Abstract
Chondrocytes in articular cartilage normally exhibit high expression of collagen II and aggrecan but rapidly dedifferentiate to a fibroblastic phenotype if passaged in culture. Previous studies have suggested that the loss of chondrocyte phenotype is associated with changes in the structure of the F-actin cytoskeleton, which also controls cell mechanical properties. In this study, we examined how dedifferentiation in monolayer influences the mechanical properties of chondrocytes isolated from different zones of articular cartilage. Atomic force microscopy was used to measure the mechanical properties of superficial and middle/deep zone chondrocytes as they underwent serial passaging and subsequent growth on fibronectin-coated, micropatterned self-assembled monolayers that restored a rounded cell shape in 2D culture. Chondrocytes exhibited significant increases in elastic and viscoelastic moduli with dedifferentiation in culture. These changes were only partially ameliorated by the restoration of a rounded shape on micropatterned surfaces. Furthermore, intrinsic zonal differences in cell mechanical properties were rapidly lost with passage. These findings indicate that cell mechanical properties may provide additional measures of phenotypic expression of chondrocytes as they undergo dedifferentiation and possibly redifferentiation in culture.
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Archer, C. W., J. McDowell, M. T. Bayliss, M. D. Stephens, and G. Bentley. Phenotypic modulation in sub-populations of human articular chondrocytes in vitro. J. Cell Sci. 97:361–371, 1990.
Aydelotte, M. B., R. R. Greenhill, and K. E. Kuettner. Differences between sub-populations of cultured bovine articular chondrocytes. II. Proteoglycan metabolism. Connect. Tissue Res. 18:223–234, 1988.
Aydelotte, M. B., and K. E. Kuettner. Differences between sub-populations of cultured bovine articular chondrocytes. I. Morphology and cartilage matrix production. Connect. Tissue Res. 18:205–222, 1988.
Bausch, A. R., F. Ziemann, A. A. Boulbitch, K. Jacobson, and E. Sackmann. Local measurements of viscoelastic parameters of adherent cell surfaces by magnetic bead microrheometry. Biophys. J. 75:2038–2049, 1998.
Benninghoff, A. Form und Bau der Gelenkknorpel in ihren beziechungen zur funktion. I. Die modellierenden und formerhalterden Faktoren des Knorpelreliefs. Z. ges. Anat. 76:43–63, 1925.
Benya, P. D., and J. D. Shaffer. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30:215–224, 1982.
Cournil-Henrionnet, C., C. Huselstein, Y. Wang, L. Galois, D. Mainard, V. Decot, P. Netter, J. F. Stoltz, S. Muller, P. Gillet, and A. Watrin-Pinzano. Phenotypic analysis of cell surface markers and gene expression of human mesenchymal stem cells and chondrocytes during monolayer expansion. Biorheology 45:513–526, 2008.
Darling, E. M., and K. A. Athanasiou. Rapid phenotypic changes in passaged articular chondrocyte subpopulations. J. Orthop. Res. 23:425–432, 2005.
Darling, E. M., and K. A. Athanasiou. Retaining zonal chondrocyte phenotype by means of novel growth environments. Tissue Eng. 11:395–403, 2005.
Darling, E. M., J. C. Y. Hu, and K. A. Athanasiou. Zonal and topographical differences in articular chondrocyte gene expression. J. Orthop. Res. 22:1182–1187, 2004.
Darling, E. M., M. Topel, S. Zauscher, T. P. Vail, and F. Guilak. Viscoelastic properties of human mesenchymally-derived stem cells and primary osteoblasts, chondrocytes, and adipocytes. J. Biomech. 41:454–464, 2008.
Darling, E. M., S. Zauscher, J. A. Block, and F. Guilak. A thin-layer model for viscoelastic, stress-relaxation testing of cells using atomic force microscopy: do cell properties reflect metastatic potential? Biophys. J. 92:1784–1791, 2007.
Darling, E. M., S. Zauscher, and F. Guilak. Viscoelastic properties of zonal articular chondrocytes measured by atomic force microscopy. Osteoarthritis Cartilage 14:571–579, 2006.
Evans, E. A., and R. M. Hochmuth. Membrane viscoelasticity. Biophys. J. 16:1–11, 1976.
Gallant, N. D., K. E. Michael, and A. J. Garcia. Cell adhesion strengthening: contributions of adhesive area, integrin binding, and focal adhesion assembly. Mol. Biol. Cell 16:4329–4340, 2005.
Grundmann, K., B. Zimmermann, H. J. Barrach, and H. J. Merker. Behaviour of epiphyseal mouse chondrocyte populations in monolayer culture. Morphological and immunohistochemical studies. Virchows Arch. A Pathol. Anat. Histol. 389:167–187, 1980.
Guck, J., S. Schinkinger, B. Lincoln, F. Wottawah, S. Ebert, M. Romeyke, D. Lenz, H. M. Erickson, R. Ananthakrishnan, D. Mitchell, J. Kas, S. Ulvick, and C. Bilby. Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence. Biophys. J. 88:3689–3698, 2005.
Guo, S., and B. B. Akhremitchev. Packing density and structural heterogeneity of insulin amyloid fibrils measured by AFM nanoindentation. Biomacromolecules 7:1630–1636, 2006.
Hauselmann, H. J., M. B. Aydelotte, B. L. Schumacher, K. E. Kuettner, S. H. Gitelis, and E. J. Thonar. Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads. Matrix 12:116–129, 1992.
Ikai, A. A review on: Atomic force microscopy applied to nano-mechanics of the cell. Adv. Biochem. Eng. Biotechnol., 2009.
