NF-κB受体激活因子配体抗体防治人工关节无菌性松动的实验研究

INHIBITION OF ASEPTIC LOOSENING BY RECEPTOR ACTIVATOR OF NUCLEAR FACTOR KAPPA B LIGAND ANTIBODY IN OSTEOLYSIS MODEL OF MOUSE

目的人工关节无菌性松动与破骨细胞激活后假体周围骨溶解有关,而NF-κB受体激活因子配体(receptor activator of NF-κB ligand,RANKL)/NF-κB受体激活因子(receptor activator of NF-κB,RANK)信号通路是激活破骨细胞的主要途径,通过建立小鼠气囊植骨模型模拟人工关节无菌性松动环境,观察RANKL抗体抑制炎性反应、减轻溶骨反应的作用。方法取8～10周龄雌性BALB/c小鼠60只(体重18～20 g),取其中20只小鼠颅骨制备骨片,作为气囊植骨供体;剩余40只小鼠随机分为阴性对照组(A组)、阳性对照组(B组)、RANKL抗体低剂量组(C组)和RANKL抗体高剂量组(D组),每组10只。于各组小鼠背部制备2 cm×2 cm大小的气囊后植入1片骨片;于植骨后1 d,A组气囊内注射0.5 mL PBS液;B、C、D组注射0.5 mL钛颗粒悬液。钛颗粒悬液注射前2 d,C、D组气囊内分别注射0.1 mL浓度为50μg/mL和500μg/mL的RANKL抗体,每天1次,连续2 d;A、B组注射0.1 mL PBS液。于植骨后14 d处死全部小鼠,取出植入颅骨骨片和周围囊壁组织,进行大体及组织学观察,并行颅骨骨溶解、骨胶原含量及破骨细胞含量分析。结果各组小鼠均存活至实验完成,切口愈合良好。大体观察见A、C、D组小鼠囊壁炎性反应较轻,偶见渗出和新生血管增生;B组炎性反应严重,见渗出以及新生血管增生。组织学观察见A、C、D组小鼠囊壁炎性细胞浸润及增厚不明显,骨胶原丢失量和破骨细胞含量少;B组大量炎性细胞浸润,囊壁增厚,骨胶原丢失量和破骨细胞含量多。B组炎性细胞数、囊壁厚度、骨胶原丢失量以及破骨细胞含量与A、C、D组比较,差异均有统计学意义(P<0.05)。结论 RANKL抗体可阻断小鼠气囊植骨模型的炎症信号传导通路,有效抑制磨损颗粒所致骨溶解。

Objective Aseptic loosening of prosthesis is associated with periprosthetical osteolysis caused by osteoclast activation.Receptor activator of nuclear factor kappa B（NF-κB） ligand（RANKL）/receptor activator of NF-κB（RANK） signal pathway is fundamental in osteoclast activation.To determine whether RANKL antibody can inhibit inflammatory osteolysis in a osteolysis model of mouse.Methods Sixty female BALB/c mice（aged 8-10 weeks,weighing 18-20 g） were selected.The skull bone piece was harvested from 20 mice as the donor of bone graft;the subcutaneous air pouches（2 cm × 2 cm） models were established on the back of the other 40 mice and the skull bone piece was inserted into the air pouches.The 40 mice were equally divided into groups A（negative control group）,B（positive control group）,C（low-dose RANKL antibody group）,and D（high-dose RANKL antibody group）.At 1 day after bone graft,0.5 mL PBS was injected into the pouch of group A,0.5 mL PBS containing titanium particle into groups B,C,and D.At 2 days before the titanium particle was injected,RANKL antibody（0.1 mL） were injected into the pouch of group C（50 μg/mL） and group D（500 μg/mL）,respectively every day for 2 days,and 0.1 mL PBS into groups A and B.At 14 days after bone implantation,the pouchmembranes containing implanted bone were harvested for gross observation and histological analyse.Results All mice survived to the end of experiment,and incisions healed well.The gross observation showed that inflammatory responses,exudation,and vascular proliferation were obvious in group B,and were inconspicuous in groups A,C,and D.The histological analysis showed that significantly more infiltration of inflammatory cells,more obvious bone resorption,more bone collagen loss,and more positive staining area were observed in group B than in groups A,C,and D.There were significant differences in inflammatory cell number,pouch membrane thickness,bone collagen loss,and osteoclast content between group B and groups A,C,and D（P 〈 0.05）.Conclusion RANKL antibody can directly block RANKL/RANK signal pathway,which is an efficient therapy to inhibit bone absorption associated with implant wearing particles.