富血小板血浆与同种异体骨复合对兔感染性骨缺损处血管化作用及成骨效果的影响

Effect of platelet-rich plasma combined with allogeneic bone on vascularization and osteogenesis of infected bone defect in rabbits

目的探讨富血小板血浆(PRP)与同种异体骨(AB)复合对兔感染性骨缺损处血管化作用及成骨效果的影响。方法选择健康SPF级新西兰大白兔140只,兔龄12~16周,体质量2.01~2.10 kg。其中20只动物用于制备PRP,通过低速离心法制备PRP。构建新西兰大白兔感染性骨缺损模型;动物实验分为4组及处理,即空白对照组(Control组,不做处理)、植入PRP组(PRP组,植入PRP)、植入AB组(AB组,植入AB)、植入PRP复合AB组(PRP+AB组,植入PRP和AB),每组30只。分别在1周、4周、12周时行X射线检查、Micro CT检查,并处死动物,采用酶联免疫吸附分析实验检测动物血清中C-反应蛋白(CRP)的水平;采用聚合酶链式反应(PCR)检测骨缺损区域中碱性磷酸酶(ALP)、骨钙素(OCN)、血管内皮生长因子(VEGF)和血管生成素1(ANGPT1)的mRNA的表达,免疫组化检测ALP和OCN的表达,免疫荧光检测ANGPT1、VEGF的表达。结果Control组样本在1、4、12周时均存在明显的不同程度的骨缺损,并且骨缺损无愈合、无好转的趋势。PRP组、AB组、PRP+AB组大白兔的骨缺损区域出现不同程度的愈合,植入材料与骨结合良好、致密,未出现明显坏死骨;尤以PRP+AB组骨缺损修复最为明显。X射线评分与同时点Control组(1、4、12周3.07分±0.15分、4.21分±0.28分、8.03分±0.23分)相比,PRP组、AB组、PRP+AB组骨缺损区域X射线评分(PRP组3.72分±0.21分、5.14分±0.37分、9.27分±0.22分;AB组4.65分±0.26分、6.49分±0.43分、10.34分±0.29分;PRP+AB组5.31分±0.35分、7.25分±0.51分、11.19分±0.64分)明显升高,且PRP+AB组X射线评分更高;差异均有统计学意义(均P<0.05)。与同时点Control组(1、4、12周0.01±0.01、0.02±0.02、0.05±0.03)相比,PRP组、AB组、PRP+AB组骨缺损区域BV/TV(PRP组1、4、12周BV/TV 0.02±0.02、0.06±0.03、0.09±0.04;AB组1、4、12周0.02±0.01、0.07±0.04、0.16±0.03;PRP+AB组1、4、12周0.04±0.03、0.13±0.05、0.24±0.07)明显升高,且PRP+AB组骨缺损区域BV/TV更高;差异均有统计学意义(均P<0.05)。病理组织学评分有同样的结果。与同时点Control组(1、4、12周18.29 mg/L±0.44 mg/L、15.04 mg/L±0.48 mg/L、13.91 mg/L±0.53 mg/L)相比,PRP组、AB组、PRP+AB组大白兔血清中CRP含量(PRP组1、4、12周17.04 mg/L±0.37 mg/L、12.32 mg/L±0.52 mg/L、10.22 mg/L±0.47 mg/L;AB组1、4、12周16.25 mg/L±0.31 mg/L、10.21 mg/L±0.36 mg/L、8.24 mg/L±0.35 mg/L;PRP+AB组1、4、12周15.41 mg/L±0.28 mg/L、8.91 mg/L±0.49 mg/L、5.16 mg/L±0.38 mg/L)明显降低,ALP、OCN、ANGPT1、VEGF mRNA和其蛋白表达水平明显升高,且PRP+AB组大白兔血清中CRP含量降低更显著,ALP、OCN、ANGPT1、VEGF mRNA和其蛋白表达水平升高更明显;差异均有统计学意义(均P<0.05)。结论PRP复合AB能明显促进兔感染性骨缺损区域中ALP和OCN、ANGPT1和VEGF的表达,推动骨缺损处的成骨和成血管效应。

