Abstract
Background: Despite recent progress in the research of nanoparticles (NPs) spanning in many scientific fields, study of NPs in dentistry is limited. This triggered us to investigate the effect of TiO2 NPs on the drug-sensitivity of oral squamous cell carcinoma and inflammation of human gingival fibroblasts (HGFs). Materials and Methods: The number of viable HGF cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Prostaglandin E2 (PGE2) was quantified by enzyme-linked immunosorbent assay. Contamination with lipopolysaccharide (LPS) was assayed by the endotoxin assay kit. Intracellular uptake and distribution of TiO2 NPs were assessed by transmission electron microscopy. Results: TiO2 NPs (0.05-3.2 mM) did not affect HGF cell viability, although TiO2 NPs clusters were dose-dependently incorporated into the vacuoles of cells. Interleukin-1β (IL-1β) (3 ng/ml) stimulated the secretion of PGE2 into the culture medium by HGF cells. TiO2 NPs also induced PGE2 production, in synergy with IL-1β. Since only a minor amount of LPS was detected in TiO2 NPs, the enhanced production of PGE2 was not simply due to LPS contamination. Conclusion: The present study demonstrates, for the first time to our knowledge, that TiO2 NPs at concentrations higher than 0.2 mM exert an pro-inflammatory action against HGF cells, regardless of the presence or absence of IL-1β.
Nanotechnology is considered a vital new technology. The use of nanoparticles (NPs) has significantly increased during the past decade in the fields of drug delivery (1), cancer targeting (2), detection (3), diagnostic (4) and therapeutic sciences (5). However, the cytotoxicity and side-effects of NPs for human health have not been well-characterized, although in vitro studies have demonstrated an intracellular uptake of NPs by endocytosis (6, 7). In dentistry, NPs in restorative and implant materials have been investigated for their safety, therapeutic and anti-bacterial activity (8). The environmental concentration of titanium dioxide (TiO2) NPs is predicted to be between 0.7 and 16 ng/ml (9), and TiO2 NPs have been widely used in sunscreens (10) due to their ability to penetrate biological barriers. Nanotoxicology is an emerging field that evaluates the toxicity of nanomaterials both in vivo and in vitro (11). Nanomaterials are easily aggregated (12, 13), and such aggregates induce less toxicity and inflammation compared to well-dispersed NPs (14). This transition between aggregation and dispersion has produced inconsistent experimental results. TiO2 NPs were non-toxic to the human squamous carcinoma cell line (OSCC) HSC-2 (0.26 mg/ml) (15), to alveolar epithelial cells (8A549), bronchial epithelial cells (16-HBE), monocytic/macrophage (THP-1), rat alveolar macrophages and peripheral blood monocyte-derived macrophages (16), while TiO2 NPs were cytotoxic against mouse fibroblast cells and human fibroblast cells (>1 mg/ml) by pro-oxidant action (17), at a concentration 100-times higher than that predicted from the environmental concentration.
The high frequency of use of NPs in dental materials suggests an importance in clarifying the mechanisms of toxicity induction against oral cells. We recently reported that TiO2 NPs did not affect the sensitivity of HSC-2 cells towards five chemotherapeutic drugs [doxorubicin (anthracycline antibiotic), melphalan (alkylating agent), fluorouracil (5-FU, pyrimidine analog), docetaxel (taxane), gefitinib (protein tyrosine kinase inhibitor)] (15). It has recently been reported that TiO2 NPs stimulated rat alveolar macrophage and human monocytic cell lines to produce interleukin-1β (IL-1β) (16). However, as far as we are aware of, no study has investigated whether TiO2 NPs stimulate the production of pro-inflammatory substances by human gingival fibroblasts (HGFs), which play an important role in inducing gingivitis.
