Titanium coin preparation
Grade IV titanium coins, Ø 6mm and height 2mm were washed in five steps as previously described,24 before the surface modification procedure. The titanium coins were divided into 6 different test groups according to the sequence of burs used for experimental implantoplasty (Table 1); carbide cutting burs (CB), CB + Arkansas stone (CB+Ark), CB + Brownie and Greenie silicone burs (CB+BG), diamond burs (DB), DB+Ark and DB+BG. Two control groups were also included; coins with a sandblasted and acid-etched surface (SLA) (kindly provided by Straumann, Straumann Holding AG, Basel, Switzerland) and polished coins (P) according to a procedure previously published.24 All burs were in contact with the titanium coin for 1 minute under copious water irrigation. By-products in the irrigation water were collected using a filter paper and vacuum suction (595 Filter Paper Circles, GE Healthcare, Merck KGaA, Darmstadt, Germany). Following in vitro experimental implantoplasty procedures, all coins were rinsed with deionized water, agitated for 60 min and then autoclaved at 121 °C.
Table 1. Explanation of burs used for implantoplasty in each sequence.
Abbreviation
|
Description
|
Manufacturer
|
Handpiece /
Rounds per minute
|
Total time bur on coin per group
|
CB
|
Carbide cutting burs, red (normal toothing) and white (fine toothing)
|
Komet Dental, Gebr. Brasseler GmbH & Co. KG, Lemgo, Germany
|
Air-driven turbine handpiece 200 000 rpm +
Contra angle handpiece 15000 rpm
|
2 min
|
CB+Ark
|
Carbide cutting burs, red (normal toothing) and white (fine toothing) +
Arkansas stone 661
|
(Komet Dental)
|
Air-driven turbine handpiece +
Contra angle handpiece 15000 rpm
|
3 min
|
CB+BG
|
Carbide cutting burs red (normal) and white (fine toothing) +
Silicone cup brownie 030 +
Silicone cup greenie 030
|
(Komet Dental) +
Shofu, Shofu Dental GmbH, Ratingen, Germany
|
Air-driven turbine handpiece +
Contra angle handpiece 15000 rpm
|
4 min
|
DB
|
Diamond sequence of decreasing coarseness 105 mm, 40 mm, 8 mm
|
105 mm (250.014, Horico Dental, Hopf, Ringleb & Co. GmbH & CIE, Berlin, Germany), 40 mm (8379EF.314, Komet Dental), 8 mm (863UF.314, Komet Dental)
|
Air-driven turbine handpiece +
Contra angle handpiece 15000 rpm
|
3 min
|
DB+Ark
|
Diamond sequence of decreasing coarseness 105 mm, 40 mm, 8 mm +
Arkansas stone 661
|
105 mm (Horico Dental), 40 mm (Komet Dental), 8 mm (Komet Dental) +
(Komet Dental)
|
Air-driven turbine handpiece +
Contra angle handpiece 15000 rpm
|
4 min
|
DB+BG
|
Diamond sequence of decreasing coarseness 105 mm, 40 mm, 8 mm +
Silicone cups brownie 030 +
Silicone cup greenie 030
|
105 mm (Horico Dental), 40 mm (Komet Dental), 8 mm (Komet Dental) +
(Shofu)
|
Air-driven turbine handpiece +
Contra angle handpiece 15000 rpm
|
5 min
|
Surface characterizations
Profilometer
A total of 48 coins (n=6 from each group) were analysed with a profilometer (Sensofar SensoSCAN 6.2, Terrassa, Spain). Topographical parameters were obtained using a blue light laser profilometer with a 150 x 0,95 DI Nikon objective. An arbitrary area of 292 mm x 220 mm was scanned for each coin. The surface amplitude parameters; arithmetical-mean-height (Sa), ten-point-height of the surface (Sz), root-mean-square deviation (Sq), and the reduced peak height (Spk) values were calculated using the SensoMap software (SensoMap Standard 7.3.7690, Sensofar, Terrassa, Spain).
