Intracellular uptake and toxicity of three different Titanium particles

Abstract

Introduction

Titanium (Ti) and its alloys are used for implants and other dental materials. In this study, cytotoxicity, DNA damage, cellular uptake and size of three kinds of Ti particles were measured.

Methods

Cytotoxicity for Ti microparticles (Ti-MPs, <44 μm), NiTi microparticles (NiTi-MPs, <44 μm), and Ti nanoparticles (Ti-NPs, <100 nm) in periodontal ligament (PDL)-hTERT cells was measured with XTT test. DNA damage was determined with comet assay. Particle size was measured with scanning electron microscope, intracellular uptake was determined with laser scanning confocal microscopy and transmission electron microscopy.

Results

The EC 50 values of investigated particles were: 2.8 mg/ml (Ti-NPs), 41.8 mg/ml (NiTi-MPs) and >999 mg/ml (Ti-MPs). The Olive Tail Moment (OTM) values at 1/10 EC 50 were: 3.2 (Ti-NPs) and 2.2 (NiTi-MPs). An OTM of 2.2 for Ti-MPs was detected at the concentration of 6666 μg/ml. Determined sizes of investigated particles were 20–250 nm (Ti-NPs), 0.7–90 μm (NiTi-MPs) and 0.3–43 μm (Ti-MPs). The highest cellular uptake efficiency was observed with Ti-NPs, followed by Ti-MPs and NiTi-MPs. Only Ti-NPs were found in the nucleus.

Conclusion

Compared to Ti-MPs and NiTi-MPs, Ti-NPs induced higher cellular uptake efficiency and higher toxic potential in PDL-hTERT cells. Ni in the alloy NiTi induced an increase in the toxic potential compared to Ti-MPs.

Introduction

Titanium (Ti) and its alloys have been used as source materials for biomedical applications especially in dentistry, since previous studies showed that these materials stand out for good mechanical properties, excellent corrosion resistance and high biocompatibility . For example, it was found that Ti is one of the most biocompatible metallic materials because of its ability to form a stable and insoluble protective oxide layer (TiO 2 ) on its surface . Ti is preferentially used for endosseous dental implant material . Besides Ti, some Titanium alloys are also used for dental applications, such as Nickel Titanium (NiTi). The alloy NiTi is used for castings of crowns and denture construction, orthodontic archwires and brackets . Recently it has been found that the properties of Ti implants can be improved by using nanostructured Ti consisting of Ti-nanoparticles (Ti-NPs) .

Even though Ti based implants are considered to be biocompatible, their induced side effects such as hypersensitivity and allergic reactions have been reported . It has also been found that Ti based materials can cause immuno-inflammatory reactions . These side effects might have been caused by the interaction between tissues and implants . Previous in vitro and in vivo studies showed that Ti ions can be released from Ti based implants, for example by corrosion, wear and electrochemical processes . The release of Ni ions from NiTi alloy also has been reported . Previous studies indicated that Ti ions and Ni ions induced cytotoxicity/DNA damage in human cells . Furthermore, Ti-particles/debris (3–250 μm) was found in the peri-implant animal tissues after application of Ti based implants . Clinical studies also showed that Ti particles in nanometer- and micrometer-size could be released into human tissues/organs of the patients with Ti based implants or replacements . The toxic effect of Ti-particles has been described in the literature: phagocytosis of Ti-particles could induce cytotoxicity in rat calvarial osteoblasts and MG63 cells ; Genotoxic effect of Ti-particles has also been detected, which induced apoptosis in mesenchym stem cells .

It was found that the particles size can influence the toxicity of metal particles . The ability of different particles entering cells may also affect the toxicity , and it is reported that particle size can impact the cellular uptake efficiency and pathway . There is less data about toxicity and cellular uptake for Ti-NPs and Ti-MPs available.

The aim of this study is to compare the toxicity and cellular uptake of nanometer-sized Ti, micrometer-sized Ti and NiTi particles, which can be released from dental implants or replacements. In this study, Ti-NPs (Ti 98.5%, <100 nm), Ti-microparticles (Ti-MPs) (Ti 99%, <44 μm) and NiTi-microparticles (NiTi-MPs) (Ni 30%, Ti 70% and <44 μm) were investigated. The cytotoxicity, genotoxicity, and cellular uptake efficiency of these investigated particles have been measured in periodontal ligament (PDL) cells.

Materials and methods

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).

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 .

Table 1
Concentrations of Ti-MPs, NiTi-MPs and Ti-NPs used in XTT viability assay, trypan blue test and comet assay.
XTT viability assay (μg/ml) Trypan blue test (μg/ml) Comet assay (μg/ml)
Ti-MPs NiTi-MPs Ti-NPs Ti-MPs NiTi-MPs Ti-NPs Ti-MPs NiTi-MPs Ti-NPs
999,000 83,400 13,080 33,300 20,900 1420 6666 4180 284
300,000 55,600 4360 6660 4180 284 3333 836 57
103,000 16,680 1308 3330 836 56 666 418 28
33,000 5560 436 666 418 28 333 209 14
1670 131

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.

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).

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).

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 .

Cellular uptake

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).

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.

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) .

Materials and methods

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).

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 .

Table 1
Concentrations of Ti-MPs, NiTi-MPs and Ti-NPs used in XTT viability assay, trypan blue test and comet assay.
XTT viability assay (μg/ml) Trypan blue test (μg/ml) Comet assay (μg/ml)
Ti-MPs NiTi-MPs Ti-NPs Ti-MPs NiTi-MPs Ti-NPs Ti-MPs NiTi-MPs Ti-NPs
999,000 83,400 13,080 33,300 20,900 1420 6666 4180 284
300,000 55,600 4360 6660 4180 284 3333 836 57
103,000 16,680 1308 3330 836 56 666 418 28
33,000 5560 436 666 418 28 333 209 14
1670 131

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.

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).

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).

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 .

Cellular uptake

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).

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.

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) .

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Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Intracellular uptake and toxicity of three different Titanium particles
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