Testing biocompatibility

Human osteoblasts were harvested from cancellous bone, which was removed from the iliac crest during routine surgery. Small bone fragments were transferred into tissue culture dishes.

The cells were cultivated using an osteogenic medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum (FCS), 100IE penicillin/ml, 100 μg streptomycin/ml, 1 mmol/l ascorbic acid and 10 nmol/l dexamethasone at 37 °C with 5% CO ₂ . The cell seeding was performed after the second passage. During passaging, the cells were detached from 75 cm ² cell culture flasks using 5 ml of a 0.05% trypsin/0.02% EDTA solution in phosphate buffered saline (PBS). After a 1:1 dilution of the cell suspension with DMEM containing 10% FCS and centrifugation at 3200 × g for 3 min, cells were resuspended in DMEM containing 10% FCS, counted and reseeded at a density of 10 ⁵ cells/75 cculturem ² cell culture flasks. The cells were cultured in the same medium used for the cell seeding in a humidified atmosphere with 5% CO ₂ at 37 °C. The medium change took place every 3 days.

Characterization of the cells

The phenotype of human osteoblasts was confirmed by detection of osteocalcin production. The cells were seeded on 8-well objectives and incubated with a monoclonal antibody directed against osteocalcin (Abcam, Cambridge, UK). The control cells were incubated with 1% bovine serum albumin (Sigma–Aldrich GmbH, Hamburg, Germany). After incubation with anti-osteocalcin antibody, the cells were washed and incubated with an enzyme-conjugated secondary antibody (Dako GmbH, Hamburg, Germany). The enzymatic detection was performed with horseradish peroxidase (Dako, GmbH, Hamburg, Germany) and counterstaining was carried out with haematoxylin–eosin (Merck, Darmstadt, Germany).

Tests of biocompatibility

The biocompatibility of six implant materials (one zirconium implant, five different titanium implants) was determined by in vitro vitality and cytotoxic tests (fluorescein diacetate-test (FDA-test), lactate dehydrogenase-test (LDH-test), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide-test (MTT-test), 5-bromo-2-deoxyuridine-test (BrdU-test) and water soluble tetrazolium-test (WST-test)). Apart from the zirconia surface, five different titanium implants with different surface characteristics were used: a modified sandblasted, acid-etched hydrophilic titanium-surface; a chemically modified micro-roughened titanium surface treated with fluoride; a highly crystalline, phosphate-enriched titanium oxide surface; a micro-roughened acid-etched titanium surface; and a dual acid-etched titanium surface with nano-scale crystals of calcium phosphate. The contact of the osteoblasts to the implant surface was determined by scanning electron microscopy (SEM).

Immediately after removal from the package, three implants from each manufacturer were eluated in cell culture media for 10 min, 1 h and 24 h, respectively. The eluates were collected and stored at 4 °C.

Assessment of cell vitality

Cell vitality was assessed by FDA-test and propidium iodide (PI) staining. 1 × 10 ⁴ cells in cell culture medium 10% FCS, 100IE penicillin/ml, 100 μg streptomycin/ml, 1 mmol/l ascorbic acid and 100 nmol/l dexamethasone were seeded on 8-well objectives (Lab-TEKII Chamber Slide w/cover RS glass slide, Nalge Nunc International, Roskilde, Denmark). After cultivation for 24 h, 200 μl of the eluate was added to the cells. After 24 h of incubation at 37 °C and 5% CO ₂ , cells were rinsed with PBS and immersed in an FDA solution prepared by diluting 30 μl × 1 mg FDA/ml acetone in 10 ml PBS. After incubation for 15 min at 37 °C in darkness, the FDA solution was removed by suction and replaced with a PI solution prepared by diluting 500 μl × 1 mg/ml PI in 10 ml PBS. After incubation for 2 min at room temperature in darkness, slides were rinsed twice in PBS. While still immersed in PBS, the slides were subjected to fluorescence microscopy with excitation at 488 nm and detection at 53 nm (FDA, green) and 62 nm (PI, red).

