2.1

Cell culture

Clinically, PDL cells are the cells growing around natural teeth, which work as the tooth anchor and sustain bone regeneration . PDL cells can provide the implants with the same mobility as natural teeth and reduce the bone loss around implants . Therefore, dental implants combined with PDL would represent a great new therapeutic tool to replace lost teeth . Studies have demonstrated the PDL formation on the surface of Ti implants .

Periodontal ligament with lentiviral gene transfer of human telomerase reverse transcriptase cells (PDL-hTERT cells) were obtained from Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU), Munich, Germany . The reason why PDL-hTERT cells were used is that PDL-hTERT cells have extended lifespan with identical morphology compared to the primary PDL cells . The extended lifespan is of great importance to the PDL engineering. 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

Particle exposure and size measurement

Ti-MPs (99%; <44 μm (−325 mesh)) and NiTi-MPs (70% Ti, 30% Ni; <44 μm (−325 mesh)) were obtained from Alfa Aesar, Karlsruhe, Germany. Ti-NPs contained 98.5% Ti < 100 nm (Sigma–Aldrich, St. Louis, USA). Fresh suspensions of Ti-MPs, NiTi-MPs and Ti-NPs were prepared for each experiment. The stock solutions were prepared by adding investigated particles (1000 mg Ti-MPs, 150 mg NiTi-MPs, and 21.8 mg Ti-NPs) into 3 ml of medium and well mixed. To determine the exact concentrations of particles, 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. The exposure concentrations of the investigated particles for each test are shown in Table 1 .

The size of investigated particles was determined with Scanning Electron Microscopy (SEM) LEO 1550 (Zeiss, Oberkochen, Germany) by measuring the minimal Feret distance . 4× 200 single particles of each investigated particle sample were used for particle size measurement. In case of spontaneous combustion Ti-NPs were suspended in PBS (phosphate buffered saline) before measurement.

2.3

XTT viability assay

XTT-based cell viability assay was applied to determine the half-maximum effect concentration (EC ₅₀ ) values for the investigated particles in PDL-hTERT cells. In this assay, a concentration of 20,000 cells/well (in 0.1 ml medium) was incubated for 24 h in the 96-well plate (BD falcon, Heidelberg, Germany). Then the cells were treated with different concentrations ( Table 1 ) of Ti-NPs, Ti-MPs and NiTi-MPs. Negative control cells received medium only. Positive control cells received 1% Triton X-100. After exposure of cells to investigated particles for 24 h, 50 μl XTT (1 mg/ml) solution (sodium 30-[1-(phenyl-aminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) labeling agent (in RPMI (Roswell Park Memorial Institute medium) 1640 without phenol red) and electron-coupling reagent PMS (N-methyldibenzopyrazine methylsulfate in PBS) (0.383 mg/ml) were added (cell proliferation kit II; Roche Diagnostics GmbH Penzberg, Germany). After 4 h incubation the photometric analysis was performed in another well plate to determine formazan values.

The formazan values were referred to positive and negative control. 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). Each experiment was repeated four times ( n = 4).

2.4

Cell death detected with trypan blue staining

Trypan blue staining reveals the ratio of live and dead cells. Non-viable cells are stained blue since the cell membrane of non-viable cells is permeable for trypan blue . The cells were treated with different concentrations ( Table 1 ) of investigated particles for 24 h. After the treatment, the cells were washed three times with PBS. To detach cells from the 12-well plate, the cells were treated with trypsin at 37 °C for 5 min. The trypsin effect was stopped by adding medium. Afterwards, the cells were stained with 0.4% trypan blue (Sigma Aldrich, Steinheim, Germany) for 3 min. Ratio of dead cells was counted in neubauer chamber (Paul Marienfeld Lauda-Königshofen, Germany) (100–120 cells). Negative control cells received medium only. Each experiment was repeated three times ( n = 3).

