人工关节磨损颗粒激活骨-假体界面巨噬细胞生物学机制探讨

Biological mechanism of macrophage activation by wear particles at the bone implant interface

目的观察无菌性松动人工关节骨假体界面磨损颗粒刺激下巨噬细胞（ＭΦ）膜Ｃａ２＋通道的变化，分析胞内游离钙离子浓度（［Ｃａ２＋］ｉ）变化在其激活机制中的作用。方法体外培养获得滑膜细胞系；以ＣＤ６８单抗免疫组化（ＳＡＢＣ法）等方法确定其中巨噬细胞样细胞（ＭＣｓ）形态及其吞噬行为发生时间；滴加１５ｍｇ／ｍｌ（Ｗ／Ｖ）Ｔｉ合金、ＣｏＣｒ合金或超高分子聚乙烯（ＵＨＭＷＰＥ）颗粒悬液等，动态监测ＭＣｓ内［Ｃａ２＋］ｉ变化。结果ＭＣｓ吞噬行为发生于接触颗粒６小时以后，而滴加三种颗粒悬液后１小时内，经不同潜伏期ＭＣｓ内［Ｃａ２＋］ｉ已出现１～６次脉冲样升高，其次数和幅度等无显著性差异（Ｐ＞００５）；ＵＨＭＷＰＥ颗粒组潜伏期较长（Ｐ＜００５），但脉冲间隔时间小于ＣｏＣｒ合金颗粒组（Ｐ＜００５）；经消炎痛孵育ＭＣｓ内仍出现［Ｃａ２＋］ｉ脉冲（Ｐ＞００５），而经尼莫地平孵育的ＭＣｓ无反应。结论骨假体界面ＭΦ的激活可能发生于吞噬磨损颗粒之前。

Objective To observe the response of membrane Ca 2+ channel of macrophage like cells (MCs) to wear particles and analyze the cytoplasmic [Ca 2+ ]i change in macrophage activation at the bone implant interface. Methods The synoviocyte system of normal hip joint was established in vitro.Immunohistochemical technique (SABC) with CD68 Mab was used to differentiate MCs and fibroblast like cells (FCs) in the system,and the time when MCs begin to perform phagocytosis was determined by SEM. 1 5 mg/ml (W/V) Ti alloy,Co Cr alloy or UHMWPE particles suspension was added into the system to monitor the [Ca 2+ ]i change in the MCs by confocal laser scanning microscope(CLSM). Results Phagocytosis of MCs happened after 6 hours, but within 1 hour the Ca 2+ channel of their cell membrane opened for 1～6 times and caused rapid and transient pulses of [Ca 2+ ]i in cytoplasm after different period of incubation. There was no significant difference between times or range of the [Ca 2+ ]i pulse( P >0 05).The group of UHMWPE particles had the longest incubation period( P <0 05),but the interval of pulses was shorter than that in the group of Co Cr alloy particles( P <0 05).[Ca 2+ ]i pulses still appeared when MCs were incubated by indomethecin ( P >0 05),but the phenomenon could be blocked by nimodipin. Conclusions Activation of macrophages at the bone implantinterface could happen before they phagocytose wear particles.The primary switch on mechanism of osteolysis mediated by cytokines at the bone implant interface may be the openning of membrane Ca 2+ channel and pulse like Ca 2+ influx of macrophages when stimulated by wear particles.