Enoxaparin sodium bone cement plays an anti-inflammatory immunomodulatory role by inducing the polarization of M2 macrophages

Abstract

Objective: The implantation of PMMA bone cement results in an immune response and the release of PMMA bone cement particles causes an inflammatory cascade. Our study discovered that ES-PMMA bone cement can induce M2 polarization of macrophages, which has an anti-inflammatory immunomodulatory effect. We also delved into the molecular mechanisms that underlie this process.

Methods: In this study, we designed and prepared samples of bone cement. These included PMMA bone cement samples and ES-PMMA bone cement samples, which were implanted into the back muscles of rats. At 3, 7, and 14 days after the operation, we removed the bone cement and a small amount of surrounding tissue. We then performed immunohistochemistry and immunofluorescence to observe the polarization of macrophages and the expression of related inflammatory factors in the surrounding tissues. The RAW264.7 cells were exposed to lipopolysaccharide (LPS) for 24 h to establish the macrophage inflammation model. Then, each group was treated with enoxaparin sodium medium, PMMA bone cement extract medium, and ES-PMMA bone cement extract medium, respectively, and cultured for another 24 h. We collected cells from each group and used flow cytometry to detect the expressions of CD86 and CD206 in macrophages. Additionally, we performed RT-qPCR to determine the mRNA levels of three markers of M1 macrophages (TNF-α, IL-6, iNOS) and two M2 macrophage markers (Arg-1, IL-10). Furthermore, we analyzed the expression of TLR4, p-NF-κB p65, and NF-κB p65 through Western blotting.

Results: The immunofluorescence results indicate that the ES-PMMA group exhibited an upregulation of CD206, an M2 marker, and a downregulation of CD86, an M1 marker, in comparison to the PMMA group. Additionally, the immunohistochemistry results revealed that the levels of IL-6 and TNF-α expression were lower in the ES-PMMA group than in the PMMA group, while the expression level of IL-10 was higher in the ES-PMMA group. Flow cytometry and RT-qPCR analyses revealed that the expression of M1-type macrophage marker CD86 was significantly elevated in the LPS group compared to the NC group. Additionally, M1-type macrophage-related cytokines TNF-α, IL-6, and iNOS were also found to be increased. However, in the LPS + ES group, the expression levels of CD86, TNF-α, IL-6, and iNOS were decreased, while the expression of M2-type macrophage markers CD206 and M2-type macrophage-related cytokines (IL-10, Arg-1) were increased compared to the LPS group. In comparison to the LPS + PMMA group, the LPS + ES-PMMA group demonstrated a down-regulation of CD86, TNF-α, IL-6, and iNOS expression levels, while increasing the expression levels of CD206, IL-10, and Arg-1. Western blotting results revealed a significant decrease in TLR4/GAPDH and p-NF-κB p65/NF-κB p65 in the LPS + ES group when compared to the LPS group. Additionally, the LPS + ES-PMMA group exhibited a decrease in TLR4/GAPDH and p-NF-κB p65/NF-κB p65 levels when compared to the LPS + PMMA group.

Conclusion: ES-PMMA bone cement is more effective than PMMA bone cement in down-regulating the expression of the TLR4/NF-κB signaling pathway. Additionally, it induces macrophages to polarize towards the M2 phenotype, making it a crucial player in anti-inflammatory immune regulation.

Keywords: Bone cement; Enoxaparin sodium; Inflammation; Macrophage.

Fig. 1

Related schematic diagram of in vitro experiment. a Enoxaparin sodium and PMMA bone cement. b Bone cement module with a length of 3 mm and a diameter of 4 mm. c Bone cement samples were examined with a C-arm X-ray machine. d Bone cement samples were surgically implanted

Fig. 2

SEM images of PMMA and ES-PMMA bone cement. a PMMA. There are many voids between the cement particles. b ES-PMMA. Enoxaparin sodium was filled on the surface and space of bone cement particles

Fig. 3

Expression and localization of CD86 and CD206 in bone cement surrounding tissues. The staining images were observed under a light microscope at ×200 magnification, red is the target protein, and the nucleus is blue. a, b are representative images of CD86 and CD206 in the two groups 7 days after surgery. c and d are the quantitative analysis of CD86 and CD206 in the tissue around bone cement (Mean ± SD. n = 6.* P ≤ 0.05; ** P ≤ 0.01; And ***P ≤ 0.001. Pictures taken 3 and 14 days after surgery are presented in Additional file 1)

Fig. 4

Expression of IL-6, IL-10, and TNF-α in the tissues surrounding bone cement. The staining images were observed under a light microscope at ×100 and ×400 magnification, and the brown-yellow color was the target protein. a, b were representative images of IL-6, IL-10, and TNF-α in the two groups 7 days after operation. c, d, and e are quantitative analyses of IL-6, IL-10, and TNF-α, respectively (Mean ± SD. n = 6. *P ≤ 0.05; **P ≤ 0.01; And ***P ≤ 0.001. Pictures taken 3 and 14 days after surgery are presented in Additional file 1)

Fig. 5

Screening of conditioned medium by CCK-8 assay: when the concentration of ES was 100AxaIU/ml and the two kinds of bone cement extracts were 25 mg/mL, the activity of RAW264.7 cells was not significantly affected (mean ± SD. n = 3. *P < 0.05 compared with control groups for each group)

Fig. 6

ES-PMMA bone cement induced polarization of macrophages to M2 phenotype. a Detection of CD86 and CD206 positive expression by flow cytometry. b, c Quantitative results of flow cytometry analysis.(Mean ± SD. n = 3. *P ≤ 0.05; **P ≤ 0.01; And ***P ≤ 0.001)

Fig. 7

ES-PMMA bone cement decreased the levels of pro-inflammatory factors and increased the levels of anti-inflammatory factors. a–e is the expression of TNF-α, IL-6,iNOS,IL-10 and Arg-1 in each group (Mean ± SD. n = 3. *P ≤ 0.05; **P ≤ 0.01; And ***P ≤ 0.001)

Fig. 8

ES-PMMA bone cement inhibited TLR4 expression and NF-κB phosphorylation compared with PMMA bone cement. a The protein expression levels of rats in each group were determined by Western blotting. b TLR4/GADPH expression levels in each group. c The expression level of p-NF-κB p65/ NF-κB p65 in each group (Mean ± SD. n = 3. *P ≤ 0.05; **P ≤ 0.01; And ***P ≤ 0.001)