Integrin β2 regulates titanium particle‑induced inflammation in macrophages: In vitro aseptic loosening model
- Authors:
- Published online on: November 11, 2024 https://doi.org/10.3892/mmr.2024.13390
- Article Number: 25
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Copyright: © Shen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Total joint arthroplasty, including total hip and total knee arthroplasties, is the most effective surgical treatment for advanced symptomatic osteoarthritis. However, the inflammatory response triggered by the byproducts of joint replacement prostheses can lead to pain, bone defects and movement disorders. In severe cases, these complications may result in aseptic loosening and periprosthetic osteolysis, which are among the most common complications limiting the lifespan of prostheses (1). This poses a challenging clinical problem because significant symptoms often do not manifest until the late stage of failure, at which point the pathological changes are irreversible, necessitating further surgical intervention (2).
A previous study identified particulate wear debris from in vivo prosthesis degradation as a crucial factor stimulating the release of proinflammatory substances, suppressing bone formation, inducing osteoclastic bone resorption, and contributing to prosthesis-related chronic inflammation (3). Therefore, a deeper understanding of the impact of prosthesis material design and particulate wear debris on the inflammatory response after joint replacement is essential for preventing aseptic loosening and periprosthetic osteolysis (4).
Among various biomaterials, titanium (Ti)-based implants are the most widely used in the medical field, including dental prostheses, hearing aids, pacemakers and joint replacements (5). It is well established that oral peri-implant and periodontal bone loss, primarily caused by peri-implantitis and periodontitis, and wear debris, particularly Ti particles near the implant site, are crucial triggers of inflammation and bone resorption (6). Among the cells involved in Ti particle-induced inflammation, macrophages are of particular interest (7). Qu et al (8) reported that the activation of mononuclear macrophages and giant cells is the primary cause of chronic inflammation induced by Ti-wear debris. These findings suggested that macrophages play a critical role in aseptic loosening and inflammation after Ti-based joint replacement. However, the underlying mechanisms, including cytokine, inflammatory expression patterns and regulatory pathways, remain poorly understood. The present study aimed to explore the influence of Ti release on macrophage-like RAW 264.7 cells in aseptic loosening and inflammation pathogenesis.
Within the integrin family, β2 integrins (CD11/CD18) are specifically expressed on the surfaces of macrophages and leukocytes and are composed of a common β2 subunit (CD18) and four different α subunits (CD11a-d) (9). Previous studies have shown that these β2 integrins, acting as adhesion receptors, are crucial in the activation of inflammatory diseases such as rheumatoid arthritis (10) and systemic lupus erythematosus (11) by mediating inflammatory cell recruitment, cell-cell contact and downstream cellular signaling (12). Han et al (13) demonstrated that β2 integrins expressed in macrophages contribute to cell activation, cytotoxicity, chemotaxis and phagocytosis in a mouse model of endotoxin shock. Lv et al (14) demonstrated that β2 integrins promote the expression of inflammatory cytokines, resulting in a pro-inflammatory transformation in macrophages in multi-organ fibrosis. Among the inside-out and outside-in signaling, Toll-like receptor (TLR) signaling has been shown to regulate β2 integrin-stimulated inflammatory responses, though the interaction between these pathways remains controversial (15,16). To further elucidate the signaling pathways involved and the role of integrin-mediated inflammation in Ti-induced osteolysis, the present study focused on TLR signaling.
The present study aimed to investigate the expression patterns of integrins and inflammatory cytokines in RAW 264.7 cells treated with Ti particles to understand the role of integrins in Ti particle-mediated inflammatory osteolysis and identify potential signaling pathways involved.
Materials and methods
Cell treatment
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.
Cell viability
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.
Gene expression analysis: Reverse transcription-quantitative PCR (RT-qPCR)
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.
 | Table I.Primer sequences used for reverse transcription-quantitative PCR analysis of gene expression. |
Protein expression analysis by western blot assay
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.
Confocal immunofluorescence
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.
Flow cytometry and intracellular cytokine staining
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.).
Enzyme-linked immunosorbent assay (ELISA)
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 assay
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.
Statistical analysis
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.
Results
Ti particles effect on RAW 264.7 cell viability
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.
Expression of integrin genes and inflammatory cytokines heatmap by RT-qPCR
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.
Promoted protein expression levels with Ti stimulation by western blot assay
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).
Ti-induced upregulation of integrin and inflammatory response by confocal immunofluorescence assay
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.
Integrin characteristics by flow cytometry assessment
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).
Ti-induced changes in RAW 264.7 cell migration features by scratch-wound healing assay
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).
Ti-induced changes in RAW 264.7 cell protein secretion by ELISA
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.
