体外冲击波联合自体细胞生长因子治疗骨不连的实验研究

Experimental Study on the Treatment of Bone Nonunion with Extracorporeal Shock Wave Combined with Autologous Growth Factor

目的:探讨体外冲击波(ESW)联合自体细胞生长因子治疗兔桡骨骨不连的效果及作用原理。方法:选用健康成年雄性新西兰大耳白兔40只,无菌条件下制作兔双侧桡骨骨不连模型,最后造模成功36只,利用随机数字表将实验动物分成4组,每组9只。其中A组为对照组,B组为单纯体外冲击波治疗组,C组为单纯自体细胞生长因子组,D组为体外冲击波联合自体细胞生长因子组。在无菌条件下将经兔耳缘静脉采血转移到15mL的无菌离心管中,在200g的条件下离心20min.小心将中上层移出并不带任何红细胞,放入15mL无菌的离心管中。采用ELISA法检测样本中各种生长因子的浓度。造模12周后对实验动物行体外冲击波及自体细胞生长因子注射治疗。分别在治疗前及治疗后第4,8,12及16周行X线检查,处死实验动物取活组织进行大体观察、骨痂钙水平测定、骨痂成骨细胞计数并进行统计学分析。结果:检测结果显示主要生长因子为VEGF和TGF-β含量分别达到(528.87±38.51)pg/mL和(162.73±21.08)pg/mL,为正常全血中含量的5倍左右。治疗后16周发现A组未愈合;B组骨不连部位开始愈合,但较C和D组薄,有少量骨痂,抗外力较弱;C组基本达到愈合,但较D组稍薄;D组达到骨性愈合,表现为骨不连部位平整光滑,颜色亮,质地硬。X线片观察治疗后16周发现,A组骨折间隙无明显改变;B组骨折间隙有改变,但较C和D两组较差。C和D两组骨不连间隙变化明显,其中D组变化较C组更为明显。A组与B,C,D组骨不连间隙4,8,12及16周差异有统计学意义(P<0.05),骨痂生成量4,8,12及16周差异有统计学意义(P<0.05)。B,C及D组骨钙水平4,8,12及16周差异均有统计学意义(P<0.05),骨痂成骨细胞计数4,8,12及16周差异有统计学意义(P<0.05)。结论:体外冲击波联合自体细胞生长因子对骨不连的治疗效果优于单纯体外冲击波和单纯自体细胞生长因子治疗,体外冲击波联合自体细胞生长因子为临床治疗骨不连提供了一种良好协同方法。

Objective：to investigate the effect and principle of extracorporeal shock wave（ESW）combined with autologous cell growth factor in the treatment of radial nonunion in rabbits.Methods：All 40 adult male New Zealand white rabbits were selected to make double radial nonunion model under aseptic condition,and 36 were successfully made.The experimental animals were divided into 4 groups（9 in each group）by random number table.Group A,B,C,D was the control group,single extracorporeal shock wave treatment group,simple autologous cytokine group,combined extracorporeal shock wave cytokine group respectively.Under aseptic condition,blood was transferred from rabbit ear margin vein to15 mL sterile centrifugation tube for 20 minutes under 200 g.Carefully remove the middle and upper layers without any red blood cells and place them in 15 mL sterile centrifuge tubes.The concentration of various growth factors in the samples was determined by ELISA.The experimental animals were treated with in vitro shock and autologous cytokine injection after 12 weeks.X-Ray examination of 4,8,12 and 16 weeks before and after treatment was carried out.The experimental animals were sacrificed to take biopsy for gross observation,osteocalcin calcium level determination,bone callus osteoblast cell count and statistical analysis.Results：The results showed that the main growth factors were VEGF and TGF-β,and their contents reached（528.87±38.51）pg/mL and（162.73±21.08）pg/mL,which were about 5 times of the normal whole blood content.At the 16 th week after treatment,group A did not heal,and group B bone nonunion began to heal,but was thinner than group C and D.There was a small amount of bone spasm and weak external resistance.Group C basically healed,but was slightly thinner than group D.In group D,bony union was achieved.The nonunion sites were smooth,bright and hard.X-ray observation showed that there was no significant change in fracture space in group A at 16 th weeks after treatment.There was a change in fracture space in group B,but it was worse in group C and D.The changes of the gaps between the two groups were significant in the C and D groups,and the change in the D group was more obvious than that in the C group.There was significant difference between group A and group B,C,D in the non-union gaps at 4 th,8 th,12 th and 16 th weeks（P〈0.05）,and there were significant differences between days 4,8,12 and 16 in osteophyte production（P〈0.05）.There were significant differences in the levels of bone calcium in groups B,C,and D at 4 th,8 th,12 th and 16 th weeks（P〈0.05）,and there were significant differences between days 4,8,12 and 16 in osteoclast osteoblast counts（P〈0.05）.Conclusion：The effect of extracorporeal shock wave combined with autologous cell growth factor on bone nonunion is superior to that of simple shock wave and autologous cell growth factor alone.Extracorporeal shock waves combined with autologous cell growth factor provide a good synergistic method for clinical treatment of nonunion.