Abstract
Objective
Ceramics are widely used materials for prosthesis, especially in dental fields. Despite multiple biomedical applications, little is known about ceramic surface modifications and the resulting cell behavior at its contact. The aim of this study is to evaluate the biological response of polished versus glazed surface treatments on lithium disilicate dental ceramic.
Methods
We studied a lithium disilicate ceramic (IPS e.max ® Press, Ivoclar Vivadent) with 3 different surface treatments: raw surface treatment, hand polished surface treatment, and glazed surface treatment (control samples are Thermanox ® , Nunc). In order to evaluate the possible modulation of cell response at the surface of ceramic, we compared polished versus glazed ceramics using an organotypic culture model of chicken epithelium.
Results
Our results show that the surface roughness is not modified as demonstrated by equivalent R a measurements. On the contrary, the contact angle θ in water is very different between polished (84°) and glazed (33°) samples. The culture of epithelial tissues allowed a very precise assessment of histocompatibility of these interfaces and showed that polished samples increased cell adhesion and proliferation as compared to glazed samples.
Significance
Lithium disilicate polished ceramic provided better adhesion and proliferation than lithium disilicate glazed ceramic. Taken together, our results demonstrate for the first time, how it is possible to use simple surface modifications to finely modulate the adhesion of tissues. Our results will help dental surgeons to choose the most appropriate surface treatment for a specific clinical application, in particular for the ceramic implant collar.
1
Introduction
Cell and tissue adhesion is a key factor in the development of new biomaterials, especially in the dental fields. Since many patients prefer dental work to be esthetically pleasing, clinicians need to find alternatives to titanium implants . To this aim, other materials such as ceramics have been developed due to their satisfactory physico-chemical and cosmetic properties, as well as their biocompatibility . Use of ceramics in the oral cavity may surpass that of metallic materials because they have improved esthetics while maintaining the same quality of oral rehabilitation . Indeed, this material offers great biological integration of fillings as well as long term quality of fixed dental prosthesis .
Among many, two specific dental applications of ceramics could be developed involving tissue–material interactions: (i) ceramics could be used in implantology to perform esthetic collar implant and (ii) in computer-aided design/computer-aided manufacturing (CAD/CAM) prosthesis to fill large cavities with bonded esthetic biomaterials.
Currently, lithium disilicate ceramic is being used more and more in place of zirconia . The fatigue behavior and reliability of lithium disilicate and zirconia all-ceramic crowns were recently described . Results from this study show that, lithium disilicate ceramic crowns in a monolithic/fully anatomical configuration were fatigue-resistant, whereas zirconia crowns were highly susceptibility to mouth-motion cyclic loading with early veneer failures . These results motivate dental clinicians to use lithium disilicate ceramic, as a more cosmetic biomaterial than zirconia without adding further clinical risks for the patient.
It is known that surface properties modify cell behavior and that clinicians can use certain surface treatments to achieve a specific cell response that is necessary to implant biomaterials in the oral cavity . Two important parameters involved in cell adhesion and proliferation are surface roughness and wettability . Here, we use a combination of interferometry and contact angle measurements to determine surface wettability together with a biological assessment of the material using organotypic culture of epithelial tissues.
The aim of this study is to evaluate for the first time, the cytocompatibility of lithium disilicate ceramic following two different surface treatments, polished versus glazed. Our study will allow the dental practitioner to finely tune the expected reaction of a tissue in order to avoid or promote its adhesion.
2
Materials and methods
Samples of ceramic were provided by Ivoclar Vivadent SAS (Saint-Jorioz, France). Ceramic is a vitro-ceramic with lithium and disilicate elements. This ceramic is nominated IPS e.max ® Press and composed of SiO 2 (57–80%), Li 2 O (11–19%), KO 2 (0–13%), P 2 O 5 (0–11%), ZrO 2 (0–8%), ZnO (0–8%), others oxides and pigments (0–10%). The crystalline phases are organized by lithium disilicate (70%) and the crystallographic design is between 3 and 6 μm (data of scientific documentation provided by Ivoclar/Vivadent in 2005).
2.1
Sample preparation
One hundred twenty square ceramic samples were separately obtained by the loose-wax technique and were completely fritted before used. The dimensions of the samples were 1.4 cm × 1.4 cm × 0.1 cm. All samples were cleaned by an acid (Invex ® , Ivoclar Vivadent) and sanded in lab with glass bead blasting before use. Sample surfaces were prepared by polishing, glazing or no treatment (forty samples per group). Control samples were cell culture treated plastic (Thermanox ® , Nunc batch # 628934).
2.1.1
Polished surface treatment
The manual mechanic polishing was realized by the same operator with the pack Optrafine ® provided by Ivoclar Vivadent (batch # NL1757). A manual dental piece was used and the sequence of polishing was always the same without water spray: 15 s with a big drill DC 83103040 provided by Komet (Paris, France) at 30,000 revs/min, 15 s with the dark blue cupule at 7000 revs/min (Polisher P), 15 s with the clear blue cupule at 7000 revs/min (Finisher F), 15 s with a little brush associated by polishing paste (Optrafine ® HP, batch # JL1606) at 7000 revs/min.