Tests of biocompatibility and proliferation: LDH and BrdU tests

The LDH-test is an indication for cell death and lysis. The cells were seeded in 96-well cell culture plates (Nunc GmbH, Langenselbold, Germany) in 100 μl DMEM with FCS, 100IE penicillin/ml, 100 μg streptomycin/ml, 1 mmol/l ascorbic acid and 100 nmol/l dexamethasone at a concentration of 5 × 10 ³ cells/well. After culture for 24 h in a humidified atmosphere with 5% CO ₂ at 37 °C, the medium was removed and replaced with 150 μl of the eluate. The cells cultured in 2% Triton-X-100 in serum-free DMEM served as high controls. The cells cultured in serum-free DMEM served as low controls. After 24 h incubation, 100 μl eluate was transferred to another 96-well cell culture plate. Extracellular LDH activity was measured with a LDH detection kit (Roche Diagnostics, Mannheim, Germany, Catalogue No. 11644793001). Absorbance was measured at 490 nm. Calibration curves of 5–0.16 × 10 ³ cells/well served as standards. The remaining 50 μl eluate per well was removed and replaced with 10 μl DMEM containing 10% FCS, 100IE penicillin/ml, 100 μg streptomycin/ml, 1 mmol/l ascorbic acid and 100 nmol/l dexamethasone. After 5 days of incubation, the proliferation was measured with a BrdU Cell Proliferation ELISA kit (Roche Diagnostics, Mannheim, Germany, Cat. No.11647229001). This method was based on the incorporation of BrdU-test instead of thymidine into the newly synthesized DNA of the proliferating cells. The absorbance was measured at 450 nm.

MTT test

The cell culturing and measurement was performed as before. After 24 h incubation with the eluates, the proliferation was assessed with the aid of an MTT Cell Proliferation Kit (Roche Diagnostics, Mannheim, Germany, Cat. No. 11465007001). The detection of cell vitality is based on the reduction of a yellow coloured dye MTT-test to blue–violet Formazan. The absorbance was measured at 550 nm.

WST test

After eluation, the implants were placed in 24-well plates. The cells were seeded on the implants at a density of 1 × 10 ⁴ cells/well. The cells were cultured in 2000 μl of the same medium used for cell seeding in a humidified atmosphere with 5% CO ₂ , at 37 °C. The medium change was carried out every 3 days and the cultures were checked microscopically. After 7 days of culturing, the proliferation was assessed with the aid of a Cell Proliferation Reagent WST-1 (Roche Diagnostics, Mannheim, Germany, Cat. No. 116446807001). The evaluation of cell proliferation is based on the cleavage of tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) by mitochondrial dehydrogenases in viable cells. Briefly, 20 μl WST-1 reagent was added to each well at a 1:10 ratio to cell culture medium. After 4 h incubation in a humidified atmosphere with 5% CO ₂ at 37 °C, the medium was transferred to 96-well plates and absorbance was measured at 460 nm. The cells culturing in wells without eluate served as controls.

Examination by SEM

The SEM investigations were carried out 1 week after cell seeding (1 × 10 ⁴ cells/well) using an XL30CP device (Phillips Electron Optics GmbH, Kassel, Germany) operating at 10–25 kV, described by Y ang et al. As preparation for the SEM investigation, the cell-seeded implants and implants without cells as control were rinsed using PBS to remove the cell culture medium. The cells were fixed using 3% glutaraldehyde in PBS at pH 7.4 for 24 h. After removal of the glutaraldehyde solution, the cells were dehydrated by incubating scaffolds in a series of ethanol solutions of increasing concentration. Scaffolds were immersed for 5 min in each of the following ethanol solutions: 50%, 60%, 70%, 80%, 90% and 100%. Subsequently, the critical point drying was performed using a K850 Critical Point Dryer (Emitech, EM Technologies Ltd., Ashford, UK). Coating with gold was carried out in a Sputter Coater (Bal-Tec SCD 500) with a 15 nm layer.

The study (AZ 118/07) was approved by the Ethics Commission of the Christian-Albrechts-University of Kiel, Germany.

Testing osseointegration in an animal model

The in vivo study of the osseointegration of zirconium and titanium implants was carried out on 8 adult female domestic pigs (>18 months, average body weight 94.5 kg). The domestic pig is an appropriate model for the simulation of human operations because of its bone metabolism and its size. The new bone formation rate of the domestic pig (1.2–1.5 μm/day) is similar to that of humans (1–1.5 μm/day) . The animals were kept in small groups in purpose-designed sties and fed a standard diet (Altromin 9023 ^® , Altromin International GmbH, Lage, Germany) and with water ad libitum.

Experimental procedure

In a split animal design, both sides of each pig were treated in exactly the same manner, except for the different implant materials (zirconium versus titanium). The osseo connected surface of the titanium implant was sandblasted and high temperature etched.