We recently established HGF cells from a extracted first premolar tooth (18) and these cells produced various pro-inflammatory substances [prostaglandin E2 (PGE2), IL-6, IL-8 and monocyte chemotactic protein-1 (MCP-1)] but not nitric oxide (NO) or tumor necrosis factor-α (TNFα), upon stimulation with IL-1β (19). This was unexpected since mouse macrophages produced very high amounts of TNFα and NO upon stimulation with LPS (20). These results suggest that regulation of the production of pro-inflammatory cytokines may be different in HGFs from that of macrophages. Using this in vitro gingivitis model system, we investigated whether contact with TiO2 NPs modifies the production of PGE2 in IL-1β-activated HGFs.
Materials and Methods
Materials. The following chemicals and reagents were obtained from the indicated companies: Dulbecco's modified Eagle's medium (DMEM) (GIBCO BRL, Grand Island, NY, USA); fetal bovine serum (FBS) (JRH Bioscience, Lenexa, KS, USA); TiO2 NPs (nanopowder, anatase phase, particle size <25 nm, purity 99.7%, trace metal basis, MW 79.87 g/mol), LPS from Escherichia coli (serotype 0111:B4), 3-(4,5-dimethyl-thyazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich (St. Louis, MO, USA); PGE2, enzyme-linked immunosorbent assay (ELISA) assay kit was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA); Endotoxin assay kit (ToxinSensor™ Chromogenic LAL) was from GenScript USA Inc. (Piscataway, NJ, USA); plastic culture dishes and plates (96-well) were purchased from Becton Dickinson (Franklin Lakes, NJ, USA).
Cell culture. HGF cells were established from the extracted first premolar tooth in the lower jaw and periodontal tissues of a 12-year-old girl, according to the guideline of Intramural Ethic Committee (No. A0808) after obtaining informed consent from the patient. These cells were cut into small pieces by surgical blade, and placed onto 80-mm plastic dishes to allow the outgrowth during 2 weeks in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin G and 100 μg/ml streptomycin sulfate at 37°C under a humidified atmosphere with 5% CO2. The outgrown cells were used as primary culture with population doubling level (PDL) zero. Cells were then harvested by treatment with 0.25% trypsin-0.025% EDTA-2Na in phosphate-buffered saline without calcium and magnesium [PBS(−)]. The sub-culture of HGF cells was carried out every week with 1:4 split ratio and medium change in between the sub-cultures. HGFs had an in vitro life span (cumulative cell population doubling number) of 47, regardless of the culture medium used (18).
Determination of viable cell number. Cells were trypsinized and inoculated at 1:3 split ratio in 96-microwell plates and incubated for 48 h to allow for complete attachment. Near-confluent cells were then incubated for 24 h in fresh culture medium without (control) or with 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mM of TiO2 NPs or 0.0001, 0.001, 0.01, 0.1, 1, 10 or 100 ng/ml of LPS. The TiO2 NPs were dissolved in distilled water, vortexed and suspended by sonication with a bath-type sonicator (Tokyo Cho-Onpa Giken Co. Tokyo, Japan) for 1 min at room temperature before application. After treatment, the viable cell number was determined by the MTT method. In brief, cells were incubated for 4 h with 0.2 mg/ml MTT in fresh culture medium. The formed formazan was dissolved with 0.1 ml of dimethyl sulfoxide (DMSO), and the absorbance at 540 nm of the lysate was determined by using a microplate reader (Multiskan, Biochromatic, Labsystem, Osaka, Japan).
PGE2 production. Near-confluent cells were treated with different concentrations of TiO2 NPs (0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mM) or LPS (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 ng/ml) for 30 min in fresh culture medium. Cells were treated with and without IL-1β (R&D Systemes Mineapolis, MN, USA) (3 ng/ml) for a further 24 h. This concentration of IL-1β has been reported to be optimal for the production of pro-inflammatory cytokine (21) and PGE2 (data not shown). The culture supernatants were then collected, and the concentration of PGE2 released into the culture medium was determined by ELISA, according to the manufacturer's instructions.
