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A comparison of biocompatibility and osseointegration of ceramic and titanium implants: an in vivo and in vitro study
Int. J. Oral Maxillofac. Surg. 2012; 41: 638–645 doi:10.1016/j.ijom.2012.02.004, available online at http://www.sciencedirect.com
Research Paper Dental Implants
A comparison of biocompatibility and osseointegration of ceramic and titanium implants: an in vivo and in vitro study
B. Y. K. E.
Mo¨ller1, H. Terheyden2, Ac¸il1, N. M. Purcz1, Hertrampf1, A. Tabakov1, Behrens1, J. Wiltfang1
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Clinic of Oral and Maxillofacial Surgery, University Hospital Schleswig-Holstein, Campus Kiel, Arnold Heller Str. 3, Haus 26, 24105 Kiel, Germany; 2Department of Oral and Maxillofacial Surgery, Red Cross Hospital, Hansteinstr. 29, 34121 Kassel, Germany
B. Mo¨ller, H. Terheyden, Y. Ac¸il, N. M. Purcz, K. Hertrampf, A. Tabakov, E. Behrens, J. Wiltfang: A comparison of biocompatibility and osseointegration of ceramic and titanium implants: an in vivo and in vitro study. Int. J. Oral Maxillofac. Surg. 2012; 41: 638–645. # 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. This study compared the biocompatibility in vitro and the osseointegration in vivo of zirconium and titanium implants regarding implant surfaces and the bone– implant contacts. The different implant surfaces and the biocompatibility of zirconium versus titanium implants were determined by vitality and cytotoxic tests in vitro. The contact of the osteoblasts to the implant surface was determined by scanning electron microscopy (SEM). The in vivo study for osseointegration was performed in domestic pigs over 4 and 12 weeks. In each animal, 4 zirconium and 4 titanium implants (WhiteSky, BlueSky, Bredent, Germany) were inserted in the os frontale and analysed by histomorphometry. Cytotoxicity and SEM showed good biocompatibility in relation to the investigated implant materials. Histological results showed direct bone–implant contact of the implant surfaces. The zirconium implants showed a slight delay in osseointegration in terms of bone–implant contact as measured by histomorphometry (after 4 weeks, zirconium (59.3 4.6%) versus titanium (64.1 3.9%); after 12 weeks, zirconium (67.1 2.3%) versus titanium (73.6 3.2%). A statistically significant difference between the two groups was not observed. The results indicated similar biocompatibility and osseointegration for zirconium compared to titanium implants.
Unobtrusive, tooth-coloured implants are needed to achieve the best possible aesthetic implant-supported restoration. Titanium implants may be visible in the event of recession or when tissue thinning occurs. In these cases, ceramic implants 0901-5027/050638 + 08 $36.00/0
may offer an aesthetic advantage and result in less plaque accumulation on the implant surface1. If there is titanium incompatibility, ceramic implants may be suitable as a substitute2. With regard to possible indications, there is a lack of
Keywords: dental implants; zirconia; biocompatibility; osseointegration; cell growth.. Accepted for publication 2 February 2012 Available online 8 March 2012
clinical experience with ceramic implants and this should be weighed against the excellent success rates documented by a large number of studies using titanium implants Titanium has been the material of choice for dental implants for about 30
# 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Zirconia versus titanium years and the success rates for various indications have remained high3. For that reason, dental implants made of other materials should not be used, unless the manufacturer can show that the results are evidence-based and at least equivalent to titanium implants4. Allergies to titanium are extremely rare and the literature describing titanium allergies has been restricted to case studies5. Disadvantages of titanium implants could include unfavourable aesthetic results due to titanium shining through or visible metal being exposed by gingival recession. An additional disadvantage has been described by SCARANO et al.1, who showed that plaque accumulation in the gingival areas was reduced by zirconium implants compared to titanium implants. The use of ceramic implants may reduce the risk of peri-implantitis1. Metallosis after the insertion of titanium implants can also be due to a proinflammatory reaction, which could lead to implant loss over time. Tooth-coloured ceramics with lower plaque accumulation and the same success rate can be regarded as a favourable alternative to the gold standard ‘titanium implants’. There have been some negative experiences with dental implants made of aluminium oxide ceramics, but yttriumstabilized tetragonal zirconium oxide (zirconia)6 has been successfully introduced for frameworks and further indications in dentistry. This material has good chemical and physical properties, such as high flexural strength (900–1200 MPa), hardness (1200 Vickers), a Weibull modulus of 10–12, fracture toughness of 8 Mpa HmKIc and a low potential for corrosion7. Ceramic materials have been used successfully in orthopaedic surgery for many years. Biocompatibility tests have produced positive results, while carcinogenicity and mutagenicity tests have shown no negative results8. The interpretation of the results of animal experiments investigating zirconium oxide in terms of its osseoconductive properties and, therefore, its ability for osseointegration, has been controversial because of the lack of long-term clinical investigations. Therefore, the use of zirconium dioxide (ZrO2) as an implant material and its potential advantages over titanium for the jaw area remain a topic for discussion. The aim of the present investigation was to compare titanium versus zirconium dioxide implants in terms of biocompatibility in cell cultures. In addition, the osseointegration in adult domestic pigs was investigated in vivo.
