Advertisement

Mechanical Loading of Chondrocytes Embedded in 3D Constructs

In Vitro Methods for Assessment of Morphological and Metabolic Response to Compressive Strain
  • David A. Lee
  • Martin M. Knight
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 100)

Abstract

Mechanical loading of chondrocytes in 3D constructs has been used to investigate mechanotransduction and its potential for stimulating tissue-engineered cartilage repair. This chapter describes the preparation of 3D agarose or alginate constructs seeded with isolated chondrocytes and specific test rigs for applying gross compressive strain to individual constructs on a confocal microscope or for longer term compression of constructs cultured within an incubator. Experimental methods are described to quantify the level of cell deformation and the elaboration of extracellular matrix. The chapter thus provides an introduction to the experimental techniques used to examine chondrocyte mechanotransduction and downstream cell function.

Key Words

Agarose bioreactor cartilage cell deformation cell mechanics cell proliferation chondrocyte confocal microscopy extracellular matrix mechanical compression mechanotransduction proteoglycan synthesis sulphate incorporation thymidine incorporation 

References

  1. 1.
    Sah, R., Kim, Y. J., Doong, J. Y. H., Grodzinsky, A. J., Plaas, A. H. K., and Sandy, J. D. (1989) Biosynthetic response of cartilage explants to dynamic compression. J. Orthop. Res. 7, 619–636.CrossRefPubMedGoogle Scholar
  2. 2.
    Kim, Y. J., Sah, R. L. Y., Grodzinsky, A. J., Plaas, A. H. K., and Sandy, J. D. (1994) Mechanical regulation of cartilage biosynthetic behaviour: physical stimuli. Arch. Biochem. Biophys. 311, 1–12.CrossRefPubMedGoogle Scholar
  3. 3.
    Vunjak-Novakovic, G., Martin, I., Obradovic, B., et al. (1999) Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue engineered cartilage. J. Orthop. Res. 17, 130–138.CrossRefPubMedGoogle Scholar
  4. 4.
    Mauck, R. L., Soltz, M. A., Wang, C. C., et al. (2000) Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J. Biomech. Eng. 122, 252–260.CrossRefPubMedGoogle Scholar
  5. 5.
    Guilak, F., Butler, D. L., and Goldstein, S. A. (2001) Functional tissue engineering: the role of biomechanics in articular cartilage repair. Clin. Orthop. 391, 295–305.CrossRefGoogle Scholar
  6. 6.
    Urban, J. P.G. (1994) The chondrocyte: A cell under pressure. Br. J. Rheumatol 33, 901–908.CrossRefPubMedGoogle Scholar
  7. 7.
    Heath, C. A. and Magari, S. R. (1996) Mechanical factors affecting cartilage regeneration in vitro. Biotechnol. Bioeng. 50, 430–437.CrossRefPubMedGoogle Scholar
  8. 8.
    Guilak, F., Sah, R., and Setton, L.A. (1997) Basic Orthopaedic Biomechanics (Mow, V. C. and Hayes, W. C., eds.), Lippincott-Raven, Philadelphia, PA, pp. 179–207.Google Scholar
  9. 9.
    Benya, P. D. and Shaffer, J. D. (1982) Dedifferentiated chondrocytes reexpress the differentiated collagen phentotype when cultured in agarose gels. Cell 30, 215–224.CrossRefPubMedGoogle Scholar
  10. 10.
    Aydelotte, M. B., Schumacher, B. L., and Kuettner, K. E. (1990) Methods in Cartilage Research. (Maroudas, A. and Kuettner, K. E., eds.), Academic, London, UK, pp. 90–92.Google Scholar
  11. 11.
    Hauselmann, H. J., Fernandes, R. J., Mok, S., et al. (1994) Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads. J. Cell Sci. 107, 17–27.PubMedGoogle Scholar
  12. 12.
    Knight, M. M., Ghori, S. A., Lee, D. A., and Bader, D. L. (1998) Measurement of the deformation of isolated chondrocytes in agarose subjected to cyclic compression. Med. Eng. Phys. 20, 684–688.CrossRefPubMedGoogle Scholar
  13. 13.
    Lee, D. A., Knight, M. M., Bolton, J. F., Idowu, B. D., Kayser, M. V., and Bader, D. L. (2000) Chondrocyte deformation within compressed agarose constructs at the cellular and sub-cellular levels. J. Biomech. 33, 81–95.CrossRefPubMedGoogle Scholar
  14. 14.
    Lee, D. A., Noguchi, T., Knight, M. M., O’Donnell, L. B., and Bader, D. L. (1997) Differential metabolic response of superficial and deep zone chondrocytes to compressive strain, in Transactions of the 42nd Annual Meeting of the Orthopaedic Research Society 22, abstract.Google Scholar
  15. 15.
    Lee, D. A., Noguchi, T., Knight, M. M., O’Donnell, L., Bentley, G., and Bader, D. L. (1998) Response of chondrocyte subpopulations cultured within unloaded and loaded agarose. J. Orthop. Res. 16, 726–733.CrossRefPubMedGoogle Scholar
  16. 16.
    Lee, D. A., Noguchi, T., Frean, S. P., Lees, P., and Bader, D. L. (2000) The influence of mechanical loading on isolated chondrocytes seeded in agarose constructs. Biorheology 37, 149–161.PubMedGoogle Scholar
  17. 17.
    Buschmann, M. D., Gluzband, Y. A., Grodzinsky, A. J., and Hunziker, E. B. (1995) Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture. J. Cell Sci. 108, 1497–1508.PubMedGoogle Scholar
  18. 18.
    Guilak, F., Ratcliffe, A., and Mow, V. C. (1995) Chondrocyte Deformation and local tissue strain in articular cartilage: A confocal microscopy study. J. Orthop. Res. 13, 410–421.CrossRefPubMedGoogle Scholar
  19. 19.
    Knight, M. M., Lee, D. A., and Bader, D. L. (1998) The influence of elaborated pericellular matrix on the deformation of isolated articular chondrocytes cultured in agarose. Biochim. Biophys. Acta 1405, 67–77.CrossRefPubMedGoogle Scholar
  20. 20.
    Knight, M. M., Ross, J. M., Sherwin, A. F., Lee, D. A., Bader, D. L., and Poole, C. A. (2001) Chondrocyte deformation within mechanically and enzymatically extracted chondrons compressed in agarose. Biochim. Biophys. Acta 1526, 141–146.PubMedGoogle Scholar
  21. 21.
    Bader, D. L., Ohashi, T., Knight, M. M., Lee, D. A., and Sato, M. (2002) Deformation properties of articular chondrocytes: a critique of three separate techniques. Biorheology 39, 69–78.PubMedGoogle Scholar
  22. 22.
    Knight, M. M., van de Breevaart Bravenboer, J., Lee, D. A., van Osch, G. J. V. M., Weinans, H., and Bader, D. L. (2002) Cell and nucleus deformation in compressed chondrocyte-alginate constructs: temporal changes and calculation of cell modulus. Biochim. Biophys. Acta 1570, 1–8.PubMedGoogle Scholar
  23. 23.
    Roberts, S. R., Knight, M. M., Lee, D. A., and Bader, D. L. (2001) Mechanical compression influences intracellular Ca2+ signaling in chondrocytes seeded in agarose constructs. J. Appl. Physiol 90, 1385–1391.PubMedGoogle Scholar
  24. 24.
    Knight, M. M., Lee, D. A., and Bader, D. L. (1996) Distribution of chondrocyte deformation in compressed agarose gel using confocal microscopy. J. Cell. Eng. 1, 97–102.Google Scholar
  25. 25.
    Bachrach, N. M., Valhmu W. B., Stazzone, E., Ratcliffe, A., Lai, W. M., and Mow, V.C. (1995) Changes in proteoglycan synthesis of chondrocytes in articular cartilage are associated with the time-dependent changes in their mechanical environment. J. Biomech. 28, 1561–1569.CrossRefPubMedGoogle Scholar
  26. 26.
    Knight, M. M. (1997) Deformation of isolated articular chondrocytes cultured in agarose constructs. PhD Thesis, University of London, London, UK.Google Scholar
  27. 27.
    Lee, D. A. and Bader, D. L. (1997) Compressive strains at physiological frequencies influence the metabolism of chondrocytes seeded in agarose. J. Orthop. Res. 15, 181–188.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • David A. Lee
    • 1
  • Martin M. Knight
    • 2
  1. 1.IRC in Biomedical Materials and Medical Engineering Division, Department of Engineering, Queen Mary CollegeUniversity of LondonLondonUK
  2. 2.Department of Engineering, Queen Mary CollegeUniversity of LondonLondonUK

Personalised recommendations