Skip to main content
SpringerLink
Log in

Analysis of Chondrocyte Functional Markers and Pericellular Matrix Components by Flow Cytometry

  • Protocol
Cartilage and Osteoarthritis

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 100))

Abstract

Flow cytometry has been used as a procedure to characterize the phenotype and function of human articular cartilage cells cultured as monolayers or in gelled artificial matrices. Procedures allowing intact cells with their cell-associated matrix, to be obtained have been described. Appropriate monoclonal antibodies have allowed plasma membrane-associated proteins, e.g., growth factors and cytokine receptors, as well as the cell-associated extracellular matrix macromolecules, to be studied. Intracellular compounds have been traced in permeabilized cells after blocking of their intracellular transport and secretion mechanisms. We report the use of fluorescent dye-labeled monoclonal antibodies or specific binding proteins against extracellular matrix compounds such as hyaluronan, aggrecan, types I and II collagen, and fibronectin. The autocrine and paracrine growth factor and cytokine pathways considered include the insulin-like growth factor-1 (IGF-1)/IGF receptor I (IGFRI), and the transforming growth factor-β1 (TGF-β1)/TGF-β receptor II (TGF-βRII) cascades, as well as the interleukin-1α/β (IL-1α/β)/interleukin-1 receptors I and II (IL-1RI and II) systems. Catabolic enzymes that mediate extracellular matrix turnover, e.g., some matrix metalloproteinases and their natural inhibitors, were also studied. Finally, flow cytometry was used to assess the results of some pharmacological interventions on the aforementioned variables in cultured chondrocytes.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Bell, R. S., Bouret, L. A., Bell, D. F., Gebhardt, M. C., Rosenberg, A., and Berrey, H. B. et al. (1988) Evaluation of fluorescein diacetate for flow cytometric determination of cell viability in orthopaedic research. J. Orthop. Res. 6, 467–74.

    Article  CAS  PubMed  Google Scholar 

  2. Vincent, F., Brun, H., Clain, E., Ronot, X., and Adolphe, M. (1989) Effects of oxygen-free radicals on proliferation kinetics of cultured rabbit articular chondrocytes. J. Cell Physiol. 141, 262–266.

    Article  CAS  PubMed  Google Scholar 

  3. Jaffray, P., Ronot, X., Adolphe, M., Fontagne, J., and Lechat, P. (1984) Effects of D-penicillamine on growth and cell cycle kinetics of cultured rabbit articular chondrocytes. Ann. Rheum. Dis. 43, 333–338.

    Article  CAS  PubMed  Google Scholar 

  4. Thuong-Guyot, M., Domarle, O., Pocidalo, J. J., and Hayem, G. (1994) Effects of fluoroquinolones on cultured articular chondrocytes flow cytometric analysis of free radical production. J. Pharmacol. Exp. Ther. 271, 1544–1549.

    CAS  PubMed  Google Scholar 

  5. Westacott, C. I., Atkins, R. M., Dieppe, P. A., and Elson, C. J. (1994) Tumor necrosis factor-alpha receptor expression on chondrocytes isolated from human articular cartilage. J. Rheumatol. 21, 1710–1715.

    CAS  PubMed  Google Scholar 

  6. Martel-Pelletier, J., McCollum, R., DiBattista, J., et al. (1992) The interleukin-1 receptor in normal and osteoarthritic human articular chondrocytes. Identification as the type I receptor and analysis of binding kinetics and biologic function. Arthritis Rheum. 35, 530–340.

    Article  CAS  PubMed  Google Scholar 

  7. Lapadula, G., Iannione, F., Zuccaro, C., et al. (1998) Chondrocyte phenotyping in human osteoarthritis. Clin. Rheumatol. 17, 99–104

    Article  CAS  PubMed  Google Scholar 

  8. Bujia, J., Behrends, U., Rotter, N., Pitzke, P., Wilmes, E., and Hammer, C. (1996) Expression of ICAM-1 on intact cartilage and isolated chondrocytes. In Vitro Cell. Dev. Biol. Anim. 32, 116–122.

    Article  CAS  PubMed  Google Scholar 

  9. Lance, E. M., Kimura, L. H., and Manibog, C. N. (1993) The expression of major histocompatibility antigens on human articular chondrocytes. Clin. Orthop. 291, 266–282

    PubMed  Google Scholar 

  10. Lapadula, G., Iannione, F., Zuccaro, C., et al. (1995) Expression of membrane-bound peptidases (CD10 and CD26) on human articular chondrocytes. Possible role of neuropeptidases in the pathogenesis of osteoarthritis. Clin. Exp. Rheumatol. 13, 143–148.

