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Phagocytosis of titanium particles and necrosis in TNF-α-resistant mouse sarcoma L929 cells
Toxicology in Vitro 17 (2003) 41–47 www.elsevier.com/locate/toxinvit
Phagocytosis of titanium particles and necrosis in TNF-a-resistant mouse sarcoma L929 cells E. Osanoa, J. Kishib, Y. Takahashic,* a
Department of Microbiology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan c Department of Dental Materials Science, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan b
Accepted 6 November 2002
Abstract In the oral cavity, titanium is an excellent biocompatible material. However, it is reported that high ratios of intracellular titanium particles can cause cell apoptosis or necrosis by as-yet unknown mechanisms. The purpose of this study was to investigate the response of tumor necrosis factor alpha (TNF-a)-resistant L929 fibroblasts to titanium particles. Cells were cultured in Eagle’s medium supplemented with fetal bovine serum and l-glutamine. Titanium particle sizes were less than 9 m. Cytotoxicity was assayed by a cell counting kit, trypan blue dye exclusion test and lactate dehydrogenase (LDH) leakage. The production of reactive oxygen species (ROS) was detected by a confocal laser scanning microscope (CLSM) using dichlorofluorescein diacetate as a fluorescent probe. Morphology was viewed by a CLSM and with an X-ray microanalyser (XMA). When titanium particles were added to cells, the viability decreased to around 50% at a particle concentration of 2.0%. The number of dead cells and LDH activity in the culture media increased significantly between 1 and 2 days. However, formation of active oxygen species did not occur, since no dichlorofluorescein fluorescence was observed. A scanning electron photomicrograph (SEM) revealed a large number of particles covering or adhering to cellular components in lysed cells compared with flattened control cells attached to the substrate. The XMA showed that the titanium accumulation was coincident with the deformed cell shape. The CLSM also confirmed that particles were within the cells. From these results it was concluded that titanium particles ingested in large quantities into the cell induced necrosis by a pathway other than by producing ROS. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Titanium; Particles; L929; Fibroblasts; TNF-a; Necrosis
1. Introduction The physical qualities of titanium, high strength, toughness, durability, low density, corrosion resistance and biocompatibility, make it useful in a variety of applications in medical and dental prosthesis. However, titanium is not highly abrasion resistant. Aseptic loosening Abbreviations: CLSM, confocal laser scanning microscopy; FBS, fetal bovine serum; LDH, lactate dehydrogenase; MEM, Eagle’s minimum essential medium; PBS, phosphate buffered saline; ROS, reactive oxygen species; SEM, scanning electron microscopy; TNF-a, tumor necrosis factor alpha; WST-8, 2-(2-methoxy-4-nitrophenyl)-3(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazorium; XMA, X-ray microanalyser. * Corresponding author. Tel.: +81-52-751-2561; fax: +81-52-7592373. E-mail address: [email protected] (Y. Takahashi).
has been reported associated with the generation of wear debris particles from the articulating surfaces of the prosthetic components. This may in turn initiate a biochemical signaling cascade resulting in osteoclast activation and bone resorption (Lombardi et al., 1989; McKellop et al., 1990). Similar titanium particles have also been reported in soft tissue surrounding nonarticulating miniplates used to repair jaw fractures (Schliephake et al., 1993) and in tissues surrounding failed titanium dental implants (Arys et al., 1998). The fate and effect of such particles is still unclear. However, Ayukawa et al. (1998) have shown that fibroblast-like cells occasionally endocytosed titanium particles, which were subsequently co-localized with lysosomal cathepsins. This would appear to be an attempt to degrade the endocytosed particles intracellularly. Such endocytosis often resulted in cell death, since high concentrations of
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particles within cultured human fibroblasts often caused apoptosis or necrosis (Maloney et al., 1993; Mostardi et al., 1999). Similar information has also been reported for rat osteoblasts (Pioletti et al., 1999). One proposed mechanism is the generation of reactive oxygen species (ROS) during attempts to degrade the titanium. To investigate this mechanism further we have attempted to look at the effect of titanium particles on L929 mouse sarcoma cells. This cell line is commonly used to evaluate medical devices (Al-Nazhan and Spangberg, 1990; Chandler et al., 1994; Rao et al., 1996; Sen et al., 1998; Yamamoto et al., 1998; Hashieh et al., 1999; Telli et al., 1999; Cimpan et al., 2000). Accumulation of ROS is also the mechanism of action by which TNF-a induces necrotic cell death (Vercammen et al., 1998). It was decided therefore to develop two types of L929 cells: one sensitive to TNF-a and therefore sensitive to reactive oxygen and the other not sensitive to TNF-a (Liddil et al., 1989; Vanhaesebroeck et al., 1991; Goossens et al., 1995). Such resistance to TNF-a is thought to be due to a decreased capacity to produce superoxide anion (Hennet et al., 1993) and/or an increase in superoxide dismutase (Polla et al., 1996). The aim of this study was to investigate the cytotoxic effect of titanium particles using TNF-a-resistant L929 fibroblasts.
