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

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. 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. 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. 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.