Modelling disc degeneration-induced neurovascular invasion on a chip for chronic low back pain therapeutic testing.

Abstract

Intervertebral disc (IVD) degeneration is the most common musculoskeletal disorder worldwide. Yet, the underlying pathological processes remain largely unclear, primarily due to the lack of suitable in vitro models that accurately replicate the complex interactions of vascular and neural growth, inflammatory cytokine secretion, and immune cell infiltration that contribute to disc degeneration. To address this gap, we developed an affordable microfluidic IVD model using a 5-axis computer numerical control (CNC) micromilling machine with a multi-chamber culture design featuring a central disc zone divided by micropillars to accommodate nucleus pulposus (NP) and annulus fibrosus (AF). The design also incorporates two peripheral zones for co-culturing neurovascular units, separated by radial microgratings to maintain the avascular and aneural milieu of healthy discs. Under healthy conditions, our microfluidic device effectively restricted the migration of neural and endothelial cells when co-cultured with NP cells. Conversely, upon IL-1β-induced inflammation, we observed significant neural (286 ± 58 µm) and endothelial (884 ± 214 µm) cell migration towards the NP cells. Beyond neurovascular invasion, our IVD-on-a-chip provided valuable insights into immune cell dynamics, revealing that M1 macrophage infiltration, not just neovascularization, plays a key role in the progression of disc degeneration. Strikingly, our findings revealed that therapeutic interventions using glycosaminoglycan supplementation and celecoxib treatment resulted in a fourfold reduction in macrophage infiltration and over a twentyfold decrease in neural invasion compared to the inflamed control group. This highlights the potential of our system for drug testing aimed at preventing neovascularization and innervation, which are typically associated with discogenic pain. STATEMENT OF SIGNIFICANCE: Most spine abnormalities are directly linked to intervertebral disc (IVD) degeneration, accounting for 40-50% of chronic lower back pain cases, often leading to lumbar disc prolapse, radiculopathy, spinal stenosis, and myelopathy. In fact, many aspects of the mechanisms behind disc degeneration remain elusive due to the lack of in vitro models replicating the complex inflammatory and catabolic processes involved. Hence, this study presents an improved micro-physiological IVD-on-a-chip platform that captures key characteristics of symptomatic disc degeneration, starting with inflammatory cytokine-induced nucleus pulposus dysregulation, neovascularization, macrophage infiltration, and sensory neuron ingrowth. The developed IVD-on-a-chip system offers new capabilities for investigating local degenerative responses that drive disc-related pain, serving as a high-throughput platform for testing potential drugs inhibiting complex neuro-immuno-vascular interactions.