Nucleus pulposus-derived mesenchymal stem cells (NP-MSCs) are suitable cell candidates for intervertebral disc (IVD) regeneration. However, little work has been done to determine the proliferation and chondrogenic differentiation of NP-MSCs in the hyperosmotic microenvironment of IVD. This study aimed to investigate the influence of the hyperosmolarity of IVD on the proliferation and chondrogenic differ-entiation of NP-MSCs. NP-MSCs were cultured in media of 300, 400, 430, and 500 mOsm/L, mimicking the osmotic pressures of serious degenerative, moderately degenerative, and healthy IVD. Cell proliferation was measured by CCK-8 assay. The expression of aggrecan, collagen I, and collagen II were measured by gene and protein expression analysis. Alcian blue and dimethylmethylene blue assay were used to investigate the accumulation of sulfate glycosaminoglycan. The regulation role of extracellular signal-regulated kinase (ERK) pathway was also analyzed. The results showed that, compared to 300 mOsm/L, hyperosmolarity of healthy IVD (430 and 500 mOsm/L) inhibited the proliferation and chondrogenic differentiation of NP-MSCs. The relative hypoosmotic condition of moderately degenerative IVD (400 mOsm/L) led to great proliferation and chondrogenic differentiation capacity. The ERK pathway was activated by the hyperosmolarity; inhibition of the ERK pathway abolished the difference in cell proliferation between the 300 mOsm/L and the hyperosmotic conditions, and enhanced chondrogenic differentiation. In conclusion, hyperosmolarity of IVD had a significant impact on the proliferation and chondrogenic differentiation of NP-MSCs. The ERK pathway was involved in the inhibition of proliferation and chondrogenic differentiation of NP-MSCs by the hyperosmolarity of IVD. The relative hypo-osmotic condition prevailing in degenerative discs offers a more permissive microenvironment for NP-MSCs.

Keywords:
Intervertebral disc, Nucleus pulposus-derived mesenchymal stem cell, Microenvironment, Osmolarity, Proliferation, Chondrogenic differentiation
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