负载柚皮苷AKE/GB30网箱在脊柱骨缺损模型修复中的作用及对BMPs-VEGF信号通路的影响

Role of AKE/GB30 cage loaded with naringin in the repair of spinal bone defect model and its effect on BMPs VEGF signaling pathway

中文关键词:  骨形成蛋白2  血管内皮生长因子  柚皮苷  脊柱  骨缺损

英文关键词:Bone morphogenetic protein 2  Vascular endothelial growth factor  Naringin  Spine  Bone defect

中文摘要:

  【摘要】 目的：观察负载柚皮苷的AKE/GB30网箱在脊柱骨缺损模型中的修复作用，并基于骨形成蛋白（BMPS）-血管内皮生长因子（VEGF）信号通路探讨该生物材料的作用机制。方法：用牙钻在30只新西兰雄兔L5与L6椎间制作一个7mm×5mm×4mm的缺损，建立脊柱骨缺损模型，制备AKE/GB30网箱。测试负载柚皮苷的AKE/GB30网箱生物力学性能，检测其体外释药行为。将造模成功的新西兰兔随机数字表法分为3组，即空白组、自体骨移植组、骨移植生物材料+柚皮苷联合组，除空白组外分别予以自体骨移植、负载柚皮苷的AKE/GB30网箱进行修复。术后6周、12周每组各取5只兔，微计算机断层扫描（Micro CT）检测骨修复情况[包括骨体积分数（BV/TV）、骨小梁厚度（Tb.Th）和骨小梁数目（Tb.N）]；实时荧光定量聚合酶链反应（RT-PCR）检测骨形成蛋白2（BMP2）、VEGF、Runt相关转录因子2（RUNX2）、碱性磷酸酶（ALP）、骨钙素（OCN）信使核糖核酸（mRNA）表达；免疫印迹法（WB）检测骨组织BMP2、VEGF、RUNX2、ALP、OCN蛋白表达。结果：AKE/GB30网箱制作成功，且其特性检测结果符合脊柱骨缺损修复要求；负载柚皮苷的AKE/GB30网箱最大抗压强度为28MPa，最大抗压力为15N，6周时其累积释药率达（98.15±1.47）%；各组术后12周时BV/TV、Tb.Th和Tb.N，骨组织BMP2、VEGF、RUNX2、ALP、OCN的mRNA与蛋白表达均高于术后6周时（P＜0.05），且自体骨移植组、骨移植生物材料+柚皮苷联合组上述指标均高于空白组（P＜0.05），自体骨移植组、骨移植生物材料+柚皮苷联合组上述指标差异均无统计学意义（P＞0.05）。结论：负载柚皮苷的AKE/GB30网箱在脊柱骨缺损模型修复的效果等同于自体骨移植，可能是通过促进BMP2、VEGF、RUNX2、ALP、OCN表达实现的。

英文摘要:

  【Abstract】 Objectives: To observe the repair effect of AKE/GB30 mesh cage loaded with naringin in spinal bone defect model, and explore the mechanism of this biomaterial based on bone morphogenetic proteins(BMPs)-vascular endothelial growth factor(VEGF) signal pathway. Methods: A dental drill was used to make a 7mm×5mm×4mm spinal bone defect model between L5 and L6 vertebrae in 30 New Zealand male rabbits, and AKE/GB30 mesh cages were prepared. The biomechanical properties of AKE/GB30 mesh cages loaded with naringin were tested, and their in vitro drug release behavior was measured. New Zealand rabbits that were successfully modeled were randomly divided into three groups using a randomly digital table method, namely, blank group, autologous bone graft group, and bone graft biomaterial+naringin combined group. Except the blank group, autologous bone transplantation and AKE/GB30 mesh cage loaded with naringin were used for repair. At 6 weeks and 12 weeks after surgery, 5 rabbits were taken from each group, and the bone repair status[including bone volume/tissue volume(BV/TV), bone trabecular thickness(Tb.Th) and bone trabecular number(Tb.N)] were detected by micro computed tomography(Micro CT). Real-time fluorescence quantitative polymerase chain reaction(RT-PCR) was used to detect the expressions of bone morphogenetic protein 2(BMP2), VEGF, Runt related transcription factor 2(RUNX2), alkaline phosphatase(ALP), and osteocalcin(OCN) messenger ribonucleic acid(mRNA). Immunoblotting assay(WB) was used to detect the expressions of BMP2, VEGF, RUNX2, ALP, and OCN proteins in bone tissues. Results: AKE/GB30 mesh cages had been successfully manufactured, and its characteristic testing results met the requirements for repairing spinal bone defects. The AKE/GB30 mesh cage loaded with naringin had a maximum compressive strength of 28MPa and a maximum resistance pressure of 15N. At 6 weeks, its cumulative release rate reached(98.15±1.47)%. After 12 weeks, the BV/TV, Tb.Th, and Tb.N, as well as the mRNA and protein expressions of BMP2, VEGF, RUNX2, ALP, and OCN in bone tissues of each group were higher than those after 6 weeks(P<0.05). The above indicators in the autologous bone graft group and the bone graft biomaterial+naringin combined group were higher than those in the blank group(P<0.05), and there were no significant differences in the above indicators between the autologous bone graft group and the bone graft biomaterial+naringin combined group(P>0.05). Conclusions: The effect of AKE/GB30 cage loaded with naringin in repairing spinal bone defect models is equivalent to that of autologous bone graft, which is presumed to achieve by promoting the expressions of BMP2, VEGF, RUNX2, ALP, and OCN.