Tension stimulation drives tissue formation in scaffold-free systems

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Abstract

Scaffold-free systems have emerged as viable approaches for engineering load-bearing tissues. However, the tensile properties of engineered tissues have remained far below the values for native tissue. Here, by using self-assembled articular cartilage as a model to examine the effects of intermittent and continuous tension stimulation on tissue formation, we show that the application of tension alone, or in combination with matrix remodelling and synthesis agents, leads to neocartilage with tensile properties approaching those of native tissue. Implantation of tension-stimulated tissues results in neotissues that are morphologically reminiscent of native cartilage. We also show that tension stimulation can be translated to a human cell source to generate anisotropic human neocartilage with enhanced tensile properties. Tension stimulation, which results in nearly sixfold improvements in tensile properties over unstimulated controls, may allow the engineering of mechanically robust biological replacements of native tissue.

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Figure 1: Large-construct generation and uniform-strain validation.
Figure 2: Tissue engineering of neocartilage with enhanced tensile properties.
Figure 3: Under tension stimulation, the TRPV4 ion channel is implicated to initiate matrix remodelling.
Figure 4: The in vivo environment results in neocartilage with morphological structure reminiscent of native articular cartilage.
Figure 5: Translation of tension stimulation to human neocartilage.

References

  1. 1

    Responte, D. J., Lee, J. K., Hu, J. C. & Athanasiou, K. A. Biomechanics-driven chondrogenesis: from embryo to adult. FASEB J. 26, 3614–3624 (2012).

    CAS  Google Scholar 

  2. 2

    Vos, T. et al. Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 386, 743–800 (2015).

    Google Scholar 

  3. 3

    Ogle, W. Aristotle’s De Partibus Animalium (Clarendon Press, 1911).

    Google Scholar 

  4. 4

    Vacanti, C. A. The history of tissue engineering. J. Cell Mol. Med. 10, 569–576 (2006).

    Google Scholar 

  5. 5

    Huey, D. J., Hu, J. C. & Athanasiou, K. A. Unlike bone, cartilage regeneration remains elusive. Science 338, 917–921 (2012).

    CAS  Google Scholar 

  6. 6

    Little, C. J., Bawolin, N. K. & Chen, X. Mechanical properties of natural cartilage and tissue-engineered constructs. Tissue Eng. B 17, 213–227 (2011).

    CAS  Google Scholar 

  7. 7

    Eleswarapu, S. V., Responte, D. J. & Athanasiou, K. A. Tensile properties, collagen content, and crosslinks in connective tissues of the immature knee joint. PLoS ONE 6, e26178 (2011).

    CAS  Google Scholar 

  8. 8

    Makris, E. A., Gomoll, A. H., Malizos, K. N., Hu, J. C. & Athanasiou, K. A. Repair and tissue engineering techniques for articular cartilage. Nat. Rev. Rheumatol. 11, 21–34 (2014).

    Google Scholar 

  9. 9

    Athanasiou, K. A., Responte, D. J., Brown, W. E. & Hu, J. C. Harnessing biomechanics to develop cartilage regeneration strategies. J. Biomech. Eng. 137, 020901 (2015).

    Google Scholar 

  10. 10

    Lee, C., Grad, S., Wimmer, M. & Alini, M. in Topics in Tissue Engineering Vol. 2 (eds Ashammakhi, N. & Reis, R. L.) Ch. 2 (Expertissues, 2006).

    Google Scholar 

  11. 11

    Chen, C. et al. Cyclic equibiaxial tensile strain alters gene expression of chondrocytes via histone deacetylase 4 shuttling. PLoS ONE 11, e0154951 (2016).

    Google Scholar 

  12. 12

    Wu, Q. Q. & Chen, Q. Mechanoregulation of chondrocyte proliferation, maturation, and hypertrophy: ion-channel dependent transduction of matrix deformation signals. Exp. Cell Res. 256, 383–391 (2000).

    CAS  Google Scholar 

  13. 13

    Huang, D., Chang, T. R., Aggarwal, A., Lee, R. C. & Ehrlich, H. P. Mechanisms and dynamics of mechanical strengthening in ligament-equivalent fibroblast-populated collagen matrices. Ann. Biomed. Eng. 21, 289–305 (1993).

