牙槽突裂组织工程骨修复的脂肪间充质干细胞体外三维培养促成骨的研究

Three-dimentional cultured adipose mesenchymal stem cells for alveolar cleft bone repair:an in vitro study

目的探讨脂肪间充质干细胞在Matrigel凝胶中三维培养条件下成骨效果,为牙槽突裂组织工程骨修复提供理论依据。方法2019年6月于广州市妇女儿童医疗中心实验室,取第4代脂肪间充质干细胞(adipose derived stem cells,ADSC),调整细胞密度为1×10^(5)/ml,分别用二维普通培养液(A组)、二维成骨诱导培养液(B组)、凝胶包裹ADSC联合成骨诱导培养液(C组)对ADSC进行培养,用CCK8法检测细胞增殖情况;用茜素红染色、碱性磷酸酶活性检测细胞矿化效果,用Western印迹法和qRT-PCR检测骨钙素和骨桥蛋白的表达量。结果ADSC包裹于水凝胶中,CCK8试剂盒检测显示ADSC在三维和二维培养条件下一样,增殖稳定。ADSC在三维培养条件下茜素红染色及碱性磷酸酶活性明显高于二维培养环境。在Western印迹法和RT-PCR检测中发现三维培养时ADSC的成骨蛋白及mRNA的表达也高于二维培养。结论ADSC包裹于水凝胶不影响干细胞的增殖能力,而3D培养干细胞可明显增强其体外的成骨能力,可作为种子细胞用于组织工程骨修复牙槽突裂。

Objective To explore the osteogenic effect of adipose-derived mesenchymal stem cells(ADSCs)in three-dimensional culture conditions in Matrigel hydrogel to provide theoretical basis for tissue engineering bone repair of alveolar process fractures.Methods This study was performed in the Laboratory of Guangzhou Women and Children Medical Center in June 2019.The fourth generation of ADSCs were used to adjust the cell density to 1×10^(5)/mL.Two-dimensional common culture media(group A)and two-dimensional osteogenic induction media(group B)were used.The ADSCs were encapsulated in hydrogel with osteogenic induction media(group C),and the proliferation of cells was detected by CCK8 method.The cell mineralization effect was detected by alizarin red staining and alkaline phosphatase activity.The expression levels of osteocalcin(OCN)and osteopontin(OPN)were detected by qRT-PCR and Western blot assay.Results ADSCs were encapsulated in hydrogel.The results of CCK8 kit showed that ADSCs could stably proliferate under the three-dimensional culture.The alizarin red staining and alkaline phosphatase activity of ADSCs under three-dimensional culture conditions were significantly higher than those of two-dimensional condition.In the detection of Western blotting and RT-PCR,we found that the expression of osteogenic protein and mRNA(OCN and OPN)of ADSCs in three-dimensional culture was also higher than that of two-dimensional culture.Conclusions The encapsulation of ADSCs in hydrogel does not affect the proliferative potential of stem cells,and 3D cultured stem cells can significantly enhance their osteogenesis in vitro.