Pre-Conditioned Bone Marrow Mesenchymal Stromal Cell-Derived Secretome Exerts an Anti-Inflammatory Effect on Degenerative Nucleus Pulposus Cells In Vitro

BACKGROUND: Intervertebral disc degeneration (IDD) is frequently associated with chronic low back pain (LBP), which contributes significantly to disability, psychological distress, and reduced work capacity. Mesenchymal stromal cell (MSC)-based treatments may offer a promising, less invasive alternative to conventional treatments for IDD. The MSC secretome has shown regenerative potential through paracrine and anti-inflammatory mechanisms. In this study, we investigated the effects of the secretome isolated from interleukin-1β (IL-1β)-preconditioned bone marrow-derived MSCs (BM-MSCs) on human nucleus pulposus cells (hNPCs) adopting a 3D in vitro culture model. METHODS: The secretome (BM-MSCsec) was collected from BM-MSCs preconditioned with 10 ng/mL IL-1β. hNPCs were isolated, expanded, encapsulated in alginate beads, and stimulated with IL-1β to mimic a pro-inflammatory environment. These cells were then treated with either standard culture media (control group), IL-1β alone, BM-MSCsec, or IL-1β + BM-MSCsec. We evaluated cell proliferation and viability via flow cytometry, nitrite and reactive oxygen species (ROS) levels using the H2DCFDA assay, glycosaminoglycan (GAG) content with the 1,9-dimethylmethylene blue assay, gene expression of extracellular matrix (ECM) components and inflammatory markers via qPCR, and cell senescence through Western blot and β-galactosidase staining. RESULTS: IL-1β stimulation increased hNPC proliferation, nitrite release, ROS production, catabolic and inflammatory gene expression, and cell senescence. Treatment with BM-MSCsec attenuated these effects, restoring proliferation to baseline levels, significantly reducing ROS and nitrite accumulation. BM-MSCsec also upregulated anabolic genes (ACAN, COL2A1, SOX9, TIMP-1/3), enhanced GAG production, and downregulated IL-1β, IL-6, IL-8, NOS2, MMP-1, and MMP-13 expression. Furthermore, senescence was markedly reduced in BM-MSCsec-treated hNPCs, with decreased β-galactosidase activity and lower p16 and p21 expression. CONCLUSIONS: Our findings support the potential of BM-MSCsec as a cell-free therapeutic strategy for IDD. The IL-1β-preconditioned secretome reduced hNPC death and senescence, mitigated inflammation and oxidative stress, and promoted ECM preservation, highlighting its potential to counteract key processes driving IDD.