Effect of Long-Term Osmotic Loading Culture on Matrix Synthesis from Intervertebral Disc Cells

The intervertebral disc is a highly hydrated tissue that acts to absorb and distribute large complex loads placed on the spine. Diurnal loading and disc degeneration causes significant changes in water volume and proteoglycan content, which alters the internal osmotic environment. Short-term osmotic loading alters disc cell gene expression; however, the long-term effect of osmotic loading on disc cell matrix synthesis is not well understood. The objective of this study was to determine the effect of long-term osmotic loading on matrix turnover and proliferation by juvenile and adult cells from the nucleus pulposus (NP) and the cartilaginous endplate (EP). Matrix synthesis was evaluated using pellets and a 3D agarose system, which has been used for developing engineered tissues. Intervertebral discs were acquired from juvenile and adult cows. Cells were acquired through enzymatic digestion and expanded in culture. Pellets were formed through centrifugation, and constructs were created by encapsulating cells within 2% w/v agarose hydrogel. Pellets and constructs were cultured up to 42 days in chemically defined medium with the osmolality adjusted to 300, 400, or 500 mOsm/kg. EP cells were evaluated as a chondrocyte comparison to chondrocyte-like NP cells. Pellet and agarose cultures of juvenile NP and EP cells demonstrated similarities with respect to cell proliferation and functional mechanical properties. Cell proliferation decreased significantly with increased osmotic loading. The final compressive Young's modulus of juvenile NP cells was 10-40× greater than initial properties (i.e., day 0) and was greater than the final Young's modulus of adult NP and juvenile EP constructs. In juvenile NP constructs, there were no significant differences in GAG content with respect to osmotic loading. However, GAG synthesis and mechanical properties were greatest for the 400 mOsm/kg group in adult NP constructs. Taken together, the results presented here suggest a tradeoff between cell proliferation and matrix production under osmotic loading conditions. In conclusion, culturing disc cells in an osmotic environment that best mimics the healthy disc environment (400 mOsm/kg) may be ideal for balancing cell proliferation, matrix production, and mechanical properties of engineered disc tissues.