比较带蒂筋膜瓣包裹接种自体红骨髓的组织工程骨修复骨缺损时促血管化成骨与膜诱导成骨作用

COMPARISON OF EFFECT BETWEEN VASCULARIZATION OSTEOGENESIS AND MEMBRANE GUIDED OSTEOGENESIS IN BONE REPAIR BY TISSUE ENGINEERED BONE WITH PEDICLED FASCIAL FLAP PACKING AUTOLOGOUS RED BONE MARROW

目的比较带蒂筋膜瓣包裹组织工程骨在修复骨缺损时促血管化成骨与膜诱导成骨的作用,为临床修复骨缺损提供参考依据。方法采用兔自体红骨髓(autologous red bone marrow,ARBM)及含重组人BMP-2的骨诱导活性材料构建组织工程骨。将4～5月龄新西兰大白兔60只(体重2.0～2.5 kg),随机分为A、B、C 3组,A组16只,B、C组各22只。制备长1.5 cm的右尺骨长段骨-骨膜完全缺损模型,A组于骨缺损处植入组织工程骨;B、C组在骨缺损邻近处制备一筋膜瓣包裹组织工程骨后植入骨缺损,B组将筋膜瓣蒂部切断形成游离筋膜瓣,C组为带蒂筋膜瓣。术后4、8、12、16周,每组随机取4只实验动物,取骨缺损区组织进行大体观察、组织学及免疫组织化学染色观察;8、12、16周B、C组各另取2只实验动物行腋动脉墨汁灌注观察。结果大体观察示,术后4、8周A组有纤维结缔组织形成,B、C组筋膜瓣形成类似骨膜样纤维组织,其中B组有软骨样组织形成,C组有新生骨形成;12、16周A组骨痂形成少,B组新生骨较多,C组骨干形成。组织学观察示,术后4、8周A、B组新生血管和骨小梁极少,C组血管丰富且成熟骨小梁及软骨组织形成;12、16周A组新生血管及骨小梁仍较少,B组血管数量较多且成熟骨小梁显著增加,髓腔结构形成但闭阻;C组血管数量减少,成熟骨结构形成,骨髓腔再通。免疫组织化学染色示,C组各时间点CD105、CD34、Ⅷ因子表达均高于A、B组。骨形态计量分析显示,术后各时间点C组新生骨小梁体积明显大于A、B组(P<0.05);A、B组间除4周差异无统计学意义(P>0.05)外,其余时间点差异均有统计学意义(P<0.05)。血管图像分析显示术后各时间点C组骨修复区内血管再生面积比值明显大于A、B组(P<0.05)。墨汁灌注检查示,各时间点B组成骨区为稀疏的墨染区;C组成骨区墨染数量多且较密集,8周达高峰,之后逐渐减少。结论 带蒂筋膜瓣包裹复合自体ARBM的组织工程骨,早期以促血管化成骨作用占主导地位,后期血管化成骨作用逐渐消失,以膜诱导成骨作用为主。

Objective To compare the effect between vascularization osteogenesis and membrane guided osteogenesis in the bone repair by the tissue engineered bone with pedicled fascial flap packing autologous red bone marrow（ARBM）,so as to provide a reference for the bone defect repair in clinic.Methods The tissue engineered bone was constructed with ARBM and the osteoinductive absorbing recombinant human materials with recombinant human bone morphogenetic protein 2.Sixty New Zealand rabbits（aged 4-5 months,weighing 2.0-2.5 kg） were randomly divided into group A（n=16）,group B（n=22）,and group C（n=22）.The complete periosteum defect model of 1.5 cm in length was prepared in right ulnar bone,then the tissue engineered bone was implanted in the bone defect area in group A,the tissue engineered bone with free fascial flap in group B,and the tissue engineered bone with pedicled fascial flap in group C.At 4,8,12,and 16 weeks,the tissue of bone defect area was harvested from 4 rabbits of each group for the general,histological,and immunohistochemical staining observations;at 8,12,and 16 weeks,2 rabbits of groups B and C,respectively were selected to perform ink perfusion experiment by axillary artery.Results The general observation showed that the periosteum-like tissues formed in the fascial flap of groups B and C,chondroid tissues formed in group B,new bone formed in group C,and the fibrous and connective tissues in group A at 4 and 8 weeks;a few porosis was seen in group A,more new bone in group B,and bone stump formation in group C at 12 and 16 weeks.Histological observation showed that there were few new blood vessels and new bone trabeculae in groups A and B,while there were large amounts of new blood vessels and mature bone trabeculae in group C at 4 and 8 weeks.There were a few new blood vessels and new bone trabeculae in group A;more blood vessels,significantly increased mature trabeculae,and the medullary cavity formation in group B;and gradually decreased blood vessels,the mature bone structure formation,and the re-opened medullary cavity in group C at 12 and 16 weeks.The immunohistochemical staining observation showed that the levels of CD105,CD34,and factor VIII were higher in group C than in groups A and B at different time points.The bone morphometry analysis showed that the trabecular volume increased gradually with time in 3 groups after operation;the trabecular volume in group C was significantly more than those in groups A and B at different time points（P 〈 0.05）;and there was significant difference between groups A and B（P 〈 0.05） except the volume at 4 weeks（P 〉 0.05）.The vascular image analysis showed that the vascular regenerative area ratio in group C was significantly higher than those in groups A and B at different time points（P 〈 0.05）.The ink perfusion experiment showed that the osteogenic zone had sparse ink area with no obvious change in group B,while the osteogenic zone had more intensive ink area and reached the peak at 8 weeks,then decreased in group C.Conclusion The tissue engineered bone with pedicled fascial flap packing ARBM has the vascularization osteogenesis effect at early stage,but the effect disappears at late stage gradually when the membrane guided osteogenesis is main.