Biocompatibility of polymer-infiltrated-ceramic-network (PICN) materials with Human Gingival Fibroblasts (HGFs)

Graphical abstract

Highlights

  • Titanium (Ti) and zirconia (Zi) gave the best results in terms of HGF behavior.

  • PICN materials and lithium disilicate glass-ceramics (eM) gave intermediate results.

  • Good results of PICNs compared to eM could be due to their polymerization mode.

  • Clinical impact of these results needs to be investigated on bone-level implants.

Abstract

Objectives

Polymer-infiltrated-ceramic-network (PICN) materials constitute an innovative class of CAD-CAM materials offering promising perspectives in prosthodontics, but no data are available in the literature regarding their biological properties. The objective of the present study was to evaluate the in vitro biocompatibility of PICNs with human gingival fibroblasts (HGFs) in comparison with materials typically used for implant prostheses and abutments.

Methods

HGF attachment, proliferation and spreading on discs made of PICN, grade V titanium (Ti), yttrium zirconia (Zi), lithium disilicate glass-ceramic (eM) and polytetrafluoroethylene (negative control), were evaluated using a specific insert-based culture system (IBS-R). Sample surface properties were characterized by XPS, contact angle measurement, profilometry and SEM.

Results

Ti and Zi gave the best results regarding HGF viability, morphology, number and coverage increase with time in comparison with the negative control, while PICN and eM gave intermediate results, cell spreading being comparable for PICN, Ti, Zi and eM. Despite the presence of polymers and their related hydrophobicity, PICN exhibited comparable results to glass-ceramic materials, which could be explained by the mode of polymerization of the monomers.

Significance

The results of the present study confirm that the currently employed materials, i.e. Ti and Zi, can be considered to be the gold standard of materials in terms of HGF behavior, while PICN gave intermediate results comparable to eM. The impact of the present in vitro results needs to be further investigated clinically, particularly in the view of the utilization of PICNs for prostheses on bone-level implants.

Introduction

Biological properties of indirect restorative materials are mandatory, especially for implant prostheses, which are in direct contact with the gingival tissue and can extend to the bone level ( Fig. 1 ). Indeed, the prosthesis connects the internal tissues with the external oral environment and a proper biological seal is needed to create a hermetic barrier able to protect peri-implant structures from bacterial penetration. This biological seal, called “biological width”, is located just above the bone level and is composed of 2 distinct parts: a connective tissue attachment of 1 or 2 mm in length and, above it, an epithelial tissue attachment (junctional epithelium) of ±2 mm in length ( Fig. 1 ). Depending on implant type, its neck can emerge from the bone (“tissue-level” implant), providing more or less space for the biological width to be created on the implant titanium surface. When the implant neck is buried within the bone (“bone-level” implant), the biological width needs to be developed either on the transgingival part of the abutment or of the screw-retained restoration, which must promote the attachment of fibroblasts and keratinocytes. Indeed, if this material does not promote cell adhesion the biological width recedes to reestablish below, i.e. on the implant neck . Consequently, a bone resorption and a gingival recession occur, which might compromise the health of the peri-implant tissues as the esthetic result . Consequently, cell attachment properties of the abutment and the prosthesis material are crucial for the stability of bone and gingival tissues and the long-term success of implant prostheses, particularly for bone-level implants.

Fig. 1
Schematic illustration of hard and soft tissue around an implant. The biological width is located above the bone level and is composed of 2 distinct parts: the connective tissue and the junctional epithelium. Depending on implant type, its neck can emerge from the bone (“tissue-level” implant), or can be buried within the bone (“bone-level” implant).