SEM and EDX
A total of 48 coins (n=6 from each group), and debris from each bur-sequence procedure were analysed with a scanning electron microscope TM3030 (Hitachi High-Technologies Europe GmbH, Krefeld, Germany). The samples were mounted on an aluminum holder with carbon tape and copper conductive tape. Scanning electron microscope (SEM) images were obtained with backscattered electrons at 15 kV voltage. Furthermore, energy dispersive x-ray spectroscopy (EDX)(Quantax 70, Bruker, Billerica, USA) was used for detection of chemical elements measured in atomic percentage on the titanium coin surfaces.25
Experimental in vitro design
HGFs from two different donors (Provitro, German type Culture Collection, Berlin, Germany, Passage 6) were cultured in fibroblast growth medium (Basal medium, Provitro) supplemented with 10% fetal bovine serum, 100U/mL penicillin, and 100mg/mL streptomycin (GE Healthcare, Utah, USA) at 37 C° in a humidified atmosphere with 5 % CO2. The coins (n=6-10 for donor 1, n=5 for donor 2 for each experimental groups) were placed into 96-well tissue culture plates (Tissue culture plates, 96 wells, VWR®, Radnor, USA). With the use of an electronic counter (Countess, Invitrogen, Carlsbad, CA, USA), cells from both donors were seeded onto the coins with a cell-number of 2000 cells/ml (~70 cells/coin) on the coins to be harvested at day 3 and 6, and a cell-number of 10000 cells/ml (~350 cells/coin) for coins harvested after 15 days and 30 days of incubation. The same number of cells was cultured on plastic in order to monitor the cell secretion.
Cell culture media was harvested from the wells cultured with the highest cell seeding density (350 cells/coin)(n=6-10 for donor 1, n=5 for donor 2) every third day for the whole study period and stored at -20°C prior to analysis of selected cytokines secreted (Luminex assay).
Luminex analysis
Multianalyte profiling of the level of the markers fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 10 (IL-10), vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), monocyte chemotactic protein-3 (MCP-3), interferon gamma-induced protein 10 (IP-10)(Human Cytokine/Chemokine Magnetic Bead Panel kit)(Billerica, MA, USA) in the harvested cell culture media was performed on the Luminex-200 (Luminex, Austin, TX, USA) using the Human Cytokine/Chemokine Magnetic Bead Panel kit (Billerica, MA, USA) according to the manufacture’s protocol.
Immunostaining
Cells cultured on coins for 3, 6, 15 and 30 days, respectively, were fixed with 4% paraformaldehyde for 20 minutes at room temperature. The cells were permeabilized with 0,02% Triton X-100 in PBS for 10 minutes at room temperature. Blocking of unspecific binding of antibodies was performed with a solution of 10% goat serum in PBS for at least one hour at room temperature. The cells were incubated overnight at 4 C with primary antibodies. Antibodies against Vinculin (1:600, #V9131, Sigma Aldrich) and Fibronectin (1:600, #F3648, Sigma Aldrich) both diluted in PBS with 2% goat serum were used. As secondary antibodies, goat-anti-mouse-Alexa647 (1:100, #A21236, Invitrogen) and goat-anti-rabbit-Alexa568 (1:100, #A11011, Invitrogen) diluted in PBS with 4% goat serum were used. To visualize the actin filaments, the cells were stained with 2,5% Phalloidin-Alexa 488 (#A12379, Invitrogen) in PBS for 20 minutes. The cell nucleus was stained using a solution of DAPI or Hoechst (0.3μM)(#33342, Thermo ScientificTM) in PBS for 30 minutes was used. The cells were stored at 4 °C for later imaging with confocal microscopy.
Confocal microscopy
Cells were imaged at minimum three non-overlapping areas (554,65 x 554,65 mm) using a 20x/0,40 HCX APO CS water-immersion objective (Leica SP8, Wetzlar, Germany). Samples were exited with lasers at 405nm, 488nm and 552nm. Confocal Z-stacks were used in every case. Image analysis, fibronectin quantification and cell counting was performed using ImageJ (Fiji software, 64 bit, Windows).26 To quantify fibronectin a dichotomous red color contrast to black threshold was arbitrarily set for each image by comparing to the original confocal images, after which the area percentage of the stain was quantified.
Statistical analysis
To enable comparison of secreted factors and cell growth for each of the donors, data were adjusted for cell number and calculated relative to the rough control (SLA) at each time point. The statistical analysis of data from each donor was performed in SigmaPlot (Systat Software, Inc, San Jose California, USA). Differences between experimental groups and control groups were determined using One-Way ANOVA on ranks. To facilitate comparison to other studies all figures are however presented with mean values ± standard deviation (SD). Correlation analyses were performed using Spearman correlation. POL < 0.05 was considered statistically significant.