Dynamic pressurization induces transition of notochordal cells to a mature phenotype while retaining production of important patterning ligands from development

Abstract

IntroductionNotochordal 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.MethodsPorcine NC-rich NP tissue was cultured and loaded with hydrostatic pressure (0.5 to 2 MPa at 0.1 Hz for 2 hours) either Daily, for 1 Dose, or Control (no pressurization) groups for up to eight days. Cell phenotype and tissue maturation was characterized with measurements of cell viability, cytomorphology, nitric oxide, metabolic activity, matrix composition, gene expression, and proteomics.ResultsDaily pressurization induced transition of NCs to small NP cells with 73.8%, 44%, and 28% NCs for Control, 1 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).DiscussionNP 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 nonvacuolated NP cells probably via differentiation as evidenced by high cell viability, lack of nitric oxide and downregulation 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.

The species-specific regenerative effects of notochordal cell-conditioned medium on chondrocyte-like cells derived from degenerated human intervertebral discs

“…Differential deposition of extracellular matrix by species-specific NCCM Canine and porcine NC-secreted factors have already shown promising potential for IVD regeneration (Abbott et al, 2012;Gantenbein et al, 2014;Korecki et al, 2010;Mehrkens et al, 2013;Purmessur et al, 2011). The impact of human NC-secreted factors and the differential effects of xenogeneic NCCM on human CLCs, however, have never been investigated.…”

“…chondrodystrophic dogs) coincides with the onset of IVD degeneration. NC-conditioned medium (NCCM, containing factors secreted by NCs) and NC:CLC co-culture stimulate in vitro differentiation of MSCs into a NP-like phenotype Korecki et al, 2010;Purmessur et al, 2011), protect the NP from apoptosis (Erwin et al, 2011;Mehrkens et al, 2013), and/or increase CLC proliferation and GAG production (Abbott et al, 2012;Aguiar et al, 1999;Gantenbein et al, 2014;Potier et al, 2014). In addition, NCs significantly stimulate CLC activity and GAG production (Gantenbein-Ritter et al, 2012) and produce more GAGs than CLCs (Saggese et al, 2014).…”

“…During IVD maturation, a transition in cell phenotype takes place from large, vacuolated notochordal cells (NCs) to smaller, non-vacuolated CLCs (Hunter et al, 2004;Purmessur et al, 2013b). During this process, the vacuolated NCs obtain a transitional phenotype, i.e.…”

“…NC-based regenerative strategies are being developed, using canine non-chondrodystrophic (NCD) and porcine donors because of easy availability of their NC-rich NP tissue (Bach et al, 2014). Although regenerative effects of canine and porcine NC-secreted factors on human CLCs/MSCs have been demonstrated in several studies (Abbott et al, 2012;Korecki et al, 2010;Mehrkens et al, 2013;Purmessur et al, 2011), the impact of human NC-secreted factors has never been determined, most probably due to limited availability of human NC-rich NP tissue. It is, however, important to define if the human CLC response to homologous human NC-secreted factors is superior to canine and porcine NC-secreted factors.…”

“…NCs have also been described to exert potential symptom modifying actions, e.g. anti-inflammatory, anti-neurogenic and antiangiogenic effects by secreting factors as sonic hedgehog, noggin, connective tissue growth factor and chondroitin sulphate (Cornejo et al, 2015;Gantenbein et al, 2014;Purmessur et al, 2013a). Thus, NCs secrete factors with auspicious potential, making them an interesting target for regenerative and/or symptom modifying therapies (Bach et al, 2014).…”