title: Dynamic pressurization induces transition of notochordal cells to a mature phenotype while retaining production of important patterning ligands from development.
abstract: BACKGROUND
  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.
  
  
  METHODS
  Porcine 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.
  
  
  RESULTS
  Daily 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).
  
  
  CONCLUSIONS
  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 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.
authors.forename: Devina
authors.initials: D
authors.lastname: Purmessur
authors.forename: Clare C
authors.initials: CC
authors.lastname: Guterl
authors.forename: Samuel K
authors.initials: SK
authors.lastname: Cho
authors.forename: Marisa C
authors.initials: MC
authors.lastname: Cornejo
authors.forename: Ying W
authors.initials: YW
authors.lastname: Lam
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Ballif
authors.forename: James C Iatridis
authors.initials: JC
authors.lastname: Laudier
authors.forename: James C
authors.initials: JC
authors.lastname: Iatridis
mesh_terms: D053674:Aggrecans / Q000235:genetics; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D018507:Gene Expression Regulation, Developmental; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D053823:Hedgehog Proteins / Q000378:metabolism; D006874:Hydrostatic Pressure; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism; D008024:Ligands; D009569:Nitric Oxide / Q000378:metabolism; D009672:Notochord / Q000166:cytology* / Q000378:metabolism; D010641:Phenotype; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D040121:Semaphorin-3A / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014746:Vimentin / Q000378:metabolism; D000099176:Noggin Protein
chemical_list: D053674:Aggrecans; D002352:Carrier Proteins; D018840:HSP70 Heat-Shock Proteins; D053823:Hedgehog Proteins; D008024:Ligands; D009569:Nitric Oxide; D020543:Proteome; D040121:Semaphorin-3A; D014746:Vimentin; D000099176:Noggin Protein