Dynamic pressurization induces transition of notochordal cells to a mature phenotype while retaining production of important patterning ligands from development
Arthritis Research & Therapy 2013, 15:R122 doi:10.1186/ar4302Published: 17 September 2013
Notochordal cells (NCs) pattern aneural and avascular intervertebral discs (IVDs), and their disappearance is associated with onset of IVD degeneration. This study induced and characterized the 'maturation' of nucleus pulposus (NP) tissue from a gelatinous NC-rich structure to a matrix rich structure populated by small NP cells using dynamic pressurization in an ex vivo culture model, and also identified soluble factors from NCs with therapeutic potential.
Porcine NC-rich NP tissue was cultured and loaded with hydrostatic pressure (0.5-2MPa@0.1Hz for 2 hours) either daily, for one dose, or control (no pressurization) for up to 8 days. Cell phenotype and tissue maturation was characterized with measurements of cell viability, cytomorphology, nitric oxide, metabolic activity, matrix composition, gene expression, and proteomics.
Daily pressurization induced transition of NCs to small NP cells with 73.8%, 44%, and 28% NCs for control, one dose and daily groups, respectively (P<0.0002) and no relevant cell death. Dynamic loading 'matured' NP tissue by significantly increasing metabolic activity and accumulating safranin-O-stained matrix. Load-induced maturation was also apparent from the significantly decreased glycolytic, cytoskeletal (vimentin) and stress-inducible (HSP70) proteins assessed with proteomics. Loading increased the production of bioactive proteins sonic hedgehog (SHH) and noggin, and maintained semaphorin3A (Sema3A).
NP tissue maturation was induced from dynamic hydrostatic pressurization in a controlled ex vivo environment without influence from systemic effects or surrounding structures. NCs transitioned into small non-vacuolated NP cells likely via differentiation as evidenced by high cell viability, lack of nitric oxide and down-regulation of stress-inducible and cytoskeletal proteins. SHH, Sema3A, and Noggin, which have patterning and neurovascular-inhibiting properties, were produced in both notochordal and 'matured' porcine NP. Results therefore provide an important piece of evidence suggesting the transition of NCs to small NP cells is a natural part of aging and not the initiation of degeneration. Bioactive candidates identified from young porcine IVDs may be isolated and harnessed for therapies to target discogenic back pain.