目的·构建可原位诱导低氧的互穿网络水凝胶（HMGL），探究其促进髓核细胞（nucleus pulposus cells，NPC）分泌细胞外基质的效果，并评估其应用于椎间盘退变治疗的可行性。方法·通过两步聚合法制备可长期原位诱导低氧的HMGL。分别构建漆酶浓度为5、10 U/mL的低氧水凝胶，即HMGL-5和HMGL-10；甲基丙烯酰化透明质酸（hyaluronic acid methacryloyl，HAMA）水凝胶作为对照组。采用核磁共振氢谱（¹H nuclear magnetic resonance spectra，¹H NMR）和傅立叶变换红外光谱（Fourier transform infrared spectroscopy，FTIR）验证HAMA和接枝香草醛的明胶（geln-van，GelVA）的分子结构。采用扫描电子显微镜（scanning electron microscope，SEM）观察水凝胶的微观结构，采用旋转流变仪和动态热机械分析仪（dynamic mechanical analyzer，DMA）测试水凝胶的力学性能，采用耗氧荧光探针检测水凝胶的诱导低氧行为。通过细胞活/死染色和细胞计数试剂盒8（cell counting kit 8，CCK-8）检测水凝胶的生物活性，通过免疫荧光染色检测NPC中缺氧诱导因子-1α（hypoxia inducible factor-1α，HIF-1α）和Ⅱ型胶原蛋白（type Ⅱ collagen，Col Ⅱ）的表达。建立大鼠椎间盘退变模型，将负载NPC的水凝胶分别注射到退变椎间盘中评估其修复效果。通过X射线和磁共振成像（magnetic resonance imaging，MRI）检测椎间盘高度和椎间盘含水量变化，通过组织学染色检测椎间盘结构完整性和蛋白聚糖水平。结果·结构表征证明材料成功制备。SEM观察显示3种水凝胶均具有疏松多孔的结构，且水凝胶的弹性模量随着孔径减小而增大。低氧测试结果显示HMGL-10水凝胶诱导低氧的能力最强。体外实验结果显示3种水凝胶均具有良好的生物相容性。相对于HAMA水凝胶，HMGL-10水凝胶中NPC的HIF-1α被显著激活。HMGL-10水凝胶组的HIF-1α和Col Ⅱ表达水平是HAMA水凝胶组的3.38和4.15倍。体内实验结果显示，低氧水凝胶组的椎间盘结构完整性、高度和含水量均优于其他治疗组。结论·在原位诱导缺氧作用下，低氧水凝胶有效激活HIF-1α以促进细胞外基质分泌，并显示出最有效的椎间盘再生作用。

Objective ·To construct an interpenetrating network hydrogel (HMGL) that can induce hypoxia in situ, investigate its effects on promoting the secretion of extracellular matrix in nucleus pulposus cells (NPCs), and evaluate its potential in the treatment of intervertebral disc degeneration. Methods ·HMGLs capable of long-term in situ hypoxiainduction were prepared by a two-step polymerization method. Hypoxic hydrogels with laccase concentrations of 5 U/mL and 10 U/mL, namely HMGL-5 and HMGL-10, were constructed, whereas a hyaluronic acid methacryloyl (HAMA) hydrogel served as the control. The molecular structures of HAMA and gelatin grafted with vanillin (GelVA) were verified by ¹H nuclear magnetic resonance spectra (¹H NMR) and Fourier transform infrared spectroscopy (FTIR). The microstructures of the hydrogels were observed by scanning electron microscope (SEM), and the mechanical properties of the hydrogels were tested by a rotational rheometer and a dynamic mechanical analyzer (DMA). The induced hypoxic behaviors of the hydrogels were detected by an oxygen consumption fluorescent probe. The biocompatibility of the hydrogels was tested by live/dead staining and the cell counting kit 8 (CCK-8) assay, and the expression of hypoxia-inducible factor-1α (HIF-1α) and type Ⅱ collagen (Col Ⅱ) in NPCs was detected by immunofluorescence staining. A rat model of intervertebral disc degeneration was established, and hydrogels loaded with NPCs were injected into degenerated intervertebral discs to evaluate their repair effects. Disc height and disc water content changes were detected by X-ray imaging and magnetic resonance imaging (MRI), and the integrity of disc structure and proteoglycan levels were detected by histological staining. Results ·Structural characterization demonstrated that the materials had been successfully prepared. SEM showed that the three hydrogels all had a loose and porous structure, and their elastic moduli increased with the decrease of pore size. Hypoxia test results indicated that the hypoxia-inducing ability of HMGL-10 hydrogel was the strongest. In vitro experimental results showed that the three hydrogels all had good biocompatibility. Compared with the HAMA hydrogel, the HIF-1α of NPCs was significantly activated in the HMGL-10 hydrogel. The expression levels of HIF-1α and Col Ⅱ in the HMGL-10 hydrogel group were 3.38 and 4.15 times higher than those in the HAMA hydrogel group. In vivo experimental results showed that the integrity, height, and water content of the intervertebral discs in the hypoxia hydrogel group were all superior to those in the other treatment groups. Conclusion ·The in situ hypoxia-inducing hydrogel effectively activates HIF-1α, promotes extracellular matrix secretion, and demonstrates superior regenerative potential for intervertebral disc repair.