2.5

Comet assay

DNA damage of investigated particles was analyzed by the alkaline single-cell microgel electrophoresis (comet) assay. This comet assay method has been described in our previous studies . Experiment was conducted in red light. Slides (26 mm × 76 mm, R. Langenbrinck, Emmendingen, Germany) were coated with a layer of 85 μl 0.5% agarose (Biozym, Oldendorf, Germany), and then dried for one week at 25 °C excluding daylight.

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 1 ) of all investigated particles for further 24 h. Then cells were washed with 1 ml PBS three times, and treated with 100 μl trypsin at 37 °C for 5 min, 300 μl medium was added to stop the effect of trypsin. The cell suspension was then centrifuged (800 rpm, 10 min), and the supernatant was discarded. The cell pellets were re-suspended in 400 μl PBS and centrifuged again. Cell pellets were suspended in 25 μl PBS, and mixed with 75 μl LMP-Agarose (low melting point) (Biozym, Oldendorf, Germany), and then transferred onto the pre-coated slides (as described above). A covering glass (24 mm × 60 mm) (R. Langenbrinck, Emmendingen, Germany) was placed on the top of the slide. The slide was then kept at 4 °C for at least 5 min, and another layer of agarose was over stacked. After incubation at 4 °C for 5 min, the covering glass was removed, and the slide was stored in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Na-lauroylsarcosinate, H ₂ O; pH = 10; before use 1% Triton X-100 and 10% dimethylsulfoxide were added) overnight.

Before electrophoresis, the slides were placed in the alkaline electrophoresis solution (6 °C) for 30 min. Electrophoresis was conducted at 6 °C with 25 V, maximum 300 mA for 30 min. The slides were then washed with 400 mM Tris buffer (Merck, Darmstadt, Germany) and stained with 50 μl 200 μg/ml ethidium bromide (Sigma–Aldrich, St. Louis, USA). The slides were evaluated using an Olympus BX 60 fluorescence microscopy (Olympus, Hamburg, Germany) with a 40× objective and the software program comet assay II (Perceptive Instrument Ltd., Haverhill, UK). Positive control cells received 100 μM methyl methanesulfonate (MMS) (Sigma–Aldrich, Steinheim, Germany) and negative control cells received medium only. For each sample, about 50 cells were investigated. The test was repeated six times ( n = 6). Olive tail moment (OTM) as a product of the tail length and the percentage of total DNA in the tail was applied to evaluate DNA damage .

2.6

Cellular uptake

2.6.1

Laser scanning confocal microscopy (LSCM) measurement

Cellular and intracellular uptake of Ti-NPs, Ti-MPs and NiTi-MPs in PDL-hTERT cells was determined with LSM 510 (Zeiss, Oberkochen, Germany), as described in a previous study . It was found that noble metals can be used as marker for cellular imaging of LSCM due to their surface plasmon resonance . Similarly, Ti-NPs, Ti-MPs and NiTi-MPs also emit intensive light in a close proximity to the wavelength (633 nm) of the laser. The nucleus was stained with Sybr green I nucleic acid gel stain (Invitrogen, Oregon, USA). For visualization of the cells the plasma membrane was stained with cell mask membrane plasma orange stain (Invitrogen, Oregon, USA). Sybr green I and cell mask membrane plasma orange were excited by 488 nm and 543 nm.

The cells were cultured for 24 h on a covering glass in 24-well plate (BD Falkon, Franklin lakes, USA) and then exposed to Ti-NPs (28 μg/ml (1/100 EC ₅₀ ), 56 μg/ml (1/50 EC ₅₀ )), NiTi-MPs (418 μg/ml (1/100 EC ₅₀ ), 4180 μg/ml (1/10 EC ₅₀ )) and Ti-MPs (418 μg/ml) for another 24 h. After that, the cells were carefully shaken, washed with PBS three times and stained with 2.5 μg/ml of Cell Mask Membrane Plasma Orange at 37 °C for 5 min. The cells were then fixed with 2% paraformaldehyde (Carl Roth, Karlsruhe, Germany), washed three times with PBS, and stained with Sybr green I (1:50,000) at 25 °C for 15 min. The covering glasses were mounted on microscopic slides using prolong gold antifade reagent with 4′,6-diamidin-2-phenylindol (DAPI) (Invitrogen, Oregon, USA) after they were washed three times with PBS. Samples were kept at 4 °C before analysis. With the LSCM, a stack of images at different points along the Z -axis was performed in the direction away from the cover slip and moving into the cells. For each experiment about 60 cells were investigated and the experiment was repeated four times ( n = 4).

