Abstract
Titanium implants are the gold standard in dentistry; however, problems such as gingival tarnishing and peri-implantitis have been reported. For zirconia to become a competitive alternative dental implant material, surface modification techniques that induce guided tissue growth must be developed.
Objectives
To develop alternative surface modification techniques to promote guided tissue regeneration on zirconia materials, for applications in dental implantology.
Methods
A methodology that combined soft lithography and sol–gel chemistry was used to obtain isotropic micropatterned silica coatings on yttria-stabilized zirconia substrates. The materials were characterized via chemical, structural, surface morphology approaches. In vitro biological behavior was evaluated in terms of early adhesion and viability/metabolic activity of human osteoblast-like cells. Statistical analysis was conducted using one-way ANOVA/Tukey HSD post hoc test.
Results
Isotropic micropatterned silica coatings on yttria-stabilized zirconia substrates were obtained using a combined approach based on sol–gel technology and soft lithography. Micropatterned silica surfaces exhibited a biocompatible behavior, and modulated cell responses (i.e. inducing early alignment of osteoblast-like cells). After 7 d of culture, the cells fully covered the top surfaces of pillar microstructured silica films.
Significance
The micropatterned silica films on zirconia showed a biocompatible response, and were capable of inducing guided osteoblastic cell adhesion, spreading and propagation. The results herein presented suggest that surface-modified ceramic implants via soft lithography and sol–gel chemistry could potentially be used to guide periodontal tissue regeneration, thus promoting tight tissue apposition, and avoiding gingival retraction and peri-implantitis.
1
Introduction
Commercially pure titanium (Ti) is the biomaterial of choice for dental implants because it is a relatively inexpensive material, promotes osseointegration, has well-documented biocompatibility, self-passivation, strength, and conformability . However, it has also been reported that Ti could exhibit problems such as potential allergenic response and poor esthetics . Since the introduction of titanium dental implants, many other materials have been explored as potential implant candidates, including zirconia, alumina, tantalum and niobium, but some have never been commercialized, and the others have achieved very limited commercial success .
Many patients present large challenges for clinicians, including those requiring implant-supported dental single crowns in the anterior segment and patients with highly demanding esthetic conditions due to the presence of high line upper lip, a thin gingival biotype, or intact natural adjacent teeth . The most common solutions include titanium-based dental implants, metallic or ceramic abutments, and all ceramic crowns (e.g., alumina, zirconia or combination) .
Zirconia has been studied due to its potential use in dentistry as inlays, onlays, full crowns, bridges, abutments, intra-root posts, scaffolds and implants . No cytotoxic or mutagenic effects have been reported, and it has been shown to exhibit osseointegration as well as low inflammatory infiltrate in gingival tissues . However, only preliminary results have been published, and long term studies are not available. Therefore, further research is required to develop an effective and competitive zirconia-based material candidate as a clinical alternative to titanium implants. Since the initial reports in 1980, dental ceramic implants have shown superior esthetic behavior, as an adequate appearance of natural teeth could be obtained; however, their mechanical properties have been questionable .
For orthopedic and dental applications, a reduction in the catastrophic failure of all-ceramic restorations was obtained with the introduction of yttria-stabilized tetragonal zirconia polycrystalline (3Y-TZP) . However, based on investigations of the use of 3Y-TZP in orthopedics, under physiological conditions, yttrium may leach from the ceramic and consequently the zirconia may lose its transformational toughening (its crystal structure changes back from tetragonal (t) to monoclinic (m) phases, known as “aging”) . After more than 20 years of research, aging still remains a key issue for zirconia implants, with aging of zirconia orthopedic devices (e.g. femoral heads) being by far the most studied example. Aging occurs by a water-assisted martensitic phase transformation, which propagates at the surface by a nucleation-and-growth mechanism and then invades the bulk structure. Therefore, zirconia could be prone to aging in the presence of human saliva. Aging results in roughening and microcracking, in the most extreme cases, it leads to catastrophic failure of the component . Considering that the use of 3Y-TZP as a dental material is still increasing and that its low temperature degradation is still latent, further extensive clinical research is necessary to achieve a risk reduction .
Advanced ceramic processing to obtain 3Y-TZP materials is a demanding process. Tailoring the microstructure by refining powder processing can result in zirconia dental structures at the top end of their strength spectrum . However, since the introduction of 3Y-TZP as a material for dental implants, only a limited number of reports concerning surface modification strategies have been produced, and the know-how available from decades of surface modifications of titanium implants is not directly applicable .
Currently, surface modification techniques are based on the removal of material through low energy processing such as etching (chemical or electrochemical), or mechanical techniques such as grinding, sandblasting or ablation by electrons or lasers . These alternatives, however, can lead to yttria leaching, phase transformation and subsequent catastrophic failure. Alternative additive approaches can be costly, present adhesion problems and introduce heterogeneities .
