Original study - ZZI 03/2009
In vitro studies to screen for implant surface properties*
To obtain a sufficient number of cells for the experiment, a second passage of the confluent cell layer was necessary. For the second subculture, the cells were again trypsinized and centrifuged, and the resulting pellet was resuspended.
Each of the seven test specimens was autoclaved at 134°C and then placed in a chamber of the Lab Tec II Chamber Slide System. The cell suspension was produced as described for the passage. The cell concentration was set at 4000 cells per ml of medium. 0.5 ml of this suspension was pipetted onto each test specimen. The thus distributed bone cells were then allowed to grow on the test specimens for three, five, seven or ten days respectively in the incubator (medium changed every three days)
The expression of BSP, osteocalcin and osteonectin was demonstrated by fluorescence immunohistochemistry . The use of HEPES buffer was necessary for labeling the osteonectin, since the primary osteonectin antibody only reacts in the presence of calcium. Otherwise it was possible to work with PBS.
After the test periods (three, five, seven and ten days), the cells were fixed for ten minutes with 4 % paraformaldehyde in PBS, rinsed four times for five minutes in PBS or HEPES and incubated for one hour with Normal Goat Serum (10 %, with 2 % bovine serum albumin [BSA] in PBS or HEPES). The dilution of the primary antibodies added subsequently for 16 hours was 1:100 in PBS with 2 % BSA for BSP and osteocalcin, and in HEPES with 2 % BSA for osteonectin. The test specimens were then rinsed four times for five minutes with the respective buffer and then incubated for 30 minutes with the secondary goat-anti-mouse, CY-3-conjugated antibody (1:200 in PBS or HEPES with 2 % BSA). After further rinsing three times for five minutes with the corresponding buffer, the cell nuclei were counterstained with DAPI (1:1000 in PBS for 1 minute). The chambers of the chamber slides were separated from the slide, the cover medium was placed on the test specimens and they were covered with a cover slip.
Quantitative analysis of protein expression
Quantitative analysis of protein expression on the test specimen surfaces was performed on the specified test days by calculating the relative gray value. Each test specimen was photographed at six specified sites with the Axiophot fluorescence microscope and the Axiocam camera installed on it using 200x magnification. The images were stored digitally. In this experiment, the UV filter was used to show the cell nuclei and the TRITC filter to show the non-collagenous proteins. The images obtained separately can be merged into one picture using suitable software (Axiovision 4.0). The relative gray value was determined individually for each of the photos and according to the same pattern. The relative gray value is defined as the ratio of the mean gray value and the exposure time. The required values were measured by the Axiovision 4.0 program. To do this, a frame with an area of 440x330 µm had to be placed around each picture. The mean gray values and exposure times for the individual photos were stored by the Excel table calculation program, and the relative gray value was calculated and shown graphically.
For the test, round titanium components with a diameter of 6 mm and a thickness of 0.5 mm were used. The surfaces of the components were treated according to the specifications of the study protocol. The study protocol involved four clinically employed and three experimentally produced surfaces. The modifications made to them are listed below.
Clinically employed surfaces
– Etched surface (Ti-et)
The surface of the titanium component (ZL-Microdent) is conditioned by subtractive methods. A mixture of concentrated sulfuric and hydrochloric acid acts on the test specimen at normal pressure (Fig. 1a).
– TICER surface (Titanium/Ceramic)
The TICER surface is used by ZL-Microdent in the clinically proven and very well investigated [7–9] ZL-Duraplant implant system. The basic implant component is made of pure titanium. The surface is produced by a conversion technique employing the anodizing oxidation with spark discharge (ANOF) described by Krysmann . The component is connected as an anode in a saturated calcium dihydrogen phosphate electrolyte solution. When a pulsed direct current is applied, an ANOF layer containing titanium, oxygen, calcium and phosphorus is formed on the surface of the titanium component (Fig. 1b).
– TiUnite-like surface (TiUniteL)
The basic implant component consists of pure titanium (ZL-Microdent). The surface (Fig. 1c) is produced by a conversion technique  and greatly resembles the original TiUnite surface both morphologically and chemically. The TiUnite surface is essentially a technical counterpart of the TICER surface. The component is connected as an anode in an electrolytic cell. The electrolyte solution consists of a mixture of sulfuric and phosphoric acid. When a direct current over 150 V is applied at room temperature and normal pressure, the surface shown in Figure 1c is produced. Titanium, oxygen, phosphorus and sulfur are included in this surface.