An-Gi Kim

Intervertebral disc (IVD) degeneration (IVDD) is a major contributor to chronic low back pain, representing a substantial burden on the spinal healthcare system and serving as a leading cause of long-term disability worldwide. Biomimetic in vitro models that accurately replicate histological characteristics, three-dimensional structures, and multicellular interactions are lacking. Consequently, monocultures of cell lines and two-dimensional culture models are still used to study the pathomechanisms of IVDD. We established functional multicellular IVD spheroid cultures using primary human annulus fibrosus and nucleus pulposus cells. The spheroids maintained the IVD-specific phenotype, including hypoxic conditions and lamellar structures. Additionally, the spheroid markedly increased the expression level of inflammatory mediators and chemokines in the presence of the pro-inflammatory cytokine IL-1β, a master regulator of IVDD. Furthermore, we implemented our microfluidic chemotaxis platform to investigate microglial neuroinflammation in response to our reconstituted IVD spheroid models. Transcriptome sequencing revealed that microglia stimulated by potential contributing factors derived from IVDD spheroids exhibited a significant upregulation of the expression levels of chemotactic factors and cytokines including CCL-2, -3, -4, -5, IL-8 and IL-6 (p < 0.05). Moreover, we observed considerable activation and infiltration of microglia induced by soluble factors derived from IVDD spheroids, which are expected to occur during IVDD. The chemotactic effects on microglia were reduced upon the neutralization of CCL-2 or IL-8 or inhibition of NF-κB signaling. These robust in vitro IVD spheroids can be used to model IVDD and provide a valuable platform for the assessment and development of IVDD therapeutics.

Degenerative cascades of the intervertebral disc (IVD) are characterized by the presence of immune cells like monocytes, macrophages, and leukocytes, which contribute to inflammation. Previous in vitro studies on monocyte chemotaxis in the presence of chemical or mechanical stimulation were unable to establish the effects of endogenous stimulating factors from resident IVD cells, or fully understand macrophage and monocyte differentiation pathways in IVD degeneration. Our study simulates monocyte extravasation using a fabricated microfluidic chemotaxis IVD organ-on-a-chip (IVD organ chip), which models the geometry of IVD, chemoattractant diffusion, and infiltration of immune cells. Additionally, the fabricated IVD organ chip mimics stepwise monocyte infiltration and differentiation into macrophages in the degenerative nucleus pulposus (NP) induced by IL-1β. We find that naïve NP cells do not recruit THP-1 monocyte-like cells, but degenerative NP cells recruit and accumulate macrophages through chemo-gradient channels. Furthermore, the differentiated and migrated THP-1 cells show phagocytic activity around inflammatory NP cells. Our in vitro model of monocyte chemotaxis with degenerative NP on an IVD organ chip depicts the sequential processes of monocyte migration/infiltration, monocyte-to-macrophage differentiation, and accumulation. Using this platform to gain a deeper understanding of monocyte infiltration and differentiation processes can provide insights into the pathophysiology of the immune response in degenerative IVD.