Highlights
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An increased surface roughness of zirconia reduces cell spreading of osteoblasts.
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An increased tetragonal phase ratio of zirconia increases cell viability of osteoblasts.
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Wettability is increased on zirconia after heat-treatment.
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Gene expression is comparable on smooth and micro-structured zirconia.
Abstract
Objectives
Different approaches are currently undertaken to structure the endosseous part of zirconia implants. The purpose of the present study was to evaluate how surface roughness and monoclinic to tetragonal phase ratio of zirconia affect cell behavior of human osteoblasts.
Methods
Zirconia discs with five different surface structures were produced: machined; machined heat-treated; polished; polished heat-treated; sandblasted, etched and heat-treated (cer.face 14, vitaclinical). The specimen surfaces were then characterized in terms of monoclinic to tetragonal phase ratio, wettability, roughness and visualized using scanning electron microscopy. To determine the reaction of the human osteoblastic cells (MG-63) to the surface roughness and monoclinic to tetragonal phase ratio of zirconia, cell spreading, morphology, actin cytoskeleton, viability and gene expression of alkaline phosphatase (ALP), collagen type I (COL) and osteocalcin (OCN) were assessed.
Results
Heat-treatment of the specimens significantly improved the surface wettability. With increased surface roughness Ra of the specimens, cell spreading was reduced. Cell viability after 24 h correlated linearly with the tetragonal phase ratio of the specimens. Gene expression after 24 h and 3 d was comparable on all specimens irrespective their surface roughness or monoclinic to tetragonal phase ratio.
Significance
Smooth zirconia surfaces with a high tetragonal phase ratio revealed best surface conditions for MG-63 osteoblastic cells and may be considered to design the endosseous part of zirconia implants.
1
Introduction
Dental implants are a valuable treatment option to replace missing teeth. Titanium is currently the material of choice. In recent years the biocompatibility of titanium implants has been critically discussed. It is still not known if periimplantitis – an inflammatory reaction associated with bone resorption around the implants – is material-induced. The risk of an implant developing a periimplantitis was estimated to range from 0.4% to 68% . There are some indications that Ti-ions released from the implant surface upregulate the expression of chemokines and cytokines in human osteoclasts and osteoblasts. Hence, osteoclastogenesis is induced that subsequently contributes to the pathomechanism of aseptic loosening . Dental implants made of zirconia can be considered a promising alternative to titanium implants with clinical data available reporting survival rates between 95.4 to 98.4% after up to 5 years in situ .
The most important requirement for the clinical success of an implant is a permanent osseointegration, meaning the formation of a direct bone-implant contact along the endosseous part of the implant . To achieve a certain primary stability after insertion, the this part of the implant is shaped as a screw. Additionally, the endosseous surface is micro-structured which is reported to enhance osseointegration . For titanium it has been shown that the implant surface should be moderately roughened (Ra ≈ 1.5 μm) to increase bone implant contact and resistance to torque . For zirconia implants a micro-structuring of the endosseous implant part was therefore also postulated. Different approaches are currently undertaken to structure zirconia such as sandblasting, acid-etching, laser structuring, additive sintering or injection molding .
Zirconia is a polymorph material and the phase transition from tetragonal to monoclinic occurring under tensile stress is used to reinforce the ceramic . A heat treatment is therefore sometimes applied at the end of the production process to retrieve the tetragonal crystal structure and thus to recover the reinforcing potential . The only implant surfaces that are commercially available providing clinical long-term data are sandblasting followed by acid etching and optionally by a heat treatment . In a further study a surface that was sandblasted and subsequently coated with porously sintered zirconia has been tested , however, this surface is currently not available on the market. Due to the variety of structuring methods it remains still unknown which surface modification technique results in the most favorable osseointegration capacity . Pre-clinical investigations with monkeys, dogs, pigs, rats, rabbits or sheep evaluated the effect of different surface modifications on the osseointegration rate . The controversial results for smooth and micro-structured endosseous surfaces that also strongly depended on the selected animal model underline the importance of further studies that are required to elucidate this topic. It has to be considered that zirconia with its differing surface characteristics may require a different surface structure to facilitate osseointegration than titanium. Therefore, also smooth surfaces may be investigated, especially because in vitro cell studies revealed that on polished zirconia surfaces, viability and initial cell spreading of osteoblast cells is increased when compared to a sand-blasted, etched and heat-treated surface . The initial spreading quality to a biomaterial is a crucial factor that will determine the subsequent cell function, proliferation, differentiation and viability . The purpose of the present study was to evaluate how surface roughness and monoclinic to tetragonal phase ratio of zirconia affect cell behavior of human osteoblastic cells.
