Responses of promyelocytic leukemia HL60 cells as an inflammatory cell lineage model to silica microparticles used to coat blood collection tubes

Abstract

Background: The preparation of platelet-rich fibrin (PRF) requires glass blood collection tubes, and thus, the shortage or unavailability of such tubes has driven clinicians to search for suitable substitutes, such as silica-coated plastic tubes. However, we have previously demonstrated the cytotoxicity of silica microparticles (MPs) used in plastic tubes to cultured human periosteal cells. To further establish the effects of silica MPs on inflammation, we examined silica MP-induced changes in a human promyelocytic cell model in vitro.

Methods: Human promyelocytic HL60 cells were used either without chemical induction or after differentiation induced using phorbol myristate acetate (PMA) or dimethyl sulfoxide. HL60 cells, osteoblastic MG63, and Balb/c mouse cells were treated with silica MPs, and their surface ultrastructure and numbers were examined using a scanning electron microscope and an automated cell counter, respectively. Differentiation markers, such as acid phosphatase, non-specific esterase, and CD11b, were visualized by cytochemical and immunofluorescent staining, and superoxide dismutase (SOD) activity was quantified.

Results: Regardless of SOD activity, silica cytotoxicity was observed in MG63 and Balb/c cells. At sub-toxic doses, silica MPs slightly or moderately upregulated the differentiation markers of the control, PMA-induced monocytic, and dimethyl sulfoxide-induced granulocytic HL60 cells. Although SOD activity was the highest (P < 0.05) in PMA-induced cells, a silica-induced reduction in cell adhesion was observed only in those cells (P < 0.05).

Conclusions: Silica MP contamination of PRF preparations can potentially exacerbate inflammation at implantation sites. Consequently, unless biomedical advantages can be identified, silica-coated plastic blood collection tubes should not be routinely used for PRF preparations.

Keywords: Differentiation; HL60 cells; Silica; Superoxide dismutase; Viability.

Fig. 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

Fig. 2

Effects of silica microparticles (MPs) on the surface microstructure of control and phorbol 12-myristate 13-acetate (PMA)-treated HL60 cells. The control cells A and those simultaneously treated with silica MPs for 3 days B were fixed and subjected to scanning electron microscopy examination. Similar data were obtained from two additional independent experiments (n = 3)

Fig. 3

Effects of silica microparticles (MPs) on cell morphology in control A and phorbol 12-myristate 13-acetate (PMA)- B and dimethyl sulfoxide (DMSO)-treated HL60 cells (C). These cells were simultaneously treated with silica MPs for 3 days and examined microscopically without fixation. Similar data were obtained from two additional independent experiments (n = 3)

Fig. 4

Effects of silica microparticles (MPs) on acid phosphatase (ACP) activity in control A and phorbol 12-myristate 13-acetate (PMA)- B and dimethyl sulfoxide (DMSO)-treated HL60 cells (C). These cells were simultaneously treated with silica MPs for 3 days. The cells were then fixed and subjected to cytochemical ACP staining. Similar data were obtained from two additional independent experiments (n = 3)

Fig. 5

Effects of silica microparticles (MPs) on non-specific esterase (NSE) activity in control A and phorbol 12-myristate 13-acetate (PMA)- B and dimethyl sulfoxide (DMSO)-treated HL60 cells (C). These cells were simultaneously treated with silica MPs for 3 days. The cells were then fixed and subjected to cytochemical NSE staining. Similar data were obtained from two additional independent experiments (n = 3)

Fig. 6

Effects of silica microparticles (MPs) on CD11b expression in control A and phorbol 12-myristate 13-acetate (PMA)- B and DMSO-treated HL60 cells (C). These cells were simultaneously treated with silica MPs for 3 days. The cells were then fixed and subjected to immunocytochemical CD11b staining followed by DAPI staining. Similar data were obtained from two additional independent experiments (n = 3). Green and blue signals indicate the presence of CD11b and nuclei, respectively

Fig. 7

Effects of silica microparticles (MPs) on the proliferation, adhesion, and superoxide dismutase (SOD) activity of HL60 cells. After 3-day treatments, the suspended HL60 cells were collected and the adherent cells enzymatically detached for cell counting. A Total cell numbers are the sum of suspended and adherent cells n = 4. B The percentage of the adherent cells was calculated by dividing the adherent cell number by the total cell number n = 4. C SOD activity of the control and differentiated HL60 cells n = 5. PMA phorbol 12-myristate 13-acetate, DMSO dimethyl sulfoxide. P < 0.05 represent a significantly difference