Hyaluronidase

NP cells were isolated from lumbar discs of 5 mice on a C57BL/6J background as described previously [30 (link)]. Briefly, the spinal columns of mice (8 weeks old) were removed whole under aseptic conditions and lumbar intervertebral discs were collected. The gel-like NP was isolated from the anulus fibrosus under a microscope and treated with 0.1% type II collagenase (Beyotime, China) and 2 U/mL hyaluronidase (Beyotime, China) for 2 ​h at 37 ​°C to digest the tissue completely. After centrifugation at 1500 ​rpm for 5 ​min, the precipitates were resuspended and placed in high glucose DMEM/F-12 (Beyotime, China) medium containing 10% FBS and 1% penicillin/streptomycin antibiotics in an incubator maintained with 5% CO₂ at 37 ​°C. The complete medium was replaced every other day. When confluent, the NP cells were harvested using trypsin–EDTA (1 ​mM, gibco, USA) solution and subcultured into suitable petri dish for later use.

GelMA and HAMA were synthesized in our laboratory. Bouin's solution was obtained from Fuzhou Phygene Biotechnology Co., LTD. Carboxyfluorescein diacetate succinimidyl ester (CFDA SE) cell proliferation assay and tracking kit, red blood cell lysis buffer, collagenase Ⅰ, collagenase Ⅱ, collagenase Ⅳ, hyaluronidase, cell counting kit 8 (CCK8), and Calcein AM/PI Live/Dead viability assay kit were bought from Shanghai Beyotime Biotechnology Co., Ltd. 1′-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR) was bought from Beijing Lablead Trading Co., LTD. Mouse TNF-α, mouse IL-6, and rat IgG ELISA kits were provided by Wuhan Elabscience Biotechnology Co., Ltd. aPD-1 (rat anti-mouse) and antibodies for flow cytometry used in this study were purchased from Biolegend Inc. (USA). Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was obtained from Shanghai Aladdin Co., Ltd. CpG ODN 1826, with the sequence 5′-tccatgacgttcctgacgtt-3′ and a phosphorothioate backbone, and CPG ODN 1826 labeling with FAM on 3′ end (named FAM-CpG ODN) were obtained from Suzhou Synbio Technologies Co.