依诺肝素钠骨水泥对其周围诱导膜血管化及生长因子表达的研究

Comparative study of enoxaparin sodium bone cement and ordinary bone cement in inducing intramembrane angiogenesis

目的探讨分析聚甲基丙烯酸甲脂(PMMA)骨水泥和最适配比的依诺肝素钠骨水泥(ES-PMMA)在兔胫骨缺损周围应用产生诱导膜组织内部血管化程度以及与血管生成相关生长因子表达水平。方法挑选24只新西兰兔随机分为4组,每组6只。A组不处理,B组制作骨缺损模型,骨缺损处不植入任何材料,C组制作骨缺损模型,骨缺损处植入PMMA骨水泥,D组制作骨缺损模型,骨缺损处植入ES-PMMA骨水泥。在术后4、6、8周3个时间点分别取出C、D组骨缺损处诱导膜组织,B组取胫骨缺损处临近结缔组织,A组取与胫骨最近的正常组织。苏木精-伊红染色法(HE)观察样本血管密集程度,免疫组织化学染色法(IHC)和蛋白质印迹法(Western blot)检测血小板-内皮细胞黏附因子(CD31)、成纤维细胞生长因子(FGF)和血管内皮生长因子(VEGF)3种生长因子表达水平。收集显微镜下观察的图像和数据。数据进行单因素方差分析比较。结果HE染色结果显示,4周时D组样本血管数量最多,且明显高于其余3组;6周时D组与C组血管密集度明显高于A组和B组;8周时D组样本血管数量最多,且明显高于其余3组。D组与C组比较在8周时微血管数量下降幅度不明显;IHC结果显示,4周时D组VEGF、FGF、CD313种蛋白表达程度均高于其余3组;6周时D组与C组表达差异无统计学意义(P>0.05),且明显高于A、B两组;8周时D组3种蛋白表达程度高于其余3组;Western blot结果显示,4周时D组VEGF(2.975±0.101)、FGF(2.461±0.147)、CD31(2.461±0.147)相对含量与B组(1.350±0.033、1.423±0.137、1.350±0.136)、C组(1.831±0.063、1.873±0.265、1.921±0.177)比较差异均有统计学意义(F=418.200、58.149、24.788,P<0.05);6周时D组VEGF(2.562±0.217)、FGF(2.767±0.274)、CD31(2.432±0.122)相对含量与C组(2.459±0.236、2.719±0.283、2.399±0.216)差异无统计学意义(F=0.331、0.001、0.045,P>0.05);8周时D组VEGF(2.780±0.165)、FGF(1.841±0.144)、CD31(2.776±0.141)相对含量与B组(1.044±0.022、0.956±0.034、1.044±0.021)、C组(1.044±0.021、1.431±0.044、1.697±0.176)比较差异均有统计学意义(F=139.478、125.483、78.046,P<0.05)。结论通过ES-PMMA组和PMMA组对比,我们发现ES-PMMA骨水泥在诱导膜早期阶段有促进诱导膜内血管形成的作用,在晚期阶段有维持诱导膜血管化水平以及相关生长因子表达的作用;证明ES-PMMA骨水泥比传统骨水泥在诱导膜形成早期和晚期阶段均有很大的优势。

Objective To analyze and compare the degree of vascularization in the induced membrane tissue and the expression of angiogenation-related growth factors around tibia defects in rabbits with polymethyl methacrylate(PMMA)bone cement and enoxaparin sodium bone cement(ES-PMMA).Methods Twenty-four New Zealand rabbits were randomly divided into 4 groups,with 6 rabbits in each group.The bone defect model was made in group B without any material implanted in the bone defect,the bone defect model was made in group C with PMMA bone cement implanted in the bone defect,and the bone defect model was made in group D with ES-PMMA bone cement implanted in the bone defect.At 4,6 and 8 weeks after operation,the induced membrane tissue was removed from the bone defect in group C and D,the connective tissue near the tibia defect in group B,and the normal tissue closest to the tibia in group A.Hematoxylin and eosin(HE)was used to observe the density of blood vessels.Immunohistochemical staining(IHC)and Western blotting were used to detect the expression levels of three growth factors,namely platelet-endothelial cell adhesion factor(CD31),fibroblast growth factor(FGF)and vascular endothelial growth factor(VEGF).Researchers collect microscopic images and data.Data were compared by one-way analysis of variance.Results HE staining results showed that the number of blood vessels in group D was the largest at 4 weeks,which was significantly higher than that in the other three groups;At 6 weeks,there was no difference in vascular intensity between groups D and C,but it was higher than that between groups A and B.At 8 weeks,the number of blood vessels in group D was the largest and significantly higher than that in the other three groups.Compared with group C,the number of microvessels in group D did not decrease significantly at 8 weeks.IHC results showed that the expressions of VEGF,FGF and CD31 in group D were higher than those in the other three groups at 4 weeks.At 6 weeks,there was no difference in the expression between groups D and C,and it was significantly higher than that between groups A and B.At 8 weeks,the expression of three proteins in group D was higher than that in the other three groups.Western blotting result:at 4 weeks,the relative contents of VEGF(2.975±0.101),FGF(2.461±0.147)and CD31(2.461±0.147)in group D were significantly higher than those in group B(1.350±0.033,1.423±0.137,1.350±0.136,P<0.05)and group C(1.831±0.063,1.873±0.265,1.921±0.177,F=418.200,58.149,24.788,P<0.05);At 6 W,the relative contents of VEGF(2.562±0.217),FGF(2.767±0.274)and CD31(2.432±0.122)in group D were not significantly different from those in group C(2.459±0.236,2.719±0.283,2.399±0.216,F=0.331,0.001,0.045,P>0.05);At 8 W,the relative contents of VEGF(2.780±0.165),FGF(1.841±0.144)and CD31(2.776±0.141)in group D were significantly higher than those in group B(1.044±0.022,0.956±0.034,1.044±0.021)and group C(1.044±0.021,1.431±0.044,1.697±0.176,F=139.478,125.483,78.046,P<0.05).Conclusion By comparing the ES-PMMA group with the PMMA group,we found that enoxaparin sodium promoted the formation of intramemmal blood vessels in the early stage of induction membrane,and maintained the level of induction membrane vascularization and the expression of related growth factors in the late stage.It was proved that ES-PMMA bone cement had great advantages over traditional bone cement in inducing membrane formation at both early and late stages.