This work explored the use of sol–gel processing as a viable approach to modify 3Y-TZP surfaces, based on its versatility to yield materials with high chemical homogeneity and fine grained or amorphous microstructure using lower temperatures, and its ease of implementation for mass production . In addition, these characteristics also allow for the combination with PDMS (polydimethylsiloxane) microstamping via soft lithography; a technique extensively used in the last decade to produce ordered (isotropic or anisotropic) or randomized features, to engineer surfaces that can modulate cell behavior. Such surfaces have the potential to induce positive cellular responses in terms of morphology, adhesion, migration, proliferation, and differentiation .
The present work describes the preparation of ordered micropatterned silica thin films on 3Y-TZP, intended to enhance guided tissue regeneration on ceramic dental implants. In order to understand how such a combination would perform, materials characterization and in vitro biological behavior in terms of early adhesion and proliferation of osteoblast-like cells were carried out.
2
Materials and methods
2.1
Substrate processing and characterization
A synthetic 3 mol% Y 2 O 3 –ZrO 2 (3Y-TZP) powder having a nearly uniform submicron particle size was used (TZ-3YS-E, Tosoh, Tokyo, Japan). Chemical analysis of the powder was carried out by X-ray fluorescence spectroscopy (XRF, Magi X; Phillips, The Netherlands) and flame emission spectroscopy for alkaline analyses (FES, 2100; Perkin Elmer) to verify the values provided by the supplier. The specific surface area of the powder was determined by the chromatographic method (Monosorb Surface Area Analyzer MS-13, Quantachrome Corporation, UK) using a BET isotherm. Particle size distribution was measured using a laser scattering size analyzer (Master Sizer S; Malvern Instruments, UK). The particle size measurements were done on slurry samples. Dolapix CE-64 (Zschimmer & Schwarz GmbH & Co KG, Germany) was used as dispersant. Dispersion and de-agglomeration of particles were further ensured through ultrasonic treatment prior to measurement.
Cylindrical specimens were produced via uniaxial pressing, and their sintering behavior was evaluated using a high resolution dilatometer (Setsys 16/18, Setaram, France), at a heating rate of 5 °C/min from 20 °C to 1550 °C. Based on the thermal expansion/contraction data (dilatometer curve, Fig. 2 a), all discs were sintered at 1480 °C for 2 h. Contraction of all specimens was calculated based in the comparison between the diameters of the “green” discs and of the full sintered discs. Maximum density was determined using the Archimedes water displacement method, while crystallography and full transformation from monoclinic to tetragonal phase was followed by XRD analysis (Siemens D5000, Germany). Visible Raman spectra (resolution = 4 cm −1 , acquisition time = 5 s, 514 nm laser) were recorded using a confocal Raman microscope (LabRAM HR800 UV, Horiba Jobin-Yvon, Villeneuve d’Ascq, France), as a complement to confirm m → t phases transformation on powders, fully sintered discs, and re-heated discs ( t → m phases transformation) .
The sintered samples were polished with a series of diamond pastes down to 1 μm. Microstructure observations were performed on polished and thermally etched surfaces via field emission scanning electron microscopy (FE-SEM, Hitachi-S4700, Tokyo, Japan, fitted with energy X-ray dispersive spectroscopy, EDS, Noran System, Japan).
2.2
Thin film production
Silica sol was produced via acid catalysis in a single step using tetraethylorthosilicate (TEOS, Aldrich, USA) and methyltriethoxysilane (MTES, Aldrich, USA) as precursors, following a previously reported method . The solution was aged for 24 h and applied via spin coating (3000 rpm for 45 s) on 3Y-TZP bare (3Y-TZP) substrates to produce flat silica coatings (fSiO 2 ).
Standard UV photolithography was used initially to create a silicon master with the pattern of interest. First, a chromium photomask was designed using L-Edit software (Tanner EDA, USA) and produced at a mail order mask-making foundry (Martin Photomask Service, USA). A negative tone SU-8 2005 (Microchem Corp, USA) photoresist was spin-coated on a silicon wafer at 3000 rpm to produce a ∼5 μm thick film ( Fig. 1 a and b ). The photoresist was exposed to ultraviolet light through the photomask, and post-processed following previously established parameters ( Fig. 1 c and d). Finally, the master was used to create PDMS (Silastic T2, Dow Corning, USA) negative molds ( Fig. 1 e and f) with an ordered texture composed of wells with 5 μm diameter, 5 μm deep, and 5 μm edge-edge spacing.
