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Mechanical confinement regulates cartilage matrix formation by chondrocytes

Nature Materials volume 16, pages 1243–1251 (2017)Cite this article

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Abstract

Cartilage tissue equivalents formed from hydrogels containing chondrocytes could provide a solution for replacing damaged cartilage. Previous approaches have often utilized elastic hydrogels. However, elastic stresses may restrict cartilage matrix formation and alter the chondrocyte phenotype. Here we investigated the use of viscoelastic hydrogels, in which stresses are relaxed over time and which exhibit creep, for three-dimensional (3D) culture of chondrocytes. We found that faster relaxation promoted a striking increase in the volume of interconnected cartilage matrix formed by chondrocytes. In slower relaxing gels, restriction of cell volume expansion by elastic stresses led to increased secretion of IL-1β, which in turn drove strong up-regulation of genes associated with cartilage degradation and cell death. As no cell-adhesion ligands are presented by the hydrogels, these results reveal cell sensing of cell volume confinement as an adhesion-independent mechanism of mechanotransduction in 3D culture, and highlight stress relaxation as a key design parameter for cartilage tissue engineering.

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Figure 1: Modulating the rate of stress relaxation or creep of alginate hydrogels independent of initial elastic modulus, swelling, and degradation.
Figure 2: Faster stress relaxation in the hydrogels promotes increased cartilage matrix production and formation of a wider volume of interconnected cartilage matrix.
Figure 3: Faster relaxation promotes proliferation and anabolic, or matrix-forming, gene expression in chondrocytes, whereas slower stress relaxation induces cell death and catabolic, or matrix degrading, gene expression in chondrocytes.
Figure 4: Slower stress relaxation induces secretion of IL-1β, which mediates strong up-regulation of catabolic activities of chondrocytes and cell death.
Figure 5: Spatial confinement against cell expansion in hydrogels with slow stress relaxation induces decreased proliferation and increases in IL-1β secretion, cell death and catabolic activities of cells.
Figure 6: Hydrogel stress relaxation regulates chondrocyte phenotype through restricting cell volume expansion and cartilage matrix formation.

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References

  1. Getgood, A., Brooks, R., Fortier, L. & Rushton, N. Articular cartilage tissue engineering: today’s research, tomorrow’s practice? J. Bone Joint Surg. Br. 91, 565–576 (2009).

    CAS  Google Scholar 

  2. Guettler, J. H. Osteochondral defects in the human knee: influence of defect size on cartilage rim stress and load redistribution to surrounding cartilage. Am. J. Sports Med. 32, 1451–1458 (2004).

    Google Scholar 

  3. Brittberg, M. et al. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. J. Med. 331, 889–895 (1994).

    CAS  Google Scholar 

  4. Gibson, A. J., McDonnell, S. M. & Price, A. J. Matrix-induced autologous chondrocyte implantation. Oper. Tech. Orthop. 16, 262–265 (2006).

    Google Scholar 

  5. Steadman, J. R., Rodkey, W. G., Briggs, K. K. & Rodrigo, J. J. The microfracture technic in the management of complete cartilage defects in the knee joint. Orthopade 28, 26–32 (1999).

    CAS  Google Scholar 

  6. Görtz, S. & Bugbee, W. D. Allografts in articular cartilage repair. Instr. Course Lect. 56, 469–480 (2007).

    Google Scholar 

  7. Roberts, S., Menage, J., Sandell, L. J., Evans, E. H. & Richardson, J. B. Immunohistochemical study of collagen types I and II and procollagen IIA in human cartilage repair tissue following autologous chondrocyte implantation. Knee 16, 398–404 (2009).

    CAS  Google Scholar 

  8. Gobbi, A., Nunag, P. & Malinowski, K. Treatment of full thickness chondral lesions of the knee with microfracture in a group of athletes. Knee Surg. Sport. Traumatol. Arthrosc. 13, 213–221 (2005).

    Google Scholar 

  9. Lee, C. S. D. et al. Integration of layered chondrocyte-seeded alginate hydrogel scaffolds. Biomaterials 28, 2987–2993 (2007).

    CAS  Google Scholar 

  10. Lima, E. G. et al. The beneficial effect of delayed compressive loading on tissue-engineered cartilage constructs cultured with TGF-β3. Osteoarthr. Cartil. 15, 1025–1033 (2007).

