摘要
目的关节软骨的病变常伴有软骨下骨缺损,其修复重建一直是骨科难题。探讨BMSCs-双相支架复合物修复骨软骨缺损的可行性,比较其与植入单纯双相支架及动物自身修复效果的差别具有重要意义。方法以聚乳酸/聚羟基乙酸共聚物(poly-lactic-co-glycolic acid,PLGA)、羟基磷灰石(hydroxyapatite,HA)为原料制备由软骨相和骨相构成的三维支架,提取天然Ⅰ型胶原(collagen typeⅠ,ColⅠ)涂于表面制成PLGA-HA-ColⅠ双相支架。取新西兰乳兔骨髓分离培养获得的第2代BMSCs,以1×106个/mL接种于双相支架,扫描电镜观察支架结构和细胞分布。取30只6月龄新西兰大白兔,建立股骨远端关节面骨软骨缺损模型,随机均分为3组,A、B组分别于缺损区植入单纯双相支架和BMSCs-双相支架复合物,C组作为空白对照组未植入支架材料。于术后1、3、6、9个月取材行大体及组织学观察,术后9个月对A、B组标本行大体评分比较、micro CT扫描定量分析及免疫组织化学染色观察。结果扫描电镜示双相支架孔隙连通性好,软骨相和骨相孔径不同,BMSCs在双相支架内生长良好。大体观察示术后9个月内A组关节表面逐渐形成类软骨样组织,部分出现塌陷或不规则缺损;B组关节面无塌陷或碎裂,新生组织质地更接近正常组织;C组缺损一直存在。大体评分显示3组修复效果比较差异均有统计学意义(P<0.001),B组优于A组,C组最差。micro CT扫描示A、B组软骨下骨得到良好的修复重建,定量分析示B组骨组织体积分数、结构模型指数及骨小梁数目均高于A组,但差异无统计学意义(P>0.05)。组织学观察示术后1个月A、B组缺损区存在炎性反应;术后3个月新生组织长入;术后6个月支架完全降解,新生组织在植入物及缺损边缘爬行生长;术后9个月形成大量胶原纤维,表面多为纤维软骨。C组观察期内缺损持续存在。术后9个月免疫组织化学染色示A、B组标本缺损区ColⅡ染色呈弱阳性,ColⅠ染色阳性。结论PLGA-HA-ColⅠ双相支架具备较适宜的一体化修复骨软骨缺损的物理特性,接种BMSCs后整体修复效果更好。
Objective To explore the preparing methods in vitro and test the clinical applicability of implantation in vivo of bone marrow stromal stem cells（BMSCs）-biphasic scaffold to repair defects of cartilage and subchondral bone and to compare the differences in repaired outcomes of composite,single biphasic scaffold and rabbits themselves.Methods The upper chondral phase and the lower osseous phase of the plugs,using poly-lactic-co-glycolic acid（PLGA）,hydroxyapatite （HA）,and other biomaterials,were fused into carrier scaffold,on which collagen type I（Col I）was coated.The surface and inner structure of biphasic scaffold were observed under scanning electron microscope（SEM）.BMSCs was isolated from the bone marrow of tibia and femurs of young New Zealand rabbits using centrifuging and washing,and their morphologies and adherences were observed everyday.Then BMSCs were inoculated on the surface of scaffold to form BMSCs-scaffold composites.Osteochondral defects were surgically created on articular surface of femoral intercondyles of 30 New Zealandrabbits,which were divided into groups A,B and C.In group A,a biphasic osteochondral composite were implanted into defect,BMSCs and biphasic cylindrical porous plug of PLGA-HA-Col I in group B,and group C was used as a control without implant.Specimens were harvested to make macroscopic and histological observations at the 1st,3rd,6th,and 9th months after operation respectively;meanwhile immunohistological and micro-computed tomography（micro CT）examinations were performed and graded at the 9th month after operation.Results SEM showed an excellent connection of holes in the biphasic scaffold infiltrated by Col I.Optical microscopy and SEM showed a good growth of BMSCs in scaffold without obvious cellular morphological changes and an accumulation in the holes.Macroscopic samples showed a resistant existence of defects of group C within 9 months;the scaffold completely degenerated and chondral-like tissue formed on articular surface with partly collapses and irregular defects in group A;and smoother surface without collapses and approach to normal with texture of new regeneration in group B.There were statistically significant differences in macroscopic results（P0.001）,group B was superior to group A,and group C was the worst.The micro CT showed good repairs and reconstruction of subchondral bone, with a acceptable integration with newborn chondral-like tissue and host bone in group B.Quantificational analysis of relevant parameters showed no significant differences.Histological results showed inflammations located in defects at the 1st month, new tissue grew into scaffold at the 3rd month;new chondral-like tissue crept on the margin of defects and biphasic scaffold degenerated completely at the 6th month,and lots of collagen formed in subchondral bone with major fibrocartilage on chondral area at the 9th month after surgery in groups A and B.In groups A and B,immunohistological observations were weak positive for Col II and positive for Col I.Conclusion Biphasic scaffold implanted in body can induce and accelerate repair of defects of articular cartilages which are mainly filled with fibrocartilage,especially for subchondral bone.Scaffold combined with BMSCs has the best repairing effects 9 months after implantation.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2010年第1期87-93,共7页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(30973049)
国家重点基础研究发展计划(973)资助项目(2005CB623901)~~
关键词
组织工程
BMSCS
双相支架
骨软骨缺损
修复
兔
Tissue engineering Bone marrow stromal stem cells Biphasic scaffold Osteochondral defect Repair Rabbit