2.1.2
Glazed surface treatment
Before glazed surface treatment, samples were cleaned with pure water and dried with air spray. A liquid glass spray (IPS e.max ® CAD crystal glazed) was sprayed from 20 to 25 cm on samples. Then, samples were cooked in an oven (P100-Programat ® by Ivoclar Vivadent) at 800 °C for 25 min. Afterwards, the area cooling had to be low in order to avoid the induction of micro-failures at the surface.
2.2
Surface characterizations
2.2.1
Contact angle measurements
Contact angles ( θ ) of water droplets were determined at ambient temperature (21–24 °C) with a drop shape analysis apparatus (DSA-10, Krüss GmbH, Hamburg, Germany). The measuring system recorded the drop shape by a CCD-camera and determined the contact angles using image analysis software taking into account the entire drop shape .
Six water droplets were deposited on each surface: values of contact angle ( θ °) represented the averages of 12 contact angle measurements (6 on the right side and 6 on the left side).
2.2.2
Scanning white light interferometry
To determine the roughness of the different treatments and the control (no treatment/raw ceramic), we used a scanning white light interferometer Zygo. The Zygo ® NewView 200 is a scanning white-light interferometer that uses Frequency Domain Analysis (FDA) to generate quantitative 3D images of surfaces . The measurements used a white light filter based on a center wavelength of 600 nm, with a bandwidth of 125 nm. The interference patterns were recorded by a CCD camera and each measurement contained 320 × 240 data points. Three objectives (magnifications 2.5×, 10×, and 50×) were used and three images were recorded at each magnification. The scan size and sampling interval were fixed by the magnification of the optical system. For the magnification of 2.5×, the sampling interval was 8.8 μm in the both directions. The lateral resolution of the microscope was limited by the numerical aperture of the objective and the vertical resolution was lower than 1 nm. This technique was used to determine the biomaterial surface roughness ( R a ), using the 10× objective.
2.2.3
Scanning electron microscopy
Samples of tissues cultivated on the different materials were rinsed in PBS, fixed in 3% glutaraldehyde in Rembaum buffer (pH 7.4) for 1 h, dehydrated in a series of graded alcohols, critical-point dried from CO 2 (Polaron Instrument Inc., Nottingham, UK), sputter-coated with gold (Polaron) and examined in Philips scanning electron microscope (ESEM FEG XL 30).
2.3
Cell study
2.3.1
Organotypic culture method
Before culture, the ceramic samples were cleaned in ultrasonic baths with acetone (10 min) and rinsed in distilled water (10 min), then autoclaved at 120 °C for 20 min.
The organotypic culture method described in Fig. 1 was used to examine the cellular response to ceramic surface treatments. For full technical description see Ref. . Culture dishes were pretreated with nutrient medium consisting of 50% Bactoagar 1% (Difco, Fisher, France) in Gey’s solution , 38.5% DMEM (Gibco, Invitrogen, Cergy Pontoise, France), 10% FCS (Gibco, Invitrogen, Cergy Pontoise, France), 1% l -glutamine (Gibco, Invitrogen, Cergy Pontoise, France) and 0.05% penicillin/streptomycin solution (Gibco, Invitrogen, Cergy Pontoise, France). Epithelial tissues were isolated from 7-day-old White Leghorn’s chicken eggs and placed in sterile PBS. The tissues were cut into 1 mm 2 pieces and put onto the bottom of a 70 cm 2 Petri dish (Dominique Dutscher, Brumath, France). Nine tissue fragments were placed in each Petri dish and a 2 cm 2 piece of ceramic material was deposited on each of those fragments.
Each dish was used for only one experiment and four dishes were prepared for each material. The cultures were incubated at 37 °C with 5% CO 2 for 7 days after which the materials were removed and stained with neutral red. Next, the total surface area covered by tissue was measured using a stereomicroscope equipped with camera and ImageJ software . This area corresponded to the total surface of the cell layer minus the initial explant surface.
Additionally, the cells were detached from the materials using 0.025% trypsin–EDTA (Gibco, Invitrogen, Cergy Pontoise, France) in Isoton ® II electrolyte solution (Beckman Coulter, Villepinte, France).
The rate of cell detachment was determined by counting the detached cells with a Multisizer ® (Beckman Coulter, Villepinte, France) after 5, 10, 20, 30 and 60 min. The rate of detachment as a function of time was used as a measure of adhesion strength, and we defined an arbitrary index.
2.3.2
Statistical analysis
All data are represented as means ± standard error of means. The data were compared using a one-way ANOVA followed by a Dunnett post hoc test (InStat), thereby comparing raw ceramic (untreated), polished ceramic, glazed ceramic and the control samples Thermanox ® .
Probabilities of P < 0.05 were considered to be statistically significant.
