骨替代材料引导骨组织再生过程中白细胞介素4对破骨细胞分化的调控

Regulation of interleukin-4 on osteoclast differentiation during bone regeneration guided by bone replacement materials

背景:研究显示,一定剂量的白细胞介素4干预可以得到适宜比例的M1/M2巨噬细胞谱,产生利于骨愈合的微环境,促进骨再生。目的:探讨在骨替代材料引导骨组织再生的过程中,白细胞介素4对NLRP3炎性小体及破骨细胞分化的影响。方法:选取6-8周龄雄性SD大鼠48只,在左侧颅骨制备直径5 mm的骨缺损并同期植入Bio-Oss骨替代材料,缝合骨膜。术后第3天,采用随机数字表法分为实验组与对照组,每组24只,实验组、对照组分别于骨缺损区局部注射白细胞介素4或PBS,1次/d,连续注射5 d。注射后1,2周,取颅骨样本,免疫组织化学染色检测裂解caspase-1和白细胞介素1β蛋白的表达,免疫荧光染色观察M1表面标志物(诱导型一氧化氮合酶)、NLRP3指标(裂解caspase-1)的表达,RT-qPCR检测caspase-1、白细胞介素1β及组织蛋白酶K基因的表达,抗酒石酸酸性磷酸酶染色观察破骨细胞分化及数量。注射后6,12周,取颅骨样本,行Micro-CT检测及苏木精-伊红染色。结果与结论:(1)免疫荧光染色显示,实验组注射后1,2周的诱导型一氧化氮合酶、裂解caspase-1双染细胞数量显著少于对照组(P<0.05);(2)免疫组织化学染色显示,实验组注射后1周的裂解caspase-1和白细胞介素1β蛋白的表达低于对照组(P<0.05);(3)RT-qPCR检测显示,实验组注射后1周的caspase-1 mRNA表达低于对照组(P<0.05),注射后1,2周的白细胞介素1β及组织蛋白酶K mRNA均低于对照组(P<0.05);(4)抗酒石酸酸性磷酸酶染色,实验组注射后1,2周的破骨细胞数量均明显少于对照组(P<0.05);(5)Micro-CT检测显示,实验组注射后6,12周的骨缺损部位骨体积分数、骨密度值均高于对照组(P<0.05);苏木精-伊红染色显示,注射后12周,实验组缺损中央形成多个骨化中心,散在成熟骨细胞及陷窝分布其中,可见材料周边成骨细胞大量排列成单层参与骨基质形成;(6)结果显示,白细胞介素4可能参与下调NLRP3炎性小体表达、抑制caspase-1的活化进而减少白细胞介素1β的分泌,减轻局部微环境炎性状态,同时抑制破骨细胞分化,发挥促进骨替代材料引导骨组织再生过程中新骨生成的作用。

BACKGROUND:Studies have shown that a certain dose of interleukin-4 intervention can yield the appropriate ratio of M1/M2 macrophage profile to generate a microenvironment conducive to bone healing and promoting bone regeneration.OBJECTIVE:To investigate the effect of interleukin-4 on NLRP3 inflammasome activation and osteoclast differentiation during bone tissue regeneration guided by bone replacement materials.METHODS:Forty-eight 6-8-week-old male SD rats were selected to establish a 5-mm diameter cranial bone defect model on the left and implanted with Bio-Oss bone replacement material at the same time,and the periosteum was sutured.Rat models were randomly divided into control and experimental groups(n=24)at postoperative 3 days.Interleukin-4 and PBS were injected locally into the cranial bone defect area for the experimental and control groups,once a day,for 5 consecutive days.SD rats were executed at 1 and 2 weeks after surgery and skull samples were taken.Immunohistochemical staining was performed to detect the expression of cleaved caspase-1 and interleukin-1βprotein.Immunofluorescence staining was performed to observe the expression of inducible nitric oxide synthase(M1 surface marker)and cleaved caspase-1(NLRP3 indicator).Real-time fluorescence quantitative PCR was performed to detect the expression of related inflammatory factors caspase-1,interleukin-1βand osteoclast factor histone K gene.The differentiation and number of osteoclasts were observed by anti-tartrate acid phosphatase staining.At 6 and 12 weeks after surgery,micro-CT and hematoxylin-eosin staining were performed to observe the osteogenesis of the skull.RESULTS AND CONCLUSION:(1)Immunofluorescence staining showed that the number of inducible nitric oxide synthase and cleaved caspase-1 double-stained cells in the experimental group was significantly lower than that in the control group at 1 and 2 weeks(P<0.05).(2)Immunohistochemical staining showed that the expression of cleaved caspase-1 and interleukin-1βin the experimental group was significantly lower than that in the control group at 1 week(P<0.05).(3)RT-qPCR suggested that the expression of caspase-1 mRNA in the experimental group was lower than that in the control group at 1 week(P<0.05).The expression intensity of interleukin-1βand osteoclast factor histone K mRNA was significantly lower in the experimental group than that in the control group at postoperative 1 and 2 weeks(P<0.05).(4)Anti-tartrate acid phosphatase staining showed that the number of osteoclasts was significantly lower in the experimental group at 1 and 2 weeks than that in the control group(P<0.05).(5)Micro-CT results showed that the bone volume fraction and bone mineral density in the bone defect area were significantly higher in the experimental group than those in the control group at 6 and 12 weeks after injection(P<0.05).Hematoxylin-eosin staining showed that 12 weeks after injection,multiple ossification centers were formed in the center of the defect in the experimental group,with scattered mature bone cells and lacunae,and a large number of osteoblasts around the material arranged into a single layer to participate in the formation of bone matrix.(6)The results suggest that interleukin-4 may be involved in downregulating NLRP3 inflammasome expression,inhibiting the activation of caspase-1 and thus reducing the secretion of interleukin-1β,reducing the inflammatory state of the local microenvironment,as well as inhibiting osteoclast differentiation and playing a role in promoting new bone production during bone tissue regeneration guided by bone replacement materials.