2. Materials and methods 2.1. Cell culture TNF-a-resistant L929, a mouse fibrosarcoma cell line, was obtained from Riken Cell Bank (Tsukuba Science City, Japan). Cells were grown in Eagle’s minimum essential medium (MEM) (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum, 2 mm l-glutamine and penicillin (100 units/ml) in a fully humidified atmosphere of 5% CO2 in air at 37 C. 2.2. Titanium particles Commercially pure titanium particles of 325-mesh nominal diameter were purchased from Aldrich (Milwaukee, WI, USA). We measured the size of titanium particles using by image analysis system and the particle sizes were 9.1 15.1 mm (n=625). The particles were autoclaved at 121 C for 20 min. 2.3. Cell viability assays The effects of titanium particles on mouse L929 cells were evaluated using three methods: WST-8 reduction by mitochondria of cells, trypan blue exclusion (Innes and Ogden, 1999), and LDH release (Sasaki and Ohno, 1994).
2.4. WST-8 assay TNFa-resistant L929 cells at a density of 5103 cells per well were cultured in 96-well tissue culture plates. After the incubation for 24 h at 37 C, titanium particles were added to wells in concentrations of 0.25, 0.5, 1.0 and 2.0%. After treatment for 24 h, 10 ml of WST-8 were added and incubated for 2 h at 37 C. WST-8 formazan production of living cells in a soluble form were measured using cell counting kit-8 (Wako Pure Chemical Industries, Ltd, Osaka, Japan). The absorbances in each well were measured at 450 nm using a microtiter plate reader (MPR-A4i (II), Tohso, Tokyo, Japan). Cell viability was calculated as a percentage of absorbance from no titanium particles to various concentrations of particles. Similar experiments were performed in triplicate. 2.5. Trypan blue exclusion and LDH release assays L929 cells were seeded in 25 cm2 culture flasks (Falcon: Becton Dickinson Labware, Tokyo, Japan) at a density of 2.0106 cells per flask in 5 ml of growth medium and allowed to attach for 24 h. Then, 1.0% titanium particles were added to the flasks and incubated further for up to 48 h at 37 C. After harvesting the media for LDH assay, cells were dissociated by treatment with 0.25% trypsin (1:250, Difco, Detroit, MI, USA). A portion of resuspended cell pellets in phosphate buffered saline (PBS) was mixed with an equal volume of 0.5% trypan blue. The cell suspension was loaded into a hemocytometer and the proportions of non-viable to viable cells were determined (Innes and Ogden, 1999). LDH contents in each media were measured using an LDH CII test kit (Wako Pure Chemical Industries, Ltd, Osaka, Japan). 2.6. Scanning electron microscope (SEM) and X-ray microanalyser (XMA) One-ml suspensions of 2.0104 cells were cultured on cell culture coverslips (Nalge Nunc International, Naperville, IL, USA) in 24-well plates for 24 h at 37 C. Following incubation for 24 h with or without 1.0% titanium, cells were washed with 5 ml of PBS for 30 s and then fixed in 1% glutaraldehyde in 0.1 m sodium cacodylate buffer, pH 7.2 for 30 min. After the cells were rinsed with the buffer for 30 min, they received a post-fixation procedure with 1% osmium tetroxide in a 0.1 m sodium cacodylate buffer. After all specimens were rinsed twice with distilled water, 5 min each time, the specimens were dehydrated through ascending ethanol solutions (50, 70, 80, 90 and 100%), for 15 min each time, and then immediately dried with hexamethyldisilazane solution (Sigma, USA) for 5 min. The samples were then coated with platinum and palladium, and carbon. These were viewed with SEM (JSM 6400 FX,
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Jeol, Tokyo, Japan) and XMA (JSM 8900 RL, Jeol, Tokyo, Japan), respectively. 2.7. Confocal laser scanning microscope (CLSM) Fluorescein isothiocyanate labelled cells in 1.0% titanium particles were observed on a Zeiss model CLSM410 (Germany) following the quenching methods of Innes and Ogden (1999) to scan cells vertically. The production of ROS was observed by the method of Ciapetti et al. (1998) using 20 ,70 -dichlorofluorescendiacetate (Sigma, USA) as fluorescent probe. 2.8. Statistical analysis The mean and standard deviation of the three tests were calculated. A comparison was performed by Student’s t-tests. Differences were considered statistically significant at P < 0.05.