Materials and methods 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 mg streptomycin/ml, 1 mmol/l ascorbic acid and 10 nmol/l dexamethasone at 37 8C with 5% CO2. The cell seeding was performed after the second passage. During passaging, the cells were detached from 75 cm2 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 105 cells/75 cculturem2 cell culture flasks. The cells were cultured in the same medium used for the cell seeding in a humidified atmosphere with 5% CO2 at 37 8C. 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 (FDAtest), lactate dehydrogenase-test (LDH-test), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium-bromide-test
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(MTT-test), 5-bromo-2-deoxyuridine-test (BrdU-test) and water soluble tetrazoliumtest (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 acidetched titanium surface; and a dual acidetched 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 8C. Assessment of cell vitality
Cell vitality was assessed by FDA-test and propidium iodide (PI) staining. 1 104 cells in cell culture medium 10% FCS, 100IE penicillin/ml, 100 mg 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 ml of the eluate was added to the cells. After 24 h of incubation at 37 8C and 5% CO2, cells were rinsed with PBS and immersed in an FDA solution prepared by diluting 30 ml 1 mg FDA/ ml acetone in 10 ml PBS. After incubation for 15 min at 37 8C in darkness, the FDA solution was removed by suction and replaced with a PI solution prepared by diluting 500 ml 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 ml DMEM with FCS, 100IE penicillin/ml, 100 mg streptomycin/ml, 1 mmol/l ascorbic acid and 100 nmol/l dexamethasone at a concentration of 5 103 cells/ well. After culture for 24 h in a humidified
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atmosphere with 5% CO2 at 37 8C, the medium was removed and replaced with 150 ml 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 ml 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 5–0.16 103 cells/well served as standards. The remaining 50 ml eluate per well was removed and replaced with 10 ml DMEM containing 10% FCS, 100IE penicillin/ml, 100 mg 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 MTTtest 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 1 104 cells/ well. The cells were cultured in 2000 ml of the same medium used for cell seeding in a humidified atmosphere with 5% CO2, at 37 8C. 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)-2H5-tetrazolio]-1,3-benzene disulfonate) by mitochondrial dehydrogenases in viable cells. Briefly, 20 ml 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% CO2 at 37 8C, the medium was transferred to 96well 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 (1 104 cells/well) using an XL30CP device (Phillips Electron Optics GmbH, Kassel, Germany) operating at 10–25 kV, described by YANG et al.9 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 ChristianAlbrechts-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 mm/day) is similar to that of humans (1–1.5 mm/ day)10. The animals were kept in small groups in purpose-designed sties and fed a standard diet (Altromin 90231, 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 (Ketavets1, Ratiopharm, Ulm, Germany). The frontal skull of the animals was selected for following properties: it provides comparable placement sites inter- and intraindividually; 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 forte1, 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 3.01, 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. forte1, Gru¨nenthal GmbH, Aachen, Germany). Postoperative pain control was one injection of 500 mg metamizol i.m. (Novalgin1, Hoechst AG, Bad Soden, Germany) and oral tramadol 2 50 mg/ day (Tramal1, Gru¨nenthal 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 AG1, 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 8C) to render the organic matrix insoluble. The specimens were dehydrated in increasing concentrations of alcohol at 21 8C in a dehydration unit (Shandon Citadel 1000, Shandon GmbH, Frankfurt, Germany). The specimens were embedded in Technovit 91001 (Heraeus Kulzer, Kulzer Division, Werheim, Germany) for histological examination by means of grounded sections using the technique described by DONATH & BREUNER11.
Zirconia versus titanium 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 mm. The microradiography of the 90 mm specimens was performed on 2 in. 2 in. plates (Microchrome Technology Inc., San Jose`, CA, USA) at 3 mAs and 25 kV using a microradiography device (Faxitron X-ray systems, Hewlett Packard GmbH, Bo¨blingen, 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 a = 0.05. For further evaluation, approximately 20 mm 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). Results Testing biocompatibility
After incubation with the eluates, all samples showed viable cells. The pronounced green colour of the cells due to FDA staining demonstrated their vitality, while the absence of a red colour despite PI staining indicated that no cells died as a result of eluates from both membranes (Fig. 1). Regarding the colorimetric tests, LDH was transformed to percentage values (%) by the distance to the low value related to the observed range between the high and low values. All other tests (MTT, WST and BrdU) were transformed to percentage values (%) in relation to control measurements. The results from the LDH-test showed a high cytotoxic effect in only one type of titanium implant. This was only seen with the 10 min and 1 h eluate and not observed with the 24 h eluate. No cytotoxicity was observed with the eluates of the zirconium implants.
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The MTT-test indicated the cellular metabolic activity and showed a high activity with the eluates from all implants; two types of titanium implants showed a slightly decreased activity. The BrdU-test showed a cell proliferation by incorporation of BrdU during DNA synthesis. A proliferation was observed with the eluates of all implant types. The WST-test indicated the metabolic activity of the cells that were cultivated on the implants after they were eluated. A reduced metabolic activity was observed with two titanium implants. The metabolic activity on the zirconium implant was slightly reduced on the implant that was eluated for 24 h prior to cultivation. This was not observed on the cells cultivated on the zirconium implants that were eluated for 10 min or 1 h prior to cultivation (Fig. 2). Regarding the examination by SEM, the cells cultivated on the implant surface showed good contact to the implant surface with all tested implant types (Fig. 3).
