"Lower back pain due to intervertebral disc (IVD) degeneration is a major health problem and often starts in the centre of the IVD, called the nucleus pulposus (NP). NP cells are considered potential candidates for cell-based therapies to induce regeneration of the IVD. However, as the availability of NP cells is limited, extensive in vitro expansion is needed prior to transplantation which results into loss of the NP phenotype hampering therapeutic use of NP cells. A promising strategy to optimize NP cell expansion is to employ physiological stimuli e.g. osmolarity, which is known to decrease during the degenerative process1. Preliminary work on human NP cells indicated that increasing medium osmolarity during expansion has beneficial effects2. Here, we present a comprehensive investigation into the effects of increasing the medium osmolarity during expansion on the phenotype of dog NP cells and their regenerative capacity after re-differentiation in a 3D culture model.
NP cells of 6 Beagle donors (mildly degenerated IVDs) were expanded for 2 passages in expansion medium with a standard osmolarity of 300 mOsm/L or adjusted to 400 or 500 mOsm/L (mimicking levels in the healthy IVD). Following expansion, cells from each condition were re-differentiated for 14 days in 3D microaggregates in chondrogenic medium with a standard osmolarity of 300 mOsm/L to mimic placing them back in the degenerated in vivo IVD environment. Readout parameters during expansion were focussed on cell morphology, phenotype and proliferation rate. After re-differentiation we assessed the phenotype and regenerative capacity of the NP cells.
NP cells expanded at an increased expansion medium osmolarity (500 mOsm/L) were able to maintain an in vivo-like, rounded (less fibroblast-like) cell morphology at the expense of a lower cell proliferation rate. Further, expansion in 500 mOsm/L lead to a significant increase of several healthy NP cell and progenitor markers at gene expression level (KRT18, ACAN, COL2, CD73, CD90) and protein level (ACAN, PAX1, CD24, TIE2, CD73) compared to NP cells expanded in standard medium osmolarity (300 mOsm/L). Immunohistochemical stainings for ACAN, PAX1, CD24 and TIE2 and qualitative and quantitative assessment of extracellular matrix deposition showed that the NP cells expanded at 500 mOsm/L were able to retain their NP cell phenotypic markers and regenerative capacity in the 3D re-differentiation culture model.
Our findings show that increasing medium osmolarity improves the NP cell phenotype during in vitro expansion, and with that, facilitates the potential of NP cells to be used for regenerative cell-based treatments.
This project has received funding from the European Union’s Horizon 2020 research and innovation programme.
- Thorpe A.A. et al., JOR spine. Sep;1(3):e1027 (2018)
- Krouwels, A et al., Tissue Engineering Part C: Methods. 24(4), 222-232 (2018)"