3. Results We assayed the effects of titanium particles on L929 cell viability using three systems. As shown in Fig. 1, when particles of different concentrations were added to cells, the viability decreased to around 50% at a particle concentration of 2.0%. Fig. 2 shows L929 cell viability as a function of time after the addition of 1.0% titanium particles. The number of viable L929 cells in contact with 1.0% titanium particles decreased or stabilized in the first 24 h after the addition of particles and then increased. In contrast, the proliferation rate of L929 cells without particles was about double for every 24 h.
Fig. 1. Cytotoxicity of titanium particles in L929 cells (meanS.D.). Cell viability was determined by WST-8 assay. Cell viability decreased with the increasing concentration of titanium particles. Significant differences were found among them. Number of samples was nine in each case. (a) Statistically significant in P<0.05; (b) statistically significant in P<0.01)
Fig. 2. Number of viable L929 cells as a function of time after the addition of 1.0% titanium particles (meanS.D.). Cell viability was determined by trypan blue dye exclusion. The number of viable L929 cells in contact with 1.0% titanium particles decreased or stabilized in the first 24 h after the addition of particles and then increased. In contrast, the proliferation rate of L929 cells without particles was about double for every 24 h.
The number of trypan blue positive dead cells after the addition of 1.0% titanium particles increased significantly 1 and 2 days later (Fig. 3). LDH activity as a function of time after the addition of 1.0% titanium particles in the culture media increased significantly one and two days later, showing cell necrosis (Fig. 4). We observed a morphological change of L929 cells in the presence of 1.0% titanium by a SEM. As shown in Plate 1(a), control L929 fibroblasts showed large, flattened cells firmly attached to the substrate, with blebs on the cell surface. A large number of particles covered or adhered to cellular components in lysed cells (Plate
Fig. 3. Number of dead L929 cells as a function of time after the addition of 1.0% titanium particles (meanS.D.). The number of trypan blue-positive dead cells increased during the first 24 h of incubation time but decreased thereafter. This phenomenon is due to melting away of the cell membrane by necrosis.
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1b). XMA analysis showed the titanium particle accumulation (Plate 1d) to be coincident with the deformed cell shape (Plate 1c). CLSM photographs showed titanium particles existing in the surface layer (Plate 2a), the middle (Plate 2b) and bottom (Plate 2c) of the cells. As large number of particles was observed in the middle of the cells, particles were within the cells (Plate 2a–d). Plate 3 shows the fluorescent images of ROS taken by CLSM. Fluorescences were negative in TNF-a-resistant L929 (a: untreated; b: treated with particles) but positive in TNF-a-sensitive L929 (c: untreated; d: treated with particles).
4. Discussion Fig. 4. LDH unit/50 ml as a function of time after the addition of 1.0% titanium particles. LDH activity increased significantly 1 and 2 days later, and significant differences (P<0.01) were found between control and 1.0% titanium particles.
Pioletti et al. (1999) reported that nearly all osteoblasts were killed at a concentration of 1.0% titanium particles. When we added 1.0% titanium particles to L929 cells, the viability almost linearly decreased to 50% to the particle concentration of 2.0% but did not reach to 100% (Fig. 1). This difference may be due to the cell type and large size of L929 cells used in our
Plate 1. Scanning electron micrographs of L929 cells (a: control L929 fibroblast cells; b and c: titanium particles covered or adhered to cellular components in lysed cells) and X-ray characteristic image of titanium (d). Arrows in the photos (c, d) point to an X-ray characteristic image of titanium to be coincident with the deformed cell shape.
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Plate 2. Confocal laser scanning micrographs (CLSM) of L929 cells after the addition of 1.0% titanium particles. (a) Differential interfacial contrast (Nomarski) image of L929 cells; (b) surface layer of cells; (c) middle of cells; (d) bottom layer of cells.