Testing osseointegration in an animal model
After the surgery, all animals showed normal eating behaviour. No signs of infection were noted on clinical examination at any time during the observation period. Neither were any signs of infection observed in the preparations. BIC increased during the observational period, both for the zirconium implants and the titanium implants. After a healing phase of 4 weeks, the mean BIC for zirconium implants was 59.3 4.6% and for titanium implants 64.1 3.9%. After a healing phase of 12 weeks, a mean BIC for zirconium implants of 67.1 2.3% and for titanium implants
Fig. 1. Fluorescence microscopy images of human osteoblast cells after incubation with the 10 min eluates of a titanium implant (a) and a zirconium implant (b) after staining with fluorescein diacetate (FDA) and propidium iodide (PI): note the green colour due to staining with FDA, indicating living cells. The lack of a red colour despite staining with PI indicates absence of dead cells.
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Fig. 2. Results of the LDH-test (top left), MTT-test (top right), BrdU-test (bottom left) and WST-test (bottom right). The x-axis shows the three different times of the eluates (1: 10 min; 2: 1 h; 3: 24 h), on the y-axis, the results in percent are described compared to the control. Cases 1–5 are titanium implants, case 6 (light blue) is the zirconium implant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 3. Close-up SEM images of a titanium implant (a) and a zirconium implant (b) seeded with human osteoblast cells. Good contact of the cells to the implant surface is shown (magnification 1000).
of 73.6 3.2% was found. The zirconium implants showed a slight delayed osseointegration compared to the titanium implants. No statistically signifidifferences between the cant osseointegration of the zirconium and titanium implants (p < 0.05) in relation to BIC after 4 or 12 weeks was detected (Fig. 4). Microradiographically and histologically, no connective tissue sheath was observed on the implant threads. After 4 weeks, newly formed osteoid and woven bone were evident on the implant threads (Figs. 5 and 6). Close BIC was seen on the titanium, as well as on the zirconium surface circumferentially. After 12 weeks of healing, the tissue was re-established as lamellar bone
Fig. 4. Increasing BIC during the experiment (4 and 12 weeks) is shown. No statistical significance was detected between the two treatment groups (p < 0.05).
Zirconia versus titanium
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Fig. 5. Microradiograph with no connective tissue sheath on the implant threads (titanium implants on the left; zirconium implants on the right). After 4 (a + b) and 12 weeks (c + d), a close BIC was observed (magnification 20).
Fig. 6. At 4 weeks after implantation, similar BIC with circumferential bone formation and osteoid on titanium (a) and zirconia implant surfaces (b) (toluidine blue, magnification 40). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
in the contact zone of zirconium and the titanium implants (Fig. 7). Discussion
The first positive experiences of replacing missing teeth with ceramic implants were based on the results of Sandhaus in 1971, and the idea was taken up by SCHULTE et al.12, with the so-called ‘Tu¨bingen immediate implant’. The implants were made of aluminium oxide, which can take pressure, but are prone to fracture at lower levels when exposed to tension, bending and torsion. Critical biomechanical
experiments have shown that aluminium oxide ceramics were of limited use as implant materials whereas ZrO2 might be the material of choice13. ZrO2 was less stiff than aluminium oxide ceramic, was tooth coloured, could be turned or ground, and the surface texture could be modified14. No chemical or physical bonding occurred between ZrO2 and plaque, consequently the unwanted accumulation of plaque, such as occurs with titanium, could be ignored and there has been no local irritation through the so-called biofilm1. Zirconia is a bio inert non-resorbable metal oxide that offers mechanical
properties that are superior to other ceramic biomaterials and seem to be integrated into bone in a similar fashion as titanium8. The aim of the present study was to compare the biocompatibility and reaction of human osteoblast to ZrO2 and titanium surfaces in vitro. The in vitro ZrO2 osseointegration and titanium dental implant data were compared to the in vivo results. Surface material, appearance and microtopography have been shown to be important factors influencing growth and differentiation of osteoblasts15. The ceramic implant material used, ZrO2, showed
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Fig. 7. After 12 weeks of healing, mature lamellar bone is evident in intimate contact with the titanium implant (a) and zirconia implant (b) (toluidine blue, magnification 50). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
good biocompatibility in the in vitro study. The biocompatibility of the ceramic implant material has also been confirmed by other studies16. No carcinogenic or mutagenic potential has been found. Recently, Rothamel et al. investigated the biocompatibility and osseointegration of structured zirconia implants in vitro and in vivo17. The growth of osteoblast-like SAOS-2 cells was significantly better on the machined zirconia surfaces compared to sand-blasted zirconia and polished titanium surfaces. The authors emphasized that manufacturing and cleaning processes may have an impact on the biocompatibility of rough zirconia surfaces. HOFFMANN et al. observed a high degree of bone apposition on zirconia and titanium implants, with comparable results for the two tested materials in a histological evaluation in rabbits18. The results of the present study showed normal cell growth on all investigated surfaces. SEM observations demonstrated appropriate adhesion and spreading of cells on both zirconia and titanium surfaces. PONADER et al.19, described higher growth rates of primary osteoblasts on compact smooth textured titanium surfaces compared to rough surfaces, but did not find effects of surface roughness on expression of osteogenic genes. FILLIES et al.20 demonstrated an increased synthesis of bone-specific matrix proteins, whereas other studies showed reduced alkaline phosphatase-specific activity in primary osteoblasts on rough surfaces21.
