犬成肌细胞复合聚乳酸聚乙酸共聚物支架在裸鼠体内异位成软骨的实验研究  

HETEROTOPIC CHONDROGENESIS OF CANINE MYOBLASTS ON POLY(LACTIDE-CO-GLYCOLIDE) SCAFFOLDS IN VIVO

作　　者：顾羊林[1] 王予彬[1] 施克勤[1] 杨玉生[1] 陈鹏[1] 朱文辉[2] 朱国兴[1] 

机构地区：[1]南京医科大学附属无锡第二医院骨科江苏无锡,214002 [2]同济大学附属东方医院运动创伤关节外科

出　　处：《中国修复重建外科杂志》2013年第6期749-754,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：上海市自然科学基金资助项目(09ZR1425500);南京医科大学科技发展基金资助项目(2012NJMU253)~~

摘　　要：目的探讨与聚乳酸聚乙酸共聚物[poly(lactide-co-glycolide),PLGA]支架复合后的成肌细胞体外经软骨来源形成蛋白2(cartilage-derived morphogenetic protein 2,CDMP-2)联合TGF-β1诱导后,能否在裸鼠体内异位构建组织工程软骨。方法取1岁龄雄性比格犬股直肌肌肉分离培养成肌细胞,取第4代成肌细胞接种于PLGA支架,加入含CDMP-2与TGF-β1的软骨诱导液体外培养2周。实验分为4组:A组为经软骨诱导的成肌细胞-PLGA复合物组,B组为未经软骨诱导的成肌细胞-PLGA复合物组,C组为经软骨诱导的单纯PLGA支架组,D组为单纯PLGA支架组。于24只裸鼠背部皮下两侧制备4个皮下腔隙,分别植入4组材料。术后8、12周处死裸鼠,取材行大体观察、HE及甲苯胺蓝染色、Ⅰ型胶原及Ⅱ型胶原免疫组织化学染色观察,RT-PCR检测Ⅰ型胶原、Ⅱ型胶原、蛋白聚糖(Aggrecan)、Sox9mRNA表达,阿利新蓝染色检测糖胺聚糖(glycosaminoglycans,GAG)含量,生物力学试验检测标本压缩弹性模量。结果 A组标本术后8周呈类软骨样,乳白色,表面光洁,有弹性;12周时外观和弹性与正常软骨相似。B组术后8周仅残余少许组织,12周已完全降解;C、D组标本术后8周均降解消失。HE染色示A组8、12周时有成熟软骨陷窝形成;B组8周时组织内未见软骨陷窝形成。甲苯胺蓝染色示A组8、12周时组织内新生软骨细胞形成,细胞椭圆,排列整齐,细胞陷窝形成,细胞质和细胞外基质均蓝染阳性;B组8周时组织内未见蓝染的细胞外基质。Ⅰ、Ⅱ型胶原免疫组织化学染色示A组8、12周时均呈阳性表达;B组8周时呈阴性表达。RT-PCR检测示术后8、12周A组均可见Ⅰ型胶原、Ⅱ型胶原、Aggrecan和Sox9 mRNA表达,B组均呈阴性。术后12周A组压缩弹性模量为(90.79±1.78)MPa,GAG含量为(10.20±1.07)μg/mL与正常半月板相比,差异有统计学意义(P<0.05)。结论体外CDMP-2联合TGF-β1软骨诱导的比格犬成肌细胞复合PLGA�Objective To explore heterotopic chondrogenesis of canine myoblasts induced by cartilage-derived morphogenetic protein 2（CDMP-2） and transforming growth factor β1（TGF-β1） which were seeded on poly（lactide-co-glycolide）（PLGA） scaffolds after implantation in a subcutaneous pocket of nude mice.Methods Myoblasts from rectus femoris of 1-year-old Beagle were seeded on PLGA scaffolds and cultured in medium containing CDMP-2 and TGF-β1 for 2 weeks in vitro.Then induced myoblasts-PLGA scaffold,uninduced myoblasts-PLGA scaffold,CDMP-2 and TGF-β1-PLGA scaffold,and simple PLGA scaffold were implanted into 4 zygomorphic back subcutaneous pockets of 24 nude mice in groups A,B,C,and D,respectively.At 8 and 12 weeks,the samples were harvested for general observation,HE staining and toluidine blue staining,immunohistochemical staining for collagen type I and collagen type II;the mRNA expressions of collagen type I,collagen type II,Aggrecan,and Sox9 were determined by RT-PCR,the glycosaminoglycans（GAG） content by Alician blue staining,and the compressive elastic modulus by biomechanics.Results In group A,cartilaginoid tissue was milky white with smooth surface and slight elasticity at 8 weeks,and had similar appearance and elasticity to normal cartilage tissue at 12 weeks.In group B,few residual tissue remained at 8 weeks,and was completely degraded at 12 weeks.In groups C and D,the implants disappeared at 8 weeks.HE staining showed that mature cartilage lacuna formed of group A at 8 and 12 weeks;no cartilage lacuna formed in group B at 8 weeks.Toluidine blue staining confirmed that new cartilage cells were oval and arranged in line,with lacuna and blue-staining positive cytoplasm and extracellular matrix in group A at 8 and 12 weeks;no blue metachromatic extracellular matrix was seen in group B at 8 weeks.Collagen type I and collagen type II expressed positively in group A,did not expressed in group B by immunohistochemical staining.At 8 weeks,the mRNA expressions of collagen type I,collagen type II,Aggre

关 键 词：组织工程软骨 成肌细胞 聚乳酸聚乙酸共聚物 犬 裸鼠 

分 类 号：R684[医药卫生—骨科学]