Plate 3. CLSM of L929 showing the fluorescence of reactive oxygen intermediates. (a) TNF-a-resistant L929 untreated cells; (b) TNFaresistant L929 cells treated with 1.0% titanium particles; (c) TNF-asensitive L929 untreated cells; (d) TNF-a-sensitive L929 cells treated with 1.0% titanium particles.
experiment, as in the report of Tomazic-Jezic et al. (2001). The viability of L929 cells in contact with titanium particles decreased somewhat or stabilized in the first 24 h after the addition of 1.0% titanium particles and then increased. After 24 h of incubation, L929 cells proliferate, thus reducing the number of particles available per cell as shown in Fig. 2. L929 cells also ingested most of the particles in the first 24 h, then due to the proliferation of cells, the ratio of particles to cells
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decreased and so did the particle intake rate. The cell population harboring a small number of particles proliferated just the same as cells that did not ingest particles (data not shown). The number of trypan blue-positive cells, which indicated dead cells, increased after 24 h and then decreased (Fig. 3). Although osteoblasts are reported to survive under low concentrations of titanium particles, they were killed under high concentrations of the particles such as 1.0% (Mostardi et al., 1999; Pioletti et al., 1999). Pioletti et al. (1999) suggested that rat osteoblasts can ingest only a restricted number of particles before damage occurs, or osteoblasts may exocytose the particles once ingested into the cell. They suggested that the association of phagocytosis between the high concentration of particles and viability might be a more important factor than composition. As to the viability of cytochalasin-D treated osteoblasts, because they inhibited phagocytosis they increased statistically more than non-treated cells. However, their report is unclear as to whether or not the shape, toxic ion or oxidation-reduction potential may influence the inhibition of the cell proliferation. Yamamoto et al. (1998) and Murthy et al. (2001) reported that some aqueous titanium such as TiCl4 is toxic. However, as for the possible role of ions released from pure titanium into the culture media, Rao et al. (1996) reported that L929 cell growth rate was almost similar to that of the control for titanium. In view of this, we followed the simple trial of Mostardi et al. (1999) culturing L929 cells with filtered media after the incubation with the titanium particles. As a result, cytotoxicity of filtered media incubated with the titanium particles was similar to that without titanium particles (data not shown). There is much evidence that reactive oxygen radicals are the key mediators of necrotic cell death in TNF-asensitive L929 cells (Liddil et al., 1989; Vanhaesebroeck et al., 1991; Goossens et al., 1995; Vercammen et al., 1998). We examined the production of ROS by CLSM using dichlorofluorescein diacetate as a fluorescent probe. As a result, production of reactive oxygen intermediates could be observed in TNF-a-sensitive cells, but not in resistant cells. Although ROS are probably the causative agent in the case of TNF-a-sensitive cells necrosis, ions released from titanium particles in cells may punch out holes in TNF-a-resistant cell membrane, leading to cellular lysis (Plate 4). This is suggested from the report (Assad et al., 1999) that titanium elements were detected after elution of pure titanium. Mu et al. (2000) showed that active oxygen species generated by macrophages induced titanium ion release, but that metal ion release was prevented by FBS, penicillin, and streptomycin. These reports showed that ions released from titanium were too small to exert harmful effects on L929 cells. However, the high concentration of particles within a TNF-a-resistant cell may be enough to make holes in the cell membrane.
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Science, Sports and Culture of Japan. We would like to thank Dr T. Hanaichi for his technical assistance with the SEM and XMA, and Dr. K. Moriguchi for his kind advice on CLSM during this study. Proofreading by Professor C. Robinson and Mr. L. Pettitt was greatly appreciated.
References
Plate 4. A scanning electron micrograph of single L929 cell after treatment with 1.0% titanium particles. Many holes in the cell membrane have been punched out.
Phagocytes such as neutrophiles, monocytes and macrophage ingest not only bacteria but also large particles such as dead cells and carbon particles. In addition to these professional phagocytes, gingival fibroblasts ingesting latex beads were observed by the electron microscope (McKeown et al., 1990). Maloney et al. (1993) observed by SEM that bovine fibroblasts ingested small titanium particle. Although there are reports that L929 necrotic or lysed cells were observed through a CLSM (Cimpan et al., 2000) and SEM (Al-Nazhan and Spangberg, 1990; Chandler et al., 1994; Sen et al., 1998), our results, which showed the necrosis of L929 cells associated with phagocytosis, are the first. LDH release from cells and morphological change observation by SEM confirmed the necrosis (Sasaki and Ohno, 1994). Two substances are known to cause L929 necrosis. One is TNF, which causes necrosis in TNF-a-sensitive L929 through the pathway of producing ROS, and the other is extracellular ATP. Pizzo et al. (1992) reported that extracellular ATP caused necrosis in TNF-a-resistant L929 more susceptible through an unknown pathway. The ingestion of particles into cells may enhance the P2z receptors of TNF-a-resistant L929 more susceptible to extracellular ATP-mediated necrosis. Our results show that fluorescences of ROS were not observed in TNF-a-resistant L929. Therefore, the ingestion of particles into cells may make the P2z receptors of TNF-a-resistant L929 more susceptible to extracellular ATP-mediated necrosis. However, we could not speculate a possible pathway by which particles cause necrosis.
