Commercially pure Ti particles (mean diameter, 3.0 µm; 93% <20 µm) were purchased from Alfa Aesar. To sterilize the particles, they were mixed with 95% ethanol and stirred magnetically for 24 h. The particles were then resuspended in sterile phosphate-buffered saline containing 6% rat serum, penicillin (100 U/ml) and streptomycin (100 U/ml) at a concentration of 1.2×10⁸/ml, stored at 4°C.

Murine macrophage-like RAW 264.7 cells were purchased from the Chinese Academy of Sciences and the Shanghai Institute of Cell Biology. Cells were cultured at 37°C in a humidified incubator with 5% CO₂ in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc.), supplemented with 10% heat-inactivated fetal bovine serum and antibiotics (100 units/ml of penicillin-G and 100 pg/ml of streptomycin). Then, RAW 264.7 cells (3×10⁵ cells/ml) were plated in 96-well plates and treated with or without Ti particles (0.1 mg/ml). Cell samples were collected at various time points (6, 12, 24, 48 and 72 h) and frozen for further analysis.

The viability of RAW 264.7 cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. At different time points (6, 12, 24, 48 and 72 h), cell samples (seeded at 3×10⁵ cells/ml) were incubated with MTT (0.5 mg/ml) at 37°C for 2 h. The culture medium was then replaced with an equal volume of dimethyl sulfoxide to dissolve formazan crystals. After centrifugation, the supernatant from each sample was transferred to a 96-well plate. The corresponding absorbance was measured at a wavelength of 570 nm using a microplate reader (Bio-Tek Instruments Inc.). Cell viability was presented as the mean ± standard deviation (SD) of the optical density values. All experiments were performed in triplicates.

mRNA expression levels in RAW 264.7 were quantified by RT-qPCR. RAW 264.7 samples treated with or without 0.1 mg/ml Ti particles collected at 48 and 72 h were homogenized in TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) using a glass/Teflon homogenizer. Total RNA was isolated according to the manufacturer's protocol. First-strand complementary DNA (cDNA) was synthesized using M-MLV reverse transcriptase (cDNA synthesis kit; Takara Bio, Inc.) according to the manufacturer's protocol. qPCR was performed using 1.0 µl cDNA from each sample with a commercial kit and a Thermal Cycler Dice TP800 (Takara Bio, Inc.). Cycling conditions began with an initial 3-min denaturation step at 95°C followed by 40 cycles of 10 sec at 95°C for denaturation and 45 sec at 60°C for annealing and extension. Primer sequences for murine integrin β1, integrin β2 (CD18), integrin β3, integrin α1, integrin α2, integrin α3, integrin α4, integrin α5, integrin α6, integrin L (CD11a), integrin M (CD11b), integrin X (CD11c), integrin D (CD11d), TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR11, interleukins (ILs; IL 1β, IL-6, IL-8, IL-10 and IL-12), tumor necrosis factor (TNF), interferons, matrix metalloproteinases (MMPs; MMP3, MMP8 and MMP11), MyD88, TIPAR and the housekeeping gene β-actin are listed in Table I. Relative expression values were normalized to β-actin and analyzed using the 2^−ΔΔCq method (17). All samples were assayed in triplicates.

RAW 264.7 macrophages were challenged with indicated concentrations of Ti particles for 6, 12, 24, 48 and 72 h, and then lysed in a buffer containing 1% Triton X-100, protease inhibitors (MilliporeSigma), and 1 mM sodium orthovanadate (MilliporeSigma). The protein concentration in the supernatant was measured using a BCA protein assay kit. Equal amounts of protein (30 µg/lane) were separated using Tris-bis SDS-PAGE gels (8 or 10% gel concentration for different molecular weight proteins; Invitrogen; Thermo Fisher Scientific, Inc.) and transferred onto polyvinylidene fluoride membranes (MilliporeSigma). After incubation in 5% skimmed milk in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 h at room temperature (to block non-specific binding), the membranes were incubated overnight at 4°C with the appropriate primary antibody (Table II). A horseradish peroxidase-conjugated donkey anti-rabbit IgG was used as a secondary antibody (1:1,000; cat. no. NA934; Cytiva). Antibody binding was detected using the Immobilon Chemiluminescence system (MilliporeSigma). Quantitative densitometry analysis was conducted and the relative densities of the protein bands were analyzed using ImageJ software (version 1.53b; National Institutes of Health). The relative expression levels of target proteins were normalized to the corresponding intensities of tubulin.