GUIZZARDI et al.22, detected no influence of surface topography on expression of characteristic osteoblast proteins. These controversial results underscored the complexity of osteoblast reactions on surface composition and topography. Hao et al. showed, that increased surface energy of magnesia partially stabilized zirconia (MgO-PSZ) bioceramic after CO2 laser treatment resulted in higher initial cell attachment and enhanced cell growth of human foetal osteoblast cells23. The high cell detachment from the zirconia surfaces could also be due to the surface topography, because the zirconia surfaces showed fewer pores and irregularities than the titanium surfaces and osteoblasts show a greater tendency to attach to deep lying areas19. In the animal experiments, a tendency to delayed osseointegration was identified. A direct comparison with titanium implants showed that the ceramic implants had a smaller BIC ratio. After a healing phase of 4 weeks, the mean BIC for zirconium implants was 59.3 4.6% and for titanium implants 64.1 3.9%. After a healing phase of 12 weeks, a mean BIC for zirconium implants of 67.1 2.3% and for titanium implants of 73.6 3.2% was found. These results were in accordance with other animal studies that also examined zirconia and titanium implant surfaces24,25. BIC values >60% were reported by several studies1,25. These differences could be caused by the type of animal model
(monkeys, rabbits, dogs and minipigs) and different implantation sites. Compared to other studies, the frontal skull base showed standardized conditions with comparable insertion sites. The bone was of desmal origin, not vascularized by a central blood vessel and with bone quality class II– III26. The tendency for delayed osseointegration of zirconium implants in the present study (59.3% after 4 weeks, 67.1% after 12 weeks) was in accordance with other studies25. It is well known that surface modifications can enhance the bone integration of titanium implants in diverse animal models27,28. Earlier experimental studies revealed that surface roughness and topography greatly influence the osseointegration of zirconia implants24. SENNERBY et al. used a coating technique to receive porous surface modifications of the zirconia implants29. In spite of evident differences in surface roughness, there were no significant differences observed in the osseointegration (BIC or bone area filling in the threads) in the investigated implants. Only the removal torque test revealed significantly lower results for the non-modified zirconia implants. These results and the results of SCARANO et al., who used unmodified zirconia implants, indicated a considerable biocompatibility of zirconia implants, even without surface treatment1. Submicrometric and nanometric topography determined cell reactions including cell orientation,
Zirconia versus titanium changes in cell motility, cell adhesion and cell shape. These topographic features played an important role in the early state of osseointegration of dental implants. In addition, differences in the physical and chemical properties of the material also affected cell responses30. The present study showed that osteoblasts were able to attach, proliferate and differentiate on zirconia surfaces and showed good biocompatibility in the in vitro study. The in vivo results revealed that the osseointegration of zirconia implants was similar to titanium implants. This implies that the ceramic material may also have beneficial effects on biocompatibility and osseointegration in patients. These results are promising for the use of zirconia implants for dental indications in the future.
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Funding
None. 12.
Competing interests
The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
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Ethical approval
The study was approved by the Ethics Commission of the Christian-AlbrechtsUniversity of Kiel, Germany (AZ 118/ 07) and the Animal Ethics Committee at the Semelweis University of Budapest, Hungary (No. 1053/eoh/2007).
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with titanium dental implants: a clinical report. J Prosthet Dent 2008;100:344–7. Kelly JR, Denry I. Stabilized zirconia as a structural ceramic: an overview. Dent Mater 2008;24:289–98. Caglar A, Bal BT, Aydin C, Yilmaz H, Ozkan S. Evaluation of stresses occurring on three different zirconia dental implants: threedimensional finite element analysis. Int J Oral Maxillofac Implants 2010;25:95–103. Piconi C, Maccauro G. Zirconia as a ceramic biomaterial. Biomaterials 1999;20:1–25. Yang B, Ludwig K, Adelung R, Kern M. Micro-tensile bond strength of three luting resins to human regional dentin. Dent Mater 2006;22:45–56. Ho¨nig JF, Merten HA. Das Go¨ttinger Miniaturschwein (GMS) als Versuchstier in der human-medizinischen osteologischen Grundlagenforschung. Z Zahna¨rztl Implantol 1993;2:244–54. Donath K, Breuner G. A method for the study of undecalcified bones and teeth with attached soft tissues. The Sage-Schliff (sawing and grinding) technique. J Oral Pathol 1982;11:318–26. Schulte W, Kleineikenscheidt H, Schareyka R, Heimke G. Concept and testing of the Tubingen immediate implant. Dtsch Zahnarztl Z 1978;33:319–25. Andreiotelli M, Wenz HJ, Kohal RJ. Are ceramic implants a viable alternative to titanium implants? A systematic literature review. Clin Oral Implants Res 2009;20:32–47. Oblak C, Jevnikar P, Kosmac T, Funduk N, Marion L. Fracture resistance and reliability of new zirconia posts. J Prosthet Dent 2004;91:342–8. Ko HC, Han JS, Bachle M, Jang JH, Shin SW, Kim DJ. Initial