摘要
目的探索3D生物打印脂肪来源干细胞(adipose-derived stem cells,ADSCs)联合甲基丙烯酰化明胶(gelatin methacryloyl,GelMA)构建组织工程软骨的可行性。方法取脂肪抽吸手术患者自愿捐赠的脂肪组织分离培养人ADSCs(human ADSCs,hADSCs),取第3代细胞与GelMA水凝胶和光引发剂混匀制成生物墨水。采用3D生物打印技术制备hADSCs-GelMA复合支架,行大体观察,于培养1 d及成软骨诱导培养14 d行扫描电镜观察;培养1、4、7 d取复合支架,行活/死细胞染色观察各时间点细胞存活率,并采用细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测细胞增殖情况;取成软骨诱导培养14 d的复合支架样本为实验组,以用完全培养基培养14 d的复合支架为对照组,行实时荧光定量PCR(real-time fluorescent quantitative PCR,qRT-PCR)检测软骨基质基因蛋白聚糖(aggrecan,ACAN)、成软骨调节因子SOX9、软骨特异性基因Ⅱ型胶原蛋白(collagen typeⅡA1,COLⅡA1)、软骨肥大标志基因Ⅹ型胶原蛋白(collagen typeⅩA1,COLⅩA1)mRNA的相对表达量。将成软骨诱导培养14 d的3D生物打印hADSCs-GelMA复合支架(实验组)和不含细胞的空白GelMA水凝胶支架(对照组)分别植入裸鼠背部皮下两侧囊袋内,4周后取材,行大体观察、番红O染色、阿利新蓝染色和Ⅱ型胶原免疫组织化学染色观察复合支架体内成软骨情况。结果大体观察和扫描电镜观察示hADSCs-GelMA复合支架形态稳定,结构规则。培养1、4、7 d细胞存活率维持在80%~90%,各时间点间差异均有统计学意义(P<0.05)。CCK-8法检测示随培养时间延长,复合支架内细胞呈持续增殖状态。对复合支架行成软骨诱导培养14 d后qRTPCR检测示,ACAN、SOX9和COLⅡA1表达显著上调,COLⅩA1表达显著下调(P<0.05)。复合支架植入裸鼠体内4周后取材,网格状形态清晰完整,组织学及免疫组织化学染色示实验组软骨基质和Ⅱ型胶原沉积,可见软骨陷窝形成,提示有软骨组织形成。结论3D生物打印的hADSCs-GelMA复合支架具备稳定的三维结构,细胞存活率高,可在体内、外诱导分化为软骨组织,可用于体内外构建组织工程软骨。
Objective To explore the feasibility of three-dimensional(3 D)bioprinted adipose-derived stem cells(ADSCs)combined with gelatin methacryloyl(GelMA)to construct tissue engineered cartilage.Methods Adipose tissue voluntarily donated by liposuction patients was collected to isolate and culture human ADSCs(hADSCs).The third generation cells were mixed with GelMA hydrogel and photoinitiator to make biological ink.The hADSCs-GelMA composite scaffold was prepared by 3 D bioprinting technology,and it was observed in general,and observed by scanning electron microscope after cultured for 1 day and chondrogenic induction culture for 14 days.After cultured for 1,4,and7 days,the composite scaffolds were taken for live/dead cell staining to observe cell survival rate;and cell counting kit 8(CCK-8)method was used to detect cell proliferation.The composite scaffold samples cultured in cartilage induction for 14 days were taken as the experimental group,and the composite scaffolds cultured in complete medium for 14 days were used as the control group.Real-time fluorescent quantitative PCR(qRT-PCR)was performed to detect cartilage formation.The relative expression levels of the mRNA of cartilage matrix gene[(aggrecan,ACAN)],chondrogenic regulatory factor(SOX9),cartilage-specific gene[collagen typeⅡA1(COLⅡA1)],and cartilage hypertrophy marker gene[collagen typeⅩA1(COLⅩA1)]were detected.The 3 D bioprinted hADSCs-GelMA composite scaffold(experimental group)and the blank GelMA hydrogel scaffold without cells(control group)cultured for 14 days of chondrogenesis were implanted into the subcutaneous pockets of the back of nude mice respectively,and the materials were taken after 4 weeks,and gross observation,Safranin O staining,Alcian blue staining,and collagen typeⅡimmunohistochemical staining were performed to observe the cartilage formation in the composite scaffold.Results Macroscope and scanning electron microscope observations showed that the hADSCs-GelMA composite scaffolds had a stable and regular structure.The cell viability could be maintained at 80%-90%at 1,4,and 7 days after printing,and the differences between different time points were significant(P<0.05).The results of CCK-8 experiment showed that the cells in the scaffold showed continuous proliferation after printing.After 14 days of chondrogenic induction and culture on the composite scaffold,the expressions of ACAN,SOX9,and COLⅡA1 were significantly upregulated(P<0.05),the expression of COLⅩA1 was significantly down-regulated(P<0.05).The scaffold was taken out at4 weeks after implantation.The structure of the scaffold was complete and clear.Histological and immunohistochemical results showed that cartilage matrix and collagen typeⅡwere deposited,and there was cartilage lacuna formation,which confirmed the formation of cartilage tissue.Conclusion The 3 D bioprinted hADSCs-GelMA composite scaffold has a stable 3 D structure and high cell viability,and can be induced differentiation into cartilage tissue,which can be used to construct tissue engineered cartilage in vivo and in vitro.
作者
穆琳
曾今实
黄元亮
林燕娴
蒋海越
滕利
MU Lin;ZENG Jinshi;HUANG Yuanliang;LIN Yanxian;JIANG Haiyue;TENG Li(Department of Craniomaxillofacial Surgery,Plastic Surgery Hospital,Peking Union Medical College,Chinese Academy of Medical Sciences,Beijing,100144,P.R.China)
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2021年第7期896-903,共8页
Chinese Journal of Reparative and Reconstructive Surgery
基金
中国医学科学院医学与健康科技创新工程(2017-I2M-I-007)。
关键词
组织工程软骨
3D生物打印
脂肪来源干细胞
生物材料
Tissue engineered cartilage
three-dimensional bioprinting
adipose-derived stem cells
biomaterials
作者简介
通信作者:滕利,Email:tenglidr@sina.com。