    CAS  Google Scholar 

  11. Mauck, R. L. et al. Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J. Biomech. Eng. 122, 252–260 (2000).

    CAS  Google Scholar 

  12. Bryant, S. J. & Anseth, K. S. Hydrogel properties influence ECM production by chondrocytes photoencapsulated in poly(ethylene glycol) hydrogels. J. Biomed. Mater. Res. 59, 63–72 (2002).

    CAS  Google Scholar 

  13. Schuh, E. et al. Chondrocyte redifferentiation in 3D: the effect of adhesion site density and substrate elasticity. J. Biomed. Mater. Res. A 100 A, 38–47 (2012).

    Google Scholar 

  14. Burdick, J. A., Chung, C., Jia, X., Randolph, M. A. & Langer, R. Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks. Biomacromolecules 6, 386–391 (2005).

    CAS  Google Scholar 

  15. Mouw, J. K., Case, N. D., Guldberg, R. E., Plaas, A.H. K. & Levenston, M. E. Variations in matrix composition and GAG fine structure among scaffolds for cartilage tissue engineering. Osteoarthr. Cartil. 13, 828–836 (2005).

    CAS  Google Scholar 

  16. Francioli, S. E. et al. Effect of three-dimensional expansion and cell seeding density on the cartilage-forming capacity of human articular chondrocytes in type II collagen sponges. J. Biomed. Mater. Res. A 95, 924–931 (2010).

    Google Scholar 

  17. Erickson, I. E. et al. High mesenchymal stem cell seeding densities in hyaluronic acid hydrogels produce engineered cartilage with native tissue properties. Acta Biomater. 8, 3027–3034 (2012).

    CAS  Google Scholar 

  18. Chaudhuri, O. et al. Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nat. Mater. 15, 326–333 (2015).

    Google Scholar 

  19. McKinnon, D. D., Domaille, D. W., Cha, J. N. & Anseth, K. S. Biophysically defined and cytocompatible covalently adaptable networks as viscoelastic 3d cell culture systems. Adv. Mater. 26, 865–872 (2014).

    CAS  Google Scholar 

  20. Nicodemus, G. D., Skaalure, S. C. & Bryant, S. J. Gel structure has an impact on pericellular and extracellular matrix deposition, which subsequently alters metabolic activities in chondrocyte-laden PEG hydrogels. Acta Biomater. 7, 492–504 (2011).

    CAS  Google Scholar 

  21. Lin, S., Sangaj, N., Razafiarison, T., Zhang, C. & Varghese, S. Influence of physical properties of biomaterials on cellular behavior. Pharm. Res. 28, 1422–1430 (2011).

    CAS  Google Scholar 

  22. Chung, C., Mesa, J., Randolph, M. A., Yaremchuk, M. & Burdick, J. A. Influence of gel properties on neocartilage formation by auricular chondrocytes photoencapsulated in hyaluronic acid networks. J. Biomed. Mater. Res. A 77A, 518–525 (2006).

    CAS  Google Scholar 

  23. Lee, K. Y. & Mooney, D. J. Hydrogels for tissue engineering. Chem. Rev. 101, 1869–1879 (2001).

    CAS  Google Scholar 

  24. Guo, J. F., Jourdian, G. W. & MacCallum, D. K. Culture and growth characteristics of chondrocytes encapsulated in alginate beads. Connect. Tissue Res. 19, 277–297 (1989).

    CAS  Google Scholar 

  25. Mhanna, R. et al. Chondrocyte culture in 3D alginate sulfate hydrogels promotes proliferation while maintaining expression of chondrogenic markers. Tissue Eng. A 20, 1–38 (2013).

    Google Scholar 

  26. Beekman, B., Verzijl, N., Bank, R. A., von der Mark, K. & TeKoppele, J. M. Synthesis of collagen by bovine chondrocytes cultured in alginate; posttranslational modifications and cell-matrix interaction. Exp. Cell Res. 237, 135–141 (1997).

    CAS  Google Scholar 

  27. Chaudhuri, O. et al. Substrate stress relaxation regulates cell spreading. Nat. Commun. 6, 6364 (2015).

    Google Scholar 

  28. Zhao, X., Huebsch, N., Mooney, D. J. & Suo, Z. Stress-relaxation behavior in gels with ionic and covalent crosslinks. J. Appl. Phys. 107, 63509 (2010).