Acknowledgements This study was supported by a Science Frontier Propulsion Enterprise from the Ministry of Education,
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Phagocytosis of titanium particles and necrosis in TNF-a-resistant mouse sarcoma L929 cells E. Osanoa, J. Kishib, Y. Takahashic,* a
Department of Microbiology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan c Department of Dental Materials Science, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan b
Accepted 6 November 2002
Abstract In the oral cavity, titanium is an excellent biocompatible material. However, it is reported that high ratios of intracellular titanium particles can cause cell apoptosis or necrosis by as-yet unknown mechanisms. The purpose of this study was to investigate the response of tumor necrosis factor alpha (TNF-a)-resistant L929 fibroblasts to titanium particles. Cells were cultured in Eagle’s medium supplemented with fetal bovine serum and l-glutamine. Titanium particle sizes were less than 9 m. Cytotoxicity was assayed by a cell counting kit, trypan blue dye exclusion test and lactate dehydrogenase (LDH) leakage. The production of reactive oxygen species (ROS) was detected by a confocal laser scanning microscope (CLSM) using dichlorofluorescein diacetate as a fluorescent probe. Morphology was viewed by a CLSM and with an X-ray microanalyser (XMA). When titanium particles were added to cells, the viability decreased to around 50% at a particle concentration of 2.0%. The number of dead cells and LDH activity in the culture media increased significantly between 1 and 2 days. However, formation of active oxygen species did not occur, since no dichlorofluorescein fluorescence was observed. A scanning electron photomicrograph (SEM) revealed a large number of particles covering or adhering to cellular components in lysed cells compared with flattened control cells attached to the substrate. The XMA showed that the titanium accumulation was coincident with the deformed cell shape. The CLSM also confirmed that particles were within the cells. From these results it was concluded that titanium particles ingested in large quantities into the cell induced necrosis by a pathway other than by producing ROS. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Titanium; Particles; L929; Fibroblasts; TNF-a; Necrosis
1. Introduction The physical qualities of titanium, high strength, toughness, durability, low density, corrosion resistance and biocompatibility, make it useful in a variety of applications in medical and dental prosthesis. However, titanium is not highly abrasion resistant. Aseptic loosening Abbreviations: CLSM, confocal laser scanning microscopy; FBS, fetal bovine serum; LDH, lactate dehydrogenase; MEM, Eagle’s minimum essential medium; PBS, phosphate buffered saline; ROS, reactive oxygen species; SEM, scanning electron microscopy; TNF-a, tumor necrosis factor alpha; WST-8, 2-(2-methoxy-4-nitrophenyl)-3(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazorium; XMA, X-ray microanalyser. * Corresponding author. Tel.: +81-52-751-2561; fax: +81-52-7592373. E-mail address: [email protected] (Y. Takahashi).
has been reported associated with the generation of wear debris particles from the articulating surfaces of the prosthetic components. This may in turn initiate a biochemical signaling cascade resulting in osteoclast activation and bone resorption (Lombardi et al., 1989; McKellop et al., 1990). Similar titanium particles have also been reported in soft tissue surrounding nonarticulating miniplates used to repair jaw fractures (Schliephake et al., 1993) and in tissues surrounding failed titanium dental implants (Arys et al., 1998). The fate and effect of such particles is still unclear. However, Ayukawa et al. (1998) have shown that fibroblast-like cells occasionally endocytosed titanium particles, which were subsequently co-localized with lysosomal cathepsins. This would appear to be an attempt to degrade the endocytosed particles intracellularly. Such endocytosis often resulted in cell death, since high concentrations of
0887-2333/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0887-2333(02)00127-3
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particles within cultured human fibroblasts often caused apoptosis or necrosis (Maloney et al., 1993; Mostardi et al., 1999). Similar information has also been reported for rat osteoblasts (Pioletti et al., 1999). One proposed mechanism is the generation of reactive oxygen species (ROS) during attempts to degrade the titanium. To investigate this mechanism further we have attempted to look at the effect of titanium particles on L929 mouse sarcoma cells. This cell line is commonly used to evaluate medical devices (Al-Nazhan and Spangberg, 1990; Chandler et al., 1994; Rao et al., 1996; Sen et al., 1998; Yamamoto et al., 1998; Hashieh et al., 1999; Telli et al., 1999; Cimpan et al., 2000). Accumulation of ROS is also the mechanism of action by which TNF-a induces necrotic cell death (Vercammen et al., 1998). It was decided therefore to develop two types of L929 cells: one sensitive to TNF-a and therefore sensitive to reactive oxygen and the other not sensitive to TNF-a (Liddil et al., 1989; Vanhaesebroeck et al., 1991; Goossens et al., 1995). Such resistance to TNF-a is thought to be due to a decreased capacity to produce superoxide anion (Hennet et al., 1993) and/or an increase in superoxide dismutase (Polla et al., 1996). The aim of this study was to investigate the cytotoxic effect of titanium particles using TNF-a-resistant L929 fibroblasts.
