同种异体间充质干细胞复合纤维蛋白修复骨缺损

1、同种异体间充质干细胞复合纤维蛋白修复骨缺损         08-07-02 10:46:00     编辑：studa20            作者：窦强兵，孔荣，朱六龙，戴永立，沈勇【关键词】  松质骨缺损摘  要：目的探讨骨髓间充质干细胞(mesenchymal stem cells，MSCs)的体外培养，以及同种异体MSCs复合纤维蛋白修

2、复兔股骨髁松质骨缺损的效果。方法在日本大耳兔双侧股骨髁制作06 cm×10 cm松质骨缺损，左侧植入同种异体MSCs纤维蛋白复合物，右侧单纯植入纤维蛋白。分别于术后2、5、8周行放射学、组织学检查，以了解骨缺损修复情况。结果单纯植入纤维蛋白的一侧不能自行愈合。植入复合物的一侧术后2周可见少量骨组织生成，5周可见大量模糊骨痂，术后8周骨缺损基本愈合。除少量炎性细胞浸润外，各期均未见明显免疫排斥反应。结论同种异体MSCs复合纤维蛋白可有效修复兔股骨髁松质骨缺损，8周内机体的免疫排斥反应较弱。关键词：松质骨缺损；  间充质干细胞；  纤维蛋白；  同种异体Ab

3、stract：ObjectiveTo discuss bone marrow derived mesenchymal stem cells (MSCs) culture in vitro and the curative effect of repairing cancellous bone defects in rabbits with the compound of allogenic MSCs and fibrin. MethodMSCs were isolated and cultureexpanted from rabbit bone marrow. Cancellous bone

4、defects were created on bilateral femur condylar in 15 Japanese big ear rabbits, the left bone defects  were  treated with allogenic MSCs and fibrin, the right were treated with fibrin only. The bone defects were examined radiologically and histoloyically at the 2nd, 5th, 8th week after op

5、eration. ResultThe defects with only fibrin implantation couldn't heal spontaneously and no bone formation were found at any timepoint. In the defects with compound of MSCs and fibrin implantation, there were a few bone formation within 2 weeks. By the 5th week plentiful callus were found, at th

6、e end of the 8th week, the defects healed. There was no immune respone except a few inflammatory cells infiltrate. ConclusionThe compound grafts of allogenic MSCs and fibrin shows low immunogenicity within 8 weeks and is an effective method for repairing cancellous bone defects.Key words: Cancellous

7、 bone defect;  Mesenchymal stem cells (MSCs);  Fibrin;  Allogenic骨缺损常发生于创伤、感染、肿瘤切除等疾病，是骨科常见难题，在治疗上近年兴起的以间充质干细胞(mesenchymal stem cells, MSCs)为种子细胞的组织工程骨修复具有广阔的临床应用前景。本实验采用同种异体MSCs与纤维蛋白复合物修复兔股骨髁骨缺损，观察异体MSCs在体内的成骨及免疫情况，为临床使用同种异体MSCs提供实验依据。1  材料和方法11  MSCs的分离培养4个月龄青紫蓝兔1只，重20 kg，

8、用含肝素30 uml的注射器于股骨粗隆抽取骨髓2 ml， 加入含10胎牛血清和青霉素及链霉素的DMEM培养基(Gibsco公司)中吹打混匀，置培养瓶中，37、5C02、95相对湿度的孵育箱内培养。5 d后半量换液，1周后全部换液，以后每周换液2次，逐步去除未贴壁细胞。14 d后贴壁细胞汇合到90生长面积时，用025的胰蛋白酶消化，按12传代，取第2代MSCs备用。12  MSCs纤维蛋白复合物的制备取第2代细胞计数后制成50×106ml的细胞悬液，按11与纤维蛋白(医用安可胶)混合，置12孔培养板，37孵育箱内聚合1 h，收缩过夜后备用。13  骨缺损模型的制作及

9、处理选用45个月龄日本大耳兔15只(安徽医科大学动物中心提供，清洁级)，重2025 kg，雌雄不限。3戊巴比妥30 mgkg耳缘静脉麻醉，取膝关节外侧切口显露股骨髁，用手钻在股骨髁外侧宽大处制作06 cm×10 cm的松质骨缺损。将骨屑冲洗干净后，左侧植入同种异体MSCs纤维蛋白复合物，右侧单纯植入纤维蛋白。术后不固定肢体，分笼饲养，连续3 d使用青霉素预防感染。14  术后观察观察动物的进食、活动和切口愈合情况。分别于术后2、5、8周摄双膝关节侧位片，以了解骨缺损的修复。各期用空气栓塞法处死动物5只，大体观察缺损区修复情况，切取股骨髁骨标本，用10中性福尔马林固定，脱钙切片后HE染色，光镜下观察新生骨痂和骨缺损愈合情况。2  结  果21  MSCs培养情况培养的原代细胞混有大量红细胞，呈圆形、椭圆形或梭形，生长较慢。传代后细胞变纯，生长加快，呈集落样螺旋状生长，成纤维细胞形态。22  实验动物情况术后34 h麻醉清醒后基本可站立，前3 d活动、进食减少，以后逐渐恢复正常。切口无感染，其中1例取标本