基于细胞外基质来源的组织工程椎间盘双相支架裸鼠皮下异位构建椎间盘  

Tissue engineering of intervertebral discs based on extracellular matrix-derived integrated annulus fibrosus-nucleus pulposus biphasic scaffolds

作　　者：祁霁舟 徐宝山[2] 张杨[2] 伍耀宏[1] 马信龙[2] 张春秋[3] 李秀兰[2] 

机构地区：[1]天津医科大学研究生院天津市和平区气象台路22号,300070 [2]天津市天津医院天津市河西区解放南路406号,300211 [3]天津理工大学机械工程学院天津市西青区宾水西道391号,300384

出　　处：《中国矫形外科杂志》2014年第21期1983-1988,共6页Orthopedic Journal of China

基　　金：国家自然科学基金课题资助项目(编号:81272046,31000432,31300798);中国博士后科学基金项目(编号:2011M500530,2012T50221);天津市卫生局科技基金项目(编号:2013KR16)

摘　　要：[目的]探讨椎间盘细胞和细胞外基质来源的一体化纤维环-髓核双相支架在裸鼠体内异位构建组织工程一体化椎间盘的可行性,并采用PKH26荧光标记和小动物活体荧光成像系统无创评估组织工程化细胞-支架复合体在体内生长情况。[方法]纤维环细胞和髓核细胞分别标记PKH26荧光,分别接种入细胞外基质来源的一体化支架不同相中,扫描电镜、Dead/Live荧光染色观察细胞粘附及活性,植入裸鼠背部皮下,6周后利用分子小动物活体荧光成像系统评价组织工程化组织在裸鼠体内生长情况,取材进行荧光显微镜下观察、组织学染色。[结果]扫描电镜观察细胞粘附在双相支架上且周围有基质分泌,Dead/Live染色示细胞在双相支架上活性良好;6周后,活体荧光成像显示椎间盘细胞在支架内生长良好,从髓核往纤维环荧光强度减弱,细胞支架复合体在裸鼠体内形成椎间盘样组织,HE、番红O染色、甲苯胺蓝染色阳性。[结论]天然骨基质明胶和软骨基质来源的一体化纤维环-髓核支架复合椎间盘细胞能够在裸鼠皮下异位构建椎间盘样组织。[ Objective ] To evaluate the feasibility of tissue engineering of nucleus pulposus in vivo by combining acellular extracellular matrix （ECM） - derived biphasic scaffolds with PKH26 - labeled goat disc cells, and to assess the cell - scaffold hybrids in vivo by using a molecule fluorescence imaging system. [ Methods] Disc cells were labeled with PKH26 and seeded into scaffolds. Cell attachment was observed by scanning electron microscopy （ SEM）, and cell viability was evaluated by LIVE/ DEAD staining. After in vitro culturing for 2 days, cell - scaffold hybrids were implanted into nude mice. After 6 weeks, an in vivo molecule fluorescence imaging system was used to evaluate the cell - scaffold hybrids, and the hybrids were analyzed by flu- orescence microscopy and histology. [ Results ] SEM imaging confirmed that cells could anchor and spread on the scaffolds. LIVE/DEAD staining showed that the cells were alive. After 6 weeks of culture in vivo, disc - like tissues formed in nude mice, which were confirmed by histological and fluorescence observations. Cells in the hybrids originated from labeled cells, which were confirmed by the molecule fluorescence imaging system. [ Conclusion ] ECM - derived biphasic scaffolds can be used for tissue engineering of intervertebral discs in vivo.

关 键 词：椎间盘 组织工程 细胞外基质 PKH26 裸鼠 

分 类 号：R318.08[医药卫生—生物医学工程]