3Y-TZP substrates were cleaned and dried. Micropatterned silica coatings (mpSiO 2 ) were prepared by pouring (40 ul) the sol on the PDMS mold and then pressing against the substrate ( Fig. 1 g). After drying for 2 h, the mold was removed ( Fig. 1 h). This technique is a modification of the pioneering work by Marzolin et al. . Afterwards, the samples were evaluated under light microscopy, and then sintered at 500 °C for 60 min to produce micropatterned silica coating on 3Y-TZP substrates. The samples were characterized post-sintering via SEM.
2.3
Cell culture
MG63 cells (ATCC, USA) were cultured in α-minimal essential medium (α-MEM, Sigma, USA) containing 10% fetal bovine serum (ATCC, USA), penicillin–streptomycin (100 IU/ml and 10 mg/ml, respectively) (Sigma), gentamicin–amphotericin B (10 μg/ml and 0.25 μg/ml, Invitrogen, USA), ascorbic acid (50 μg/ml, Sigma), and l -glutamine (2 mM, Sigma). The cells were seeded at a density of 2 × 10 4 cells/cm 2 on bare and silica coated (micropatterned and flat) zirconia and control group samples (tissue culture-treated polystyrene, TCP). All cultures were incubated for 3 different time points (1, 4 and 7 d). Then, the cells were washed twice with 37 °C PBS, fixed in 10% (v/v) neutral buffered formalin (Ted Pella, USA) for 15 min, and prepared for SEM and epifluorescence microscopy.
2.4
Morphology and proliferation
For morphology evaluation via MIF (Eclipse TE2000-5, Nikon, Japan), the cells were washed and permeabilized with 0.1% (v/v) Triton X-100 for 30 min. F-actin filaments were stained using alexafluor phalloidin (Invitrogen, USA) for 30 min. Nuclei were stained with a buffer of propidium iodide and RNase (BD Pharmigen, USA) at 4 °C for 15 min and washed with PBS.
For morphology evaluation via SEM, the cells were dehydrated in graded ethanol and hexamethyldisilazane (HMDS, Ted Pella, USA) solutions (50–100%), respectively . The samples were then gold-coated and visualized by SEM.
2.5
Viability/proliferation
Proliferation was evaluated using the alamar blue assay, a simple, non-reactive assay based on the oxidation–reduction of resuzarim, a non-fluorescent blue component that is reduced by living cells to a fluorescent pink component. Briefly, fresh medium with 10% alamar blue (v/v) (Sigma) was added, and incubated for 3 h. 100 μl of the medium was then transferred to a 96-well plate and a microplate reader (Infinite F-500, Tecan Trading AG, Switzerland) was used to quantify the fluorescence (at 535 nm excitation wavelength and 590 emission wavelength).
All cell culture experiments were carried out in duplicated ( n = 3) and results were expressed in relative to TCP Statistical analysis of the viability/metabolic activity was performed using one-way ANOVA/Tukey HSD post hoc test. Levels of p < 0.05 were considered to be statistically significant.
2
Materials and methods
2.1
Substrate processing and characterization
A synthetic 3 mol% Y 2 O 3 –ZrO 2 (3Y-TZP) powder having a nearly uniform submicron particle size was used (TZ-3YS-E, Tosoh, Tokyo, Japan). Chemical analysis of the powder was carried out by X-ray fluorescence spectroscopy (XRF, Magi X; Phillips, The Netherlands) and flame emission spectroscopy for alkaline analyses (FES, 2100; Perkin Elmer) to verify the values provided by the supplier. The specific surface area of the powder was determined by the chromatographic method (Monosorb Surface Area Analyzer MS-13, Quantachrome Corporation, UK) using a BET isotherm. Particle size distribution was measured using a laser scattering size analyzer (Master Sizer S; Malvern Instruments, UK). The particle size measurements were done on slurry samples. Dolapix CE-64 (Zschimmer & Schwarz GmbH & Co KG, Germany) was used as dispersant. Dispersion and de-agglomeration of particles were further ensured through ultrasonic treatment prior to measurement.
Cylindrical specimens were produced via uniaxial pressing, and their sintering behavior was evaluated using a high resolution dilatometer (Setsys 16/18, Setaram, France), at a heating rate of 5 °C/min from 20 °C to 1550 °C. Based on the thermal expansion/contraction data (dilatometer curve, Fig. 2 a), all discs were sintered at 1480 °C for 2 h. Contraction of all specimens was calculated based in the comparison between the diameters of the “green” discs and of the full sintered discs. Maximum density was determined using the Archimedes water displacement method, while crystallography and full transformation from monoclinic to tetragonal phase was followed by XRD analysis (Siemens D5000, Germany). Visible Raman spectra (resolution = 4 cm −1 , acquisition time = 5 s, 514 nm laser) were recorded using a confocal Raman microscope (LabRAM HR800 UV, Horiba Jobin-Yvon, Villeneuve d’Ascq, France), as a complement to confirm m → t phases transformation on powders, fully sintered discs, and re-heated discs ( t → m phases transformation) .