2
Materials and methods
Samples of ceramic were provided by Ivoclar Vivadent SAS (Saint-Jorioz, France). Ceramic is a vitro-ceramic with lithium and disilicate elements. This ceramic is nominated IPS e.max ® Press and composed of SiO 2 (57–80%), Li 2 O (11–19%), KO 2 (0–13%), P 2 O 5 (0–11%), ZrO 2 (0–8%), ZnO (0–8%), others oxides and pigments (0–10%). The crystalline phases are organized by lithium disilicate (70%) and the crystallographic design is between 3 and 6 μm (data of scientific documentation provided by Ivoclar/Vivadent in 2005).
2.1
Sample preparation
One hundred twenty square ceramic samples were separately obtained by the loose-wax technique and were completely fritted before used. The dimensions of the samples were 1.4 cm × 1.4 cm × 0.1 cm. All samples were cleaned by an acid (Invex ® , Ivoclar Vivadent) and sanded in lab with glass bead blasting before use. Sample surfaces were prepared by polishing, glazing or no treatment (forty samples per group). Control samples were cell culture treated plastic (Thermanox ® , Nunc batch # 628934).
2.1.1
Polished surface treatment
The manual mechanic polishing was realized by the same operator with the pack Optrafine ® provided by Ivoclar Vivadent (batch # NL1757). A manual dental piece was used and the sequence of polishing was always the same without water spray: 15 s with a big drill DC 83103040 provided by Komet (Paris, France) at 30,000 revs/min, 15 s with the dark blue cupule at 7000 revs/min (Polisher P), 15 s with the clear blue cupule at 7000 revs/min (Finisher F), 15 s with a little brush associated by polishing paste (Optrafine ® HP, batch # JL1606) at 7000 revs/min.
2.1.2
Glazed surface treatment
Before glazed surface treatment, samples were cleaned with pure water and dried with air spray. A liquid glass spray (IPS e.max ® CAD crystal glazed) was sprayed from 20 to 25 cm on samples. Then, samples were cooked in an oven (P100-Programat ® by Ivoclar Vivadent) at 800 °C for 25 min. Afterwards, the area cooling had to be low in order to avoid the induction of micro-failures at the surface.
2.2
Surface characterizations
2.2.1
Contact angle measurements
Contact angles ( θ ) of water droplets were determined at ambient temperature (21–24 °C) with a drop shape analysis apparatus (DSA-10, Krüss GmbH, Hamburg, Germany). The measuring system recorded the drop shape by a CCD-camera and determined the contact angles using image analysis software taking into account the entire drop shape .
Six water droplets were deposited on each surface: values of contact angle ( θ °) represented the averages of 12 contact angle measurements (6 on the right side and 6 on the left side).
2.2.2
Scanning white light interferometry
To determine the roughness of the different treatments and the control (no treatment/raw ceramic), we used a scanning white light interferometer Zygo. The Zygo ® NewView 200 is a scanning white-light interferometer that uses Frequency Domain Analysis (FDA) to generate quantitative 3D images of surfaces . The measurements used a white light filter based on a center wavelength of 600 nm, with a bandwidth of 125 nm. The interference patterns were recorded by a CCD camera and each measurement contained 320 × 240 data points. Three objectives (magnifications 2.5×, 10×, and 50×) were used and three images were recorded at each magnification. The scan size and sampling interval were fixed by the magnification of the optical system. For the magnification of 2.5×, the sampling interval was 8.8 μm in the both directions. The lateral resolution of the microscope was limited by the numerical aperture of the objective and the vertical resolution was lower than 1 nm. This technique was used to determine the biomaterial surface roughness ( R a ), using the 10× objective.
2.2.3
Scanning electron microscopy
Samples of tissues cultivated on the different materials were rinsed in PBS, fixed in 3% glutaraldehyde in Rembaum buffer (pH 7.4) for 1 h, dehydrated in a series of graded alcohols, critical-point dried from CO 2 (Polaron Instrument Inc., Nottingham, UK), sputter-coated with gold (Polaron) and examined in Philips scanning electron microscope (ESEM FEG XL 30).
2.3
Cell study
2.3.1
Organotypic culture method
Before culture, the ceramic samples were cleaned in ultrasonic baths with acetone (10 min) and rinsed in distilled water (10 min), then autoclaved at 120 °C for 20 min.
The organotypic culture method described in Fig. 1 was used to examine the cellular response to ceramic surface treatments. For full technical description see Ref. . Culture dishes were pretreated with nutrient medium consisting of 50% Bactoagar 1% (Difco, Fisher, France) in Gey’s solution , 38.5% DMEM (Gibco, Invitrogen, Cergy Pontoise, France), 10% FCS (Gibco, Invitrogen, Cergy Pontoise, France), 1% l -glutamine (Gibco, Invitrogen, Cergy Pontoise, France) and 0.05% penicillin/streptomycin solution (Gibco, Invitrogen, Cergy Pontoise, France). Epithelial tissues were isolated from 7-day-old White Leghorn’s chicken eggs and placed in sterile PBS. The tissues were cut into 1 mm 2 pieces and put onto the bottom of a 70 cm 2 Petri dish (Dominique Dutscher, Brumath, France). Nine tissue fragments were placed in each Petri dish and a 2 cm 2 piece of ceramic material was deposited on each of those fragments.