2. Materials and methods 2.1. Cell culture TNF-a-resistant L929, a mouse fibrosarcoma cell line, was obtained from Riken Cell Bank (Tsukuba Science City, Japan). Cells were grown in Eagle’s minimum essential medium (MEM) (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum, 2 mm l-glutamine and penicillin (100 units/ml) in a fully humidified atmosphere of 5% CO2 in air at 37 C. 2.2. Titanium particles Commercially pure titanium particles of 325-mesh nominal diameter were purchased from Aldrich (Milwaukee, WI, USA). We measured the size of titanium particles using by image analysis system and the particle sizes were 9.1 15.1 mm (n=625). The particles were autoclaved at 121 C for 20 min. 2.3. Cell viability assays The effects of titanium particles on mouse L929 cells were evaluated using three methods: WST-8 reduction by mitochondria of cells, trypan blue exclusion (Innes and Ogden, 1999), and LDH release (Sasaki and Ohno, 1994).
2.4. WST-8 assay TNFa-resistant L929 cells at a density of 5103 cells per well were cultured in 96-well tissue culture plates. After the incubation for 24 h at 37 C, titanium particles were added to wells in concentrations of 0.25, 0.5, 1.0 and 2.0%. After treatment for 24 h, 10 ml of WST-8 were added and incubated for 2 h at 37 C. WST-8 formazan production of living cells in a soluble form were measured using cell counting kit-8 (Wako Pure Chemical Industries, Ltd, Osaka, Japan). The absorbances in each well were measured at 450 nm using a microtiter plate reader (MPR-A4i (II), Tohso, Tokyo, Japan). Cell viability was calculated as a percentage of absorbance from no titanium particles to various concentrations of particles. Similar experiments were performed in triplicate. 2.5. Trypan blue exclusion and LDH release assays L929 cells were seeded in 25 cm2 culture flasks (Falcon: Becton Dickinson Labware, Tokyo, Japan) at a density of 2.0106 cells per flask in 5 ml of growth medium and allowed to attach for 24 h. Then, 1.0% titanium particles were added to the flasks and incubated further for up to 48 h at 37 C. After harvesting the media for LDH assay, cells were dissociated by treatment with 0.25% trypsin (1:250, Difco, Detroit, MI, USA). A portion of resuspended cell pellets in phosphate buffered saline (PBS) was mixed with an equal volume of 0.5% trypan blue. The cell suspension was loaded into a hemocytometer and the proportions of non-viable to viable cells were determined (Innes and Ogden, 1999). LDH contents in each media were measured using an LDH CII test kit (Wako Pure Chemical Industries, Ltd, Osaka, Japan). 2.6. Scanning electron microscope (SEM) and X-ray microanalyser (XMA) One-ml suspensions of 2.0104 cells were cultured on cell culture coverslips (Nalge Nunc International, Naperville, IL, USA) in 24-well plates for 24 h at 37 C. Following incubation for 24 h with or without 1.0% titanium, cells were washed with 5 ml of PBS for 30 s and then fixed in 1% glutaraldehyde in 0.1 m sodium cacodylate buffer, pH 7.2 for 30 min. After the cells were rinsed with the buffer for 30 min, they received a post-fixation procedure with 1% osmium tetroxide in a 0.1 m sodium cacodylate buffer. After all specimens were rinsed twice with distilled water, 5 min each time, the specimens were dehydrated through ascending ethanol solutions (50, 70, 80, 90 and 100%), for 15 min each time, and then immediately dried with hexamethyldisilazane solution (Sigma, USA) for 5 min. The samples were then coated with platinum and palladium, and carbon. These were viewed with SEM (JSM 6400 FX,
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Jeol, Tokyo, Japan) and XMA (JSM 8900 RL, Jeol, Tokyo, Japan), respectively. 2.7. Confocal laser scanning microscope (CLSM) Fluorescein isothiocyanate labelled cells in 1.0% titanium particles were observed on a Zeiss model CLSM410 (Germany) following the quenching methods of Innes and Ogden (1999) to scan cells vertically. The production of ROS was observed by the method of Ciapetti et al. (1998) using 20 ,70 -dichlorofluorescendiacetate (Sigma, USA) as fluorescent probe. 2.8. Statistical analysis The mean and standard deviation of the three tests were calculated. A comparison was performed by Student’s t-tests. Differences were considered statistically significant at P < 0.05.
