Cartilage damage is one of the main causes of disability, and 3D bioprinting technology can produce complex structures that are particularly suitable for constructing a customized and irregular tissue engineering scaffold for cartilage repair. Alginate is an attractive biomaterial for bioinks because of its good biological safety profile and fast ionic gelation. However, ionically crosslinked alginate hydrogels are recognized as lacking enough mechanical property and long-term stability due to ion exchange. Here, we developed a double crosslinked alginate (DC-Alg) hydrogel for 3D bioprinting, and human umbilical cord mesenchymal stem cells (huMSCs) could differentiate into chondrocytes on its printed 3D scaffold after 4 weeks’ culture. We performed sequential modification of alginate with L-cysteine and 5-norbornene-2-methylamine, and the DC-Alg hydrogels were obtained in the presence of CaCl₂ and ultraviolet light with stronger mechanical properties than those of the single ionic crosslinked alginate hydrogels, which was similar to natural cartilage. They also had better stability and could be maintained in DMEM medium for over 1 month, as well good viability for huMSCs. Moreover, the DC-Alg as 3D printing inks demonstrated a better printing accuracy (∼200 m). After 4 weeks culture of huMSCs in the 3D printed DC-Alg scaffolds, the expressions of chondrogenic genes such as aggrecan (agg), collagen II (col II), and SRY-box transcription factor 9 (sox-9) were obviously observed, indicating the differentiation of huMSCs into cartilage. Immumohistochemical staining analysis further exhibited cartilage tissue developed well in the 3D printed scaffolds. Our study is the first demonstration of DC-Alg in 3D printing for MSC differentiation into cartilage, which shows a potential application in cartilage defect repair.

软骨损伤是致残的主要原因之一，3D生物打印技术可以产生复杂的结构，特别适合构建定制的、不规则的组织工程支架进行软骨修复。海藻酸盐因其良好的生物安全性和快速的离子凝胶化而成为一种有吸引力的生物墨水生物材料。然而，离子交联的海藻酸盐水凝胶由于离子交换而被认为缺乏足够的机械性能和长期稳定性。在这里，我们开发了一种用于 3D 生物打印的双交联海藻酸盐 (DC-Alg) 水凝胶，人脐带间充质干细胞 (huMSCs) 在其打印的 3D 支架上培养 4 周后可以分化成软骨细胞。我们用 L-半胱氨酸和 5-降冰片烯-2-甲胺对海藻酸盐进行了连续修饰，₂和紫外光具有比单离子交联海藻酸盐水凝胶更强的机械性能，类似于天然软骨。它们还具有更好的稳定性，可以在 DMEM 培养基中维持 1 个月以上，对 huMSCs 具有良好的生存能力。此外，DC-Alg 作为 3D 打印墨水表现出更好的打印精度（~200 m）。在 3D 打印的 DC-Alg 支架中培养 4 周 huMSCs 后，软骨形成基因如聚集蛋白聚糖 (agg)、胶原蛋白 II (col II) 和 SRY-box 转录因子9 (sox-9)明显观察到，表明huMSCs向软骨分化。免疫组织化学染色分析进一步表明软骨组织在 3D 打印支架中发育良好。我们的研究首次展示了 DC-Alg 在 3D 打印中用于 MSC 分化为软骨，这显示了在软骨缺损修复中的潜在应用。