Biocompatibility of human amniotic mesenchymal stem cells with human acellular amniotic membrane scaffold

doi:10.3969/j.issn.2095-4344.0445

Abstract

Abstract:

BACKGROUND: Anterior cruciate ligament (ACL) injury is commonly seen, and mainly treated with ACL reconstruction. Ligament tissue engineering treatment for ACL injury has become the current focus due to the extensive use of tissue engineering techniques in clinical practice.
OBJECTIVE: To investigate the biocompatibility of human amniotic mesenchymal stem cells (hAMSCs) and human acellular amniotic membrane (HAAM) scaffolds in vitro.
METHODS: hAMSCs were isolated using enzyme digestion and then identified. The fresh human amniotic membrane was subjected to decellularization using enzymatic digestion and chemical decontamination. Hematoxylin-eosin staining and immunofluorescence were used to detect the removal of cell components and destruction of the extracellular matrix. hAMSCs were cultured with HAAM extract (experimental group) and normal L-DMEM/F12 medium (control group). Cell proliferation was detected by cell counting kit-8. Cell adhesion and growth were observed by scanning electron microscope and inverted microscope at 14 days after co-culture with HAAM.
RESULTS AND CONCLUSION: hAMSCs exhibited a spiral, adherent growth under the inverted phase contrast microscope; and the cells highly expressed vimentin and lowly expressed CK-19, with osteogenic, adipogenic, and chondrogenic potential. Hematoxylin-eosin staining showed that human acellular amniotic epithelial cells and mesenchymal stem cells were completely removed and the extracellular matrix had no obvious damage. Immunofluorescence staining showed positive expression of type I collagen and type III collagen. The HAAM extract had no cytotoxicity to hAMSCs and exerted no effect on its proliferative activity. HAAM could promote the proliferation and adhesion of hAMSCs after incubation under the inverted microscope and scanning electron microscope. These findings indicate that HAAM has good biocompatibility and is favorable for the proliferation of hAMSCs.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Amnion, Mesenchymal Stem Cells, Biocompatible Materials, Tissue Engineering

CLC Number:

R394.2

Yang Ji-bin, Zhu Xi-zhong, Xiong Hua-zhang, Li Yu-wan, Jin Ying, Liu Yi. Biocompatibility of human amniotic mesenchymal stem cells with human acellular amniotic membrane scaffold[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(5): 742-747.

Figures/Tables 2

2.1 倒置相差显微镜下hAMSCs的形态及鉴定采用酶消化法进行hAMSCs原代培养，镜下可见原代hAMSCs多呈类圆形，部分纺锤形或多角状，贴壁生长；经过传代培养后，细胞逐渐变为长梭形，漩涡状贴壁生长，见图1。第3代hAMSCs经成骨诱导分化21 d后行茜素红染色，细胞透光性变弱，细胞密度增加，可见斑块状褐色物质，部分细胞变成小圆形矿化结节；成脂诱导21 d后行油红O染色，可见细胞排列散乱，细胞形状改变，类圆形细胞中出现脂滴样物质；成软骨诱导21 d后行甲苯胺蓝染色，可见细胞部分消失，细胞核增大，偏心，见图2。第3代hAMSCs免疫荧光结果显示波形蛋白呈阳性表达，CK-19呈阴性表达，见图3。 2.2 倒置相差显微镜下HAAM组织结构变化苏木精-伊红染色示新鲜人羊膜具有明显的分层结构，主要包括单独的上皮层、基底膜以及基质部分。经过脱细胞处理后，可见上皮层的细胞成分以及基质中的hAMSCs全部被移除，并且羊膜基质部分无明显破坏，见图4。 2.3 倒置荧光显微镜下人羊膜及人脱细胞羊膜的组织学观察结果免疫荧光染色显示Ⅰ型胶原、Ⅲ型胶原在新鲜人羊膜以及人脱细胞羊膜中均呈阳性表达(红色)，DAPI染色可见人脱细胞羊膜呈阴性表达，人羊膜呈阳性(蓝色)表达，见图5。 2.4 CCK-8检测细胞活性第3代细胞在经过7 d的培养过程中，两组细胞均生长良好，分别在每天同一时间测定A450值，实验组与阴性对照组相比在第3-7天时出现显著差异，见图6。 2.5 hAMSCs在HAAM上的黏附及生长情况在体外将第3代hAMSCs与HAAM共培养14 d后可见hAMSCs与HAAM黏附良好，广泛分布于HAAM中，hAMSCs呈长梭状，排列整齐，与HAAM具有良好的生物相容性，见图7。"

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