The animals were divided into two groups, which were killed after 4 and 12 weeks. For all surgical procedures, the animals were anesthetized by an intravenous injection of ketamine HCl (Ketavets ^® , Ratiopharm, Ulm, Germany). The frontal skull of the animals was selected for following properties: it provides comparable placement sites inter- and intra-individually; the bone is of desmal origin and not vascularized by a central blood vessel; and the bone quality is class II–III.

After applying local anaesthesia to the area of the frontal skull (Ultracain D-S forte ^® , Hoechst GmbH, Frankfurt, Germany), a sagittal incision was made, and the soft tissue and periosteum were mobilized. In each animal, 4 zirconium (WhiteSky, Bredent, Germany) and 4 titanium implants (BlueSky, Bredent, Germany) (diameter 4 mm; length 12 mm) were inserted with primary stability ( n = 64). The implants were inserted following the protocol in two rows of four implants and executed according to the guidelines of the manufacturer without any additional measures. They were inserted 1 cm apart to avoid biological interimplant action. After the insertion of the implants, the soft tissues were readapted and the wounds were closed with resorbable sutures (Vicryl 3.0 ^® , Ethicon GmbH & Co KG, Norderstedt, Germany). Perioperative antibiosis was achieved with a preoperative i.m. injection of 1 g clemizol-penicillin (Clemizol-Penicillin i.m. forte ^® , Grünenthal GmbH, Aachen, Germany). Postoperative pain control was one injection of 500 mg metamizol i.m. (Novalgin ^® , Hoechst AG, Bad Soden, Germany) and oral tramadol 2 × 50 mg/day (Tramal ^® , Grünenthal GmbH, Aachen, Germany).

The animals were killed after 4 ( n = 4) and 12 ( n = 4) week’s observational period. The animals were sedated with a mixture of azaperone and midazolam (1 mg/kg, i.m.), then 20% pentobarbital solution (Dermocal AG ^® , Buenos Aires, Argentina) was delivered into the ear vein until cardiac arrest occurred.

The ossa frontalia were harvested and specimens were fixed by immersion in 1.4% paraformaldehyde (41 °C) to render the organic matrix insoluble. The specimens were dehydrated in increasing concentrations of alcohol at 21 °C in a dehydration unit (Shandon Citadel 1000, Shandon GmbH, Frankfurt, Germany). The specimens were embedded in Technovit 9100 ^® (Heraeus Kulzer, Kulzer Division, Werheim, Germany) for histological examination by means of grounded sections using the technique described by D onath & B reuner .

Histology

The preparations were prepared as undecalcified hard sections, stained with toluidine blue and examined microradiographically and histologically. After gradual dehydration in ethyl alcohol, the block was embedded in acrylic resin (Fluka Chemie AG, Buchs, Switzerland) and sectioned in 0.5 mm slices. One section per implant was fixed on an acrylic carrier and ground and polished down to approximately 90 μm. The microradiography of the 90 μm specimens was performed on 2 in. × 2 in. plates (Microchrome Technology Inc., San Josè, CA, USA) at 3 mAs and 25 kV using a microradiography device (Faxitron X-ray systems, Hewlett Packard GmbH, Böblingen, Germany). At a magnification of 4×, the plates were photographed under the microscope in approximately 100 slides. These were composed into a total view of the specimens and subjected to qualitative evaluation.

The histomorphometric evaluation was performed after one central slice was chosen. The percentage of the bone–implant contact (BIC) was analysed in the threaded area. The analysis was carried out with the computerized morphometric program of a digital image analysis camera (Q500MC, Leica( Cambridge Ltd., Cambridge, UK). The microscopic image was digitalized and then a 10-fold enlargement was transferred to the computer screen. Within the test groups, the BIC of the zirconium implant side was compared to that of the titanium implant side and statistically evaluated by means of a paired t test. The significance level was defined as α = 0.05.

For further evaluation, approximately 20 μm thin sections were stained with toluidine blue including histometric analysis. The threads of the screw type implants allowed easy orientation. The surface of the implant was traced by hand using the computer mouse. The length of this line was measured by computer after calibration with a known distance (total length). The contact areas of bone and metal were traced and the lengths of the line segments were added (bone–metal interface). By the division of bone–metal interface through total length the percentage of BIC was calculated. The BIC of the experimental sites was compared with the control sites and compared statistically to the paired t test at a significance level of 0.05.

The investigation was approved by the Animal Ethics Committee at the Semmelweis University of Budapest, Hungary (No.: 1053/eoh/2007).