Integrin β2 regulates titanium particle‑induced inflammation in macrophages: In vitro aseptic loosening model

Abstract

Aseptic loosening is a major complication of joint replacement surgery, characterized by periprosthetic osteolysis and chronic inflammation at the bone‑implant interface. Cells release chemokines, cytokines and other pro‑inflammatory substances that perpetuate inflammation reactions, while other particle‑stimulated macrophages promote osteoclastic bone resorption and impair bone formation. The present study investigated integrin and inflammatory cytokine expression patterns in RAW 264.7 cells treated with titanium (Ti) particles to elucidate the role of integrins in Ti particle‑mediated inflammatory osteolysis. Assessment was performed by reverse transcription‑quantitative PCR, western blotting, confocal immunofluorescence, flow cytometry and enzyme‑linked immunosorbent assays. Cell migration was evaluated by wound healing assay. It was found that Ti particles significantly induced integrin expression in RAW 264.7 cells, including upregulation of integrins β2 (CD18), aL (CD11a), aM (CD11b) and aX (CD11c). Ti particles also enhanced the expression of Toll‑like receptors (TLRs; TLR1, TLR2, TLR3 and TLR4) and triggered the release of inflammatory cytokines such as tumor necrosis factor α, interleukin (IL)‑1β, IL‑8 and IL‑12. Proteomics showed higher expression and activity levels of TLR2 and TLR4, along with their downstream signaling adaptors myeloid differentiation primary response protein 88 (MyD88) and Mal/TIR‑domain‑containing adapter protein (TIRAP), following Ti treatment. Additionally, Ti treatment significantly enhanced the migration rate of RAW 264.7 cells. The present findings indicated that Ti particles regulate the inflammatory response of RAW 264.7 cells in an in vitro aseptic loosening model by activating the TLR/TIRAP/MyD88 signaling pathway.

Keywords: aseptic loosening; integrin β2; macrophages; myeloid differentiation primary response protein 88/Mal/TIR‑domain‑containing adapter protein; titanium.

Figure 1.

Effects of Ti particles on the viability of RAW 264.7 cells and expression heatmap of integrin genes and inflammatory cytokines. (A) RAW 264.7 cells co-cultured with 0.1 mg/ml Ti particles. (B and C) Heatmap of integrins and cytokines expression levels (red: high expression; green: low expression) (magnification, left: ×100; right: ×40). Ti, titanium; TIRAP, Mal/TIR-domain-containing adapter protein. (D) Viability of RAW 264.7 cells after co-culture with Ti particles.

Figure 2.

Western blotting and quantitative analysis of integrin levels and inflammation response proteins. (A) Western blot immunoblots results showing CD18, CD11a, CD11b, CD11c and MyD88 TIRAP. (B-D) Quantification of immunoblots using ImageJ software. *P<0.05. Ti, titanium; TIRAP, TIR-domain-containing adapter protein.

Figure 3.

Confocal immunofluorescence of integrins and inflammatory response proteins with/without Ti treatment. (A-C) Localization of CD18, CD11b and CD11a after Ti co-culture. (D and E) Confocal images of Myd88 and TIRAP localizations. Non-linear adjustments. (F) Statistical analysis was performed among multiple marks comparing fluorescence ratio. Green represents anti-IgA staining. Scale bar, 10 µm. *P<0.05. Ti, titanium; TIRAP, TIR-domain-containing adapter protein.

Figure 4.

Flow cytometry and wound healing assay results of RAW 264.7 cells. (A) Flow cytometric analysis of integrin surface protein expression (CD18, CD11a, CD11b and CD11c) after 72 h. (B and C) Migration assessment of RAW 264.7 cells using wound healing assay. Scale bar, 500 µm. *P<0.05. Ti, titanium.

Figure 5.

Time-dependent expression of CD18, CD11a, CD11b, CD11c, MyD88 and TIRAP in RAW 264.7 cells following Ti particle treatment using ELISA at (A) 6 h; (B) 24 h; (C) 48 h and (D) 72 h. Data are presented as the mean ± SD; *P<0.05 compared with control. Ti, titanium; TIRAP, TIR-domain-containing adapter protein.