Discussion
Previous studies have shown that macrophage-like cells are involved in the chronic inflammation induced by Ti particles. Vallés et al (18) found that primary macrophages released increased amounts of TNF-α, IL-6 and IL-1β after incubation with Ti particles. Pettersson et al (19) demonstrated that Ti particles activate lipopolysaccharide-primed macrophages and induce the release of interleukin-1β. Pajarinen et al (20) showed that Ti particles influence the local cytokine microenvironment through foreign body reactions, thereby modulating macrophage polarization. Lee et al (21) demonstrated that conditioned media of macrophages challenged with Ti particles (Ti-CM) suppresses early and late differentiation markers of osteoprogenitors. Bi et al (22) proved that conditioned media of murine marrow cells challenged with Ti particles stimulate in vitro bone resorption primarily by inducing osteoclast differentiation with minor effects on osteoclast activity or survival. In the present study, the impact of Ti particles on RAW 264.7 macrophage cells was investigated, focusing on cell viability, inflammatory response, migratory behavior, and particularly the underlying regulatory mechanisms of integrins.
Extensive research has focused on reducing the inflammatory response and toxicity of implant wear particles in local tissues. Studies indicate that the cytotoxic effects of chemical components depend on factors such as dose, duration, particle size, temperature and cell type (23). For Ti-based implants, macrophage cell viability is primarily influenced by the concentration of Ti particles and the surface characteristics of the implant (24). The present results demonstrated that Ti particles did not compromise the viability of RAW 264.7 cells at any of the examined time points, suggesting that Ti particles do not exert cytotoxic effects under the conditions tested. This is consistent with previous studies reporting minimal cytotoxicity of Ti particles on macrophages at ~0.1 mg/ml (25). Therefore, the Ti particles used in the present study were considered non-toxic and suitable for further experiments. The present study also provided insight into the safe application of Ti-based implants under well-controlled concentrations and surface modifications.
β2 integrins are crucial in macrophage functions, including cell adhesion, migration, phagocytosis and immune responses (26). The current integrin expression analysis revealed that Ti particles significantly upregulated the expression of CD18, CD11b and CD11c at both the mRNA and protein levels, as shown by RT-qPCR, western blotting and confocal immunofluorescence assays. In macrophages, CD18, which mediates cell binding to the extracellular matrix (27), was upregulated in response to Ti particles, indicating increased cell adhesion and proliferation, as depicted in Fig. 1. Previous studies have reported that CD11/CD18 is essential for polymorphonuclear neutrophils recruitment and activation, particularly later stages of immune cell activation through β2 integrin-mediated Syk activation (28). The delayed but significant increase in CD18 expression at 48 and 72 h depicted in Fig. 2 was likely due to the time required for its upregulation as part of the immune response, including cell activation, migration and adhesion, which becomes more prominent at later stages. For example, Ebnet et al (29) found that CD11/CD18 were involved in the regulation of leukocyte-endothelial cell interaction. Through their cytoplasmic domains, junctional adhesion molecules directly associate with various tight junction-associated proteins including ZO-1, AF-6, MUPP1 and PAR-3. CD11b and CD11c have close physical and functional relationships (30) but exhibit different expression levels and functions in various macrophages and show cell-type-specific regulation under inflammatory conditions (31). CD11c expression increased as early as 24 h after Ti treatment in RAW 264.7 cells, indicating its potential role in macrophage activation. By contrast, CD11b, critical for macrophage recruitment during the inflammatory response (32), showed significant increases at 48 h, reflecting the need for additional signaling events to mediate macrophage migration and phagocytosis. The delayed expression aligns with the role of macrophages in clearing debris and promoting tissue remodeling, processes that typically follow the initial inflammatory response. Sándor et al (33) reported that blocking CD11b significantly enhanced the attachment of MDDCs and MDMs to fibrinogen, demonstrating a competition between CD11b and CD11c for this ligand.
Notably, differences in cell morphology revealed by confocal immunofluorescence may be attributed to macrophage activation induced by Ti particles. Flow cytometry showed that CD18 expression increased significantly on the cell surface, while CD11b and CD11c exhibited moderate increases following Ti-particle treatment. These results align with findings from western blotting and immunofluorescence. However, while CD11a mRNA levels were elevated, its surface expression decreased, indicating potential post-transcriptional regulation or protein degradation mechanisms. Further ELISA results depicted in Fig. 5 indicated that CD11a, CD11b and CD11c are involved in early recognition with Ti particles. The delayed but significant increase in CD18 at 24 and 48 h may indicate that CD18 plays a critical role in sustaining and amplifying the immune response once upregulated.
TLRs are pivotal in mediating inflammatory responses in macrophages, primarily through the MyD88-dependent and MyD88-independent pathways (34). Previous research investigated the crosstalk between β2 integrins (CD11/CD18) and TLRs in macrophages and other immune cells (12), suggesting β2 integrin regulates TLR signaling positively or negatively depending on cell types and inflammatory status (35). Bai et al (36) demonstrated that CD11b enhances the TIRAP enrichment in the plasma membrane, exerting a positive regulatory effect via the MyD88-dependent pathway. Saitoh et al (37) also found that MyD88 is required for the interplay between TLR7 and integrin CD11a/CD18.