Implant abutments and prostheses are often made of metal alloys and ceramic materials, mainly titanium and yttria-tetragonal-zirconia-polycrystal (Y-TZP), while glass-ceramics reinforced with lithium disilicate (Li 2 Si 2 O 5 ) can also be used, notably for abutments cemented on a titanium base. Yet, over the last few years, new computer-aided design and computer-aided manufacturing (CAD-CAM) composite materials have flooded the market of indirect restorative materials. Recent advances introduced with CAD-CAM composite blocks are related to the development of new polymerization modes using high temperature (HT) or a combination of high temperature and high pressure (HT-HP). CAD-CAM composites are also associated with innovative microstructures and two classes are distinguished: those with dispersed fillers and polymer-infiltrated-ceramic-network materials (PICNs) . Also called hybrid ceramics by the manufacturer, PICNs differ significantly from other composite materials with dispersed fillers classically incorporated by mixing, since they are the result of the infiltration of a pre-sintered glass-ceramic scaffold with urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) monomers, which are secondarily polymerized. The interpenetrating network technology associated with the HT-HP polymerization process (300 MPa, 180 °C) yield materials with significantly enhanced mechanical properties in comparison with light-cured composites . Moreover, a recent in vitro study showed that HT-HP UDMA exhibits very low monomer release in comparison with light-cured UDMA . PICNs reached the market in 2013 (Enamic, Vita, Bad Säckingen, Germany) for use as dental prostheses on natural teeth, and more recently on implants ( Fig. 2 ). Indeed, they exhibit significant advantages in comparison with ceramics, notably in terms of elasticity modulus, which is closer to dental tissues, and in terms of machinability with CAD-CAM systems since they are not as brittle, exhibit a higher damage tolerance and resistance to marginal chipping, and can be milled more rapidly and to a very low thickness .

Fig. 2
PICN dental implant crown to restore a mandibular molar (tooth #46) (Photo courtesy of A. Mainjot.).

  • (a)

    Tissue-level implant (Institut Straumann AG, Basel, Switzerland) with its titanium healing cap, just after the surgery and before immediate loading with a PICN crown. Note that in this case the biological width can be created totally or partially on the implant titanium neck, contrary to bone-level implants. In this pilot case, the immediate loading choice was promoted by the favorable elasticity modulus of PICN in comparison with metallic or ceramic materials. Optical impression was taken with the Omnicam intra-oral scanner (Sirona, Salzburg, Austria).

  • (b)

    PICN CAD-CAM block (Vita Enamic, Vita Zahnfabrik, Bad Säckingen, Germany) before milling.

  • (c)

    Crown just after chair-side manufacturing with the Cerec MCXL CAD-CAM machine (Sirona, Salzburg, Austria) (Video 1).

  • (d)

    Crown 3 months after placement, showing a nice gingival tissue healing around the prosthesis.

  • (e)

    Picture of the final crown design: the PICN crown is cemented on a titanium base.

  • (f)

    Radiograph after 3-month follow-up.

Many studies have focused on the biocompatibility properties of titanium and Y-TZP, several authors having shown a good in vitro cytocompatibility of zirconia and titanium with Human Gingival Fibroblasts (HGFs) in terms of attachment, proliferation, and expression of integrin, of collagen and of focal adhesion linker proteins. If the two materials have given comparable results in most studies in terms of fibroblast attachment and proliferation , some authors have shown a better HGF attachment on titanium and others a better proliferation on zirconia . Unfortunately, a comparison of these results is not possible since material roughness and evaluation times varied considerably from one study to the other. In vivo , a recent review selected nine relevant clinical studies and highlighted better esthetic results for zirconia, but no statistically significant difference between zirconia and titanium regarding gingival and bone tissue response . Regarding lithium disilicate glass-ceramics, HGF behavior on this material has been poorly studied either in vitro or in vivo . Tete et al. reported a lower type I collagen secretion of HGFs on lithium disilicate glass-ceramic compared to zirconia, which could cause a slower growth of HGFs on this substrate . Other authors observed cytotoxic effects . Finally, to the authors’ knowledge, there are still no data in the literature about the biocompatibility of PICNs.

Consequently, the objective of the present study was to evaluate the biocompatibility of PICNs in comparison with metallic and ceramic materials used for dental implant prostheses, evaluating attachment, proliferation and HGF spreading.

Materials and methods

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.

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.

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 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.

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.

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.

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).

Cell study

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.

Fig. 3
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.

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).

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.

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.

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).

Materials and methods

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.

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.

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 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.

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.

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.

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).

Cell study

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.

Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Biocompatibility of polymer-infiltrated-ceramic-network (PICN) materials with Human Gingival Fibroblasts (HGFs)
Premium Wordpress Themes by UFO Themes