2.6.2

Transmission electron microscopy (TEM) measurement

TEM Libra 120 (Zeiss, Oberkochen, Germany) was used to confirm the cellular uptake of Ti-NPs. Ti-MPs and NiTi-MPs were not investigated due to the technical difficulties obtaining ultra thin sections from large particles. PDL-hTERT cells were treated with Ti-NPs (28 μg/ml) for 24 h. After the incubation with Ti-NPs, the cells were washed with PBS three times and fixed with 2% glutaraldehyde in 0.1 M PBS buffer at 25 °C for at least 2 h. Fixed cells were washed with PBS three times and then treated with 1% osmium tetroxide (Merck, Darmstadt, Germany) at 25 °C for 1 h. The dehydration was performed with ascending concentrations of ethanol: 30%, 50%, 70%, 90% and 100% (ethanol purity ≥ 99.8%, Carl Roth, Karlsruhe, Germany). Afterwards, cells were embedded in epon 812 (Serva, Heidelberg, Germany). After the resin blocks were hardened, they were cut into ultra thin sections (70–90 nm) with an ultra-microtome (Zeiss, Oberkochen, Germany), and then contrasted with 2% uranyl acetate (Merck, Darmstadt, Germany) followed by Pb-citrate (Merck, Darmstadt, Germany). The ultra thin sections were analysed.

To obtain morphology and size, suspensions of Ti-NPs in water were evaporated to dryness on TEM grids and observed with TEM.

2.7

Statistic analysis

The results were presented as mean ± standard deviation (SD). To analyze the effect of particles on cytotoxicity, DNA damage, and cellular uptake, 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) .

2.1

Cell culture

Clinically, PDL cells are the cells growing around natural teeth, which work as the tooth anchor and sustain bone regeneration . PDL cells can provide the implants with the same mobility as natural teeth and reduce the bone loss around implants . Therefore, dental implants combined with PDL would represent a great new therapeutic tool to replace lost teeth . Studies have demonstrated the PDL formation on the surface of Ti implants .

Periodontal ligament with lentiviral gene transfer of human telomerase reverse transcriptase cells (PDL-hTERT cells) were obtained from Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU), Munich, Germany . The reason why PDL-hTERT cells were used is that PDL-hTERT cells have extended lifespan with identical morphology compared to the primary PDL cells . The extended lifespan is of great importance to the PDL engineering. 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

Particle exposure and size measurement

Ti-MPs (99%; <44 μm (−325 mesh)) and NiTi-MPs (70% Ti, 30% Ni; <44 μm (−325 mesh)) were obtained from Alfa Aesar, Karlsruhe, Germany. Ti-NPs contained 98.5% Ti < 100 nm (Sigma–Aldrich, St. Louis, USA). Fresh suspensions of Ti-MPs, NiTi-MPs and Ti-NPs were prepared for each experiment. The stock solutions were prepared by adding investigated particles (1000 mg Ti-MPs, 150 mg NiTi-MPs, and 21.8 mg Ti-NPs) into 3 ml of medium and well mixed. To determine the exact concentrations of particles, 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. The exposure concentrations of the investigated particles for each test are shown in Table 1 .

The size of investigated particles was determined with Scanning Electron Microscopy (SEM) LEO 1550 (Zeiss, Oberkochen, Germany) by measuring the minimal Feret distance . 4× 200 single particles of each investigated particle sample were used for particle size measurement. In case of spontaneous combustion Ti-NPs were suspended in PBS (phosphate buffered saline) before measurement.