2
Materials and methods
Zirconia discs were machined overdimensioned in the green state, sintered and hot isostatically pressed in order to get disc shaped specimens with final dimensions of 13 mm in diameter and 2 mm in height. The zirconia that was used was composed of 93.0 wt% ZrO 2 , 5.0 wt% Y 2 O 3 , 0.1 wt% Al 2 O 3 , 1.9 wt% HfO 2 ; grain size was 0.3 μm (MZ111, CeramTec, Plochingen, Germany). Five different surfaces were investigated: Zp: polished; Zpt: polished and heat-treated 1 h 1250 °C; Zm: as machined; Zmt: as machined and heat-treated 1 h 1250 °C; Z14: sandblasted Al 2 O 3 105 μm, etched 1 h in hydrofluoric acid 38–40%, heat-treated 1 h 1250 °C. Z14 is the endosseous surface of a clinically tested dental implant (cer.face 14, vitaclinical, Vita, Bad Säckingen, Germany) and was provided by the manufacturer of the implant as control. Prior to use, all specimens were cleaned in an ultrasonic bath, 70% ethanol for 5 min, distilled water for 5 min and sterilized in a heating chamber at 200 °C for 2 h (FED-240, Binder, Tuttlingen, Germany). The specimen surfaces were then characterized in terms of monoclinic to tetragonal phase ratio, wettability and roughness and visualized using scanning electron microscopy (SEM). To determine the reaction of human osteoblasts to the surface roughness and monoclinic to tetragonal phase ratio of zirconia, viability, cell spreading, morphology, actin cytoskeleton, and gene expression were assessed.
2.1
Specimen characterization
2.1.1
Monoclinic to tetragonal phase ratio
The monoclinic to tetragonal phase ratio present at the surface of the specimens was obtained using X-Ray Diffraction (D8 Advance, Bruker, Billerica, MA, USA) on 5 specimens per group. Diffractograms were measured using angles ranging from 22° to 65° with 40 kV and 40 mA. For measurements, best-fit for tetragonal and monoclinic structure was chosen and used for refinement with Rietveld Analysis (Bruker Diffrac.eva, V 3.2 and Topas, V 4.2, Bruker AXS, Karlsruhe, Germany).
2.1.2
Surface wettability
The contact angle of water and diiodomethane was measured on 5 specimens per group using a drop shape analyzer (DSA100, Krüss, Hamburg, Germany). Five drops of 0.5 μl of each liquid were measured per specimen with the sessile drop technique. Surface-free energy as well as dispersive and polar part were calculated using the method of Owens, Wendt, Rabel & Kaelble . The surface free energy can be divided into polar and dispersive interaction fractions. Polar interactions are caused by a permanent asymmetry of electron density in molecules (molecules with a dipole moment such as water have high polar interactions). Dispersive interactions are usually weaker and are due to fluctuations of the electron density distribution in molecules that cause temporary charge differences (electrostatic attraction).
2.1.3
Surface roughness
The roughness parameters arithmetical mean height (Ra) and maximum height of profile (Rz) was measured with a contact profilometer (T1000/TKK50, Hommelwerke, Schwenningen, Germany). For each group 6 specimens were analyzed with 5 contact measurements over a distance of 4.8 mm (T1E, tip 5 μm 90°, 1.6 mN, Hommel-Etamic/Jenoptik, Jena, Germany).
2.1.4
SEM imaging
The specimens’ surfaces were sputtered with a 20 nm layer of gold (Leica EM ACE600 – double sputter coater, Leica Microsystems, Heerbrugg, Switzerland) and visualized with SEM (ESEM XL30, Philips, Eindhoven, the Netherlands). Images were taken with aperture 4, 15 kV using both, SE (secondary electrons) and BSE (back scattered electrons) detectors.
2.2
Cell behavior
2.2.1
Cell cultivation
Human osteoblastic cells MG-63 (American Type Culture Collection ATCC, CRL1427) were cultivated in Dulbecco’s modified eagle medium (DMEM + GlutaMAX-l + 4.5 g/l DGlucose + Pyruvate; Gibco, Thermo Fisher Scientific, Waltham, USA) with 10% fetal calf serum (FBS superior standardized S0615 0879F, Biochrom, Berlin, Germany) and 1% antibiotic (gentamicin, ratiopharm, Ulm, Germany). Specimen surfaces were seeded with MG-63 cells passages 8–23 , 70–80% confluent and incubated in 24-well plates at 37 °C in a humidified atmosphere with 5% CO 2 for the respective time intervals. All cell experiments were performed 3 times using different cell passages.
2.2.2
Cell viability
Mitochondrial dehydrogenase activity was measured by MTS assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to investigate viability of cells growing on specimens. A drop of 120 μl cell culture medium containing 5 × 10 4 MG-63 cells was carefully placed on each specimen ( n = 2 per group) and incubated for 20 min to ensure cell attachment on the specimens. Afterwards 1 ml of cell culture was added per well and the specimens were incubated for 24 h. Specimens were transferred to a new well-plate and MTS solution (CellTiter 96 ONE-Solution Cell Proliferation Assay, Promega, Madison, USA) with culture medium was added (1:5) to each specimen. A blank group containing a specimen with culture medium without cells and a control group with cells growing on well bottom were additionally tested. After 80 min, supernatants were transferred to a 96-well plate (for each specimen 3 × 80 μl were analyzed). The optical density (OD) was recorded at 490 nm with a micro-plate reader (Anthos, Mikrosysteme, Krefeld, Germany). Relative cell viability was calculated using the following equation:
Relativecellviability=(ODspecimen−ODblankspecimen)/(ODcontrol−ODblankcontrol).
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