    CAS  Google Scholar 

  14. 14

    Connelly, J. T., Vanderploeg, E. J. & Levenston, M. E. The influence of cyclic tension amplitude on chondrocyte matrix synthesis: experimental and finite element analyses. Biorheology 41, 377–387 (2004).

    CAS  Google Scholar 

  15. 15

    Vanderploeg, E. J., Wilson, C. G. & Levenston, M. E. Articular chondrocytes derived from distinct tissue zones differentially respond to in vitro oscillatory tensile loading. Osteoarthritis Cartilage 16, 1228–1236 (2008).

    CAS  Google Scholar 

  16. 16

    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 

  17. 17

    Nims, R. J., Cigan, A. D., Albro, M. B., Hung, C. T. & Ateshian, G. A. Synthesis rates and binding kinetics of matrix products in engineered cartilage constructs using chondrocyte-seeded agarose gels. J. Biomech. 47, 2165–2172 (2014).

    Google Scholar 

  18. 18

    Elder, B. D. & Athanasiou, K. A. Systematic assessment of growth factor treatment on biochemical and biomechanical properties of engineered articular cartilage constructs. Osteoarthritis Cartilage 17, 114–123 (2009).

    CAS  Google Scholar 

  19. 19

    Responte, D. J., Arzi, B., Natoli, R. M., Hu, J. C. & Athanasiou, K. A. Mechanisms underlying the synergistic enhancement of self-assembled neocartilage treated with chondroitinase-ABC and TGF-β1. Biomaterials 33, 3187–3194 (2012).

    CAS  Google Scholar 

  20. 20

    Makris, E. A., Responte, D. J., Paschos, N. K., Hu, J. C. & Athanasiou, K. A. Developing functional musculoskeletal tissues through hypoxia and lysyl oxidase-induced collagen cross-linking. Proc. Natl Acad. Sci. USA 111, E4832–E4841 (2014).

    CAS  Google Scholar 

  21. 21

    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 

  22. 22

    Hu, J. C. & Athanasiou, K. A. A self-assembling process in articular cartilage tissue engineering. Tissue Eng. 12, 969–979 (2006).

    CAS  Google Scholar 

  23. 23

    Yaeger, P. C. et al. Synergistic action of transforming growth factor-β and insulin-like growth factor-I induces expression of type II collagen and aggrecan genes in adult human articular chondrocytes. Exp. Cell Res. 237, 318–325 (1997).

    CAS  Google Scholar 

  24. 24

    Blunk, T. et al. Differential effects of growth factors on tissue-engineered cartilage. Tissue Eng. 8, 73–84 (2002).

    CAS  Google Scholar 

  25. 25

    Asanbaeva, A., Masuda, K., Thonar, E. J., Klisch, S. M. & Sah, R. L. Mechanisms of cartilage growth: modulation of balance between proteoglycan and collagen in vitro using chondroitinase ABC. Arthritis Rheum. 56, 188–198 (2007).

    CAS  Google Scholar 

  26. 26

    Williamson, A. K., Chen, A. C., Masuda, K., Thonar, E. J. M. A. & Sah, R. L. Tensile mechanical properties of bovine articular cartilage: variations with growth and relationships to collagen network components. J. Orthop. Res. 21, 872–880 (2003).

    CAS  Google Scholar 

  27. 27

    Atsawasuwan, P. et al. Lysyl oxidase binds transforming growth factor-β and regulates its signaling via amine oxidase activity. J. Biol. Chem. 283, 34229–34240 (2008).

    CAS  Google Scholar 

  28. 28

    Steele, J. A. et al. Combinatorial scaffold morphologies for zonal articular cartilage engineering. Acta Biomater. 10, 2065–2075 (2013).

    Google Scholar 

  29. 29

    Kevorkian, L. et al. Expression profiling of metalloproteinases and their inhibitors in cartilage. Arthritis Rheum. 50, 131–141 (2004).

    CAS  Google Scholar 

  30. 30

    Wachsmuth, L. et al. ADAMTS-1, a gene product of articular chondrocytes in vivo and in vitro, is downregulated by interleukin 1β. J. Rheumatol. 31, 315–320 (2004).

    CAS  Google Scholar 

  31. 31

    Iftikhar, M. et al. Lysyl oxidase-like-2 (LOXL2) is a major isoform in chondrocytes and is critically required for differentiation. J. Biol. Chem. 286, 909–918 (2011).