osteoblast-like cell response to pure titanium and zirconia/alumina ceramics. Dent Mater 2007;23:1349–55. Depprich R, Ommerborn M, Zipprich H, Naujoks C, Handschel J, Wiesmann HP, Ku¨bler NR, Meyer U. Behavior of osteoblastic cells cultured on titanium and structured zirconia surfaces. Head Face Med 2008;4:29. Rothamel D, Ferrari D, Herten M, Schwarz F, Becker J. Biokompatibilita¨t und Hartgewebsintegration einphasiger oberfla¨chenstrukturierter Zirkoniumoxidimplantate – Eine kombinierte in vitro – und in vivoStudie. Implantologie 2007;15:405–14. Hoffmann O, Angelov N, Gallez F, Jung RE, Weber FE. The zirconia implant–bone interface: a preliminary histologic evaluation in rabbits. Int J Oral Maxillofac Implants 2008;23:691–5. Ponader S, Vairaktaris E, Heinl P, Wilmowsky CV, Rottmair A, Korner C, Singer RF, Holst S, Schlegel KA, Neukam FW, Nkenke E. Effects of topographical surface modifications of electron beam melted Ti– 6Al–4V titanium on human fetal osteoblasts. J Biomed Mater Res A 2008;84:1111–9. Fillies T, Wiesmann HP, Sommer D, Joos U, Meyer U. Osteoblast reaction on SLA and
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microgrooved implant surfaces. Mund Kiefer Gesichtschir 2005;9:24–8. Lohmann CH, Bonewald LF, Sisk MA, Sylvia VL, Cochran DL, Dean DD, Boyan BD, Schwartz Z. Maturation state determines the response of osteogenic cells to surface roughness and 1,25-dihydroxyvitamin D3. J Bone Miner Res 2000;15:1169–80. Guizzardi S, Galli C, Martini D, Belletti S, Tinti A, Raspanti M, Taddei P, Ruggeri A, Scandroglio R. Different titanium surface treatment influences human mandibular osteoblast response. J Periodontol 2004;75:273–82. Hao L, Lawrence J, Chian KS. Osteoblast cell adhesion on a laser modified zirconia based bioceramic. J Mater Sci Mater Med 2005;16:719–26. Gahlert M, Gudehus T, Eichhorn S, Steinhauser E, Kniha H, Erhardt W. Biomechanical and histomorphometric comparison between zirconia implants with varying surface textures and a titanium implant in the maxilla of miniature pigs. Clin Oral Implants Res 2007;18:662–8. Sennerby L, Gottlow J. Clinical outcomes of immediate/early loading of dental implants. A literature review of recent controlled prospective clinical studies. Aust Dent J 2008;53:S82–8. Schlegel KA, Lang FJ, Donath K, Kulow JT, Wiltfang J. The monocortical critical size bone defect as an alternative experimental model in testing bone substitute materials. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:7–13. Gotfredsen K, Berglundh T, Lindhe J. Anchorage of titanium implants with different surface characteristics: an experimental study in rabbits. Clin Implant Dent Relat Res 2000;2:120–8. Triplett RG, Frohberg U, Sykaras N, Woody RD. Implant materials, design, and surface topographies: their influence on osseointegration of dental implants. J Long Term Eff Med Implants 2003;13:485–501. Sennerby L, Dasmah A, Larsson B, Iverhed M. Bone tissue responses to surface-modified zirconia implants: a histomorphometric and removal torque study in the rabbit. Clin Implant Dent Relat Res 2005;7:S13–20. Schwartz Z, Kieswetter K, Dean DD, Boyan BD. Underlying mechanisms at the bone– surface interface during regeneration. J Periodontal Res 1997;32:166–71.
Address: Katrin Hertrampf Clinic of Oral and Maxillofacial Surgery University Hospital Schleswig-Holstein Campus Kiel Arnold Heller Str. 3 Haus 26 24105 Kiel Germany Tel.: +49 431 5972896 fax: +49 431 597 4084 E-mail: [email protected]
Research Paper Dental Implants
A comparison of biocompatibility and osseointegration of ceramic and titanium implants: an in vivo and in vitro study
B. Y. K. E.
Mo¨ller1, H. Terheyden2, Ac¸il1, N. M. Purcz1, Hertrampf1, A. Tabakov1, Behrens1, J. Wiltfang1
1
Clinic of Oral and Maxillofacial Surgery, University Hospital Schleswig-Holstein, Campus Kiel, Arnold Heller Str. 3, Haus 26, 24105 Kiel, Germany; 2Department of Oral and Maxillofacial Surgery, Red Cross Hospital, Hansteinstr. 29, 34121 Kassel, Germany
B. Mo¨ller, H. Terheyden, Y. Ac¸il, N. M. Purcz, K. Hertrampf, A. Tabakov, E. Behrens, J. Wiltfang: A comparison of biocompatibility and osseointegration of ceramic and titanium implants: an in vivo and in vitro study. Int. J. Oral Maxillofac. Surg. 2012; 41: 638–645. # 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. This study compared the biocompatibility in vitro and the osseointegration in vivo of zirconium and titanium implants regarding implant surfaces and the bone– implant contacts. The different implant surfaces and the biocompatibility of zirconium versus titanium implants were determined by vitality and cytotoxic tests in vitro. The contact of the osteoblasts to the implant surface was determined by scanning electron microscopy (SEM). The in vivo study for osseointegration was performed in domestic pigs over 4 and 12 weeks. In each animal, 4 zirconium and 4 titanium implants (WhiteSky, BlueSky, Bredent, Germany) were inserted in the os frontale and analysed by histomorphometry. Cytotoxicity and SEM showed good biocompatibility in relation to the investigated implant materials. Histological results showed direct bone–implant contact of the implant surfaces. The zirconium implants showed a slight delay in osseointegration in terms of bone–implant contact as measured by histomorphometry (after 4 weeks, zirconium (59.3 4.6%) versus titanium (64.1 3.9%); after 12 weeks, zirconium (67.1 2.3%) versus titanium (73.6 3.2%). A statistically significant difference between the two groups was not observed. The results indicated similar biocompatibility and osseointegration for zirconium compared to titanium implants.