Jones, W. R., H. P. Ting-Beall, G. M. Lee, S. S. Kelley, R. M. Hochmuth, and F. Guilak. Alterations in the Young’s modulus and volumetric properties of chondrocytes isolated from normal and osteoarthritic human cartilage. J. Biomech. 32:119–127, 1999.
Kemp, M. W., and K. E. Davies. The role of intermediate filament proteins in the development of neurological disease. Crit. Rev. Neurobiol. 19:1–27, 2007.
Lee, D. A., T. Noguchi, M. M. Knight, L. O’Donnell, G. Bentley, and D. L. Bader. Response of chondrocyte subpopulations cultured within unloaded and loaded agarose. J. Orthop. Res. 16:726–733, 1998.
Leipzig, N. D., S. V. Eleswarapu, and K. A. Athanasiou. The effects of TGF-beta1 and IGF-I on the biomechanics and cytoskeleton of single chondrocytes. Osteoarthritis Cartilage 14:1227–1236, 2006.
Lin, Z., J. B. Fitzgerald, J. Xu, C. Willers, D. Wood, A. J. Grodzinsky, and M. H. Zheng. Gene expression profiles of human chondrocytes during passaged monolayer cultivation. J. Orthop. Res. 26:1230–1237, 2008.
Makale, M. Cellular mechanobiology and cancer metastasis. Birth Defects Res. C Embryo Today 81:329–343, 2007.
Mallein-Gerin, F., R. Garrone, and M. van der Rest. Proteoglycan and collagen synthesis are correlated with actin organization in dedifferentiating chondrocytes. Eur. J. Cell Biol. 56:364–373, 1991.
McConnaughey, W. B., and N. O. Petersen. Cell poker: an apparatus for stress-strain measurements on living cells. Rev. Sci. Instrum. 51:575–580, 1980.
Mehlhorn, A. T., P. Niemeyer, S. Kaiser, G. Finkenzeller, G. B. Stark, N. P. Sudkamp, and H. Schmal. Differential expression pattern of extracellular matrix molecules during chondrogenesis of mesenchymal stem cells from bone marrow and adipose tissue. Tissue Eng. 12:2853–2862, 2006.
Murphy, C. L., and A. Sambanis. Effect of oxygen tension and alginate encapsulation on restoration of the differentiated phenotype of passaged chondrocytes. Tissue Eng. 7:791–803, 2001.
Pritchard, S., and F. Guilak. The role of F-actin in hypo-osmotically induced cell volume change and calcium signaling in anulus fibrosus cells. Ann. Biomed. Eng. 32:103–111, 2004.
Shin, D., and K. Athanasiou. Cytoindentation for obtaining cell biomechanical properties. J. Orthop. Res. 17:880–890, 1999.
Siczkowski, M., and F. M. Watt. Subpopulations of chondrocytes from different zones of pig articular cartilage. Isolation, growth and proteoglycan synthesis in culture. J. Cell Sci. 97:349–360, 1990.
Suresh, S. Biomechanics and biophysics of cancer cells. Acta Biomater. 3:413–438, 2007.
Tim O’Brien, E., J. Cribb, D. Marshburn, R. M. Taylor, 2nd, and R. Superfine. Chapter 16: Magnetic manipulation for force measurements in cell biology. Methods Cell Biol. 89:433–450, 2008.
Trickey, W. R., F. P. Baaijens, T. A. Laursen, L. G. Alexopoulos, and F. Guilak. Determination of the Poisson’s ratio of the cell: recovery properties of chondrocytes after release from complete micropipette aspiration. J. Biomech. 39:78–87, 2006.
Trickey, W. R., G. M. Lee, and F. Guilak. Viscoelastic properties of chondrocytes from normal and osteoarthritic human cartilage. J. Orthop. Res. 18:891–898, 2000.
Trickey, W. R., T. P. Vail, and F. Guilak. The role of the cytoskeleton in the viscoelastic properties of human articular chondrocytes. J. Orthop. Res. 22:131–139, 2004.
van Susante, J. L., P. Buma, G. J. van Osch, D. Versleyen, P. M. van der Kraan, W. B. van der Berg, and G. N. Homminga. Culture of chondrocytes in alginate and collagen carrier gels. Acta Orthop. Scand. 66:549–556, 1995.
Weiss, L., and K. Clement. Studies on cell deformability. Some rheological considerations. Exp. Cell Res. 58:379–387, 1969.
Zanetti, M., A. Ratcliffe, and F. M. Watt. Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate. J. Cell Biol. 101:53–59, 1985.
Zanetti, N. C., and M. Solursh. Induction of chondrogenesis in limb mesenchymal cultures by disruption of the actin cytoskeleton. J. Cell Biol. 99:115–123, 1984.
Acknowledgments
This work was supported in part by NIH grants AR53448 (EMD), EB01630, AG15768, AR48182, AR50245, and EB002025 (RS) and NSF grant CMS-0507151 (RS). The authors would like to thank Drs. Ashutosh Chilkoti, Dominic Chow, David Dumbauld, and Andrés Garcia for discussions and advice on the development of micropatterned substrates. The authors would also like to acknowledge the Shared Materials Instrumentation Facility at Duke University for providing resources for SEM imaging.
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Darling, E.M., Pritchett, P.E., Evans, B.A. et al. Mechanical Properties and Gene Expression of Chondrocytes on Micropatterned Substrates Following Dedifferentiation in Monolayer. Cel. Mol. Bioeng. 2, 395–404 (2009). https://doi.org/10.1007/s12195-009-0077-3
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DOI: https://doi.org/10.1007/s12195-009-0077-3