RAW 264.7 cells at a density of 1×10⁵ cells/ml were seeded on coverslips using six-well plates and stained for immunofluorescence detection under confocal fluorescence microscopy. Filamentous actin was used as a housekeeping marker and stained with rhodamine-phalloidin (Molecular Probes; Thermo Fisher Scientific, Inc.) for 1 h at room temperature in the dark. Samples were stained with the following primary antibodies overnight at 4°C: CD11a (1:100; cat. no. ab186873; Abcam), CD11b (1:500; cat. no. ab184308; Abcam), CD11c (1:100; cat. no. 97585; Cell Signaling Technology, Inc.), CD18 (1:500; cat. no. 102259; SinoBiological), MyD88 (1:50; cat. no. PA5-19919; Thermo Fisher Scientific, Inc.) and TIRAP (1:100; cat. no. ab17218; Abcam). A secondary antibody of Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1,000; cat. no. A-11034; Invitrogen; Thermo Fisher Scientific, Inc.) was then added and incubated for 1 h at room temperature in the dark. Nuclei were visualized using Hoechst 33342 (cat. no. H1399; Thermo Fisher Scientific, Inc.) staining for 1 h at room temperature. The images were captured with a 633 or 320 objective lens using appropriate laser excitation (wavelengths of 540, 488 and 360 nm) on a laser-scanning confocal microscope (Carl Zeiss AG) or an Olympus IX81-FV1000 (Olympus Corporation) laser microscope system. Detector gain was initially optimized by sampling several regions of the coverslip and then fixed for each corresponding channel. Once set, the detector gain value was kept stable throughout the image acquisition process. Images were analyzed using Zeiss LSM Image Examiner Software and FV10-ASW 3.0 Viewer. Immunofluorescence was used to quantitatively analyze CD18, CD11b, CD11c, myeloid differentiation primary response protein 88 (MyD88) and TIR-domain-containing adapter protein (TIRAP) expression levels. Specific antibodies were used and detected using green fluorescence. Simultaneously, the cytoskeletal protein actin was labeled with red fluorescence as an internal reference standard. Relative fluorescence intensity was calculated as the ratio of the average green fluorescence intensity (target proteins) to the red fluorescence intensity (actin), yielding the fluorescence ratio. Each experiment was repeated three times to ensure reliability. Multiple group statistical analyses were performed to evaluate the significance of differences between groups. All images were captured under identical magnification and microscopy conditions.

RAW 264.7, macrophages were treated with Ti particles for 6, 12, 24, 48 and 72 h. For the final 2 h of stimulation, macrophage Fc receptors were blocked with 2.4G2 for 10 min at 4°C, followed by surface staining with anti-F4/80 (1:100; cat. no. 14-4801-85; BM8/eBioscience; Thermo Fisher Scientific, Inc.). Peritoneal cells were surface stained with anti-Siglec F (1:100; cat. no. 564514; E50-2440/BD Biosciences) and anti-Gr1 (1:200; cat. no. MA1-10402; eBioscience; Thermo Fisher Scientific, Inc.). Macrophages were then fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) for 20 min at 4°C. Flow cytometric analysis was conducted on an LSR2 (BD Biosciences) using FACS Diva software (BD Biosciences). The gating strategy involved using Forward Scatter (FSC) and Side Scatter (SSC) to isolate viable, intact cells while excluding debris or dead cells. Fluorescence marker gating was applied to quantify integrin expression with negative controls setting baseline fluorescence and positive controls verifying accuracy. The scale bars on histograms were adjusted to ensure clear separation between marker-positive and marker-negative populations. All flow cytometry analyses and procedures were conducted using FlowJo software (version 10.8.1; Tree Star, Inc.).

RAW 264.7 cells were cultured and treated with Ti particles to induce an inflammatory response. After 6, 24, 48 and 72 h of treatment, the cells were harvested and lysed to obtain cellular extracts. Surface adhesion molecules and signaling protein levels were quantitatively assessed using specific ELISA kits according to the manufacturer's protocol. Briefly, cell lysates were added to microtiter plates pre-coated with antibodies specific to each target protein. Following incubation, the plates were washed to remove unbound proteins, and a secondary enzyme-linked antibody specific to the target protein was added. The enzyme-substrate reaction produced a colorimetric signal, measured at 450nm using a microplate reader. The concentrations of CD11a (cat. no. SEB572Mu; Cloud-Clone Corp.), CD11b (cat. no. JM-12253M2; Jiangsu Jingmei Biological Technology Co., Ltd.), CD11c (cat. no. SEB159Mu; Cloud-Clone Corp.), CD18 (cat. no. JM-13347M2; Jiangsu Jingmei Biological Technology Co., Ltd.), MyD88 (cat. no. SEB707Mu; Cloud-Clone Corp.) and TIRAP (cat. no. JM-13352M2; Jiangsu Jingmei Biological Technology Co., Ltd.) were subsequently calculated based on the appropriate standard curve.

Wound healing assays were performed to assess collective cell migration. After reaching 90% cell confluence, RAW 264.7 cells were scratched with a sterile 100-µl pipette tip and then treated with serum-free medium. The distance between the wound edges was measured using an IX71 inverted light microscope (Olympus Corporation). Images were captured at 0-, 6-, 12-, 24- and 36-h time points. The experiments were performed three times for each group, and three measurements were taken for each sample using ImageJ software.

