Dicalcium Silicate Induced Proinflammatory Responses through TLR2-Mediated NF-κB and JNK Pathways in the Murine RAW 264.7 Macrophage Cell Line.

Shixiang Lai,Wenchao Zhong,Wei Cao,Shiman Cui,Qingbin Zhang,Mingyu Ma,Xingyang Li,Liangjiao Chen

doi:10.1155/2018/8167932

Abstract

Proinflammatory responses are important aspects of the immune response to biomaterials, which may cause peri-implantitis and implant shedding. The purpose of this study was to test the cytotoxicity and proinflammatory effects of dicalcium silicate particles on RAW 264.7 macrophages and to investigate the proinflammatory response mechanism induced by C2S and tricalcium phosphate (TCP). C2S and TCP particles were characterized using scanning electron microscopy (SEM), energy spectrum analysis (EDS) and X-ray diffraction (XRD). Cytotoxicity and apoptosis assays with C2S and TCP in the murine RAW 264.7 cell line were tested using the cell counting kit-8 (CCK-8) assay and flow cytometry (FCM). The detection results showed that C2S and TCP particles had no obvious toxicity in RAW 264.7 cells and did not cause obvious apoptosis, although they both caused an oxidative stress response by producing ROS when the concentrations were at 100 μg/mL. C2S particles are likely to induce a proinflammatory response by inducing high TLR2, TNF-α mRNA, TNF-α proinflammatory cytokine, p-IκB, and p-JNK1 + JNK2 + JNK3 expression levels. When we added siRNA-TLR2-1, a significant reduction was observed. These findings support the theory that C2S particles induce proinflammatory responses through the TLR2-mediated NF-κB and JNK pathways in the murine RAW 264.7 macrophage cell line.

Highlights

An important factor of biological material safety evaluation is to explore the toxicity effects of biological materials on cells and their proinflammatory effects
We previously reported that following coculture with the murine RAW 264.7 cell line, dicalcium silicate coating material and silicon, calcium, and other released ions showed no obvious cytotoxicity effects on RAW 264.7 cells; there was a potential proinflammatory response, and it increased the release of inflammatory factors [1]
Observed under scanning electron microscopy (SEM) (3000x), the C2S particles presented as circular particles, but most of the particles aggregated together as a block mass, while the tricalcium phosphates (TCP) hydrate under SEM had two different shapes: strip and grainy shapes (Figures 1(a) and 1(b))

Summary

Introduction

An important factor of biological material safety evaluation is to explore the toxicity effects of biological materials on cells and their proinflammatory effects. We previously reported that following coculture with the murine RAW 264.7 cell line, dicalcium silicate coating material and silicon, calcium, and other released ions showed no obvious cytotoxicity effects on RAW 264.7 cells; there was a potential proinflammatory response, and it increased the release of inflammatory factors [1]. Dicalcium silicate (Ca2SiO4) is an important material in the calcium-silica system, which is frequently identified as an important constituent in Portland cement [2, 3]. Previous studies have indicated that γ-Ca2SiO4 ceramic possesses good bioactivity, biocompatibility, and mechanical properties and that it might be a promising bone implant material [4, 5]. Γ-Ca2SiO4 ceramic might be suitable for a potential application in the biomedical field, preferentially as materials for bone repair [6]. Dicalcium silicate (C2S) and tricalcium phosphates (TCP) can both develop desirable physical properties as a result of undergoing hydration reactions [9], both of which were studied in this report

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