Objective: To explore the feasibility of three-dimensional (3D) 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 3D 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, and 7 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 type Ⅱ A1 (COLⅡA1)], and cartilage hypertrophy marker gene [collagen type ⅩA1 (COLⅩA1)] were detected. The 3D 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 type Ⅱ immunohistochemical 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 up-regulated ( P<0.05), the expression of COLⅩA1 was significantly down-regulated ( P<0.05). The scaffold was taken out at 4 weeks after implantation. The structure of the scaffold was complete and clear. Histological and immunohistochemical results showed that cartilage matrix and collagen type Ⅱ were deposited, and there was cartilage lacuna formation, which confirmed the formation of cartilage tissue.
Conclusion: The 3D bioprinted hADSCs-GelMA composite scaffold has a stable 3D 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.
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 qRT-PCR 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。.
Keywords: Tissue engineered cartilage; adipose-derived stem cells; biomaterials; three-dimensional bioprinting.