Assay for endotoxin contamination. LPS present in TiO2 NPs or that released from TiO2 NPs was quantified using a kinetic-chromogenic endotoxin-specific LAL reagent. Briefly, 6.4 and 0.64 mM of TiO2 NPs were first suspended in LAL water reagent, and then stood at room temperature or centrifuged at 21,880× g for 5 min to collect the supernatant that are free from TiO2 NPs. These uncentrifuged (that contained TiO2 NPs) and centrifuged samples (that did not contain TiO2 NPs) were incubated for 30 min at 37°C and changes in the absorbance at 540 were measured using a microplate reader, according to manufacturer's instructions. The contamination of LPS in TiO2 NPs was expressed per g TiO2 NPs.
Intracellular uptake of TiO2 NPs. HGF cells were treated without or with 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mM of TiO2 NPs for 3 h. The cells were washed three times with cold PBS(−) and fixed for 1 h with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4°C, scraped with a rubber policemen, post-fixed for 90 min with 1% osmium tetroxide 0.1M cacodylate buffer (pH 7.4), dehydrated and then embedded in Araldite M (Ciba-Geigy Swiss; NISSHIN EN Co., Ltd., Tokyo Japan). Thin sections were stained with uranyl acetate and lead citrate, and were then observed under a JEM-1210 transmission electron microscope (JEOL) at an accelerating voltage of 80 kV (22).
Statistical analysis. Each reported value represents the mean±standard deviation (S.D.) The data were subject to Kolmogorov-Smirnov normality test and paired t-test using Statistical Package for Social Science (Chicago, IL, USA). Differences were considered significant at p<0.05.
Results
Intracellular uptake of TiO2 NPs. Homogeneous TiO2 NP suspension was prepared by sonication in water. TiO2 NPs were found to be easily aggregated during culture, confirming previous reports (12, 13) and some of the aggregates were incorporated into vacuoles as clusters, in a dose-dependent fashion (Figure 1).
Intracellular uptake of TiO2 nanoparticles (NPs). Near-confluent human gingival fibroblast (HGF) cells were incubated for 3 h without (A) or with 0.05 (B), 0.1 (C), 0.2 (D), 0.4 (E), 0.8 (F), 1.6 (G) or 3.2 (H) mM TiO2 NPs. The cells were then washed three times with cold PBS(−) and fixed for 1 h with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4°C for observation under transmission electron microscopy.
Synergistic stimulation of prostaglandin E2 (PGE2) production by TiO2 nanoparticles (NPs) and interleukin-1β (IL-1β) in human gingival fibroblast (HGF) cells. Near-confluent HGF cells were incubated for 24 h without (control) or with 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 or 3.2 mM TiO2 NPs, in the presence or absence of 3 ng/ml of IL-1β. The viable cell number was then determined by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay (A). The concentration of PGE2 in the culture medium was determined by ELISA, according to the manufacturer's instruction (B). Each value represents the mean±S.D. of three independent experiments (each experiment was performed in triplicate). *p<0.05 paired t-test.
Cytotoxicity. TiO2 NPs exhibited essentially no cytotoxicity against HGFs cell over the concentration range from 0.05 to 3.2 mM, regardless of presence or absence of IL-1β (Figure 2A), supporting previous reports (12). Slight, but significant (p<0.05) increase of absorbance at 540 nm at higher TiO2 NP concentration may have been due to mitochondrial activation (Figure 2A). It has been reported that many toxicants can stimulate the growth of cultured cells at lower concentrations (so-called ‘hormesis’) (23). However, TiO2 NPs did not induce such hormetic growth stimulation at lower concentrations.
Effect of lipopolysaccharide (LPS) on viable cell number and prostaglandin E2 (PGE2) production. Near-confluent human gingival fibroblast (HGF) cells were incubated for 24 h without (control) or with 0.0001, 0.001, 0.01, 0.1, 1, 10 or 100 ng/ml LPS, in the presence or absence of 3 ng/ml interleukin-1β (IL-1β). The viable cell number was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay (A). PGE2 concentration in the culture medium was assayed by ELISA (B). Each value represents the mean±S.D. of three independent experiments (each experiment was performed in triplicate). *p<0.05 paired t-test.
