Dynamic Hydrostatic Pressure Regulates Nucleus Pulposus Phenotypic Expression and Metabolism in a Cell Density-Dependent Manner

Dynamic hydrostatic pressure (HP) loading can modulate nucleus pulposus (NP) cell metabolism, extracellular matrix (ECM) composition, and induce transformation of notochordal NP cells into mature phenotype. However, the effects of varying cell density and dynamic HP magnitude on NP phenotype and metabolism are unknown. This study examined the effects of physiological magnitudes of HP loading applied to bovine NP cells encapsulated within three-dimensional (3D) alginate beads. Study 1: seeding density (1 M/mL versus 4 M/mL) was evaluated in unloaded and loaded (0.1 MPa, 0.1 Hz) conditions. Study 2: loading magnitude (0, 0.1, and 0.6 MPa) applied at 0.1 Hz to 1 M/mL for 7 days was evaluated. Study 1: 4 M/mL cell density had significantly lower adenosine triphosphate (ATP), glycosaminoglycan (GAG) and collagen content, and increased lactate dehydrogenase (LDH). HP loading significantly increased ATP levels, and expression of aggrecan, collagen I, keratin-19, and N-cadherin in HP loaded versus unloaded groups. Study 2: aggrecan expression increased in a dose dependent manner with HP magnitude, whereas N-cadherin and keratin-19 expression were greatest in low HP loading compared to unloaded. Overall, the findings of the current study indicate that cell seeding density within a 3D construct is a critical variable influencing the mechanobiological response of NP cells to HP loading. NP mechanobiology and phenotypic expression was also found to be dependent on the magnitude of HP loading. These findings suggest that HP loading and culture conditions of NP cells may require complex optimization for engineering an NP replacement tissue.