    CAS  Google Scholar 

  32. 32

    Ito, H. et al. Molecular cloning and biological activity of a novel lysyl oxidase-related gene expressed in cartilage. J. Biol. Chem. 276, 24023–24029 (2001).

    CAS  Google Scholar 

  33. 33

    Lahiji, K., Polotsky, A., Hungerford, D. & Frondoza, C. Cyclic strain stimulates proliferative capacity, α2 and α5 integrin, gene marker expression by human articular chondrocytes propagated on flexible silicone membranes. In Vitro Cell. Dev. Biol. Anim. 40, 138–142 (2004).

    CAS  Google Scholar 

  34. 34

    Nakao, A. et al. Identification of Smad7, a TGF[β]-inducible antagonist of TGF-[β] signalling. Nature 389, 631–635 (1997).

    CAS  Google Scholar 

  35. 35

    Iwai, T., Murai, J., Yoshikawa, H. & Tsumaki, N. Smad7 inhibits chondrocyte differentiation at multiple steps during endochondral bone formation and down-regulates p38 MAPK pathways. J. Biol. Chem. 283, 27154–27164 (2008).

    CAS  Google Scholar 

  36. 36

    Eleswarapu, S. V. & Athanasiou, K. A. TRPV4 channel activation improves the tensile properties of self-assembled articular cartilage constructs. Acta Biomater. 9, 5554–5561 (2013).

    CAS  Google Scholar 

  37. 37

    Lee, J. K., Gegg, C. A., Hu, J. C., Kass, P. H. & Athanasiou, K. A. Promoting increased mechanical properties of tissue engineered neocartilage via the application of hyperosmolarity and 4α-phorbol 12,13-didecanoate (4αPDD). J. Biomech. 47, 3712–3718 (2014).

    Google Scholar 

  38. 38

    Liu, H. et al. Enhanced tissue regeneration potential of juvenile articular cartilage. Am. J. Sports Med. 41, 2658–2667 (2013).

    Google Scholar 

  39. 39

    Temple, M. M. et al. Age- and site-associated biomechanical weakening of human articular cartilage of the femoral condyle. Osteoarthritis Cartilage 15, 1042–1052 (2007).

    CAS  Google Scholar 

  40. 40

    Temple-Wong, M. M. et al. Biomechanical, structural, and biochemical indices of degenerative and osteoarthritic deterioration of adult human articular cartilage of the femoral condyle. Osteoarthritis Cartilage 17, 1469–1476 (2009).

    CAS  Google Scholar 

  41. 41

    Below, S., Arnoczky, S. P., Dodds, J., Kooima, C. & Walter, N. The split-line pattern of the distal femur: a consideration in the orientation of autologous cartilage grafts. Arthroscopy 18, 613–617 (2002).

    Google Scholar 

  42. 42

    Butler, D. L. et al. Functional tissue engineering for tendon repair: a multidisciplinary strategy using mesenchymal stem cells, bioscaffolds, and mechanical stimulation. J. Orthop. Res. 26, 1–9 (2008).

    Google Scholar 

  43. 43

    Henshaw, D. R., Attia, E., Bhargava, M. & Hannafin, J. A. Canine ACL fibroblast integrin expression and cell alignment in response to cyclic tensile strain in three-dimensional collagen gels. J. Orthop. Res. 24, 481–490 (2006).

    CAS  Google Scholar 

  44. 44

    Moreau, J. E., Bramono, D. S., Horan, R. L., Kaplan, D. L. & Altman, G. H. Sequential biochemical and mechanical stimulation in the development of tissue-engineered ligaments. Tissue Eng. A 14, 1161–1172 (2008).

    CAS  Google Scholar 

  45. 45

    Rodrigues, M. T., Reis, R. L. & Gomes, M. E. Engineering tendon and ligament tissues: present developments towards successful clinical products. J. Tissue Eng. Regen. Med. 7, 673–686 (2013).

    CAS  Google Scholar 

  46. 46

    Connelly, J. T., Vanderploeg, E. J., Mouw, J. K., Wilson, C. G. & Levenston, M. E. Tensile loading modulates bone marrow stromal cell differentiation and the development of engineered fibrocartilage constructs. Tissue Eng. A 16, 1913–1923 (2010).