    CAS  PubMed  Google Scholar 

  11. Vivien, D., Boumedienne, K., Galera, P., Lebrun, E., and Pujol, J. P. (1992) Flow cytometric detection of transforming growth factor-β expression in rabbit articular chondrocytes (RAC) in culture-association with S-phase traverse. Exp. Cell. Res. 203, 56–61.

    Article  CAS  PubMed  Google Scholar 

  12. Adolphe, M., Froger, B., Ronot, X., Corvol, M. T., and Forest, N. (1984) Cell multiplication and type II collagen production by rabbit articular chondrocytes cultivated in a defined medium. Exp. Cell. Res. 155, 527–536.

    Article  CAS  PubMed  Google Scholar 

  13. Mok, S. S., Masuda, K., Häuselmann, H. J., Aydelotte, M.B., and Thonar, E. J.-M. A. (1994) Aggrecan synthesized by mature bovine chondrocytes suspended in alginate. J. Biol. Chem. 269, 33,021–33,027.

    CAS  PubMed  Google Scholar 

  14. Malfait, A. M. (1994) Development of an in vitro model to study cytokine-mediated interactions between inflammatory cells and human chondrocytes. Ghent University Thesis: pp. 45.

    Google Scholar 

  15. Almqvist, K.F., Wang, L., Wang, J., et al. (2001) Culture of chondrocytes in alginate surrounded by fibrin gel: characteristics of the cells over a period of 8 weeks. Ann. Rheum. Dis. 60, 781–790.

    Article  CAS  PubMed  Google Scholar 

  16. Platt, D., Wells, T., and Bayliss, M. T. (1997) Proteoglycan metabolism of equine articular chondrocytes cultured in alginate beads. Res. Vet. Sci. 62, 39–47.

    Article  CAS  PubMed  Google Scholar 

  17. Bayliss, M. T. (1992) Metabolism of animal and human osteoarthritic cartilage, in Articular Cartilage and Osteoarthritis. (Kuettner, K. E., Schleyerbach, R., Peyron, J. G., Hascall, V. C., eds.), Raven, New York, NY, pp. 487–500.

    Google Scholar 

  18. Wang, J., Elewaut, D., Veys, E. M., and Verbruggen, G. (2003) IGF-1-induced IL-1RII overrules IL-1 activity and controls the homeostasis of the extracellular matrix of cartilage. Arthritis Rheum. 48, 1281–1291.

    Article  CAS  PubMed  Google Scholar 

  19. Knudson, C. B. and Knudson, W. (1993) Hyaluronan-binding proteins in development, tissue homeostasis, and disease. FASEB J. 7, 1231–1241.

    Google Scholar 

  20. Von der Mark, K., Mollenhauer, J., Pfaeffle, M., van Menxel, M., and Mueller, P. K. (1986) Role of anchorin CII in the interaction of chondrocytes with extracellular collagen, in Articular Cartilage Biochemistry. (Kuettner, K.E., Schleyerbach, R., and Hascall, V.C., eds.) Raven, New York, NY, pp.125–141.

    Google Scholar 

  21. Repraeger, A. and Bernfield, M. (1982) An integral membrane proteoglycan can bind the extracellular matrix directly to the cytoskeleton. J. Cell Biol. 95(Abstract), A125.

    Google Scholar 

  22. Hewitt, A. T., Varner, H. H., Silver, M. H., Dessau, W., Wilkes, C. M., and Martin, G.R. (1982) The isolation and partial characterization of chondronectin, an attachment factor for chondrocytes. J. Biol. Chem. 257, 2330–2334.

    CAS  PubMed  Google Scholar 

  23. Wang, L., Almqvist, K. F., Veys, E. M., and Verbruggen, G. (2002) Control of extracellular matrix homeostasis of normal cartilage by a TGFβ autocrine pathway. Validation of flow cytometry as a tool to study chondrocyte metabolism in vitro. Osteoarthritis Cartilage 10, 188–198.

    Article  CAS  PubMed  Google Scholar 

  24. Wood, B. T., Thompson, S. H., and Goldstein, G. (1965) Fluorescent antibody staining. III. Preparation of fluorescein-isothiocyanate-labeled antibodies. J. Immunol. 95, 225–229.

    CAS  PubMed  Google Scholar 

  25. Kronick, M. N. and Grossman, P. D. (1983) Immunoassay techniques with fluorescent phycobiliprotein conjugates. Clin. Chem. 29, 1582–1586.

    CAS  PubMed  Google Scholar 

  26. Gysen, P. and Franchimont, P. (1984) Radioimmunoassay of proteoglycan. J. Immunoassay 5, 221–243.

    Article  CAS  PubMed  Google Scholar 

  27. Heinegård, D., and Oldberg, A. (1989) Structure and biology of cartilage and bone matrix noncollagenous macromolecules. FASEB J. 3, 2042–2051.

