Cell culture and morphology

For the purposes of this study, we used the human promyelocytic leukemia HL60 cells, human osteosarcoma-derived osteoblastic MG63 cells [6], and Balb/c mouse 3T3 cells [7], originally obtained from American Type Culture Collection (ATCC) (Gaithersburg, MD, USA) and stored in a laboratory liquid nitrogen tank. In our previous study [1], we used primary cultures of human periosteum-derived cells for cytotoxicity assays. Here, we used similar human osteogenic, but malignant MG63 cells as a reference. Although Balb/c cells are derived from mouse embryos, a preliminary study found that this cell line displays higher superoxide dismutase (SOD) activity than that of the other cells used here. Thus, we used Balb/c cells to test the involvement of SOD in silica cytotoxicity.

Fetal bovine serum (FBS), used to supplement culture media, was obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA) and was heat-inactivated at 56 °C for 30 min prior to use. HL60 cells were cultured in RPMI 1640 medium (FUJIFILM Wako Pure Chemical Co., Osaka, Japan), supplemented with 1% heat-inactivated (hi)-FBS, in a humidified atmosphere containing 5% CO2 at 37 °C. MG63 and Balb/c cells were cultured under the same conditions in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% hi-FBS.

HL60 and the other cells (MG63 and Balb/c) were seeded into 40-mm culture dishes (TPP, Trasadingen, Switzerland) at a density of 7 and 4.5 × 104 cells/dish, respectively, and cultured in the aforementioned growth media for 3 days. With regard to HL60 cell differentiation, 1 μM phorbol 12-myristate 13-acetate (PMA) (Adipogen Corporation, San Diego, CA, USA) and 1.2% dimethyl sulfoxide (DMSO) (FUJIFILM Wako Pure Chemical) were used for monocytic and granulocytic differentiation, respectively [8,9,10,11].

Preparation of silica microparticle suspensions

Plastic vacuum tubes, the inner walls of which were coated with silica in the form of cerite (Neotube; Nipro, Otsu, Japan) were filled with 2 mL of RPMI supplemented with 1% hi-FBS or DMEM supplemented with 10% hi-FBS and vortexed to fully harvest the silica MPs from the tube. The resulting silica suspensions were stored at 4 °C until use (< 1 week). Prior to use, the silica suspensions were well vortexed and diluted with the same respective media [1].

Scanning electron microscopy

Having carefully aspirated the medium from cultured cells to minimize cell detachment, cell morphology was examined using an inverted microscope (Eclipse Ti–U; Nikon, Tokyo, Japan) or a scanning electron microscope (TM-1000; Hitachi, Tokyo, Japan). Photomicrographs of living HL60 cells were obtained without fixation. With regard to sample preparation for scanning electron microscopy (SEM), the culture medium was carefully aspirated to minimize cell loss, and cultured cells tightly or weakly bound on plastic dishes were fixed with 2.5% neutralized glutaraldehyde, dehydrated, and freeze-dried, as described previously [12, 13]. The rim of the dish was removed, and thereafter, the fixed cells were subjected to magnetron sputtering and examined using SEM at an accelerating voltage of 15 kV.

Determination of cell numbers

Following treatment, the media used for culturing HL60 cells was gently mixed 15 times with a dropper, and the floating and weakly bound cells suspended in the medium were counted using a MOXI Z automated cell counter (ORFLO, Ketchum, ID, USA). Moderately and tightly bound cells, which were resistant to initial gentle agitation, were treated with 0.05% w/v trypsin–0.53 mmol/L EDTA (FUJIFILM Wako Pure Chemical Co.) and detached by pipetting for adherent cell counts.

The MG63 and Balb/c cells tightly adhered to the bottom surface of the dish. The cells were not detached using gentle agitation. Thus, the culture media was aspirated without agitation, and the cells were enzymatically detached for cell counting, as described above.

Cytochemical examination (acid phosphatase and non-specific esterase)

To determine the expression of acid phosphatases (ACP) and non-specific esterase (NSE), HL60 cells cultured in plastic dishes were fixed for 1 h with 10% neutralized formalin (FUJIFILM Wako Pure Chemical Co.) and stained for 30–60 min in a CO2 incubator using commercial kits (Muto Chemical Co., Tokyo, Japan), as described previously [14]. To enhance staining, we eliminated the counterstaining step and examined cells directly under an Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan) connected to a cooled VB-7000 CCD camera (Keyence, Osaka, Japan).

Immunocytochemical fluorescence staining (CD11b)

Following incubation, HL60 cells were fixed for 1 h with 10% neutralized formalin (FUJIFILM Wako Pure Chemical Co.), washed twice with phosphate-buffered saline (PBS), and blocked for 30 min with 0.1% Block Ace (Sumitomo Dainippon Pharma Co. Ltd., Osaka, Japan) in 0.1% Tween 20-containing PBS. The samples were then treated for 45 min at room temperature (22–25 °C) with FITC-conjugated mouse monoclonal anti-CD11b antibodies (1:25 dilution; BioLegend, San Diego, CA, USA). For nuclear staining, the samples were further treated for 15 min with 1 μg/mL 4ʹ,6-diamidino-2-phenylindole (DAPI) (Dojin Chemical Lab., Kumamoto, Japan). Having washed with PBS, the samples were mounted using an antifade mounting medium (Vectashield®; Vector Laboratories, Burlingame, CA, USA), and examined under a fluorescence microscope (Nikon) connected to a cooled CCD camera (Keyence) [3].

Determination of superoxide dismutase activity

Cultured cells were harvested using cell scrapers, washed with PBS, collected by centrifugation, and subsequently resuspended in PBS at higher densities (HL60: 2–10 × 106/mL, MG63: 3–10 × 106/mL, Balb/c: 1.8–10 × 106/mL). Following sonication for 10 s, the cells were centrifuged at 10,000 × g for 15 min at 4 °C and the resulting supernatants were collected and stored at – 80 °C until use. Enzyme activity assays were performed using a SOD assay kit (Dojin Chemical Lab.) according to the manufacturer’s instructions.

Statistical analysis

Data are expressed as the mean ± standard deviation of four or five independent cultures. For multi-group comparisons, when the data satisfied both normality (Shapiro–Wilk) and equal variance (Brown–Forsythe) assumptions, one-way analysis of variance (ANOVA) was performed, followed by a Bonferroni test (vs. control in Fig. 1A) or Tukey test (all pairwise comparisons in the remainder of the figures) as a post hoc test (SigmaPlot 13.0; Systat Software, Inc., San Jose, CA, USA). A P-value < 0.05 was considered to be indicative of a statistically significant difference.

Fig. 1
figure 1

Dose-dependent effects of silica microparticles on the proliferation A and the superoxide dismutase (SOD) activity B of HL60, MG63, and Balb/c cells. n = 4 A and n = 5 B in independent cultures. The raw cell numbers at 100% represent 7.49 ± 9.84 × 105 (HL60), 2.68 ± 0.16 × 105 (MG63), and 3.09 ± 0.33 × 105/dish (Balb/c), respectively. aP < 0.05 represents a significant difference from the respective controls, to which no silica suspension was added

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