3. Results We assayed the effects of titanium particles on L929 cell viability using three systems. As shown in Fig. 1, when particles of different concentrations were added to cells, the viability decreased to around 50% at a particle concentration of 2.0%. Fig. 2 shows L929 cell viability as a function of time after the addition of 1.0% titanium particles. The number of viable L929 cells in contact with 1.0% titanium particles decreased or stabilized in the first 24 h after the addition of particles and then increased. In contrast, the proliferation rate of L929 cells without particles was about double for every 24 h.
Fig. 1. Cytotoxicity of titanium particles in L929 cells (meanS.D.). Cell viability was determined by WST-8 assay. Cell viability decreased with the increasing concentration of titanium particles. Significant differences were found among them. Number of samples was nine in each case. (a) Statistically significant in P<0.05; (b) statistically significant in P<0.01)
Fig. 2. Number of viable L929 cells as a function of time after the addition of 1.0% titanium particles (meanS.D.). Cell viability was determined by trypan blue dye exclusion. The number of viable L929 cells in contact with 1.0% titanium particles decreased or stabilized in the first 24 h after the addition of particles and then increased. In contrast, the proliferation rate of L929 cells without particles was about double for every 24 h.
The number of trypan blue positive dead cells after the addition of 1.0% titanium particles increased significantly 1 and 2 days later (Fig. 3). LDH activity as a function of time after the addition of 1.0% titanium particles in the culture media increased significantly one and two days later, showing cell necrosis (Fig. 4). We observed a morphological change of L929 cells in the presence of 1.0% titanium by a SEM. As shown in Plate 1(a), control L929 fibroblasts showed large, flattened cells firmly attached to the substrate, with blebs on the cell surface. A large number of particles covered or adhered to cellular components in lysed cells (Plate
Fig. 3. Number of dead L929 cells as a function of time after the addition of 1.0% titanium particles (meanS.D.). The number of trypan blue-positive dead cells increased during the first 24 h of incubation time but decreased thereafter. This phenomenon is due to melting away of the cell membrane by necrosis.
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1b). XMA analysis showed the titanium particle accumulation (Plate 1d) to be coincident with the deformed cell shape (Plate 1c). CLSM photographs showed titanium particles existing in the surface layer (Plate 2a), the middle (Plate 2b) and bottom (Plate 2c) of the cells. As large number of particles was observed in the middle of the cells, particles were within the cells (Plate 2a–d). Plate 3 shows the fluorescent images of ROS taken by CLSM. Fluorescences were negative in TNF-a-resistant L929 (a: untreated; b: treated with particles) but positive in TNF-a-sensitive L929 (c: untreated; d: treated with particles).
4. Discussion Fig. 4. LDH unit/50 ml as a function of time after the addition of 1.0% titanium particles. LDH activity increased significantly 1 and 2 days later, and significant differences (P<0.01) were found between control and 1.0% titanium particles.
Pioletti et al. (1999) reported that nearly all osteoblasts were killed at a concentration of 1.0% titanium particles. When we added 1.0% titanium particles to L929 cells, the viability almost linearly decreased to 50% to the particle concentration of 2.0% but did not reach to 100% (Fig. 1). This difference may be due to the cell type and large size of L929 cells used in our
Plate 1. Scanning electron micrographs of L929 cells (a: control L929 fibroblast cells; b and c: titanium particles covered or adhered to cellular components in lysed cells) and X-ray characteristic image of titanium (d). Arrows in the photos (c, d) point to an X-ray characteristic image of titanium to be coincident with the deformed cell shape.
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Plate 2. Confocal laser scanning micrographs (CLSM) of L929 cells after the addition of 1.0% titanium particles. (a) Differential interfacial contrast (Nomarski) image of L929 cells; (b) surface layer of cells; (c) middle of cells; (d) bottom layer of cells.
Plate 3. CLSM of L929 showing the fluorescence of reactive oxygen intermediates. (a) TNF-a-resistant L929 untreated cells; (b) TNFaresistant L929 cells treated with 1.0% titanium particles; (c) TNF-asensitive L929 untreated cells; (d) TNF-a-sensitive L929 cells treated with 1.0% titanium particles.