    CAS  Google Scholar 

  47. 47

    Chen, J.-P., Liao, H.-T. & Cheng, T.-H. Cultivation of chondrocytes and meniscus cells in thermo-responsive hydrogels containing chitosan and hyaluronic acid under mechanical tensile stimulation. J. Mech. Med. Biol. 11, 1003–1015 (2011).

    Google Scholar 

  48. 48

    McMahon, L., Reid, A., Campbell, V. & Prendergast, P. Regulatory effects of mechanical strain on the chondrogenic differentiation of MSCs in a collagen-gag scaffold: experimental and computational analysis. Ann. Biomed. Eng. 36, 185–194 (2008).

    Google Scholar 

  49. 49

    Hung, C. T., Mauck, R. L., Wang, C. C., Lima, E. G. & Ateshian, G. A. A paradigm for functional tissue engineering of articular cartilage via applied physiologic deformational loading. Ann. Biomed. Eng. 32, 35–49 (2004).

    Google Scholar 

  50. 50

    Bryant, S. J. & Anseth, K. S. Controlling the spatial distribution of ECM components in degradable PEG hydrogels for tissue engineering cartilage. J. Biomed. Mater. Res. A 64, 70–79 (2003).

    Google Scholar 

  51. 51

    Demirbag, B., Huri, P. Y., Kose, G. T., Buyuksungur, A. & Hasirci, V. Advanced cell therapies with and without scaffolds. Biotechnol. J. 6, 1437–1453 (2011).

    CAS  Google Scholar 

  52. 52

    Lee, J. K., Gegg, C. A., Hu, J. C., Reddi, A. H. & Athanasiou, K. A. Thyroid hormones enhance the biomechanical functionality of scaffold-free neocartilage. Arthritis Res. Ther. 17, 28–38 (2015).

    Google Scholar 

  53. 53

    Murphy, M. K., Huey, D. J., Hu, J. C. & Athanasiou, K. A. TGF-β1, GDF-5, and BMP-2 stimulation induces chondrogenesis in expanded human articular chondrocytes and marrow-derived stromal cells. Stem Cells 33, 762–773 (2015).

    CAS  Google Scholar 

  54. 54

    Guilak, F. et al. Mechanically induced calcium waves in articular chondrocytes are inhibited by gadolinium and amiloride. J. Orthop. Res. 17, 421–429 (1999).

    CAS  Google Scholar 

  55. 55

    Phan, M. N. et al. Functional characterization of TRPV4 as an osmotically sensitive ion channel in porcine articular chondrocytes. Arthritis Rheum. 60, 3028–3037 (2009).

    CAS  Google Scholar 

  56. 56

    Acerbi, I. et al. Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration. Integr. Biol. 7, 1120–1134 (2015).

    CAS  Google Scholar 

  57. 57

    Maas, S. A., Ellis, B. J., Ateshian, G. A. & Weiss, J. A. FEBio: finite elements for biomechanics. J. Biomech. Eng. 134, 011005 (2012).

    Google Scholar 

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Acknowledgements

This work was made possible with the support of the National Institutes of Health (NIH) awards R01 AR067821 (National Institute of Arthritis and Musculoskeletal and Skin Diseases), R01 DE015038 (National Institute of Dental and Craniofacial Research) and T32 GM00799 (National Institute of General Medical Sciences) for J.K.L. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Microarray analysis was made possible by the UC Davis Comprehensive Cancer Center Genomics Shared Resource (NCI P30 CA93373). We also thank L. Cassereau and the Weaver laboratory for assistance with second harmonic generation imaging.

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J.K.L., L.W.H., J.C.H. and K.A.A. were responsible for the design and execution of InTenS and CoTenS studies. N.P. and J.K.L. together conducted all animal work, while N.P., A.A. and L.W.H. performed the human articular chondrocyte experiments. C.A.G. assisted in the design and fabrication of the tensile loading device. J.K.L., L.W.H., N.P., A.A. and C.A.G. collected all data. A.A. performed finite element modelling and analysis. J.K.L. and L.W.H. performed the data analysis. J.K.L., L.W.H., J.C.H. and K.A.A. prepared the manuscript.

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Correspondence to Kyriacos A. Athanasiou.

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Lee, J., Huwe, L., Paschos, N. et al. Tension stimulation drives tissue formation in scaffold-free systems. Nature Mater 16, 864–873 (2017). https://doi.org/10.1038/nmat4917

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