Unobtrusive, tooth-coloured implants are needed to achieve the best possible aesthetic implant-supported restoration. Titanium implants may be visible in the event of recession or when tissue thinning occurs. In these cases, ceramic implants 0901-5027/050638 + 08 $36.00/0
may offer an aesthetic advantage and result in less plaque accumulation on the implant surface1. If there is titanium incompatibility, ceramic implants may be suitable as a substitute2. With regard to possible indications, there is a lack of
Keywords: dental implants; zirconia; biocompatibility; osseointegration; cell growth.. Accepted for publication 2 February 2012 Available online 8 March 2012
clinical experience with ceramic implants and this should be weighed against the excellent success rates documented by a large number of studies using titanium implants Titanium has been the material of choice for dental implants for about 30
# 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Zirconia versus titanium years and the success rates for various indications have remained high3. For that reason, dental implants made of other materials should not be used, unless the manufacturer can show that the results are evidence-based and at least equivalent to titanium implants4. Allergies to titanium are extremely rare and the literature describing titanium allergies has been restricted to case studies5. Disadvantages of titanium implants could include unfavourable aesthetic results due to titanium shining through or visible metal being exposed by gingival recession. An additional disadvantage has been described by SCARANO et al.1, who showed that plaque accumulation in the gingival areas was reduced by zirconium implants compared to titanium implants. The use of ceramic implants may reduce the risk of peri-implantitis1. Metallosis after the insertion of titanium implants can also be due to a proinflammatory reaction, which could lead to implant loss over time. Tooth-coloured ceramics with lower plaque accumulation and the same success rate can be regarded as a favourable alternative to the gold standard ‘titanium implants’. There have been some negative experiences with dental implants made of aluminium oxide ceramics, but yttriumstabilized tetragonal zirconium oxide (zirconia)6 has been successfully introduced for frameworks and further indications in dentistry. This material has good chemical and physical properties, such as high flexural strength (900–1200 MPa), hardness (1200 Vickers), a Weibull modulus of 10–12, fracture toughness of 8 Mpa HmKIc and a low potential for corrosion7. Ceramic materials have been used successfully in orthopaedic surgery for many years. Biocompatibility tests have produced positive results, while carcinogenicity and mutagenicity tests have shown no negative results8. The interpretation of the results of animal experiments investigating zirconium oxide in terms of its osseoconductive properties and, therefore, its ability for osseointegration, has been controversial because of the lack of long-term clinical investigations. Therefore, the use of zirconium dioxide (ZrO2) as an implant material and its potential advantages over titanium for the jaw area remain a topic for discussion. The aim of the present investigation was to compare titanium versus zirconium dioxide implants in terms of biocompatibility in cell cultures. In addition, the osseointegration in adult domestic pigs was investigated in vivo.
Materials and methods 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 mg streptomycin/ml, 1 mmol/l ascorbic acid and 10 nmol/l dexamethasone at 37 8C with 5% CO2. The cell seeding was performed after the second passage. During passaging, the cells were detached from 75 cm2 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 105 cells/75 cculturem2 cell culture flasks. The cells were cultured in the same medium used for the cell seeding in a humidified atmosphere with 5% CO2 at 37 8C. 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 (FDAtest), lactate dehydrogenase-test (LDH-test), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium-bromide-test
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(MTT-test), 5-bromo-2-deoxyuridine-test (BrdU-test) and water soluble tetrazoliumtest (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 acidetched titanium surface; and a dual acidetched 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 8C. Assessment of cell vitality
Cell vitality was assessed by FDA-test and propidium iodide (PI) staining. 1 104 cells in cell culture medium 10% FCS, 100IE penicillin/ml, 100 mg 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 ml of the eluate was added to the cells. After 24 h of incubation at 37 8C and 5% CO2, cells were rinsed with PBS and immersed in an FDA solution prepared by diluting 30 ml 1 mg FDA/ ml acetone in 10 ml PBS. After incubation for 15 min at 37 8C in darkness, the FDA solution was removed by suction and replaced with a PI solution prepared by diluting 500 ml 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 ml DMEM with FCS, 100IE penicillin/ml, 100 mg streptomycin/ml, 1 mmol/l ascorbic acid and 100 nmol/l dexamethasone at a concentration of 5 103 cells/ well. After culture for 24 h in a humidified
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atmosphere with 5% CO2 at 37 8C, the medium was removed and replaced with 150 ml 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 ml 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 5–0.16 103 cells/well served as standards. The remaining 50 ml eluate per well was removed and replaced with 10 ml DMEM containing 10% FCS, 100IE penicillin/ml, 100 mg 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 MTTtest 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 1 104 cells/ well. The cells were cultured in 2000 ml of the same medium used for cell seeding in a humidified atmosphere with 5% CO2, at 37 8C. 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)-2H5-tetrazolio]-1,3-benzene disulfonate) by mitochondrial dehydrogenases in viable cells. Briefly, 20 ml 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% CO2 at 37 8C, the medium was transferred to 96well 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 (1 104 cells/well) using an XL30CP device (Phillips Electron Optics GmbH, Kassel, Germany) operating at 10–25 kV, described by YANG et al.9 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 ChristianAlbrechts-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 mm/day) is similar to that of humans (1–1.5 mm/ day)10. The animals were kept in small groups in purpose-designed sties and fed a standard diet (Altromin 90231, 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 (Ketavets1, Ratiopharm, Ulm, Germany). The frontal skull of the animals was selected for following properties: it provides comparable placement sites inter- and intraindividually; 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 forte1, 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 3.01, 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. forte1, Gru¨nenthal GmbH, Aachen, Germany). Postoperative pain control was one injection of 500 mg metamizol i.m. (Novalgin1, Hoechst AG, Bad Soden, Germany) and oral tramadol 2 50 mg/ day (Tramal1, Gru¨nenthal 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 AG1, 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 8C) to render the organic matrix insoluble. The specimens were dehydrated in increasing concentrations of alcohol at 21 8C in a dehydration unit (Shandon Citadel 1000, Shandon GmbH, Frankfurt, Germany). The specimens were embedded in Technovit 91001 (Heraeus Kulzer, Kulzer Division, Werheim, Germany) for histological examination by means of grounded sections using the technique described by DONATH & BREUNER11.