experiment, as in the report of Tomazic-Jezic et al. (2001). The viability of L929 cells in contact with titanium particles decreased somewhat or stabilized in the first 24 h after the addition of 1.0% titanium particles and then increased. After 24 h of incubation, L929 cells proliferate, thus reducing the number of particles available per cell as shown in Fig. 2. L929 cells also ingested most of the particles in the first 24 h, then due to the proliferation of cells, the ratio of particles to cells
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decreased and so did the particle intake rate. The cell population harboring a small number of particles proliferated just the same as cells that did not ingest particles (data not shown). The number of trypan blue-positive cells, which indicated dead cells, increased after 24 h and then decreased (Fig. 3). Although osteoblasts are reported to survive under low concentrations of titanium particles, they were killed under high concentrations of the particles such as 1.0% (Mostardi et al., 1999; Pioletti et al., 1999). Pioletti et al. (1999) suggested that rat osteoblasts can ingest only a restricted number of particles before damage occurs, or osteoblasts may exocytose the particles once ingested into the cell. They suggested that the association of phagocytosis between the high concentration of particles and viability might be a more important factor than composition. As to the viability of cytochalasin-D treated osteoblasts, because they inhibited phagocytosis they increased statistically more than non-treated cells. However, their report is unclear as to whether or not the shape, toxic ion or oxidation-reduction potential may influence the inhibition of the cell proliferation. Yamamoto et al. (1998) and Murthy et al. (2001) reported that some aqueous titanium such as TiCl4 is toxic. However, as for the possible role of ions released from pure titanium into the culture media, Rao et al. (1996) reported that L929 cell growth rate was almost similar to that of the control for titanium. In view of this, we followed the simple trial of Mostardi et al. (1999) culturing L929 cells with filtered media after the incubation with the titanium particles. As a result, cytotoxicity of filtered media incubated with the titanium particles was similar to that without titanium particles (data not shown). There is much evidence that reactive oxygen radicals are the key mediators of necrotic cell death in TNF-asensitive L929 cells (Liddil et al., 1989; Vanhaesebroeck et al., 1991; Goossens et al., 1995; Vercammen et al., 1998). We examined the production of ROS by CLSM using dichlorofluorescein diacetate as a fluorescent probe. As a result, production of reactive oxygen intermediates could be observed in TNF-a-sensitive cells, but not in resistant cells. Although ROS are probably the causative agent in the case of TNF-a-sensitive cells necrosis, ions released from titanium particles in cells may punch out holes in TNF-a-resistant cell membrane, leading to cellular lysis (Plate 4). This is suggested from the report (Assad et al., 1999) that titanium elements were detected after elution of pure titanium. Mu et al. (2000) showed that active oxygen species generated by macrophages induced titanium ion release, but that metal ion release was prevented by FBS, penicillin, and streptomycin. These reports showed that ions released from titanium were too small to exert harmful effects on L929 cells. However, the high concentration of particles within a TNF-a-resistant cell may be enough to make holes in the cell membrane.
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Science, Sports and Culture of Japan. We would like to thank Dr T. Hanaichi for his technical assistance with the SEM and XMA, and Dr. K. Moriguchi for his kind advice on CLSM during this study. Proofreading by Professor C. Robinson and Mr. L. Pettitt was greatly appreciated.
References
Plate 4. A scanning electron micrograph of single L929 cell after treatment with 1.0% titanium particles. Many holes in the cell membrane have been punched out.
Phagocytes such as neutrophiles, monocytes and macrophage ingest not only bacteria but also large particles such as dead cells and carbon particles. In addition to these professional phagocytes, gingival fibroblasts ingesting latex beads were observed by the electron microscope (McKeown et al., 1990). Maloney et al. (1993) observed by SEM that bovine fibroblasts ingested small titanium particle. Although there are reports that L929 necrotic or lysed cells were observed through a CLSM (Cimpan et al., 2000) and SEM (Al-Nazhan and Spangberg, 1990; Chandler et al., 1994; Sen et al., 1998), our results, which showed the necrosis of L929 cells associated with phagocytosis, are the first. LDH release from cells and morphological change observation by SEM confirmed the necrosis (Sasaki and Ohno, 1994). Two substances are known to cause L929 necrosis. One is TNF, which causes necrosis in TNF-a-sensitive L929 through the pathway of producing ROS, and the other is extracellular ATP. Pizzo et al. (1992) reported that extracellular ATP caused necrosis in TNF-a-resistant L929 more susceptible through an unknown pathway. The ingestion of particles into cells may enhance the P2z receptors of TNF-a-resistant L929 more susceptible to extracellular ATP-mediated necrosis. Our results show that fluorescences of ROS were not observed in TNF-a-resistant L929. Therefore, the ingestion of particles into cells may make the P2z receptors of TNF-a-resistant L929 more susceptible to extracellular ATP-mediated necrosis. However, we could not speculate a possible pathway by which particles cause necrosis.
Acknowledgements This study was supported by a Science Frontier Propulsion Enterprise from the Ministry of Education,
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