2.3

XTT viability assay

XTT-based cell viability assay was applied to determine the half-maximum effect concentration (EC ₅₀ ) values for the investigated particles in PDL-hTERT cells. In this assay, a concentration of 20,000 cells/well (in 0.1 ml medium) was incubated for 24 h in the 96-well plate (BD falcon, Heidelberg, Germany). Then the cells were treated with different concentrations ( Table 1 ) of Ti-NPs, Ti-MPs and NiTi-MPs. Negative control cells received medium only. Positive control cells received 1% Triton X-100. After exposure of cells to investigated particles for 24 h, 50 μl XTT (1 mg/ml) solution (sodium 30-[1-(phenyl-aminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) labeling agent (in RPMI (Roswell Park Memorial Institute medium) 1640 without phenol red) and electron-coupling reagent PMS (N-methyldibenzopyrazine methylsulfate in PBS) (0.383 mg/ml) were added (cell proliferation kit II; Roche Diagnostics GmbH Penzberg, Germany). After 4 h incubation the photometric analysis was performed in another well plate to determine formazan values.

The formazan values were referred to positive and negative control. 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). Each experiment was repeated four times ( n = 4).

2.4

Cell death detected with trypan blue staining

Trypan blue staining reveals the ratio of live and dead cells. Non-viable cells are stained blue since the cell membrane of non-viable cells is permeable for trypan blue . The cells were treated with different concentrations ( Table 1 ) of investigated particles for 24 h. After the treatment, the cells were washed three times with PBS. To detach cells from the 12-well plate, the cells were treated with trypsin at 37 °C for 5 min. The trypsin effect was stopped by adding medium. Afterwards, the cells were stained with 0.4% trypan blue (Sigma Aldrich, Steinheim, Germany) for 3 min. Ratio of dead cells was counted in neubauer chamber (Paul Marienfeld Lauda-Königshofen, Germany) (100–120 cells). Negative control cells received medium only. Each experiment was repeated three times ( n = 3).

2.5

Comet assay

DNA damage of investigated particles was analyzed by the alkaline single-cell microgel electrophoresis (comet) assay. This comet assay method has been described in our previous studies . Experiment was conducted in red light. Slides (26 mm × 76 mm, R. Langenbrinck, Emmendingen, Germany) were coated with a layer of 85 μl 0.5% agarose (Biozym, Oldendorf, Germany), and then dried for one week at 25 °C excluding daylight.

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 1 ) of all investigated particles for further 24 h. Then cells were washed with 1 ml PBS three times, and treated with 100 μl trypsin at 37 °C for 5 min, 300 μl medium was added to stop the effect of trypsin. The cell suspension was then centrifuged (800 rpm, 10 min), and the supernatant was discarded. The cell pellets were re-suspended in 400 μl PBS and centrifuged again. Cell pellets were suspended in 25 μl PBS, and mixed with 75 μl LMP-Agarose (low melting point) (Biozym, Oldendorf, Germany), and then transferred onto the pre-coated slides (as described above). A covering glass (24 mm × 60 mm) (R. Langenbrinck, Emmendingen, Germany) was placed on the top of the slide. The slide was then kept at 4 °C for at least 5 min, and another layer of agarose was over stacked. After incubation at 4 °C for 5 min, the covering glass was removed, and the slide was stored in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Na-lauroylsarcosinate, H ₂ O; pH = 10; before use 1% Triton X-100 and 10% dimethylsulfoxide were added) overnight.

Before electrophoresis, the slides were placed in the alkaline electrophoresis solution (6 °C) for 30 min. Electrophoresis was conducted at 6 °C with 25 V, maximum 300 mA for 30 min. The slides were then washed with 400 mM Tris buffer (Merck, Darmstadt, Germany) and stained with 50 μl 200 μg/ml ethidium bromide (Sigma–Aldrich, St. Louis, USA). The slides were evaluated using an Olympus BX 60 fluorescence microscopy (Olympus, Hamburg, Germany) with a 40× objective and the software program comet assay II (Perceptive Instrument Ltd., Haverhill, UK). Positive control cells received 100 μM methyl methanesulfonate (MMS) (Sigma–Aldrich, Steinheim, Germany) and negative control cells received medium only. For each sample, about 50 cells were investigated. The test was repeated six times ( n = 6). Olive tail moment (OTM) as a product of the tail length and the percentage of total DNA in the tail was applied to evaluate DNA damage .

