Effect of in situ hypoxia-inducing hydrogelon extracellular matrix secretion in the nucleus pulposus

doi:10.3969/j.issn.1674-8115.2025.08.006

Abstract

Abstract:

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.

Key words: hypoxic hydrogels, oxygen tension, nucleus pulposus, extracellular matrix, disc degeneration

CLC Number:

R318.08

ZHOU Xingdie, CHEN Zehao, LÜ Zhendong, ZHANG Yuhui, LIU Li. Effect of in situ hypoxia-inducing hydrogelon extracellular matrix secretion in the nucleus pulposus[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(8): 990-1000.

Figures/Tables 7

Fig 1 Schematic illustration and structural characterization of HAMA

Fig 2 Schematic illustration and structural characterization of GelVA

Fig 3 Morphological characterization of different hydrogels

Fig 4 Mechanical properties and hypoxia-inducing behavior of hypoxic hydrogels

Fig 5 Biocompatibility of hypoxic hydrogels

Fig 6 Expression of HIF-1α and Col Ⅱ in nucleus pulposus cells cultured on different hydrogel matrices

Fig 7 Repair of degenerated intervertebral discs using different hydrogel matrices loaded with nucleus pulposus cells

TrendMD

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