人脂肪干细胞与软骨脱细胞基质3D打印构建三维细胞生物支架的研究

Construction of three-dimensional cellular scaffolds by 3D printing of human adipose derived stem cells and cartilage acellular matrix

目的人脂肪干细胞(hASCs)与软骨脱细胞基质(CAM)3D打印构建适合细胞生长的三维细胞生物支架。方法从郑州大学第二附属医院10例抽脂患者的脂肪组织中提取脂肪干细胞进行体外培养,取第3代hASCs进行软骨诱导,用阿利新蓝染色检测hASCs的软骨分化。通过脱细胞方法对猪耳软骨进行脱细胞,后对脱细胞效果进行检测。利用制备的CAM和海藻酸钠(SA)按(CAM∶SA 7∶3、6∶4、5∶5、4∶6)进行混合,用3D打印机打印成个性化三维生物支架,通过细胞计数试剂盒(CCK-8)进行支架细胞毒性检测,对不同比例支架的生物学特性如孔隙率、力学性能、降解率进行比较,找出最优的支架混合比例。用上述最优比例接种hASCs(1×10^(6)个)行3D打印,三维细胞生物支架体外培养1周后在倒置显微镜下观察细胞存活情况。组间采用t检验。结果hASCs传代后在显微镜下观察呈梭形、多角形,胞质丰富,胞核清楚。hASCs经软骨诱导剂诱导后,hASCs可向软骨分化。猪耳软骨经脱细胞后效果良好,内部无细胞残留,DNA残留量和未脱细胞软骨比较,差异有统计学意义(t=65.420,P<0.05)。支架浸提液和完全培养液分别培养hASCs比较,培养后第2、4、6天时间点,细胞增殖数量比较,差异无统计学意义(t=1.750、2.365、2.106、P>0.05)。3D打印不同比例的支架,CAM含量越多,降解速率越快,力学强度和孔隙率越小。其中5∶5和4∶6混合比例形成的支架生物学性能更优。hASCs(1×10^(6)个)混合这两种比例支架经3D打印形成个性化三维支架,活死细胞染色显示,第3天和第7天时间点,5∶5支架混合的hASCs活细胞更多。细胞增殖比较,第3天和第7天时间点,5∶5支架混合的hASCs增殖数量多余4∶6比例组,差异有统计学意义(t=4.350、4.922,P<0.05)。结论比例为5∶5的CAM-SA经3D构建的三维生物支架最适合细胞生长。

Objective To construct a three-dimensional cell biological scaffold suitable for cell growth using 3D printing of human adipose derived stem cells(hASCs)and cartilage acellular matrix(CAM).Methods ASCs were extracted from the adipose tissue of 10 liposuction patients in the Second Affiliated Hospital of Zhengzhou University for in vitro culture,and the third generation hASCs were taken for cartilage induction.The cartilage differentiation of hASCs was detected by alcian blue staining.The porcine ear cartilage was decellularized by the decellularization method,and then the decellularization effect was tested.The prepared cam and sodium alginate(SA)were mixed according to(CAM∶SA 7∶3,6∶4,5∶5,4∶6)and printed into personalized three-dimensional biological scaffolds by 3D printer.The cytotoxicity of scaffolds was detected by cell counting kit-8(CCK-8).The biological characteristics such as porosity,mechanical properties and degradation rate of different proportions of scaffolds were compared to find the optimal scaffold mixing ratio.HASCs(1×106 cells)were printed in 3D,and the cell survival was observed under an inverted microscope after the three-dimensional cell biological scaffold was cultured in vitro for 1 week.T test was used between groups.Results After passage,hASCs were spindle shaped and polygonal,with abundant cytoplasm and clear nuclei.HASCs could differentiate into cartilage after being induced by cartilage inducer.After decellularization,the effect of porcine ear cartilage was good,and there was no cell residue inside.There was a significant difference in DNA residue compared with that of non decellularized cartilage(t=65.420,P<0.05).There was no significant difference in cell proliferation at the time points of day 2,day 4 and day 6 after culture(t=1.750,2.365,2.106,P>0.05).3D printing scaffolds with different proportions showed the more cam content,the faster the degradation rate,and the smaller the mechanical strength and porosity.The scaffolds with the ratio of 5∶5 and 4∶6 had better biological performance.The hASCs(1×106 cells)were mixed to form personalized three-dimensional scaffolds after 3D printing.The staining of live and dead cells showed that at the time points of day 3 and day 7,5∶5 scaffolds mixed more hASCs live cells.In comparison of cell proliferation,at the time points of day 3 and day 7,the number of proliferation of hASCs mixed with 5∶5 scaffolds was more than that in the 4∶6 ratio group,and the difference was statistically significant(t=4.350,4.922,P<0.05).Conclusion The 3D biological scaffold constructed by cam-sa with a ratio of 5∶5 is the most suitable for cell growth.