Zirconia versus titanium 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 mm. The microradiography of the 90 mm specimens was performed on 2 in. 2 in. plates (Microchrome Technology Inc., San Jose`, CA, USA) at 3 mAs and 25 kV using a microradiography device (Faxitron X-ray systems, Hewlett Packard GmbH, Bo¨blingen, 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 a = 0.05. For further evaluation, approximately 20 mm 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). Results Testing biocompatibility
After incubation with the eluates, all samples showed viable cells. The pronounced green colour of the cells due to FDA staining demonstrated their vitality, while the absence of a red colour despite PI staining indicated that no cells died as a result of eluates from both membranes (Fig. 1). Regarding the colorimetric tests, LDH was transformed to percentage values (%) by the distance to the low value related to the observed range between the high and low values. All other tests (MTT, WST and BrdU) were transformed to percentage values (%) in relation to control measurements. The results from the LDH-test showed a high cytotoxic effect in only one type of titanium implant. This was only seen with the 10 min and 1 h eluate and not observed with the 24 h eluate. No cytotoxicity was observed with the eluates of the zirconium implants.
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The MTT-test indicated the cellular metabolic activity and showed a high activity with the eluates from all implants; two types of titanium implants showed a slightly decreased activity. The BrdU-test showed a cell proliferation by incorporation of BrdU during DNA synthesis. A proliferation was observed with the eluates of all implant types. The WST-test indicated the metabolic activity of the cells that were cultivated on the implants after they were eluated. A reduced metabolic activity was observed with two titanium implants. The metabolic activity on the zirconium implant was slightly reduced on the implant that was eluated for 24 h prior to cultivation. This was not observed on the cells cultivated on the zirconium implants that were eluated for 10 min or 1 h prior to cultivation (Fig. 2). Regarding the examination by SEM, the cells cultivated on the implant surface showed good contact to the implant surface with all tested implant types (Fig. 3).
Testing osseointegration in an animal model
After the surgery, all animals showed normal eating behaviour. No signs of infection were noted on clinical examination at any time during the observation period. Neither were any signs of infection observed in the preparations. BIC increased during the observational period, both for the zirconium implants and the titanium implants. After a healing phase of 4 weeks, the mean BIC for zirconium implants was 59.3 4.6% and for titanium implants 64.1 3.9%. After a healing phase of 12 weeks, a mean BIC for zirconium implants of 67.1 2.3% and for titanium implants
Fig. 1. Fluorescence microscopy images of human osteoblast cells after incubation with the 10 min eluates of a titanium implant (a) and a zirconium implant (b) after staining with fluorescein diacetate (FDA) and propidium iodide (PI): note the green colour due to staining with FDA, indicating living cells. The lack of a red colour despite staining with PI indicates absence of dead cells.
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Fig. 2. Results of the LDH-test (top left), MTT-test (top right), BrdU-test (bottom left) and WST-test (bottom right). The x-axis shows the three different times of the eluates (1: 10 min; 2: 1 h; 3: 24 h), on the y-axis, the results in percent are described compared to the control. Cases 1–5 are titanium implants, case 6 (light blue) is the zirconium implant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 3. Close-up SEM images of a titanium implant (a) and a zirconium implant (b) seeded with human osteoblast cells. Good contact of the cells to the implant surface is shown (magnification 1000).
of 73.6 3.2% was found. The zirconium implants showed a slight delayed osseointegration compared to the titanium implants. No statistically signifidifferences between the cant osseointegration of the zirconium and titanium implants (p < 0.05) in relation to BIC after 4 or 12 weeks was detected (Fig. 4). Microradiographically and histologically, no connective tissue sheath was observed on the implant threads. After 4 weeks, newly formed osteoid and woven bone were evident on the implant threads (Figs. 5 and 6). Close BIC was seen on the titanium, as well as on the zirconium surface circumferentially. After 12 weeks of healing, the tissue was re-established as lamellar bone
Fig. 4. Increasing BIC during the experiment (4 and 12 weeks) is shown. No statistical significance was detected between the two treatment groups (p < 0.05).
Zirconia versus titanium
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Fig. 5. Microradiograph with no connective tissue sheath on the implant threads (titanium implants on the left; zirconium implants on the right). After 4 (a + b) and 12 weeks (c + d), a close BIC was observed (magnification 20).
