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Background: Daily physiologic loading causes fluctuations in hydration of the intervertebral disc (IVD); thus, the embedded cells experience cyclic alterations to their osmotic environment. These osmotic fluctuations have been described as a mechanism linking mechanics and biology, and have previously been shown to promote biosynthesis in chondrocytes. However, this phenomenon has yet to be fully interrogated in the IVD. Additionally, the specialized extracellular matrix surrounding the cells, the pericellular matrix (PCM), transduces the biophysical signals that cells ultimately experience. While it is known that the PCM is altered in disc degeneration, whether it disrupts normal osmotic mechanotransduction has yet to be determined. Thus, our objectives were to assess: (1) whether dynamic osmotic conditions stimulate biosynthesis in nucleus pulposus cells, and (2) whether pericellular heparan sulfate proteoglycans (HSPGs) modulate the biosynthetic response to osmotic loading.Methods: Bovine nucleus pulposus cells isolated with retained PCM were encapsulated in 1.5% alginate beads and treated with or without heparinase III, an enzyme that degrades the pericellular HSPGs. Beads were subjected to 1 h of daily isoosmotic, hyper-osmotic, or hypo-osmotic loading for 1, 2, or 4 weeks. At each timepoint the total amount of extracellular and pericellular sGAG/DNA were quantified.Additionally, whether osmotic loading triggered alterations to HSPG sulfation was assessed via immunohistochemistry for the heparan sulfate 6-O-sulfertransferase 1 (HS6ST1) enzyme.Results: Osmotic loading significantly influenced sGAG/DNA accumulation with a hyper-osmotic change promoting the greatest sGAG/DNA accumulation in the pericellular region compared with iso-osmotic conditions. Heparanase-III treatment significantly reduced extracellular sGAG/DNA but pericellular sGAG was not affected. HS6ST1 expression was not affected by osmotic loading. Conclusion:Results suggest that hyper-osmotic loading promotes matrix synthesis and that modifications to HSPGs directly influence the metabolic responses of cells to osmotic fluctuations. Collectively, results suggest degeneration-associated