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STRUCTURED CO-CULTURE OF STEM CELLS AND DISC CELLS ENHANCES MATRIX SYNTHESIS

Intervertebral disc tissue engineering holds the promise of a minimally-invasive low-back pain treatment, but many of the biological processes remain unclear. Our goal is to identify mechanisms involved in disc regeneration to optimize tissue engineering strategies. We explored the benefits of co-culturing nucleus pulposus cells (NPC) and adult mesenchymal stem cells (MSC) using a 3D system that exploits embryonic processes such as tissue induction and condensation. Our hypothesis is that signaling interactions between MSC and NPC regulate cell differentiation and matrix production. If this proves true, modulation of their structural organization and cell number can enhance therapeutic benefits beyond using MSC alone. Human MSC and bovine NPC were cultured using a novel spherical bi-layer where one cell type forms an inner sphere enclosed within a shell of the other cell type. The inner versus outer cell type and ratio of cell density were varied with pellets cultured in growth medium for up to three weeks. Pellets were harvested and proteoglycan content (GAG) was quantified. After 14 days, a spherical bi-layer pellet with MSC on the inside and NPC on the outside at a ratio of 25/75 produced up to 48% more GAG then all other co-culture conformations and ratios. After 21 days, the spherical bi-layer arrangement still produced the most GAG but it no longer mattered which cell was on the inside vs outside. At this stage, it is likely that all MSC have differentiated independent of their position in the bi-layer. However, there was still a difference between pellets with different ratios, where MSC/NPC at 25/75 produced the most GAG. Our data show that both organization and relative ratio of the cell types play important roles at different MSC differentiation stages, and that structured communication between MSC and NPC enhances the efficacy of stem-cell-based strategies for nucleus regeneration. I N T E R N A T I O N A L S O C I E T Y F O R T H E S T U D Y O F L U M B A R S P I N E