The sintered samples were polished with a series of diamond pastes down to 1 μm. Microstructure observations were performed on polished and thermally etched surfaces via field emission scanning electron microscopy (FE-SEM, Hitachi-S4700, Tokyo, Japan, fitted with energy X-ray dispersive spectroscopy, EDS, Noran System, Japan).
2.2
Thin film production
Silica sol was produced via acid catalysis in a single step using tetraethylorthosilicate (TEOS, Aldrich, USA) and methyltriethoxysilane (MTES, Aldrich, USA) as precursors, following a previously reported method . The solution was aged for 24 h and applied via spin coating (3000 rpm for 45 s) on 3Y-TZP bare (3Y-TZP) substrates to produce flat silica coatings (fSiO 2 ).
Standard UV photolithography was used initially to create a silicon master with the pattern of interest. First, a chromium photomask was designed using L-Edit software (Tanner EDA, USA) and produced at a mail order mask-making foundry (Martin Photomask Service, USA). A negative tone SU-8 2005 (Microchem Corp, USA) photoresist was spin-coated on a silicon wafer at 3000 rpm to produce a ∼5 μm thick film ( Fig. 1 a and b ). The photoresist was exposed to ultraviolet light through the photomask, and post-processed following previously established parameters ( Fig. 1 c and d). Finally, the master was used to create PDMS (Silastic T2, Dow Corning, USA) negative molds ( Fig. 1 e and f) with an ordered texture composed of wells with 5 μm diameter, 5 μm deep, and 5 μm edge-edge spacing.
3Y-TZP substrates were cleaned and dried. Micropatterned silica coatings (mpSiO 2 ) were prepared by pouring (40 ul) the sol on the PDMS mold and then pressing against the substrate ( Fig. 1 g). After drying for 2 h, the mold was removed ( Fig. 1 h). This technique is a modification of the pioneering work by Marzolin et al. . Afterwards, the samples were evaluated under light microscopy, and then sintered at 500 °C for 60 min to produce micropatterned silica coating on 3Y-TZP substrates. The samples were characterized post-sintering via SEM.
2.3
Cell culture
MG63 cells (ATCC, USA) were cultured in α-minimal essential medium (α-MEM, Sigma, USA) containing 10% fetal bovine serum (ATCC, USA), penicillin–streptomycin (100 IU/ml and 10 mg/ml, respectively) (Sigma), gentamicin–amphotericin B (10 μg/ml and 0.25 μg/ml, Invitrogen, USA), ascorbic acid (50 μg/ml, Sigma), and l -glutamine (2 mM, Sigma). The cells were seeded at a density of 2 × 10 4 cells/cm 2 on bare and silica coated (micropatterned and flat) zirconia and control group samples (tissue culture-treated polystyrene, TCP). All cultures were incubated for 3 different time points (1, 4 and 7 d). Then, the cells were washed twice with 37 °C PBS, fixed in 10% (v/v) neutral buffered formalin (Ted Pella, USA) for 15 min, and prepared for SEM and epifluorescence microscopy.
2.4
Morphology and proliferation
For morphology evaluation via MIF (Eclipse TE2000-5, Nikon, Japan), the cells were washed and permeabilized with 0.1% (v/v) Triton X-100 for 30 min. F-actin filaments were stained using alexafluor phalloidin (Invitrogen, USA) for 30 min. Nuclei were stained with a buffer of propidium iodide and RNase (BD Pharmigen, USA) at 4 °C for 15 min and washed with PBS.
For morphology evaluation via SEM, the cells were dehydrated in graded ethanol and hexamethyldisilazane (HMDS, Ted Pella, USA) solutions (50–100%), respectively . The samples were then gold-coated and visualized by SEM.
2.5
Viability/proliferation
Proliferation was evaluated using the alamar blue assay, a simple, non-reactive assay based on the oxidation–reduction of resuzarim, a non-fluorescent blue component that is reduced by living cells to a fluorescent pink component. Briefly, fresh medium with 10% alamar blue (v/v) (Sigma) was added, and incubated for 3 h. 100 μl of the medium was then transferred to a 96-well plate and a microplate reader (Infinite F-500, Tecan Trading AG, Switzerland) was used to quantify the fluorescence (at 535 nm excitation wavelength and 590 emission wavelength).
All cell culture experiments were carried out in duplicated ( n = 3) and results were expressed in relative to TCP Statistical analysis of the viability/metabolic activity was performed using one-way ANOVA/Tukey HSD post hoc test. Levels of p < 0.05 were considered to be statistically significant.
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