    PubMed  Google Scholar 

  28. Kumagai, J., Sarkar, K., Uhthoff, H. K., Okawara, Y., and Coshima, A. (1994) Immunohistochemical distribution of type I, II and III collagen in the rabbit supraspinous tendon insertion. J. Anat. 185, 279–284.

    CAS  PubMed  Google Scholar 

  29. Green, W. T. (1971) Behavior of articular chondrocytes in cell culture. Clin. Orthop. 75, 248–260.

    Article  PubMed  Google Scholar 

  30. Kuettner, K. E., Pauli, B. U., Gall, G., McMemoli, V. A., and Schenk, R. K. (1982) Synthesis of cartilage matrix by mammalian chondrocytes in vitro. Isolation, culture characteristics and morphology. J. Cell Biol. 93, 743–750.

    Article  CAS  PubMed  Google Scholar 

  31. Benya, P. D. and Shaffer, J.D. (1982) Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30, 215–224.

    Article  CAS  PubMed  Google Scholar 

  32. Cornelissen, M., Verbruggen, G., Malfait, A. M., Veys, E. M., Broddelez, C., and De Ridder, L. (1993) The study of representative populations of native aggrecan aggregates synthesized by human chondrocytes in vitro. J. Tissue Culture Methods 15, 139–146.

    Article  Google Scholar 

  33. Verbruggen, G., Veys, E. M., Wieme, N., et al. (1990) The synthesis and immobilisation of cartilage-specific proteoglycan by human chondrocytes in different concentrations of agarose. Clin. Exp. Rheumatol. 8, 371–378.

    CAS  PubMed  Google Scholar 

  34. Guo, J., Jourdian, G.W., and MacCallum, D.K. (1989) Culture and growth charecteristics of chondrocytes encapsulated in alginate beads. Connect. Tissue Res. 9, 277–297.

    Article  Google Scholar 

  35. Wang, L., Verbruggen, G., Almqvist, K. F., Elewaut, D., Broddelez, C., and Veys, E. M. (2001) Flow cytometric analysis of the human articular chondrocyte phenotype in vitro. Osteoarthritis Cartilage 9, 73–84.

    Article  CAS  PubMed  Google Scholar 

  36. Cornelissen, M., Dewulf, M., Verbruggen, G., et al. (1993) Size distribution of native aggrecan aggregates of human articular chondrocytes in agarose. In Vitro Cell. Dev. Biol. Anim. 29A, 356–358.

    Article  CAS  PubMed  Google Scholar 

  37. Wang, L., Almqvist, K. F., Broddelez, C., Veys, E. M., and Verbruggen, G. (2001) Evaluation of chondrocyte cell-associated matrix metabolism by flow cytometry. Osteoarthritis Cartilage 9, 454–462.

    Article  CAS  PubMed  Google Scholar 

  38. Wang, L., Wang, J., Almqvist, K. F., Veys, E. M., and Verbruggen, G. (2002) Influence of polysulphated polysaccharides and hydrocortisone on the extracellular matrix metabolism of human articular chondrocytes in vitro. Clin. Exp. Rheumatol. 20, 669–676.

    CAS  PubMed  Google Scholar 

  39. Martineau, L. C. and Gardiner, P. F. (2001) Insight into skeletal muscle mechanotransduction: MAPK activation is quantitatively related to tension. J. Appl. Physiol. 91, 693–702.

    CAS  PubMed  Google Scholar 

  40. Hung, C. T., Henshaw, D. R., Wang, C. C., et al. (2000) Mitogen-activated protein kinase signaling in bovine articular chondrocytes in response to fluid flow does not require calcium mobilization. J. Biomech. 33, 73–80.

    Article  CAS  PubMed  Google Scholar 

  41. Honda, K., Ohno, S., Tanimoto, K., et al. (2000) The effects of high magnitude cyclic tensile load on cartilage matrix metabolism in cultured chondrocytes. Eur. J. Cell Biol. 79, 601–609.

    Article  CAS  PubMed  Google Scholar 

  42. Fujiwara, K., Masuda, M., Osawa, M., Kano, Y., and Katoh, K. (2001) Is PECAM-1 a mechanoresponsive molecule? Cell. Struct. Funct. 26, 11–17.

    Article  CAS  PubMed  Google Scholar 

  43. Verschure, P. J., van Marle, J., Joosten, L. A., and van den Berg, W. B. (1995) Chondrocyte IGF-1 receptor expression and responsiveness to IGF-1 stimulation in mouse articular cartilage during various phases of experimentally induced arthritis. Ann. Rheum. Dis. 54, 645–653.

    Article  CAS  PubMed  Google Scholar 

  44. Guenther, H. L., Guenther, H. E., Froesch, E. R., and Fleisch, H. (1982) Effect of insulin-like growth factor on collagen and glycosaminoglycan synthesis by rabbit articular chondrocytes in culture. Experientia 38, 979–981.

