Biocompatibility of Genipin cross-linked type I collagen with human adipose-derived stem cells in vitro

doi:10.3969/j.issn.2095-4344.2014.34.003

Abstract

Abstract:

BACKGROUND: Low toxicity of Genipin has certain species and cell specificity. Biocompatibility of Genipin cross-linked type I collagen with human adipose-derived stem cells is essential for construction of tissue-engineered adipose.

OBJECTIVE: To investigate the bbiocompatibility of Genipin cross-linked type I collagen with human adipose-derived stem cells.

METHODS: Human adipose-derived stem cells were isolated and cultured to the third generation, and the cells were seeded on Genipin cross-linked type I collagen scaffold. MTT assay was used to evaluate the adhesion and proliferation of cells on the scaffold, and the toxic effects of Genipin cross-linked type I collagen on human adipose-derived stem cells. Optical microscopy and scanning electron microscopy were utilized to observe the adhesion and growth process of human adipose-derived stem cells on the scaffold as well as the morphological changes of cells.

RESULTS AND CONCLUSION: Human adipose-derived stem cells could adhere to the scaffold immediately after seeded and increase gradually on the scaffold, with the average adhesion rate of 86.5%. Optical microscopy and scanning electron microscopy showed that human adipose-derived stem cells adhered well on the scaffold. The cells increased gradually over time, and could migrate into the scaffold, and distribute evenly with the passage of time when observed with optical microscopy. The result showed Genipin possesses very low cytotoxicity to the cells, and the outstanding biocompatibility is found between the cells and scaffold in vitro after cross-linked with Genipin.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: adipose tissue, mesenchymal stem cells, collagen type I, cross-linking reagents, biocompatible materials

CLC Number:

R318

Wang Gang, Kang Ting, Liu Yi, Liu Gang-qiang. Biocompatibility of Genipin cross-linked type I collagen with human adipose-derived stem cells in vitro[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(34): 5423-5428.

Figures/Tables 2

2.1 人脂肪间充质干细胞的分离培养结果倒置显微镜下可见第3代人脂肪间充质干细胞以梭形为主，细胞核居中，呈平行、漩涡或者辐射状排列，CM-DIL标记后，细胞膜呈红色(图1A，B)。MTT测定生长曲线，显示分离、培养的人脂肪间充质干细胞符合干细胞的生长特性(图1C)，且用CM-DIL标记后，对细胞生长的影响差异无显著性意义(P > 0.05)。 2.2 支架材料大体及电镜检查结果京尼平交联后，Ⅰ型胶原蛋白材料由白色变为蓝色(图2A)。扫描电镜显示：京尼平交联的Ⅰ型胶原蛋白支架材料呈海绵状，内部布满三维网状孔隙，孔径为60-130 μm，孔壁相互连接(图2B，C)。经测定支架材料的孔隙率约为87.3%。 2.3 细胞-支架复合物光镜观察人脂肪间充质干细胞刚接种到支架材料时，细胞呈圆形分布于支架材料表面。随着培养时间的延长，细胞数量逐渐增多，可以迁移进入支架内部并均匀分布(图3A，B)；荧光显微镜下亦可更加清楚地观察到：细胞在支架材料上的数量随着培养时间的延长而逐渐增多(图3C，D)。 2.4 MTT法检测细胞在支架材料上的黏附情况在各检测时间点，实验组的吸光度值均显著小于对照组(P=0.00)；但随时间推移，实验组与对照组吸光度值的差值逐渐缩小，实验组的吸光度值愈加趋近对照组(图4A)。Kasten改进法测得人脂肪间充质干细胞在支架材料上的黏附率平均为86.5%。 2.5 MTT法检测细胞在支架材料上的增殖情况细胞接种于支架材料后的前3 d，实验组的吸光度值小于对照组 (P < 0.05)，两组之间差异有显著性意义；随着培养时间的延长，两组的吸光度值逐渐增加(图4B)，且在第5天之后，实验组的吸光度值明显优于对照组(P < 0.05)，且两组之间吸光度值的差值逐渐增大。 2.6 扫描电镜观察细胞-支架复合物细胞接种于支架材料3 d后，多呈圆形，黏附于材料表面；7 d后，细胞数量显著增多，并可迁移进入孔隙内部，细胞伸出伪足平铺于材料表面或呈圆形黏附于材料孔隙内，此时细胞形态变为梭形或多角形，在材料表面呈聚集性分布，并分泌细胞外基质成分(图5)。"

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