2.1

Analysis of Ti and Zr release from Ti-implants and ZrO ₂ -implants in mini pig maxillae

2.1.1

Animals

In this study, 18 female mini pigs (Goettinger mini pig) with an average age of 23.7 months and weight between 31 kg and 51 kg were studied. These animals were kept in cages and fed with a standard diet. The animals were not supplied with food 12 h before and after surgery, but with water accessible ad libitum. The protocol of the animal experiment was approved by the Swedish authorities in Malmö (ethical approval number: M 66/07).

2.1.2

Implant and study design

Threaded implants with a 6-cornered shaft, a diameter of 4.1 mm and length of 10 mm were manufactured using different techniques for Ti (Ti-SLA) and ZrO ₂ to generate nearly identical implant surfaces on the two different materials. Details of the implant manufacturing processes were given in a previous study [ ]. Briefly, Ti and ZrO ₂ grade 4 implants were manufactured using an injection molding technique followed by a chemical treatment with hydrofluoric acid, according to a proprietary process of Institute Straumann AG (Basel, Switzerland). The treatment resulted in a comparable mean surface roughness of 1.26 μm (SD ± 0.02 μm) for the Ti and 0.63 μm (SD ± 0.05 μm) for the ZrO ₂ implants. Six months prior to implant insertion, the positions where the implants would be inserted in the maxillae were created as edentulous areas. During the second operation the implants were inserted in the edentulous parts of the maxilla and allowed to heal submerged under the buccalmucoperiostal covering. Every pig received two different implants in the anterior regions of the maxilla. For the present investigation animals were sacrificed after 12 weeks of healing. During that time interval the implants were not loaded and no dental superstructure (i.e. tooth replacement) was created. Surgical procedures and implant placement were described in detail in a previous study [ ]. Each jawbone received both types of implants with an average distance of 1 cm between implants. After sacrifice, the jawbones were dissected and smaller specimens containing the individual implants were fixed in 4% buffered paraformaldehyde. For removal of the paraformaldehyde the specimens were rinsed in tap water, and afterwards were dehydrated in ascending ethanol fractions (50%, 70%, 96% and 100%), cleared in xylene (Merck, Darmstadt, Germany) and embedded in methylmethacrylate (MMA) (Fluka, Switzerland) [ ]. For the present study 9 implants from 5 different animals were collected. Implants were collected from right and left maxillae and details regarding location and animal number are given in Table 3 . The study design allowed two implants of the same or different type in the anterior region of the maxilla. As a control tissue bone slices 2 cm away from the nearest Ti-/ZrO ₂ -implant were taken. Unfortunately, no other bone tissue from the animals was available.

2.1.3

Ti/Zr content measurements by inductively coupled plasma optical emission spectrometry (ICP-OES)/inductively coupled plasma mass spectrometry (ICP-MS)

2.1.3.1

Sample preparation

Maxilla slices in the sagittal plane were prepared by cutting the MMA-blocks with a diamond band saw (cut-grinder macro, patho-service GmbH, Oststeinbek, Germany). One or two bone slices (approximately 1 mm thick) immediately adjacent to the implant surface were cut from each embedded specimen from the 12-week group.

The bone slices (0−1 mm and 1−2 mm distance from the implant) were placed into 1 ml Acetone for 24 h to remove the embedding resin. The bone slices were then put onto a glass dish, allowed to dry and then were weighed. The bone slices without MMA had a weight between 0.09 and 0.19 g. After determination of their dry weight, the bone slices were dissolved in 1 ml sub-boiled distilled nitric acid at 170 °C for 12 h. The solution was subsequently diluted (1:3) in Milli-Q Water. The contents of the elements Ti and Zr in each slice were first analysed by ICP-MS. The content of elemental Zr was additionally measured by ICP-OES (Ciros Vision, Spectro-Ametec, Kleve, Germany), to verify Zr concentrations since the measurement with ICP-MS could be hampered by interferences.

2.1.3.2

Calculations and statistics

The results were presented as mean ± standard deviation (mean ± SD). Independent two-sample t-test was performed for statistical analysis. Differences were considered statistically significant if the p-value was less than 0.05 (p < 0.05) [ ].

2.1.4

Content distribution measured by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) on resin embedded sections

2.1.4.1

Sample preparation

After the cutting procedure in Section 2.1.3 , the polymerized methacrylate blocks were cut parallel to the long axis of the implants in the mesio-distal plane using a Leica SP1600 saw-microtome (Leica, Wetzlar, Germany). The obtained methacrylate blocks with implants in the center were then analyzed by LA-ICP-MS–NWR-213® (New Wave Research Co., Ltd.) coupled to a NexION®300 ICP-MS (PerkinElmer) in order to determine the spatial distribution of Ti and Zr in the bone and soft tissues adjacent to the implant surface.

Above used method/principle of LA-ICP-MS is described in detail in previous publications [ , ].

2.1.4.2

Analytical procedure

The laser ablation started from the tissue region at a distance of around 4 mm from the implant surface and stopped immediately adjacent to the implant itself (the laser did not touch the implant surface in order to protect the detector from being flooded with implant elements). In this manner, the laser ablation in an area of approximately 4 mm × 2.5 mm was performed line by line, the interval between two lines being 50 μm, resulting in an average of 50 lines that were ablated.

Instrument settings and parameters are shown in detail in Table 1 .

After the laser ablations, the data were analyzed with the software Iolite/IGOR PRO 6. A map of the ablated area exhibiting the elemental distribution of Ti, Zr and Ca was built from ICP-MS data.