Data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using SPSS 28 software (IBM Corp.). Differences between unpaired data groups were analyzed using the unpaired Student's two-tail t-test after normality was confirmed with the Kolmogorov-Smirnov test. A non-parametric Mann-Whitney test was conducted for data that did not follow a normal distribution. P<0.05 was considered to indicate a statistically significant difference. Data analysis was conducted by Prism 8.0 (GraphPad Software; Dotmatics) software.

To evaluate the impact of Ti particles on cell viability, RAW 264.7 cells were cultured with micron-sized pure Ti particles at a concentration of 0.1 mg/ml. The viability of RAW 264.7 cells was not negatively affected by Ti particles (Fig. 1A and D). Compared with the control group, no significant decrease in cell viability was observed at any of the examined time points, including 6, 12, 24, 48 and 72 h post-treatment.

The expression levels of integrin family members, TLRs, and inflammation response gene classes in RAW 264.7 cells, with and without Ti particle treatment, were detected by RT-qPCR (detailed results are presented in Fig. S1) and are presented as heat maps in Fig. 1B and C.

The expression levels of integrin subunits CD18, CD11a, CD11b and CD11c significantly increased following Ti particle treatment (Fig. 1B). Additionally, there was a notable upregulation in the gene expression of TLRs (1–4) at 48 and 72 h after Ti treatment. The relative expression folds of MyD88 and TIRAP, downstream molecules in the TLR signaling pathway, were also elevated simultaneously (P<0.05).

The expression patterns of inflammatory cytokines in RAW 264.7 cells treated with Ti particles are shown in Fig. 1C. There was a significant increase in the expression of TNF-α, IL 1β, IL-8 and IL-12 in cells cultured with Ti particles compared with the blank control group at 72 h (P<0.05). Furthermore, the matrix metalloproteinase-3 (MMP-3), MMP-8 and MMP-11 levels significantly increased following Ti treatment (P<0.05). Notably, the expression of IL 1β and IL 12 was upregulated earlier than that of the other inflammatory cytokines.

Protein expression levels in the Ti co-culture group vs. the blank control group were analyzed by western blotting at the 24-, 48- and 72-h time points (Fig. 2A). Higher protein expression levels were observed in the Ti particle co-culture group, consistent with the mRNA levels of the corresponding genes, CD18, CD11b, CD11c and MyD88. However, discrepancies were noted where protein levels did not align with mRNA expression profiles, such as CD11a and TIRAP.

Quantitative analysis of western blot protein levels was normalized to the housekeeping protein tubulin. The results showed markedly increased protein levels of CD18 (at 48 and 72 h), CD11b (at 48h) and CD11c (at 24 and 48 h) in RAW 264.7 cells cultured with Ti particles compared with the control group (P<0.05) (Fig. 2B-D). The protein level of MyD88 was also significantly elevated in the Ti-treated group compared with the control group at the 24-h checking point (P<0.05).

Immunofluorescent staining of RAW 264.7 cells was conducted with and without Ti treatment and observed under a confocal microscope (Fig. 3). Consistent with the immunoblotting results, the confocal immunofluorescent images demonstrated upregulation of CD18, CD11b and CD11c in response to Ti particle co-culture (Fig. 3A-C). Additionally, MyD88 and TIRAP exhibited significant changes in immunofluorescent intensity following Ti co-culture (Fig. 3D and E). Statistical analysis was performed in term of fluorescence ratio (Fig. 3F). The observed increase in the intensity of MyD88 and TIRAP aligned with the protein and gene expression levels depicted in Figs. 1 and 2.

Flow cytometry was used to characterize and quantify the impact of Ti particles on integrin protein expression on the surface of RAW 264.7 cells (Fig. 4A). The analysis revealed enhanced CD18, CD11b and CD11c expression in the Ti co-culture group compared with the control group. The most notable increase was observed for CD18 (from 0.2–6.2%), while CD11b and CD11c showed slight increases. However, CD11a expression decreased by over 10% (Fig. 4A).

A wound healing assay was performed to assess the migratory characteristics of RAW 264.7 cells co-cultured with Ti particles. Representative images of cell migration at different time points are revealed in Fig. 4B, with quantification presented in Fig. 4C. The results indicated that Ti particles significantly enhanced the migration of RAW 264.7 compared with the control group at 24 and 36 h (P<0.05).

The expression levels of CD18, CD11a, CD11b, CD11c, MyD88 and TIRAP were analyzed in RAW 264.7 cells following treatment with Ti particles at 6, 24, 48 and 72 h (Fig. 5A-D). At the 6-h mark, a significant increase was observed in the expression of CD11a, CD11b and CD11c but not CD18. However, at 24 and 48 h, there was significant elevation in mostly all target molecules detected, including CD18, CD11a, CD11b, CD11c and TIRAP, indicating a pronounced response. At the 72-h time point, CD18, CD11b and TIRAP continued to show significant increases in expression. These results suggested a time-dependent activation of these markers in response to Ti particle exposure.