2.6

Cellular uptake

2.6.1

Laser scanning confocal microscopy (LSCM) measurement

Cellular and intracellular uptake of Ti-NPs, Ti-MPs and NiTi-MPs in PDL-hTERT cells was determined with LSM 510 (Zeiss, Oberkochen, Germany), as described in a previous study . It was found that noble metals can be used as marker for cellular imaging of LSCM due to their surface plasmon resonance . Similarly, Ti-NPs, Ti-MPs and NiTi-MPs also emit intensive light in a close proximity to the wavelength (633 nm) of the laser. The nucleus was stained with Sybr green I nucleic acid gel stain (Invitrogen, Oregon, USA). For visualization of the cells the plasma membrane was stained with cell mask membrane plasma orange stain (Invitrogen, Oregon, USA). Sybr green I and cell mask membrane plasma orange were excited by 488 nm and 543 nm.

The cells were cultured for 24 h on a covering glass in 24-well plate (BD Falkon, Franklin lakes, USA) and then exposed to Ti-NPs (28 μg/ml (1/100 EC ₅₀ ), 56 μg/ml (1/50 EC ₅₀ )), NiTi-MPs (418 μg/ml (1/100 EC ₅₀ ), 4180 μg/ml (1/10 EC ₅₀ )) and Ti-MPs (418 μg/ml) for another 24 h. After that, the cells were carefully shaken, washed with PBS three times and stained with 2.5 μg/ml of Cell Mask Membrane Plasma Orange at 37 °C for 5 min. The cells were then fixed with 2% paraformaldehyde (Carl Roth, Karlsruhe, Germany), washed three times with PBS, and stained with Sybr green I (1:50,000) at 25 °C for 15 min. The covering glasses were mounted on microscopic slides using prolong gold antifade reagent with 4′,6-diamidin-2-phenylindol (DAPI) (Invitrogen, Oregon, USA) after they were washed three times with PBS. Samples were kept at 4 °C before analysis. With the LSCM, a stack of images at different points along the Z -axis was performed in the direction away from the cover slip and moving into the cells. For each experiment about 60 cells were investigated and the experiment was repeated four times ( n = 4).

2.6.2

Transmission electron microscopy (TEM) measurement

TEM Libra 120 (Zeiss, Oberkochen, Germany) was used to confirm the cellular uptake of Ti-NPs. Ti-MPs and NiTi-MPs were not investigated due to the technical difficulties obtaining ultra thin sections from large particles. PDL-hTERT cells were treated with Ti-NPs (28 μg/ml) for 24 h. After the incubation with Ti-NPs, the cells were washed with PBS three times and fixed with 2% glutaraldehyde in 0.1 M PBS buffer at 25 °C for at least 2 h. Fixed cells were washed with PBS three times and then treated with 1% osmium tetroxide (Merck, Darmstadt, Germany) at 25 °C for 1 h. The dehydration was performed with ascending concentrations of ethanol: 30%, 50%, 70%, 90% and 100% (ethanol purity ≥ 99.8%, Carl Roth, Karlsruhe, Germany). Afterwards, cells were embedded in epon 812 (Serva, Heidelberg, Germany). After the resin blocks were hardened, they were cut into ultra thin sections (70–90 nm) with an ultra-microtome (Zeiss, Oberkochen, Germany), and then contrasted with 2% uranyl acetate (Merck, Darmstadt, Germany) followed by Pb-citrate (Merck, Darmstadt, Germany). The ultra thin sections were analysed.

To obtain morphology and size, suspensions of Ti-NPs in water were evaporated to dryness on TEM grids and observed with TEM.

2.7

Statistic analysis

The results were presented as mean ± standard deviation (SD). To analyze the effect of particles on cytotoxicity, DNA damage, and cellular uptake, 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) .