    Article  CAS  PubMed  Google Scholar 

  45. McQuillan, D. L., Handley, C. J., Campbell, M. A., Bolis, S., Milway, V. E., and Herington, A. C. (1986) Stimulation of proteoglycan synthesis by serum and insulin-like growth factor-1 in cultured bovine articular cartilage. Biochem. J. 240, 423–430.

    CAS  PubMed  Google Scholar 

  46. Tesch, G. H., Handley, C. J., Cornell, H. J., and Herington, A. C. (1992) Effects of free and bound insulin-like growth factors on proteoglycan metabolism in articular cartilage explants. J. Orthop. Res. 10, 14–22.

    Article  CAS  PubMed  Google Scholar 

  47. Tyler, J. A. (1989) Insulin-like growth factor 1 can decrease degradation and promote synthesis of proteoglycan in cartilage exposed to cytokines. Biochem. J. 260, 543–548.

    CAS  PubMed  Google Scholar 

  48. Kollewe, C., Neumann, D., and Martin, M. U. (2000) The first two N-terminal immunoglobulin-like domains of soluble human IL-1 receptor type II are sufficient to bind and neutralize IL-1β. FEBS Lett. 487, 189–193.

    Article  CAS  PubMed  Google Scholar 

  49. Colotta, F., Saccani, S., Giri, J. G., et al. (1996) Regulated expression and release of the IL-1 decoy receptor in human mononuclear phagocytes. J. Immunol. 156, 2534–2541.

    CAS  PubMed  Google Scholar 

  50. Wang, J., Verdonk, P., Elewaut, D., Veys, E. M., and Verbruggen, G. (2003) Homeostasis of the extracellular matrix of normal and osteoarthritic human articular cartilage chondrocytes in vitro. Osteoarthritis Cartilage 11, 801–809.

    Article  CAS  PubMed  Google Scholar 

  51. Lee, S. W., Tsou, A. P., Chan, H., et al. (1988) Glucocorticoids selectively inhibit the transcription of the interleukin 1β gene and decrease the stability of interleukin 1β mRNA. Proc. Natl. Acad. Sci. USA 85, 1204–1208.

    Article  CAS  PubMed  Google Scholar 

  52. Pelletier, J. P., Mineau, F., Raynauld, J. P., Woessner, J. F., Gunja-Smith, Z., and Martel-Pelletier, J. (1994) Intraarticular injections with methylprednisolone acetate reduce osteoarthritic lesions in parallel with chondrocyte stromelysin synthesis in experimental osteoarthritis. Arthritis Rheum. 37, 414–423.

    Article  CAS  PubMed  Google Scholar 

  53. Pelletier, J. P., Martel-Pelletier, J., Cloutier, J. M., and Woessner, J. F. (1987) Proteoglycan-degrading acid metalloproteinase activity in human osteoarthritic cartilage, and the effects of intraarticular steroid injections. Arthritis Rheum. 30, 541–548.

    Article  CAS  PubMed  Google Scholar 

  54. Pelletier, J. P. and Martel-Pelletier, J. (1985) Cartilage degradation by neutral proteoglycanases in experimental osteoarthritis. Suppression by steroids. Arthritis Rheum. 28, 1393–1401.

    Article  CAS  PubMed  Google Scholar 

  55. McGuire, M. B., Murhy, M., Reynolds, J. J., and Russel, R. G. G. (1981) Production of collagenase and inhibitor (TIMP) by normal, rheumatoid and osteoarthritic synovium in vitro: effects of hydrocortisone and indomethacine. Clin. Biol. 61, 703–710.

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Rhumatology, Ghent University Hospital, Ghent University, Ghent, Belgium

    Gust Verbruggen, Jun Wang, Lai Wang, Dirk Elewaut & Eric M. Veys

Authors
  1. Gust Verbruggen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jun Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lai Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dirk Elewaut
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Eric M. Veys
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Division de Rhumatologie, Institut de Recherches Servier, Suresnes, France

    Massimo Sabatini , Philippe Pastoureau  & Frédéric De Ceuninck ,  & 

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Verbruggen, G., Wang, J., Wang, L., Elewaut, D., Veys, E.M. (2004). Analysis of Chondrocyte Functional Markers and Pericellular Matrix Components by Flow Cytometry. In: Sabatini, M., Pastoureau, P., De Ceuninck, F. (eds) Cartilage and Osteoarthritis. Methods in Molecular Medicine™, vol 100. Humana Press. https://doi.org/10.1385/1-59259-810-2:183

Download citation

  • DOI: https://doi.org/10.1385/1-59259-810-2:183

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-247-6

  • Online ISBN: 978-1-59259-810-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation