title: Loss of notochordal cell phenotype in 3D-cell cultures: implications for disc physiology and disc repair.
abstract: BACKGROUND
  Embryonic notochordal disc nucleus cells (NC) have been identified to protect disc tissue against disc degeneration but in human beings NC phenotype gets lost with aging and the pathophysiological mechanisms are poorly understood. NC may stimulate other cells via soluble factors, and NC-conditioned medium can be used to stimulate matrix production of other disc cells and mesenchymal stem cells and thus may be of special interest for biological disc repair. As this stimulatory effect is associated with the NC phenotype, we investigated how cell morphology and gene-expression of the NC phenotype changes with time in 3D-cell culture.
  
  
  METHODS
  NC and inner annulus chondrocyte-like cells (CLC) from immature pigtails (freshly isolated cells/tissue, 3D-alginate beads, 3D-clusters) were cultured for up to 16 days under normoxia and hypoxia. Protein-expression was analysed by immunohistology and gene-expression analysis was carried out on freshly isolated cells and cultured cells. Cell morphology and proliferation were analysed by two-photon-laser-microscopy.
  
  
  RESULTS
  Two-photon-laser-microscopy showed a homogenous and small CLC population in the inner annulus, which differed from the large vacuole-containing NC in the nucleus. Immunohistology found 93 % KRT8 positive cells in the nucleus and intracellular and pericellular Col2, IL6, and IL12 staining while CLC were KRT8 negative. Freshly isolated NC showed significantly higher KRT8 and CAIII but lower Col2 gene-expression than CLC. NC in 3D-cultures demonstrated significant size reduction and loss of vacuoles with culture time, all indicating a loss of the characteristic NC morphology. Hypoxia reduced the rate of decrease in NC size and vacuoles. Gene-expression of KRT8 and CAIII in NC fell significantly early in culture while Col2 did not decrease significantly within the culture period. In CLC, KRT8 and CAIII gene-expression was low and did not change noticeably in culture, whereas Col2 expression fell with time in culture.
  
  
  CONCLUSIONS
  3D-culture caused a rapid loss of NC phenotype towards a CLC phenotype with disappearance of vacuoles, reduced cell size, increased proliferation, and gene-expression changes. These findings may be related to NC nutritional demands and support the latest hypothesis of NC maturation into CLC opposing the idea that NC get lost in human discs by cell death or apoptosis to be replaced by CLC from the inner annulus.
authors.affiliation: Department of Orthopaedic Surgery and Trauma Surgery, Heidelberg University Hospital, 69118, Heidelberg, Germany.
authors.forename: G W
authors.initials: GW
authors.lastname: Omlor
authors.forename: A G
authors.initials: AG
authors.lastname: Nerlich
authors.forename: U K
authors.initials: UK
authors.lastname: Tirlapur
authors.forename: J P
authors.initials: JP
authors.lastname: Urban
authors.forename: T
authors.initials: T
authors.lastname: Guehring
mesh_terms: D000818:Animals; D017209:Apoptosis; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002478:Cells, Cultured; D015870:Gene Expression; D007403:Intervertebral Disc / Q000293:injuries / Q000473:pathology / Q000502:physiology* / Q000601:surgery*; D055959:Intervertebral Disc Degeneration; D059630:Mesenchymal Stem Cells; D018613:Microscopy, Confocal; D009672:Notochord / Q000166:cytology*; D010641:Phenotype; D013552:Swine