Liangjiao Chen, Ping Zhu, Ruoyu Liu, Yanli Zhang, Ting Sun, Yanjun Liu, Longquan Shao
Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China.
J Mech Behav Biomed Mater. 2015 Apr;44:10-22. doi: 10.1016/j.jmbbm.2014.12.012. Epub 2014 Dec 26.
Due to their biocompatibility and bioactivity, dicalcium silicate (C2S) and hydroxyapatite (HA) are used as coating materials for prosthetic orthopedic and dental implants or as bone substitute materials to fill bone defects. However, prostheses and bone substitutes can release particles that trigger an immune response in the recipient. The immunological effects of C2S particles have not yet been studied.
The aim of this study was to determine the cytotoxic effects of C2S particles on primary human monocytes, a human monocyte cell line (THP-1) and an osteoblast-like cell line (MG-63). The proinflammatory effects of C2S particles on THP-1 were also detected. Moreover, the osteogenic effects of C2S and HA on MG-63 cells were investigated.
Characterization of C2S and HA was performed using scanning electron microscopy (SEM), energy dispersive analysis (EDS), X-ray diffraction (XRD), Brunner-Emmett-Teller (BET) measurements and laser diffraction. The cytotoxic effect of C2S on primary human monocytes as well as THP-1 and MG-63 cells was measured using Trypan blue assays, Cell Counting Kit-8 (CCK-8) assays and flow cytometry to detect apoptosis. THP-1 human monocytes with or without lipopolysaccharide (LPS) stimulation were exposed to C2S and HA for 6 and 24h. Thereafter, the mRNA expression and protein concentrations of MMP-2, MMP-9, TIMP-2, TIMP-1 and TNF-α were evaluated using real-time PCR and ELISA, respectively. RANKL and OPG mRNA expression levels in MG-63 cells were examined using real-time PCR.
No significant cytotoxicity was recorded when cells were directly cultured with C2S/HA particles. After THP-1 cells were cultured with C2S/HA for 24h, MMP-2, MMP-9 and TNF-α expression increased, whereas TIMP-2 and TIMP-1 expression decreased. Compared with HA, C2S slightly increased MMP-9 expression and slightly decreased TIMP-1 expression. The MMP: TIMP ratio increased in the C2S and HA groups; however, HA significantly increased the MMP-9: TIMP-1 ratio compared with C2S. Compared with HA, C2S caused less TNF-α production. C2S/HA did not modify the expression of proinflammatory mediators in LPS-stimulated cells. Furthermore, C2S/HA significantly increased OPG expression and slightly increased RANKL expression in MG-63 cells. C2S and HA decreased the RANKL: OPG ratio.
Our in vitro data suggest that C2S is relatively safe when directly cultured with cells. In addition, C2S may exert proinflammatory effects; however, compared with HA, C2S had fewer proinflammatory effects on THP-1. C2S and HA did not alter the LPS-induced production of proinflammatory mediators and had similar osteogenic effects on MG-63 cells.
由于具有生物相容性和生物活性，硅酸二钙（C2S）和羟基磷灰石（HA）被用作骨科和牙科假体植入物的涂层材料，或作为填充骨缺损的骨替代材料。然而，假体和骨替代物会释放颗粒，从而在受体中引发免疫反应。C2S颗粒的免疫效应尚未得到研究。
本研究旨在确定C2S颗粒对原代人单核细胞、人单核细胞系（THP-1）和成骨样细胞系（MG-63）的细胞毒性作用。还检测了C2S颗粒对THP-1的促炎作用。此外，研究了C2S和HA对MG-63细胞的成骨作用。
使用扫描电子显微镜（SEM）、能量色散分析（EDS）、X射线衍射（XRD）、布鲁诺-埃米特-泰勒（BET）测量和激光衍射对C2S和HA进行表征。使用台盼蓝测定法、细胞计数试剂盒-8（CCK-8）测定法和流式细胞术检测凋亡，以测量C2S对原代人单核细胞以及THP-1和MG-63细胞的细胞毒性作用。将有或无脂多糖（LPS）刺激的THP-1人单核细胞暴露于C2S和HA 6小时和24小时。此后，分别使用实时PCR和ELISA评估MMP-2、MMP-9、TIMP-2、TIMP-1和TNF-α的mRNA表达和蛋白浓度。使用实时PCR检测MG-63细胞中RANKL和OPG的mRNA表达水平。
当细胞与C2S/HA颗粒直接培养时，未记录到明显的细胞毒性。THP-1细胞与C2S/HA培养24小时后，MMP-2、MMP-9和TNF-α表达增加，而TIMP-2和TIMP-1表达降低。与HA相比，C2S使MMP-9表达略有增加，TIMP-1表达略有降低。C2S和HA组的MMP：TIMP比值增加；然而，与C2S相比，HA显著增加了MMP-9：TIMP-1比值。与HA相比，C2S引起的TNF-α产生较少。C2S/HA未改变LPS刺激细胞中促炎介质的表达。此外，C2S/HA显著增加了MG-63细胞中OPG的表达，并使RANKL表达略有增加。C2S和HA降低了RANKL：OPG比值。
我们的体外数据表明，C2S与细胞直接培养时相对安全。此外，C2S可能发挥促炎作用；然而，与HA相比，C2S对THP-1的促炎作用较小。C2S和HA未改变LPS诱导的促炎介质产生，并且对MG-63细胞具有相似的成骨作用。