    Google Scholar 

  29. Wilusz, R. E., Sanchez-Adams, J. & Guilak, F. The structure and function of the pericellular matrix of articular cartilage. Matrix Biol. 39, 25–32 (2014).

    CAS  Google Scholar 

  30. Schuh, E. et al. The influence of matrix elasticity on chondrocyte behavior in 3D. J. Tissue Eng. Regen. Med. 6, 31–42 (2012).

    Google Scholar 

  31. Lefrebvre, V. & de Crombrugghe, B. Toward understanding S0X9 function in chondrocyte differentiation. Matrix Biol. 16, 529–540 (1998).

    Google Scholar 

  32. Sandell, L. J. & Aigner, T. Articular cartilage and changes in arthritis: cell biology of osteoarthritis. Arthritis Res. 3, 107–113 (2001).

    CAS  Google Scholar 

  33. Daheshia, M. & Yao, J. Q. The interleukin 1β pathway in the pathogenesis of osteoarthritis. J. Rheumatol. 35, 2306–2312 (2008).

    CAS  Google Scholar 

  34. Pelletier, J. P., DiBattista, J. A., Roughley, P., McCollum, R. & Martel-Pelletier, J. Cytokines and inflammation in cartilage degradation. Rheum. Dis. Clin. North Am. 19, 545–568 (1993).

    CAS  Google Scholar 

  35. Buckwalter, J. A., Mower, D., Ungar, R., Schaeffer, J. & Ginsberg, B. Morphometric analysis of chondrocyte hypertrophy. J. Bone Joint Surg. Am. 68, 243–255 (1986).

    CAS  Google Scholar 

  36. Desrochers, J., Amrein, M. W. & Matyas, J. R. Viscoelasticity of the articular cartilage surface in early osteoarthritis. Osteoarthr. Cartil. 20, 413–421 (2012).

    CAS  Google Scholar 

  37. Guilak, F., Alexopoulos, L., Nielsen, R., Ting-Beall, H. P. & Haider, M. The biomechanical properties of the chondrocyte pericellular matrix: micropipette aspiration of mechanically isolated chondrons. InTrans. 48th Annual Meeting of the Orthopaedic Research Soc. 405 (ORS, 2002).

    Google Scholar 

  38. Nguyen, B. V. et al. Biomechanical properties of single chondrocytes and chondrons determined by micromanipulation and finite-element modelling. J. R. Soc. Interface 7, 1723–1733 (2010).

    Google Scholar 

  39. Leipzig, N. D. & Athanasiou, K. A. Unconfined creep compression of chondrocytes. J. Biomech. 38, 77–85 (2005).

    Google Scholar 

  40. Darling, E. M., Zauscher, S. & Guilak, F. Viscoelastic properties of zonal articular chondrocytes measured by atomic force microscopy. Osteoarthr. Cartil. 14, 571–579 (2006).

    CAS  Google Scholar 

  41. Wong, M., Siegrist, M., Wang, X. & Hunziker, E. Development of mechanically stable alginate/chondrocyte constructs: effects of guluronic acid content and matrix synthesis. J. Orthop. Res. 19, 493–499 (2001).

    CAS  Google Scholar 

  42. Cooper, K. L. et al. Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions. Nature 495, 375–378 (2013).

    CAS  Google Scholar 

  43. Bush, P. G. & Hall, A. C. The volume and morphology of chondrocytes within non-degenerate and degenerate human articular cartilage. Osteoarthr. Cartil. 11, 242–251 (2003).

    CAS  Google Scholar 

  44. O’ Conor, C. J., Leddy, H. A., Benefield, H. C., Liedtke, W. B. & Guilak, F. TRPV4-mediated mechanotransduction regulates the metabolic response of chondrocytes to dynamic loading. Proc. Natl Acad. Sci. USA 111, 1316–1321 (2014).

    Google Scholar 

  45. Discher, D. E., Janmey, P. & Wang, Y.-L. Tissue cells feel and respond to the stiffness of their substrate. Science 310, 1139–1143 (2005).

    CAS  Google Scholar 

  46. Huebsch, N. et al. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat. Mater. 9, 518–526 (2010).

    CAS  Google Scholar 

  47. Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7, 265–275 (2006).