In the present study, increased expressions of TLR1, TLR2, TLR3 and TLR4 were observed at 48 and 72 h, along with elevated MyD88 and TIRAP expression levels following Ti particle treatment, indicating the involvement of TLRs downstream MyD88-dependent signaling in RAW 264.7 cell activation and pro-inflammatory cytokine production. MyD88 expression may peak at 24 h owing to early activation. It was reported that MyD88 can be regulated by upstream Src kinase activated by Rac1-induced F-actin formation in macrophages (38). MyD88 expression may not show significance as the signaling cascade progresses or due to negative feedback mechanisms. Avbelj et al (39) found a negative feedback regulation of MyD88 by inflammasome-activated caspase-1. Zhang and Ghosh (40) also found that the negative regulation of MyD88-mediated signaling through the suppression of IRAK1 and TRAF6 contributes to the attenuation of the inflammatory response over time. Additionally, the significant upregulation of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-8 and IL-12, along with MMP-3, MMP-8 and MMP-11, indicated that Ti particles induce a robust inflammatory response in RAW 267.4 cells.
As a well-known pro-inflammatory factor, LPS-induced biomarker pattern in RAW 264.7 cells has been well studied (41). As early as 1–4 h period, LPS caused TLR4 and MyD88 levels to increase to more than twice those of the control group (42), while CD14 and TNF-alpha levels increased nearly 10-fold (43,44). Additionally, IL-6, MMP-9 and IL-1β were significantly elevated more than 3-fold compared with the untreated group (43). Among the integrin family, LPS-induced RAW 264.7 cells were characterized by an increase in CD11b/CD18, several-fold compared with the untreated group after 24 h (44). By contrast, Ti particles induced a slower and more moderate inflammatory response in RAW 264.7 cells, which was markedly different from that caused by LPS, both in terms of pattern and magnitude.
The role of β2 integrins in macrophage migration is complex, depending on the specific context and cellular environment (45). A previous study suggested that β2 integrins inhibit macrophage migration by increasing adhesion, leading to their retention at inflammation sites (46). However, other research suggested that the migration of human monocytes in vitro is inhibited when integrin β2 function is blocked (47). The activation of TLR signaling can upregulate integrin expression and other molecules, further facilitating macrophage migration toward inflammation sites (48). In the present study, Ti particles significantly promoted RAW 264.7 cell migration at all examined time points, as indicated by the wound healing assay, accompanied by increased levels of pro-inflammatory cytokines and MMPs, indicating that Ti particles enhance the inflammatory response in macrophages. This effect is likely associated with the activation of β2 integrins and the TLR signaling pathway.
The current study explored the role of integrin mediated TLR signaling in Ti particle-induced inflammation in RAW 264.7 macrophage cells, and provided valuable insights for developing strategies to mitigate aseptic loosening and periprosthetic osteolysis in patients with Ti-based implants. The limitations of the current study include the incomplete elucidation of the crosstalk between integrins and TLRs in the response of RAW 264.7 macrophages induced by Ti particles. Additionally, the effects of conditioned media from macrophages were not further investigated on osteogenic and osteoclastic cells, which would clarify the indirect effect of Ti particles.
In summary, Ti particles induced an inflammatory response in RAW 264.7 macrophages, characterized by enhanced integrin expression, TLR signaling activation, pro-inflammatory cytokine production and increased cell migration, as illustrated in Fig. S2. These findings elucidated the molecular mechanisms underlying Ti particle-induced inflammation and provided valuable insights for developing strategies to mitigate aseptic loosening and periprosthetic osteolysis in patients with Ti-based implants. Future studies should identify specific regulatory pathways and potential therapeutic targets to modulate macrophage activity and improve implant longevity. Further study should also investigate the mechanisms underlying the differences in inflammatory responses to Ti particles and LPS, with the goal of identifying more targeted therapeutic strategies.
Supplementary Material
Supporting Data
Acknowledgements
The authors would like to thank Professor Dai Xuesong (Department of Orthopedic Surgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China) and Professor Ye Zhaoming (Department of Orthopedic Surgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China) for their assistance with statistical advice and suggestions on the web interface.
Funding
The present study was supported by the National Natural Science Foundation of China (grant nos. 81800782 and 81902279), the Medical and Health Research Project of Zhejiang Association (grant no. 2022KY808) and the Zhejiang Traditional Chinese Medicine Administration Science and Technology Project (grant no. 2024ZL574).
Availability of data and materials
The data generated in the present study may be requested from the corresponding author.
Authors' contributions
YS and WGW conceptualized the study. YS and HN acquired the data. HN and JFZ analyzed the data. YS wrote the original draft. HN, JFZ and GWW wrote, reviewed and edited the manuscript. All authors read and approved the final version of the manuscript. YS, JFZ and WGW confirm the authenticity of all the raw data.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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