组织工程骨修复山羊负重骨大段缺损

目的研究组织工程骨结合带锁髓内钉修复成年山羊大段负重骨缺损的可行性，探索更可行的技术路径。方法将24只成年山羊，通过骨髓穿刺法获取山羊骨髓间充质干细胞（BMSC），将体外扩增及成骨定向诱导的第2代BMSC与同种异体脱钙骨基质（DBM）通过双相接种法构建组织工程骨。24只成年山羊，以带锁髓内钉构建股骨中段3cm骨缺损模型。随机分为3组，每组8只。实验组以组织工程骨修复骨缺损，对照组单独使用DBM和空白组旷置。术后1、12、24周行X线片观察及评分，12、24周每组各处死4只动物行组织学观察和生物力学检测。结果标本大体观察示实验组和对照组术后12周骨缺损部位被骨痂连接，髓腔贯通，24周全部愈合；实验组24周恢复正常解剖形态，对照组外形仍然粗糙、不规则；空白组术后12周及24周缺损部位均为纤维组织充填。术后1周各组X线评分无明显差异（P〉0．05），实验组术后12周及24周X线评分均优于对照组和空白组，对照组优于空白组，各组24周X线评分均高于12周时，差异均有统计学意义（P〈0．05）。实验组术后12、24周的最大抗扭强度分别达正常侧的47．07％±5．05％和83．73％士2．33％，显著高于对照组和空白组（P〈O．05）；空白组2个时间点最大抗扭强度均不超过正常的15％，与骨不连时的纤维连接相符。组织学检查示术后12周实验组和对照组骨缺损区DBM支架材料基本被吸收，有典型的同心圆排列的哈弗系统形成，周围偶见淋巴细胞；术后24周，实验组和对照组股骨缺损均被修复，但实验组较对照组的新骨更多、骨塑形更好；空白组术后24周骨缺损区中央仍为纤维组织填充。结论组织工程骨结合带锁髓内钉能够更有效修复成年山羊负重骨大段骨缺损，满足负重骨的生物力学要求。

The Experimental Study on Healing of Large Segmental Defects by Tissue Engineering Bone in a Goat Model

Objective To explore the feasibility of healing large segmental load-bearing bone defects by tissue engineering bone combined with locked intramedullary nail in a large animal model,and explore the feasible technical route.Methods Twenty-four goats were randomly divided into 3 groups with 8 in each group.Mesenchymal stem cells(MSCs) aspirated from the goat were cultured and induced into osteoblasts in vitro.Then the induced MSCs were seeded to deminerilized bone matrix(DBM) by biphasic seeding technique to construct tissue engineering bones.The 3 cm femoral diaphyseal defect was made in every goat and fixed with inter-locking medullary nail,then filled with tissue engineering bones as experimental group,with DBM alone as control group,and left empty as blank group.Four goats of each group were sacrificed for radiological,histological and biomechanical examination at 12 and 24 weeks postoperatively.Results On gross examination,defects in the experimental group and control group were filled by bone-like tissue with marrow cavity formation at 12 weeks postoperatively,and were all healed at 24 weeks.In the experimental group,new bone formation was more,and the contour was almost completely restored at 24 weeks,while defects in the control group remained irregular.The blank group were filled with fibrous tissue and appeared mechanically unstable at 12 and 24 weeks.The radiological investigation revealed no radiographic evidence of bone formation immediately after surgery.At 12 weeks and 24 weeks postoperatively,the radiographic score in the experimental group was significantly superior compared with those in the control group and blank group(P < 0.05),and in each group the score at 24 weeks was higher than at 12 weeks with a significant difference(P < 0.05).The ultimate torsional strength of the femur in the experimental group was 47.07% ± 5.05% and 83.73% ± 2.33% of the normal side at 12 weeks and 24 weeks,which was higher than those in the control group and blank group(P < 0.05).The biomechanical test results in the blank group were below 15% of the normal side,which was consistent with a nonunion.Histological analysis showed that the grafted DBM was mostly degraded by 12 weeks both in the experimental group and the control group,and woven bone was observed with some concentrically arranged lamellar bone and few lymphocytes.By 24 weeks,remodeling of the newly formed woven bone into lamellar bone was present in both groups.Comparatively,the amount of newly formed bone in the experimental group was more and the remodeling was better.In the blank group,fibrous tissue still filled the whole defect.Conclusion The tissue engineering bone combined with locked intramedullary nail can improve healing of large segmental load-bearing bone defects,and also recover the bio-mechanical strength earlier.