    CAS  Google Scholar 

  48. Khetan, S. et al. Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels. Nat. Mater. 12, 458–465 (2013).

    CAS  Google Scholar 

  49. Nicodemus, G. D. & Bryant, S. J. Cell encapsulation in biodegradable hydrogels for tissue engineering applications. Tissue Eng. B. Rev. 14, 149–165 (2008).

    CAS  Google Scholar 

  50. Kim, J.-H. et al. Matrix cross-linking-mediated mechanotransduction promotes posttraumatic osteoarthritis. Proc. Natl Acad. Sci. USA 112, 9424–9429 (2015).

    CAS  Google Scholar 

  51. Nam, S., Hu, K. H., Butte, M. J. & Chaudhuri, O. Strain-enhanced stress relaxation impacts nonlinear elasticity in collagen gels. Proc. Natl Acad. Sci. USA 113, 201523906 (2016).

    Google Scholar 

  52. Lai, J. H., Kajiyama, G., Smith, R. L., Maloney, W. & Yang, F. Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels. Sci. Rep. 3, 3553 (2013).

    Google Scholar 

  53. Farndale, R. W., Buttle, D. J. & Barrett, A. J. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim. Biophys. Acta 883, 173–177 (1986).

    CAS  Google Scholar 

  54. Enobakhare, B. O., Bader, D. L. & Lee, D. A. Quantification of sulfated glycosaminoglycans in chondrocyte/alginate cultures, by use of 1,9-dimethylmethylene blue. Anal. Biochem. 243, 189–191 (1996).

    CAS  Google Scholar 

  55. Stegemann, H. & Stalder, K. Determination of hydroxyproline. Clin. Chim. Acta. 18, 267–273 (1967).

    CAS  Google Scholar 

  56. Im, H. J. et al. Inhibitory effects of insulin-like growth factor-1 and osteogenic protein-1 on fibronectin Fragment- and interleukin-1β-stimulated matrix metalloproteinase-13 expression in human chondrocytes. J. Biol. Chem. 278, 25386–25394 (2003).

    CAS  Google Scholar 

  57. Smith, L. J. et al. Nucleus pulposus cells synthesize a functional extracellular matrix and respond to inflammatory cytokine treatment following long term agarose culture. Eur. Cells Mater. 20, 291–301 (2011).

    Google Scholar 

  58. Lagerwerff, J. V., Ogata, G. & Eagle, H. E. Control of osmotic pressure of culture solutions with polyethylene glycol. Science 133, 1486–1487 (1961).

    CAS  Google Scholar 

  59. Stanley, C. B. & Strey, H. H. Measuring osmotic pressure of poly(ethylene glycol) solutions by sedimentation equilibrium ultracentrifugation. Macromolecules 36, 6888–6893 (2003).

    CAS  Google Scholar 

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Acknowledgements

The authors acknowledge the help of R. Stowers, J. Lee, A. Arvayo, J. Lai, G. Baylon, M. Aliyeh, K. Wisdom, S. Nam, S. Lee, D. No, J. Yu, K. Kim, K. Han and all members of the Chaudhuri lab, and thank D. Weitz (Harvard University) for helpful discussions. This work was supported by the Jeongsong Cultural Foundation (to H.L.), NIH grant R01 DE013033 (to D.J.M.), and DARPA grant D14AP00044 (to O.C.).

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Authors and Affiliations

  1. Department of Mechanical Engineering, Stanford University, Stanford, California, 94305, USA

    Hong-pyo Lee, Marc E. Levenston & Ovijit Chaudhuri

  2. School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, 02138, USA

    Luo Gu & David J. Mooney

  3. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, 02138, USA

    Luo Gu & David J. Mooney

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  1. Hong-pyo Lee
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Contributions

H.L., M.E.L. and O.C. designed the experiments. H.L. conducted all experiments and analysed the data. L.G. and D.J.M. contributed to the materials design and materials. H.L. and O.C. wrote the manuscript.

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Correspondence to Ovijit Chaudhuri.

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Lee, Hp., Gu, L., Mooney, D. et al. Mechanical confinement regulates cartilage matrix formation by chondrocytes. Nature Mater 16, 1243–1251 (2017). https://doi.org/10.1038/nmat4993

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