同种异体脂肪源性间充质干细胞复合β-磷酸三钙支架修复兔桡骨大段骨缺损

Use of rADSCs/β-TCP bone tissue engineering complex for repairing of large segmental radial bone defect in rabbits

目的探讨以β-磷酸三钙(β-tricalcium phosphate,β-TCP)陶瓷为支架材料复合兔脂肪源性间充质干细胞(rabbit adipose-derived mesenchymal stem cells,rADSCs)构建组织工程骨修复骨缺损的可行性。方法将体外培养的rADSCs接种于β-TCP支架上,构建rADSCs/β-TCP骨组织工程复合体,并进行体外培养。将24只新西兰大白兔在双侧桡骨中下段造成2cm长度的节段性骨缺损模型,随机平均分为3组,A组:空白对照组,不植入任何材料;B组:单β-TCP人工骨对照组;C组:rADSCs/β-TCP复合体实验组。在手术后2周、4周、6周和8周时进行X线检查,然后每组各选2只兔处死后取出标本,对标本进行大体观察以及常规HE染色,光镜下观察骨修复情况,并观察是否发生免疫排斥反应。结果 rADSCs在适当的诱导条件下具有成骨细胞分化潜能,复合β-TCP后对其生长分化无影响。X线检查结果发现,A组有稍多的骨组织形成,未见髓腔及正常骨影像学表现;B组虽然骨连接性基本恢复,但骨髓腔尚未完全再通;C组骨连接性完全恢复,骨缺损基本修复,骨髓腔再通。术后4周,C组可观察到骨缺损周围开始形成新生骨,并随着时间的延长新生骨量逐渐增多。术后6周,材料逐渐降解,材料孔隙内有新生骨长入。术后8周,C组材料降解明显,材料孔隙基本消失,髓腔已基本再通;B组部分骨髓腔再通,而A组未发现骨髓腔再通。新生骨组织面积比较,各时间点C组的面积最大,且其差异有统计学意义。组织学检测未见免疫排斥现象。结论 rADSCs在适当的诱导条件下具有成骨细胞分化潜能。ADSCs有望成为理想的修复骨缺损、促进骨折愈合的组织工程骨所需要的种子细胞,为大段骨缺损组织工程的修复提供可行的技术方案,为临床治疗大段骨缺损提供新的方法。

Objective To investigate the possibility of repairing large segmental radial bone defect in rabbit by using rabbit adipose-derived mesenchymal stem cells （rADSCs）/β-tricalcium phosphate （β-TCP） composite bone tissue engineering.Methods In vitro cultured rADSCs was inoculated in β-TCP,rADSCs/β-TCP bone tissue engineering complex was co-culture in vitro.All rabbits with 2 cm bone defect of bilateral radius on middle and lower level were divided into three groups：control group with no implant （group A）,control group with β-TCP （group B） implant,and rADSCs/β-TCP composite bone tissue as experimental group （group C）.Radiographic examination was done 2,4,6 and 8 weeks after surgery.From each group,2 rabbits were sacrificed,and specimens were harvested for gross observation and HE staining at the same time period mentioned above.The stained samples were observed for bone repair and allograft rejection under light microscopy （LM）.Results The rADSCs were pluripotent with the potential to differentiate into osteoblast.β-TCP scaffold had good biocompatibility without cytotoxicity.The X-ray examination result on the 8th week showed that group A had a small amount of new bones growth around the defect.Theboneconnectivity recovered was observed in group B,however bone marrow cavity had not yet recanalized.In addition,the bone connectivity and bone marrow cavity was completely recovery in group C.Group C had new bone growth around the defect and there was a gradual increase in bone mass with increase postoperative period 4 weeks after surgery.There was a gradual degradation of the material,and new bone growth was observed in material pores 6 weeks after surgery.Material degradation was significant 8 weeks after surgery with disappearence of pores,and recanalization of the bone marrow.But at 8 weeks after surgery,impaired bone defect in A and B group was not completely healed,bone marrow was not recanalized.There were significant differences when compared with the experimental group.The allografts showed no histological signs of acute rejection.Conclusions rADSCs/β-TCP bone tissue engineering complex had healing property,and the large segmental radial defect in rabbit was successfully repaired.Thus it supported the fact that ADSCs is expected to become the ideal repair substrate of bone defects,which can be used as seed cells for bone tissue engineering.After implantation,ADSCs do not provoke an immune response