Fig. 6. At 4 weeks after implantation, similar BIC with circumferential bone formation and osteoid on titanium (a) and zirconia implant surfaces (b) (toluidine blue, magnification 40). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
in the contact zone of zirconium and the titanium implants (Fig. 7). Discussion
The first positive experiences of replacing missing teeth with ceramic implants were based on the results of Sandhaus in 1971, and the idea was taken up by SCHULTE et al.12, with the so-called ‘Tu¨bingen immediate implant’. The implants were made of aluminium oxide, which can take pressure, but are prone to fracture at lower levels when exposed to tension, bending and torsion. Critical biomechanical
experiments have shown that aluminium oxide ceramics were of limited use as implant materials whereas ZrO2 might be the material of choice13. ZrO2 was less stiff than aluminium oxide ceramic, was tooth coloured, could be turned or ground, and the surface texture could be modified14. No chemical or physical bonding occurred between ZrO2 and plaque, consequently the unwanted accumulation of plaque, such as occurs with titanium, could be ignored and there has been no local irritation through the so-called biofilm1. Zirconia is a bio inert non-resorbable metal oxide that offers mechanical
properties that are superior to other ceramic biomaterials and seem to be integrated into bone in a similar fashion as titanium8. The aim of the present study was to compare the biocompatibility and reaction of human osteoblast to ZrO2 and titanium surfaces in vitro. The in vitro ZrO2 osseointegration and titanium dental implant data were compared to the in vivo results. Surface material, appearance and microtopography have been shown to be important factors influencing growth and differentiation of osteoblasts15. The ceramic implant material used, ZrO2, showed
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Fig. 7. After 12 weeks of healing, mature lamellar bone is evident in intimate contact with the titanium implant (a) and zirconia implant (b) (toluidine blue, magnification 50). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
good biocompatibility in the in vitro study. The biocompatibility of the ceramic implant material has also been confirmed by other studies16. No carcinogenic or mutagenic potential has been found. Recently, Rothamel et al. investigated the biocompatibility and osseointegration of structured zirconia implants in vitro and in vivo17. The growth of osteoblast-like SAOS-2 cells was significantly better on the machined zirconia surfaces compared to sand-blasted zirconia and polished titanium surfaces. The authors emphasized that manufacturing and cleaning processes may have an impact on the biocompatibility of rough zirconia surfaces. HOFFMANN et al. observed a high degree of bone apposition on zirconia and titanium implants, with comparable results for the two tested materials in a histological evaluation in rabbits18. The results of the present study showed normal cell growth on all investigated surfaces. SEM observations demonstrated appropriate adhesion and spreading of cells on both zirconia and titanium surfaces. PONADER et al.19, described higher growth rates of primary osteoblasts on compact smooth textured titanium surfaces compared to rough surfaces, but did not find effects of surface roughness on expression of osteogenic genes. FILLIES et al.20 demonstrated an increased synthesis of bone-specific matrix proteins, whereas other studies showed reduced alkaline phosphatase-specific activity in primary osteoblasts on rough surfaces21.
GUIZZARDI et al.22, detected no influence of surface topography on expression of characteristic osteoblast proteins. These controversial results underscored the complexity of osteoblast reactions on surface composition and topography. Hao et al. showed, that increased surface energy of magnesia partially stabilized zirconia (MgO-PSZ) bioceramic after CO2 laser treatment resulted in higher initial cell attachment and enhanced cell growth of human foetal osteoblast cells23. The high cell detachment from the zirconia surfaces could also be due to the surface topography, because the zirconia surfaces showed fewer pores and irregularities than the titanium surfaces and osteoblasts show a greater tendency to attach to deep lying areas19. In the animal experiments, a tendency to delayed osseointegration was identified. A direct comparison with titanium implants showed that the ceramic implants had a smaller BIC ratio. After a healing phase of 4 weeks, the mean BIC for zirconium implants was 59.3 4.6% and for titanium implants 64.1 3.9%. After a healing phase of 12 weeks, a mean BIC for zirconium implants of 67.1 2.3% and for titanium implants of 73.6 3.2% was found. These results were in accordance with other animal studies that also examined zirconia and titanium implant surfaces24,25. BIC values >60% were reported by several studies1,25. These differences could be caused by the type of animal model
(monkeys, rabbits, dogs and minipigs) and different implantation sites. Compared to other studies, the frontal skull base showed standardized conditions with comparable insertion sites. The bone was of desmal origin, not vascularized by a central blood vessel and with bone quality class II– III26. The tendency for delayed osseointegration of zirconium implants in the present study (59.3% after 4 weeks, 67.1% after 12 weeks) was in accordance with other studies25. It is well known that surface modifications can enhance the bone integration of titanium implants in diverse animal models27,28. Earlier experimental studies revealed that surface roughness and topography greatly influence the osseointegration of zirconia implants24. SENNERBY et al. used a coating technique to receive porous surface modifications of the zirconia implants29. In spite of evident differences in surface roughness, there were no significant differences observed in the osseointegration (BIC or bone area filling in the threads) in the investigated implants. Only the removal torque test revealed significantly lower results for the non-modified zirconia implants. These results and the results of SCARANO et al., who used unmodified zirconia implants, indicated a considerable biocompatibility of zirconia implants, even without surface treatment1. Submicrometric and nanometric topography determined cell reactions including cell orientation,
Zirconia versus titanium changes in cell motility, cell adhesion and cell shape. These topographic features played an important role in the early state of osseointegration of dental implants. In addition, differences in the physical and chemical properties of the material also affected cell responses30. The present study showed that osteoblasts were able to attach, proliferate and differentiate on zirconia surfaces and showed good biocompatibility in the in vitro study. The in vivo results revealed that the osseointegration of zirconia implants was similar to titanium implants. This implies that the ceramic material may also have beneficial effects on biocompatibility and osseointegration in patients. These results are promising for the use of zirconia implants for dental indications in the future.
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Funding
None. 12.
Competing interests
The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
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Ethical approval
The study was approved by the Ethics Commission of the Christian-AlbrechtsUniversity of Kiel, Germany (AZ 118/ 07) and the Animal Ethics Committee at the Semelweis University of Budapest, Hungary (No. 1053/eoh/2007).
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