An siCDKN2A-loaded biomimetic hydrogel for dual modulation of oxidative stress and the degenerative microenvironment in intervertebral disc repair.

Abstract

Intervertebral disc degeneration (IVDD) is driven by sustained inflammatory microenvironment activation and dysfunction of nucleus pulposus cells (NPC). Excessive ROS induce oxidative damage, restrict NPC proliferation/migration, accelerate senescence, and disrupt ECM metabolic balance. Thus, concurrently targeting both endogenous oxidative stress in NPC and the exogenous inflammatory microenvironment of the degenerated intervertebral disc (IVD) is a key strategy for IVDD intervention. In this study, we found that CDKN2A was significantly upregulated in degenerated nucleus pulposus (NP) tissues and NPC, and its expression level correlated with degeneration severity. Functional experiments demonstrated that silencing CDKN2A with siCDKN2A did not act through regulating cuproptosis but rather improved mitochondrial function and reduced ROS accumulation in NPC, restored cell proliferation and migration capabilities, suppressed senescence phenotypes, and reestablished ECM homeostasis involving COLII, ACAN, MMP3, MMP13, and inflammatory factors. To address siRNA delivery bottlenecks (degradation, insufficient retention, limited single-target efficacy), we constructed an injectable biomimetic hydrogel (OP) via dynamic crosslinking of sodium hyaluronic acid (SHA) with polyamidoamine (PAMAM). This system enables sustained siCDKN2A release, ECM supplementation, and anti-inflammatory/antioxidant properties, coupling gene silencing with microenvironment remodeling. OP@siCDKN2A sustained siCDKN2A release, facilitated NPC uptake, reduced ROS levels, delayed senescence, and enhanced proliferation, migration, and ECM synthesis in vitro. In vivo, compared to OP or siCDKN2A alone, OP@siCDKN2A better maintained disc height and T2 signal, reduced histological degeneration scores, suppressed IL-6/IL-1β elevation, and restored COLII/ACAN expression. This work establishes an "internal-external synergistic" strategy coupling CDKN2A-targeted silencing with OP-mediated microenvironment remodeling, offering a new framework for gene-material combination therapy against IVDD.

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