Chinese Journal of Tissue Engineering Research ›› 2020, Vol. 24 ›› Issue (31): 4934-4940.doi: 10.3969/j.issn.2095-4344.2129
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Chen Jia1, Yang Yiqiang1, Hu Chen1, Chen Qi1, Zhao Tian1, Yong Min1, Ma Dongyang2, Ren Liling3
1Stomatology Hospital of General Hospital, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 2Department of Craniomaxillofacial Surgery, The 940th Hospital of Joint Logistics Support Force of Chinese PLA, Lanzhou 730052, Gansu Province, China; 3School of Stomatology, Lanzhou University, Lanzhou 730000, Gansu Province, China
Received:
2020-02-24
Revised:
2020-02-29
Accepted:
2020-04-03
Online:
2020-11-08
Published:
2020-09-03
Contact:
Ma Dongyang, MD, PhD, Associate professor, Department of Craniomaxillofacial Surgery, The 940th Hospital of Joint Logistics Support Force of Chinese PLA, Lanzhou 730052, Gansu Province, China
Ren Liling, MD, PhD, Professor, School of Stomatology, Lanzhou University, Lanzhou 730000, Gansu Province, China
About author:
Chen Jia, Master, Attending physician, Stomatology Hospital of General Hospital, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
Yang Yiqiang, Master, Attending physician, Stomatology Hospital of General Hospital, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
Supported by:
CLC Number:
Chen Jia, Yang Yiqiang, Hu Chen, Chen Qi, Zhao Tian, Yong Min, Ma Dongyang, Ren Liling. Fabrication of prevascularized osteogenic differentiated cell sheet based on human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(31): 4934-4940.
2.1 人骨髓间充质干细胞膜片表征 人骨髓间充质干细胞高密度接种后连续培养2周,培养皿底可见呈现乳白色半透明的薄膜样物质,见图1A,将该膜状物夹起后自然收缩,具有一定弹性,见图1B。镜下观察显示,高密度接种的人骨髓间充质干细胞迅速融合,5 d后细胞融合并持续生长形成致密的细胞膜片;继续培养,单个细胞形态消失,细胞逐渐融合成片,成骨诱导细胞膜片钙盐逐渐沉积,11-14 d时可见白色的矿化结节形成,见图1C。碱性磷酸酶染色发现,成骨诱导细胞膜片内有较多的大小不等的橘黄色沉淀,见图1E,茜素红染色发现数个大小不一的深红色结节,见图1G,而未分化对照组膜片碱性磷酸酶染色和茜素红染色均呈阴性,见图1F,H。 "
2.2 内皮血管网的形成 人骨髓间充质干细胞以9×104/cm2密度接种后第2天即迅速融合,经过14 d的连续培养及成骨诱导形成均匀致密的成骨诱导细胞膜片,见图2A,而人脐静脉内皮细胞膜片则成“铺路石”样排列,见图2B。将人脐静脉内皮细胞以5×104/cm2密度种在上述成骨诱导细胞膜片上,倒置显微镜下观察细胞形貌变化, 0 h时膜片未见明显变化,见图2C,3 d时人脐静脉内皮细胞逐渐发生迁移,排列变得有序,并且膜片中的细胞外基质随之发生重排, 可以观察到膜片上有整齐的微槽结构出现,见图2D;随培养时间的延长,人脐静脉内皮细胞进一步迁移,微槽结构也更加典型,见图2E,并开始形成小的空泡样结构,见图2F。 "
2.3 进行性管腔的形成 倒置相差荧光显微镜下观察成骨诱导细胞膜片接种人脐静脉内皮细胞(5×104/cm2密度)后血管网络结构的进行性形成过程。接种0 h时,内皮细胞呈单个的、形似带足突的圆形,见图3A;接种2 h后,内皮细胞逐渐开始变得细长,见图3B;接种24 h时,人脐静脉内皮细胞在膜片上发生迁移,排列变得有序,见图3C;随时间延长,人脐静脉内皮细胞进一步发生迁移,到第3天时内皮细胞之间互相连接、局部融合,并形成小的空泡样结构,见图3D;接种第7天时,可见细长的分支和细胞间管腔样结构,见图3F。然而, 在培养皿内单纯培养24 h的人脐静脉内皮细胞组则表现出内皮细胞“铺路石样”随机排列,并未发现有类似的改变,见图3G。 "
2.4 CD31免疫荧光染色结果 CD31免疫荧光染色检测显示成骨诱导细胞膜片接种人脐静脉内皮细胞后2,24 h以及3,7 d时进行性管腔的形成过程。接种人脐静脉内皮细胞至成骨诱导细胞膜片2 h时,人脐静脉内皮细胞变得细长,见图4A;24 h后,人脐静脉内皮细胞在成骨诱导细胞膜片上发生迁移并动态排列,见图4B;培养至3 d时,人脐静脉内皮细胞发生融合,形成空泡样结构,见图4C,此时形成的管腔样结构数量达最大,见图4I;随时间延长,更多的人脐静脉内皮细胞延长并且动态排列;培养至7 d时,一些空泡开始聚集,见图4D,形成的管腔样结构数量反而下降(P < 0.05)。单纯的成骨诱导细胞膜片(未接种人脐静脉内皮细胞)CD31表达阴性,见图4G;单纯在培养皿内的人脐静脉内皮细胞CD31表达阳性,但是该内皮细胞随机排列,无管网状结构形成,见图4H。 "
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