title: Osteoarthritic cartilage explants affect extracellular matrix production and composition in cocultured bone marrow-derived mesenchymal stem cells and articular chondrocytes.
abstract: BACKGROUND
  In the present study, we established a novel in vitro coculture model to evaluate the influence of osteoarthritis (OA) cartilage explants on the composition of newly produced matrix and chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs) and the phenotype of OA chondrocytes. In addition, we included a "tri-culture" model, whereby a mixture of BMSCs and chondrocytes was cultured on the surface of OA cartilage explants.
  
  
  METHODS
  Gene expression analysis, protein and glycosaminoglycan (GAG) assays, dot-blot, immunofluorescence, and biomechanical tests were used to characterize the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiated BMSCs and a mix thereof. We compared articular cartilage explant cocultures with BMSCs, chondrocytes, and mixed cultures (chondrocytes and BMSCs 1:1) embedded in fibrin gels with fibrin gel-embedded cells cultured without cartilage explants (monocultures).
  
  
  RESULTS
  In general, co- and tri-cultured cell regimens exhibited reduced mRNA and protein levels of collagens I, II, III, and X in comparison with monocultures, whereas no changes in GAG synthesis were observed. All co- and tri-culture regimens tended to exhibit lower Young's and equilibrium modulus compared with monocultures. In contrast, aggregate modulus and hydraulic permeability seemed to be higher in co- and tri-cultures. Supernatants of cocultures contained significant higher levels of interleukin-1 beta (IL-1β), IL-6, and IL-8. Stimulation of monocultures with IL-1β and IL-6 reduced collagen gene expression in BMSCs and mixed cultures in general but was often upregulated in chondrocytes at late culture time points. IL-8 stimulation affected BMSCs only.
  
  
  CONCLUSIONS
  Our results suggest an inhibitory effect of OA cartilage on the production of collagens. This indicates a distinct modulatory influence that affects the collagen composition of the de novo-produced ECM from co- and tri-cultured cells and leads to impaired mechanical and biochemical properties of the matrix because of an altered fibrillar network. We suggest that soluble factors, including IL-1β and IL-6, released from OA cartilage partly mediate these effects. Thus, neighbored OA cartilage provides inhibitory signals with respect to BMSCs' chondrogenic differentiation and matrix composition, which need to be accounted for in future cell-based OA treatment strategies.
authors.forename: Michaela
authors.initials: M
authors.lastname: Leyh
authors.forename: Andreas
authors.initials: A
authors.lastname: Seitz
authors.forename: Lutz
authors.initials: L
authors.lastname: Dürselen
authors.forename: Hans-Robert
authors.initials: HR
authors.lastname: Springorum
authors.forename: Peter
authors.initials: P
authors.lastname: Angele
authors.forename: Anita
authors.initials: A
authors.lastname: Ignatius
authors.forename: Joachim
authors.initials: J
authors.lastname: Grifka
authors.forename: Susanne
authors.initials: S
authors.lastname: Grässel
mesh_terms: D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002358:Cartilage, Articular / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D018920:Coculture Techniques / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D010003:Osteoarthritis / Q000503:physiopathology*; D060888:Real-Time Polymerase Chain Reaction; D059467:Transcriptome