摘要
目的评估体外构建组织工程髓核的可行性。方法从成年新西兰大白兔体内提纯兔滑膜干细胞(synovium-derived stem cells,SDSCs),接种到预制备的脱细胞细胞外基质(decellularized stem cell matrix,DSCM)支架中。实验分为3组:分别为空白组(空白DSCM支架)、实验组(SDSCsDSCM复合体)和对照组(正常兔髓核组织)。肉眼以及光镜下观察材料和细胞的形态特征,扫描电镜(SEM)观察各组内部结构和细胞粘附情况,HE染色观察各组细胞分布情况,荧光免疫组织化学染色观察细胞外Ⅱ型胶原(CollagenⅡ)、聚集蛋白聚糖(Aggrecan)蛋白表达情况,实时定量PCR(qRT-PCR)测定两组CollagenⅡ、Aggrecan相关基因的变化,压缩载荷检测各组抗压缩性能。结果肉眼下,组织工程髓核形态接近正常髓核;SEM显示细胞在支架内部粘附、生长良好;HE染色表明,随时间的延长,实验组材料内部细胞的分布逐渐均匀;免疫组化显示CollagenⅡ、Aggrecan分泌量随时间递增;qRT-PCR测定结果提示:实验组CollagenⅡ、Aggrecan mRNA表达量随时间递增,但均低于正常对照组(P <0. 05);支架的压缩载荷检测结果显示:在相同位移下,SDSCs-DSCM与正常髓核的压缩载荷差异无统计学意义。结论采用DSCM支架复合SDSCs可在体外成功构建组织工程髓核。
Objective To evaluate the feasibility of constructing tissue engineering nucleus pulposus in vitro. Methods After synovial-derived stem cells (SDSCs) were isolated from adult New Zealand white rabbit and purified, the cells were seeded into the prefabricated scaffolds of decellularized stem cell matrix (DSCM). So , the experiment included 3 groups, blank group (blank DSCM scaffolds), experimental group (SDSCs-DSCM complex) and control group (normal rabbit nucleus pulpous). Gross observation and scanning electron microscopy (SEM) were performed to observe the morphology and internal structure of materials and adhesion of cells. Hematoxylin and eosin (HE) staining was used to observe the distribution of cells in each group, and immunofluorescence histochemical staining was also used to detect the expression of CollagenⅡ protein and Aggrecan. Meanwhile, their mRNA levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Compression loading test was used to detect the compressive properties of each group. Results Gross observation found that the obtained tissue engineering nucleus pulposus was close to normal nucleus pulpous in morphology. SEM showed that the cells were adhered and grew well on the inner surface of the scaffolds. HE staining displayed that the cells in the experimental group presented uniform distribution gradually with the elapse of time. Immunofluorescence histochemical staining and qRT-PCR indicated that the expression of CollagenⅡand Aggrecan at protein and mRNA levels were increased with time, and all above levels were significantly lower than those in the control group (P<0.05). The results of compression loading test showed that there was no statistical difference in compression load between the experience group and control group under the same displacement conditions. Conclusion DSCM scaffold combined with SDSCs can be successfully built into tissue engineering nucleus pulposus in vitro.
作者
朱水涛
闵竞
王群波
郑华
郭华
ZHU Shuitao;MIN Jing;WANG Qunbo;ZHENG Hua;GUO Hua(Department of Orthopedics,Yongchuan Hospital,Chongqing Medical University,Chongqing,402160,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2019年第1期71-76,共6页
Journal of Third Military Medical University
基金
重庆市永川区科委资助项目(YCSTC2016nc5007)~~
关键词
细胞外基质
滑膜干细胞
组织工程髓核
decellularized stem cell matrix
synovial-derived stem cells
tissue engineering nucleus pulpous
