK study

2.3.1

Cell culture and seeding

All manipulations were made 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 Keratinocytes 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). The epithelial portion of the explant was separated from the conjunctival part using a scalpel. The epithelial portion was placed in 0.25% trypsin without EDTA (Gibco) at 4 °C overnight. The sample was then scrubed with a scalpel in a Petri dish and cells were harvested with FBS (Gibco). The suspension was centrifuged at 200 g for 5 min, the supernatant was removed and the pellet resuspended in a complete medium composed of Keratinocyte Serum Free Medium (KSFM Supplement: Human Recombinant Epidermal Growth Factor (EGF 1-53) & Bovine Pituitary Extract (BPE)) (Gibco) supplemented with 200 IU/ml penicillin-200 μg/ml streptomycin (Lonza). The solution was then filtered in a 100 μm filter (cell strainer BD Faclcon TM, Dutscher, Brumath, France) and the filter was rinsed with additional medium. Wells of a 12-well plate culture were filled with 2 ml of this solution and the plate was placed in the incubator. 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 detachment was performed with 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 ). 135 “disk-insert” systems were assembled: 3 (technical triplicate) for each material (PICN, Ti, Zi, eM, PTFE) and for each incubation time (24 h, 48 h and 72 h), the test being triplicated with cells coming from 3 different patients (biological triplicate). The systems were distributed in 24-well plate cultures and 2.10 ⁴ cells in 1 ml of culture medium were seeded on each disk. 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, 2016; 68:2437-48. With permission of Springer.

2.3.2

Cell viability assay

The living cell population on the different materials was evaluated by a viability test (MTS assay, Promega, Madison, USA) directly on discs at 24 h, 48 h and 72 h. Cellular viability of 100% was attributed to the HGK 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, 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 covering on discs and their morphology were determined from microscopic fluorescent images. Cell permeabilization was performed 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. Vinculin was stained with mouse anti-vinculin (Sigma) and rabbit anti-mouse alexa fluor 568 (Life Technologies, Carlsbad, California, United States), the incubation was performed in 0.05% Tween-20 (Sigma) in PBS solution with corresponding dilutions (1:200), at 37 °C for 1 h for mouse anti-vinculin and at room temperature for 30 min for rabbit anti-mouse alexa fluor 568. 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) at 37 °C for 1 h. A nuclear stain dye DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) (D8417-1MG, Sigma)/PBS 1:5000 was 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 a 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× objective) 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. A qualitative analysis of cellular morphology on discs was conducted with the fluorescent actin and DAPI staining at the three time points with the 10× objective and with the fluorescent actin, DAPI and vinculin staining at 72 h with a UPLSAPO 20× oil objective (Olympus). It must be noted that the observed activated form of vinculin, which mediates cell adhesion, appears at the cell periphery in well-organized focal adhesion plaques while the vinculin present in the cytosol is the inactivated form, which does not contribute to adhesion .

2.3.4

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).

2.4

Analysis of monomer release using 
ultra high-performance liquid chromatography coupled to mass tandem spectrometry (UPLC–MS/MS)

The UDMA release from PICN samples was quantified by mass spectrometry. Because they do not contain monomers, eM samples were used as negative controls. 3 PICN and 3 eM discs were incubated at 37 °C for 72 h with 1 ml of complete medium used for HGK culture (KSFM). 20 μl of a 10 mg/l internal standard solution (benzophenone-3 d5 98% chemical purity from C/D/N Isotopes Inc., Quebec, Canada) and 1 ml of Milli-Q water (>18 MΩ) were added to 0.5 ml of KSFM, and extracted twice with 2 ml of ethyl acetate. Combined organic phases were evaporated until dryness under a gentle stream of nitrogen at 30 °C, and reconstituted in 100 μl of a 70:30 (v:v) water–acetonitrile solution prior analysis by UPLC–MS/MS. The Acquity UPLC system (Waters, Milford, MA, USA) was equipped with an Acquity HSST3 column (2.1 × 100 mm, 1.8 μm) from Waters, maintained at 35 °C. The injection volume was 10 μl. The mobile phases A and B consisted, respectively in water acidified by 0.1% of formic acid and acetonitrile. The gradient started at a constant flow of 0.4 ml/min with 70% A held for 1 min, then linearly decreased to 10% A in 9 min, held for 2 min, returned to the initial conditions in 0.5 min and finally maintained for 2.5 min prior to the next injection. The Quattro Premier XE mass spectrometer (Waters) operated in positive electrospray mode with the capillary and cone voltages set at 3.2 kV and 30 V respectively. The source and desolvatation temperatures were set at 120 °C and 350 °C, with nitrogen used as cone and desolvatation gas at a flow of 50 l/h and 800 l/h. The collision gas was argon (99.99997%, Air Liquide, Liege, Belgium) settled at a flow of 0.15 ml/min. Two transitions for UDMA were monitored (493.2 > 407.2 and 113.2 > 69.3) in multiple reactions monitoring (MRM) to check ion ratios and ensure proper compound identification. The transition 234.16 > 151.2 was selected for the internal standard (benzophenone-3 d5). The collision energy was set at 30 V for both UDMA transitions and 21 V for benzophenone-3 d5. The quantification was performed using a matrix-matched calibration curve built by spiking KSFM with increasing levels of UDMA to obtain 8 points from 0.5 to 100 ng/ml. The limit of quantification was set at 0.5 ng/ml. Moreover, a home-made quality control was also analyzed, consisting in KSFM sample spiked at 50 ng/ml.

2.5

Indirect cell contact cytotoxicity assay

L929 mouse fibroblasts were grown in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin (Lonza) under standard cell culture conditions (37 °C and 5% CO ² ). The cytotoxicity of PICN and eM materials was tested as follows using a 24-well plate: 6 wells were filled with 1 ml of DMEM complete medium covering a PICN disk (DMEM + PICN), 6 with 1 ml of DMEM complete medium covering an eM disk (DMEM + eM), and 6 with 1 ml of DMEM complete medium without a disk (DMEM). The plate was placed in the incubator. The conditioned DMEM was collected after 24 h for half of the wells, and after 72 h for the other half. For the cytotoxicity assay, L929 fibroblasts were seeded into 96-well plates at 2000 cells/well and incubated for 24 h to allow cell adhesion (n = 21 wells). The medium was then replaced by 200 μl of conditioned DMEM from the 3 different groups (n = 18 wells). Moreover, three wells served as a control (Control), with cells exposed to non-conditioned (fresh) DMEM complete medium. After an incubation of 72 h, cell viability was evaluated using MTS assays (Promega), as described previously. Cell viability was calculated as a percentage of the control group. The data were treated by a variance analysis with respect to fixed and random effects. The level of significance was set at 5% (p < 0.05).