2.1.5

Histological analysis

The bone sections with implants were glued (Cyanolit 201, Panacol LTD., Zürich, Switzerland) on opaque plastic slides, ground thinner, polished (EXAKT® 400CS grinding system, EXAKT Vertriebs GmbH, Norderstedt, Germany) and stained with Giemsa-Eosin stain (Sigma Aldrich, Steinheim, Germany). The stained sections were examined in transmitted light mode with an Axiophot microscope (Zeiss, Goettingen, Germany) that was equipped with Zeiss Plan-Neofluar objectives (5× and 10×). Images were recorded with am Axciocam HRc digital camera (Zeiss, Goettingen, Germany).

2.2

Assessment of the toxicity of ZrO ₂ -particles (cytotoxicity and DNA damage) in PDL-hTERT cells

2.2.1

Cell culture

Periodontal ligament cells with lentiviral gene transfer of human telomerase reverse transcriptase (PDL-hTERT) were obtained from the Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians University (LMU), Munich, Germany [ ].

PDL-hTERT cells were cultured in a 250 ml tissue culture flask (BD falcon, Franklin Lakes, USA) at 37 °C and 100% humidity with 5% CO ₂ . The VLE (very low endotoxin) Dulbecco’s Minimum Essential Medium (MEM) with 4.5 g/l d -glucose (Biochrom, Berlin, Germany) was supplemented with 1% penicillin/streptomycin (Biochrom, Berlin, Germany) and 10% Fetal Bovine Serum (Sigma–Aldrich, Munich, Germany) [ ].

2.2.2

Particle exposure and size measurement

ZrO ₂ nanoparticles (ZrO ₂ -NPs, <100 nm) and ZrO ₂ microparticles (ZrO ₂ -MPs, <5 μm) were obtained from Sigma–Aldrich (St. Louis, USA). Fresh suspensions of investigated particles were prepared for each experiment. The stock solutions were prepared by adding investigated particles (batches of: 300 mg ZrO ₂ -NPs, 30 mg ZrO ₂ -NPs and 3 mg ZrO ₂ -NPs; 1000 mg ZrO ₂ -MPs and 150 mg ZrO ₂ -MPs) into 3 ml of medium and mixed well. To determine the exact concentrations of particles in the resulting stock solutions, 200 μl of stock solution was evaporated at 70 °C to complete dryness and the average net weight of the particles was measured six times. The final exposure concentrations (particle weight/0.1 ml) were obtained by adding different volumes of stock solution. Medium was then added or removed to obtain the equal volume of 0.1 ml in XTT test and 1 ml in comet assay. The exposure concentrations of the investigated particles for each test are shown in Table 2 .

The size (ferret diameter) of investigated particles was determined with Scanning Electron Microscopy (SEM) (Hitachi S-4700 II FESEM, Hitachi High Technologies, Germany) at an acceleration voltage of 3 kV with a current of 40 μA.

Tests exhibited only slight agglomerations/aggregations of particles in cell medium using Laser Scanning Microscope (LSM) for visualization [ ].

2.2.3

XTT viability assay

XTT-based cell viability assay was applied to determine the half- maximum effect concentration (EC ₅₀ ) values of ZrO ₂ -NPs and ZrO ₂ -MPs in PDL-hTERT cells. In the present study, 20,000 cells per well were incubated with 0.1 ml medium for 24 h on a 96-well plate (BD falcon, Heidelberg, Germany). Then the cells were treated with different concentrations of ZrO ₂ -NPs and ZrO ₂ -MPs ( Table 2 ). Negative control cells received only medium, while positive control cells were treated with 1% Triton X-100.

After exposure to ZrO ₂ -NPs and ZrO ₂ -MPs for 24 h, the XTT-test was performed according to our previous study [ ]. The formazan formation was quantified spectrophotometrically at a wavelength of 450 nm (reference wavelength 670 nm) by using a microplate reader (Thermo Fisher Scientific (Shanghai) Instruments Co., Ltd., China). The formazan production in percentage was calculated referring to the negative and positive controls. EC ₅₀ values were obtained by fitting the data to a dose–effect sigmoidal curve using GraphPad prism 4 (GraphPad Software, Inc. La Jolla, USA). Four independent replicates were performed (n = 4).

2.2.4

Comet assay

DNA damage of ZrO ₂ -NPs and ZrO ₂ -MPs was analyzed by the alkaline single-cell microgel electrophoresis (comet) assay. In this experiment, PDL-hTERT cells (2 × 10 ⁵ ) were cultured in 1 ml medium in a 12-well plate (BD falcon, Heidelberg, Germany) for 24 h. Afterwards, the cells were exposed to different concentrations ( Table 2 ) of ZrO ₂ -NPs and ZrO ₂ -MPs for further 24 h. Positive control cells were treated with 100 μM methyl methanesulfonate (MMS) (Sigma–Aldrich, Steinheim, Germany) and negative control cells were treated only with medium. Before the comet assay was performed, the cells were stained with trypan blue and a threshold level for cytotoxicity after exposure was set at 75% viable cells.

Further experimental details of comet assay were described in our previous study [ ]. For each sample, about 50 cells were investigated in each test. Five independent replicates were performed (n = 5). Olive tail moment (OTM), as a product of the tail length and the fraction of total DNA in the tail, was taken to evaluate the DNA damage [ , ].

The results were presented as mean ± standard deviation (SD). To analyse the effect of particles on cytotoxicity and DNA damage, a one-way ANOVA analysis followed by Tukey’s test was applied. Differences were considered statistically significant only when the p-value was less than 0.05 (p < 0.05) [ ].