title: Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 in vitro.
abstract: BACKGROUND
  Chondrocytes experience a hypertonic environment compared with plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to nonphysiological conditions. During in vitro expansion, chondrocytes quickly lose their specialized phenotype, making them inappropriate for cell-based regenerative strategies. We aimed to elucidate the effects of tonicity during isolation and in vitro expansion on chondrocyte phenotype.
  
  
  METHODS
  Human articular chondrocytes were isolated and subsequently expanded at control tonicity (280 mOsm) or at moderately elevated, physiological tonicity (380 mOsm). The effects of physiological tonicity on chondrocyte proliferation and chondrogenic marker expression were evaluated. The role of Tonicity-responsive Enhancer Binding Protein in response to physiological tonicity was investigated using nuclear factor of activated T-cells 5 (NFAT5) RNA interference.
  
  
  RESULTS
  Moderately elevated, physiological tonicity (380 mOsm) did not affect chondrocyte proliferation, while higher tonicities inhibited proliferation and diminished cell viability. Physiological tonicity improved expression of chondrogenic markers and NFAT5 and its target genes, while suppressing dedifferentiation marker collagen type I and improving type II/type I expression ratios >100-fold. Effects of physiological tonicity were similar in osteoarthritic and normal (nonosteoarthritic) chondrocytes, indicating a disease-independent mechanism. NFAT5 RNA interference abolished tonicity-mediated effects and revealed that NFAT5 positively regulates collagen type II expression, while suppressing type I.
  
  
  CONCLUSIONS
  Physiological tonicity provides a simple, yet effective, means to improve phenotypical characteristics during cytokine-free isolation and in vitro expansion of human articular chondrocytes. Our findings will lead to the development of improved cell-based repair strategies for chondral lesions and provides important insights into mechanisms underlying osteoarthritic progression.
authors.affiliation: Department of Orthopaedics, Erasmus MC, University Medical Center Rotterdam, Dr, Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.
authors.forename: Anna E
authors.initials: AE
authors.lastname: van der Windt
authors.forename: Esther
authors.initials: E
authors.lastname: Haak
authors.forename: Ruud H J
authors.initials: RH
authors.lastname: Das
authors.forename: Nicole
authors.initials: N
authors.lastname: Kops
authors.forename: Tim J M
authors.initials: TJ
authors.lastname: Welting
authors.forename: Marjolein M J
authors.initials: MM
authors.lastname: Caron
authors.forename: Niek P
authors.initials: NP
authors.lastname: van Til
authors.forename: Jan A N
authors.initials: JA
authors.lastname: Verhaar
authors.forename: Harrie
authors.initials: H
authors.lastname: Weinans
authors.forename: Holger
authors.initials: H
authors.lastname: Jahr
mesh_terms: D053674:Aggrecans / Q000378:metabolism; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D024042:Collagen Type I / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology*; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D009994:Osmolar Concentration; D010641:Phenotype
chemical_list: D053674:Aggrecans; D024042:Collagen Type I; D024043:Collagen Type II; D006982:Hypertonic Solutions; D050778:NFATC Transcription Factors