Synchronous restoration of intervertebral disc structure and biological function utilizing cartilage microtissue-based active hydrogels

Objective Intervertebral disc degeneration (IVDD) constitutes a primary cause of low back pain, with existing clinical interventions often failing to facilitate effective regeneration of degenerated nucleus pulposus tissue. This study seeks to develop an active injectable hydrogel composite system incorporating cartilage microtissues (CM) (CM-Gel) and to assess its potential and underlying mechanisms for repairing degenerated intervertebral discs through two approaches, in vitro maintenance of cellular phenotype and in vivo molecular regulation. Methods CM containing cartilage-specific extracellular matrix (ECM) were generated in vitro via three-dimensional culture and subsequently integrated with hydrogel to form CM-Gel. Immunofluorescence double staining was conducted on days 1, 4, and 7 of in vitro culture to assess the dynamic expression of ACAN and COL-II. An animal model of intervertebral disc degeneration was established, and tissue samples were collected 4 weeks post-surgery. qRT-PCR was employed to measure the mRNA expression levels of anabolic genes, catabolic genes, and the fibrosis marker gene within the nucleus pulposus tissue. Additionally, the reparative effects were evaluated through histological and imaging analyses. Results The expression levels of ACAN and COL-II in CM-Gel increased in a time-dependent manner, indicating progressive maturation of the microtissue phenotype. In vivo experiments demonstrated that CM-Gel effectively preserved intervertebral disc height and facilitated ECM regeneration. Furthermore, the expression of ACAN, COL-II, and SOX9 was significantly upregulated in the CM-Gel group, whereas the expression of MMP13 and ADAMTS5, as well as COL-I, was significantly downregulated. These molecular changes suggest that CM-Gel promotes matrix synthesis, inhibits degradation, and mitigates fibrosis. Conclusion CM-Gel not only preserves the mature phenotype of CM in vitro but also reverses the degenerative microenvironment of intervertebral discs in vivo , facilitating structural repair and functional restoration by reestablishing the balance between synthesis and catabolism. This composite system presents a novel strategy with potential for clinical translation in the biological treatment of intervertebral disc degeneration.