Novel composite bone cement modulates inflammatory response in vitro.

1. To evaluate the anti-inflammatory properties of enoxaparin sodium polymethylmethacrylate bone cement within the indirect co-culture model comprising endothelial cells and macrophages. 2. To investigate the impacts of inflammatory factors IL-6 and IL-10 on macrophage M2 polarisation and endothelial cell apoptosis. An indirect co-culture system of endothelial cells and macrophages was established by utilizing 1 µg/mL of lipopolysaccharide (LPS) to trigger an inflammatory response model. The experiment was categorized into 4 groups: blank control group, LPS-indicated group, PMMA + LPS group, and ES-PMMA + LPS group. Flow cytometry was performed to ascertain the apoptosis rate of endothelial cells and macrophage polarisation trend in the co-culture system. Meanwhile, ELISA, Western blotting, and immunofluorescence were adopted to measure the expression levels of Interleukin-6(IL-6), Tumour Necrosis Factor-α(TNF-α), Intercellular Cell Adhesion Molecule (ICAM), and Interleukin-10(IL-10) in cells and supernatants. In the detection of two typical polarisation proteins, CD86 and CD206, it was observed that the expression level of the CD86 protein, which indicates M1 polarisation, was elevated in the LPS-induced group in comparison to the blank control group (**P < 0.01). The expression level was found to be down-regulated in the ES-PMMA + LPS group (*P < 0.05). In contrast, the expression level of CD206 protein, which indicates the trend of M2-polarisation, was observed to be down-regulated in the LPS-induced group compared to the blank control group (***P < 0.001). Conversely, this expression level was up-regulated in the ES-PMMA + LPS group in comparison to the LPS-induced group (**P < 0.01). The expression of IL-6, TNF-α, IL-10, and ICAM was investigated in cell culture supernatants using the Elisa assay. The results showed that the LPS-induced group had higher levels of IL-6, TNF-α, and ICAM compared to the blank control group (***P < 0.001), while the LPS-induced group had lower levels of IL-10 (***P < 0.001). Additionally, the ES-PMMA + LPS-induced group showed lower levels of the aforementioned cytokines (**P < 0.01 or *P < 0.05) and higher levels of IL-10 (*P < 0.05). Western Blot and immunofluorescence results revealed that the expression of IL-6, TNF-α, and ICAM was up-regulated (***P < 0.001) and IL-10 was down-regulated (***P < 0.001) in the LPS-induced group compared with the blank control group. Compared with the LPS-induced group and PMMA + LPS group, in the ES-PMMA + LPS group, the expression of all three proteins was down-regulated (*P < 0.05 or **P < 0.01), whereas the expression of the IL-10 protein was up-regulated (***P < 0.001). The inflammatory proteins IL-6, TNF-α, and ICAM were shown to have higher fluorescence intensity in the LPS-induced group compared to the blank control group (***P < 0.001), the intensity of IL-10 was observed to be diminished (***P < 0.001). In contrast, the fluorescence intensity of IL-6, TNF-α, and ICAM was reduced in the ES-PMMA + LPS group relative to the LPS-induced group (***P < 0.001), the intensity of IL-10 was enhanced (***P < 0.001). In terms of endothelial cell apoptosis rate detection, the rate of apoptosis considerably reduced in the ES-PMMA + LPS-induced group when compared to the LPS-induced group (***P < 0.001) and rose noticeably in the LPS-induced group when compared to the blank control group (***P < 0.001). (1) In the co-culture system, ES-PMMA bone cement fulfills anti-inflammatory functions by impeding the expression of inflammatory factor IL-6 and promoting IL-10. (2) ES-PMMA bone cement facilitates the M2 polarisation response of macrophages and declines endothelial cell apoptosis within a co-culture system. (3) ES-PMMA bone cement can modify the local inflammatory environment by modulating the expression of inflammatory factors, which is potentially valuable for the application of cement-related surgery.