Objective To investigate the effect of platelet-rich plasma(PRP)combined with allogeneic bone(AB)on vascularization and osteogenesis of infectious bone defects in rabbits.Methods Atotal of 140 healthy SPFNew Zealand white rabbits were sacrificed,which aged 12-16 weeks with body mass of 2.01-2.10 kg.Among them,20 were used to prepare PRP,and PRP was prepared by low-speed centrifugation.The New Zealand white rabbitmodel of infectious bone defect was constructed,all of themodel rabbits were divided into 4 groups:blank control group(Control group,non-treatment),implanted PRP group(PRP group),implanted AB group(AB group)and implanted PRP combined with AB group(PRP+AB group),with 30 rabbits in each group.The X-ray examination and Micro CT examination were performed at 1st week,4th week and 12th week,respectively,and rabbits were sacrificed.The level of C-reactive protein(CRP)in serum was detected by enzyme-linked immunosorbent assay,themRNAexpressions of alkaline phosphatase(ALP),osteocalcin(OCN),vascular endothelial growth factor(VEGF)and angiopoietin 1(ANGPT1)in bone defect area were detected by polymerase chain reaction(PCR).The expressions of ALP and OCN were detected by immunohistochemistry,and expressions of ANGPT1 and VEGF were detected by immunofluorescence.Results There were obvious different degrees of bone defects in Control group at 1st week,4th week and 12th week,and bone defects were not healed and there was no improvement trend.In PRP group,AB group and PRP+AB group,the bone defect area healed to varying degrees,the implant material was well combined with bone,and there was no obvious necrotic bone,especially in PRP+AB group,the bone defect repair was the most obvious.Compared with X-ray scores of Control group at the same time point(3.07 scores±0.15 scores,4.21 scores±0.28 scores,8.03 scores±0.23 scores at 1st week,4th week and 12th week),the bone defect areas of PRP group(3.72 scores±0.21 scores,5.14 scores±0.37 scores,9.27 scores±0.22 scores at 1st week,4th week and 12th week),AB group(4.65 scores±0.26 scores,6.49 scores±0.43 scores,10.34 scores±0.29 scores at 1st week,4th week and 12th week)and PRP+AB group(5.31 scores±0.35 scores,7.25 scores±0.51 scores,11.19 scores±0.64 scores at 1st week,4th week and 12th week)were significantly in creased,the X-ray score of PRP+AB group was higher,and the differences were statistically significant(P<0.05).Compared with BV/TV of control group at the same time point(0.01±0.01,0.02±0.02,0.05±0.03 at 1st week,4th week and 12th week),the bone defect area of PRP group(0.02±0.02,0.06±0.03,0.09±0.04 at 1st week,4th week and 12th week),AB group(0.02±0.01,0.07±0.04,0.16±0.03 at 1st week,4th week and 12th week)and PRP+AB group(0.04±0.03,0.13±0.05,0.24±0.07 at 1st week,4th week and 12th week)were significantly increased,the BV/TV of PRP+AB group was higher,and the differences were statistically significant(P<0.05).The histopathological score showed the same results,compared with CRP content in serum of Control group at the same time point(18.29 mg/L±0.44 mg/L,15.04 mg/L±0.48 mg/L,13.91 mg/L±0.53 mg/L at 1st week,4th week and 12th week),PRP group(17.04 mg/L±0.37 mg/L,12.32 mg/L±0.52 mg/L,10.22 mg/L±0.47 mg/L at 1st week,4th week and 12th week),AB group(16.25 mg/L±0.31 mg/L,10.21 mg/L±0.36 mg/L,8.24 mg/L±0.35 mg/L at 1st week,4th week and 12th week)and PRP+AB group(15.41 mg/L±0.28 mg/L,8.91 mg/L±0.49 mg/L,5.16 mg/L±0.38 mg/L at 1st week,4th week and 12th week)decreased significantly.The mRNA and protein expression levels of ALP,OCN,ANGPT1 and VEGF were significantly increased,and serum CRP content in PRP+AB group decreased more significantly,the mRNA and protein expression levels of ALP,OCN,ANGPT1 and VEGF were significantly increased,and the differences were statistically significant(P<0.05).Conclusion It is demonstrated that PRP combined with AB could significantly promote expression of ALP,OCN,ANGPT1 and VEGF in infected bone defect area of rabbits,and promote osteogenic and angiogenic effects at bone defect.