PGE2 production. IL-1β (3 ng/ml) stimulated the production of PGE2 in HGF cells. TiO2 NPs alone also induced PGE2 production, and furthermore synergistically enhanced IL-1β-stimulated PGE2 production (Figure 2B).
Assessment of lipopolysaccharide (LPS) contamination in TiO2 nanoparticles (NP) sample.
LPS contamination. There was a possibility that the apparent increase of PGE2 production by TiO2 NPs may simply be due to contamination by LPS. We tested this possibility using an endotoxin-specific detection kit. TiO2 NPs were found to contain approximately 29-100 ng LPS per gram, when the relative activity was assumed to be 1 EU=0.1 ng, 22-48% of which was released or detached from NPs during incubation (Table I). It was impossible for us to investigate the possibility that TiO2 NPs may have interfered with the measurement of LPS.
TiO2 NPs at 0.2 mM (that should contain only 9.0-31.2 ng LPS/g TiO2 NPs) effectively stimulated the IL-1β-stimulated PGE2 production. This concentration of LPS was not enough to stimulate IL-1β-stimulated PGE2 production (Figure 3B), reducing the possibility that LPS contamination is the reason why TiO2 NPs enhanced PGE2 production. LPS at a higher concentration (100 ng/ml) slightly, but significantly (p<0.05), reduce HGF viability, regardless of the presence (Experiment 1) or absence of IL-1β (Figure 3A).
Discussion
The present study demonstrated for the first time that TiO2 NPs at concentrations above 0.2 mM significantly increased the PGE2 production by HGF cells, and when IL-1β was present, the PGE2 production was synergistically enhanced. This was not due to LPS contamination, since much higher concentrations of LPS were required to induce similar magnitude of PGE2 production. This finding leads us to recommend application of TiO2 NPS at 0.1 mM or less for safe dental application.
It has been reported that TLRs (toll-like receptors) may be involved in the uptake of TiO2 NPs and in the promotion of the associated inflammatory responses (24, 25). One study has investigated the cellular uptake of TiO2 NPs and the inflammatory response in human hepatocellular carcinoma (HepG2) and human chronic myelogenous leukemia (K562) cell lines, and found that TLR4 acted as the signaling receptor without protein complex of LPS, LPS-binding protein and CD14 (26). However, no study has been performed with OSCC cell lines or normal oral cells.
Although TiO2 NPs have been classified as a biological inert substance for animals and humans, ultrafine TiO2 NPs induced inflammation and bronchiolar damage by producing reactive oxygen species (27). Furthermore, the effects of NPs are known to be very variable depending on experimental conditions: the size (27) and physicochemical properties (such tendency of aggregation) of TiO2 NPs (28) and the type of target cells (29).
We previously reported that TiO2 NPs did not alter the sensitivity of an OSCC cell line (HSC-2) towards five popular antitumor agents (15) when HGF cells are not present. However, the experimental conditions we had used previously were not physiological in the sense that there were no oral components such as cells derived from gingiva and periodontal tissues, mucosa and saliva. It is possible that in the presence of HGF cells that produce many inflammatory cytokines, TiO2 NPs may alter the sensitivity not only of cancer cells but also of normal oral cells to chemotherapeutic agents. This type of experiment is very important when we consider the application of NPs to the stomatognathic system.
At present, the mechanism of the pro-oxidant action of TiO2 NPs is unknown in the presence of HGF cells. It remains to be investigated whether nuclear factor-κB is involved in TiO2 NP-induced inflammation, and what type of anti-inflammatory agents (either synthetic or natural products) are effective at alleviating the pro-inflammatory action of TiO2 NPs.
Footnotes
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↵* Ph.D. student of Health Science, UAEM.
- Received November 13, 2013.
- Revision received December 24, 2013.
- Accepted December 27, 2013.
- Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
