体外诱导聚氧乙烯-聚氧丙烯醚共聚物水凝胶内兔牙髓干细胞成骨向分化的研究  

Experimental study on the in vitro osteogenic differentiation of dental pulp stem cells encapsulated in Pluronic F-127 hydrogel

作　　者：帕尔哈提·阿布肚热合曼 木合塔尔·霍加[1] 多力昆·吾甫尔[1] 麦麦提依明·哈力克 刘晓文[1] 

机构地区：[1]新疆维吾尔自治区人民医院口腔科,乌鲁木齐830001

出　　处：《中华口腔医学杂志》2016年第7期420-425,共6页Chinese Journal of Stomatology

基　　金：国家自然科学基金（81560180）

摘　　要：目的 探讨牙髓干细胞（dental pulp stem cells,DPSC）与聚氧乙烯-聚氧丙烯醚嵌段共聚物（Pluronic F-127）水凝胶的生物相容性,以及DPSC在Pluronic F-127水凝胶支架内的成骨活性,以期为颌骨缺损的组织工程修复提供依据.方法 酶解组织块儿法分离DPSC,有限稀释法进行纯化,镜下观察细胞形态.制备质量浓度为20％的Pluronic F-127水凝胶,扫描电镜观察水凝胶支架材料的微形态.用Pluronic F-127水凝胶包封DPSC进行三维培养,并以普通培养皿的二维培养作为对照组,光学显微镜下观察细胞生长情况.培养第1、3、5、7天甲基噻唑基四唑（methyl thiazolyl terazolium,MTT）法检测支架对细胞增殖活性的影响.常规成骨矿化液诱导14d进行茜素红、免疫细胞化学染色,荧光定量PCR检测碱性磷酸酶、Runt相关转录因子2（Runt-related transcription factor 2,RUNX2）、Ⅰ型胶原、骨钙蛋白,以评价牙髓干细胞的成骨活性.结果 成功分离并纯化DPSC,二维及三维培养的细胞镜下观察均生长良好,扫描电镜图显示多孔管状结构.培养第1、3、5、7天时A570值：二维培养DPSC分别为0.30±0.06、0.30±0.17、0.35±0.04、0.25±0.06,三维培养的DPSC分别为0.36±0.06、0.54±0.18、0.70±0.10、0.32±0.10,三维培养DPSC的增殖率显著高于二维培养（P＜0.05）,DPSC的细胞活性均在培养第5天时达峰值.常规矿化液诱导14d后茜素红染色结果显示：对照组及三维培养组矿化结节计数分别为（6.8±1.3）和（7.0±1.2）个,两组差异无统计学意义（P＞0.05）.免疫细胞化学染色显示,对照组及三维培养组DPSC中RUNX2、Ⅰ型胶原呈强阳性,骨钙蛋白染色呈弱阳性.荧光定量PCR结果显示：三维培养组Ⅰ型胶原相对表达量显著高于对照组（P=0.015）,其余基因相对表达水平组间差异均无统计学意义（P＞0.05）.结论 Pluronic F-127具有良好的生物相容性,可支持DPSC的成骨向分化;Pluronic F-t27可�Objective To evaluate the biocompatibility and viability of nonionic triblock copolymer Pluronic F-127 as a cell scaffold for osteogenic differentiation of dental pulp stem cells（DPSC）.Methods DPSC were obtained via enzymatic digestion method and purified bylimited dilution method.The freeze dried hydrogel of 20% Pluronic F-127 was prepared and itsstructurewas observed usingscanning electron microscopy（SEM）.After the encapsulation of cells of passage 3 in Pluronic F-127,the effects of hydrogel on the proliferations of DPSC were assessed with methyl thiazolyl terazolium（MTT） after one day and 3,5,7 days of incubations,respectively.On day 14,osteogenic abilities of DPSC encapsulated in the hydrogel were estimated by means of alizarin red S,immunocytochemical staining and real-time quantitative PCR（RT-qPCR）.Results DPSC were isolated and cultured successfully in the present study.SEM observations showed that porous structures which might be suitable for cell culture.A570 values of MTT were then normalized.A570 values of the cells in 2D cultures were 0.30±0.06,0.30±0.17,0.35±0.04 and 0.25±0.06and A570 values of DPSC in 3D cultures were 0.36±0.06,0.54±0.18,0.70±0.10 and 0.32±0.10 on day 1,3,5and 7,respectively.A570 value peaks were found on day 5 in both groups.The proliferation of 3D cultured DPSC was higher than that of 2D cultured cells（P〈0.05）.After 14 days of osteogenic induction,there were no calcium nodules observed in the control group and the numbers of calcium nodulesin the 2D and 3D groups had no significant difference（P〉0.05）.Inmmunocytochemical staining demonstrated strong expression of osteoblast marker Runt-related transcription factor 2（RUNX2）,type Ⅰ collagen（Col-Ⅰ） and relatively low expression of osteocalcin（OCN）.Moreover,RT-qPCR showed no differences between the relative expression of ALP,RUNX-2,OCN in the 2D and 3D groups （P〉0.05）,but a higher relative expression of Col-Ⅰ was observed in the 3D group（P=0.023）.Conclusions Pluronic F-127

关 键 词：水凝胶 组织工程 成骨细胞 牙髓干细胞 

分 类 号：R782.2[医药卫生—口腔医学]