Abstract
Early clinical studies investigating the effects of delivery of mesenchymal stromal cells (MSCs) to degenerated intervertebral discs have shown promising results, but with an incomplete understanding of the therapeutic mechanism(s) of action. To address this, we have developed a 3D co-culture system to unravel the biological interaction between MSCs and nucleus pulposus (NP) cells. Alginate constructs were created using a biphasic configuration consisting of a cylindrical shell with an encapsulated bead. Human NP cells were seeded in monolayer or encapsulated within alginate and cultured in hypoxia with variations of pH, osmolarity and growth factors (n = 6) to replicate healthy or degenerative conditions. Wells and gels were fixed and stained for ECM content, and retrieved cells and media were analysed for ECM and inflammatory factor expression. Encapsulated hNPCs showed no migration from either alginate structure and full bead separation was achieved over 14 days, reinforcing the construct as a separable 3D co-culture method. Addition of the degenerative growth factors TNFα and IL-1β as well as the adjustment of media pH to degenerative levels (pH 6.8) caused the hNPCs to decrease in size and proliferate significantly higher than control levels. TGF-β3 addition showed higher incidence of aggrecan deposition over addition of IL-1β. Addition of FGF2 altered cell morphology and ECM deposition including formation of pseudo lamellae, indicating a phenotype shift toward annulus fibrosis cells as shown in late-stage degenerative disc disease. The data from this study will be used in future MSC:NPC co-cultures to determine immunoregulatory interactions in a degenerative environment.
