复合血管生成素1基因及富血小板血浆的组织工程骨修复桡骨骨缺损

REPAIR OF RADIAL SEGMENTAL BONE DEFECTS BY COMBINED ANGIOPOIETIN 1 GENE TRANSFECTED BONE MARROW MESENCHYMAL STEM CELLS AND PLATELET-RICH PLASMA TISSUE ENGINEERED BONE IN RABBITS

目的探讨以β磷酸三钙(β tricalcium phosphate,β-TCP)为支架,复合富血小板血浆(platelet-rich plasma,PRP)和转染血管生成素1(angiopoietin1,Ang-1)基因的组织工程骨修复节段性骨缺损的能力。方法取兔股骨骨髓分离培养BMSCs,将Ang-1基因通过慢病毒转染至第2代BMSCs,转染后48h接种于β-TCP上培养,然后复合0.5mLPRP,制备复合PRP的组织工程骨。取20只3月龄新西兰大白兔制备双侧桡骨15mm骨缺损模型,采用自身对照,分别于每只动物左侧和右侧骨缺损处植入未转染Ang-1基因的组织工程骨(对照组)及转染Ang-1基因的组织工程骨(实验组)。术后行大体观察,于2、4、8、12周行X线片观察,取材行HE染色、免疫组织化学染色及生物力学检测。结果细胞转染48h后转染效率>90%,通过RT-PCR检测到转染细胞内Ang-1基因表达显著增高。术后所有动物患肢活动良好。X线片观察示实验组术后4周骨缺损处已有骨痂形成,8周有部分骨性愈合,12周骨缺损处完全骨性愈合;对照组至术后12周时无骨性愈合。HE染色示实验组术后8周有毛细血管出现,12周大量毛细血管生长于新生骨中;对照组8周未见新生毛细血管,12周见少量毛细血管。术后8、12周实验组微血管密度分别为(50.1±7.8)个/mm2和(66.1±3.5)个/mm2,对照组分别为0和(30.3±7.2)个/mm2;术后12周两组间比较差异有统计学意义(Z=—2.107,P=0.031)。免疫组织化学染色观察示术后8周实验组仍有Ang-1分泌。生物力学测试结果显示术后12周实验组三点弯曲试验和压缩试验的最大负荷均显著高于对照组(P<0.05)。结论复合PRP和转染Ang-1基因的组织工程骨修复兔桡骨节段性骨缺损后,组织工程骨内可形成新生血管,促进骨愈合。

Objective To investigate the osteogenesis effects of angiopoietin 1（Ang-1） gene transfected bone marrow mesenchymal stem cells（BMSCs） seeded on β tricalcium phosphate（β-TCP） scaffolds（tissue engineered bone） with platelet-rich plasma（PRP）.Methods BMSCs were isolated from bone marrow tissue of rabbits.The Ang-1 gene was transfected into the BMSCs at passage 2 by lentivector,which were seeded on β-TCP scaffolds with PRP（0.5 mL） after 48 hours of transfection.Bilateral radial segmental bone defects（15 mm in length） were created in 20 3-month-old New Zealand rabbits.Then the tissue engineered bone with the Ang-1 gene transfected BMSCs（experimental group） and untransfected BMSCs（control group） were implanted into the defects in the right and left radius,respectively.X-ray,histology,immunohistochemistry,and biomechanics observations were done at 2,4,8,and 12 weeks after operation.Results In vitro,the transfected rate was over 90% and RT-PCR showed that the Ang-1 expression were significantly increased after transfection.The X-ray films showed that some callus formed at 4 weeks,partial bony union was observed at 8 weeks,and complete union at 12 weeks in experimental group;and bone union was not observed at 12 weeks in control group.HE staining showed that capillary appeared at 8 weeks and more capillaries were observed in new bone at 12 weeks in experimental group;only a few capillaries were observed at 12 weeks in control group.At 8 and 12 weeks,the microvascular density were（50.1 ± 7.8） /mm2 and（66.1 ± 3.5） /mm2 in experimental group and were 0 and（30.3 ± 7.2）/mm2 in control group,showing significant differences between 2 groups at 12 weeks（Z= —2.107,P=0.031）.Immunohistochemistry examination showed that the positive cells can be found at 8 weeks in experimental group.And the biomechanical analysis showed that maximum loads of experimental group were significantly higher than those of control group in three-point bending test and compression test at 12 weeks（P 〈 0.05）.Conclusion The tissue engineered bone with PRP and Ang-1 can increase the osteogenic properties by enhancing capillary regeneration,thus it can be used to repair radial segmental bone defects of rabbit