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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
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).
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).
Ogle, W. Aristotle’s De Partibus Animalium (Clarendon Press, 1911).
Vacanti, C. A. The history of tissue engineering. J. Cell Mol. Med. 10, 569–576 (2006).
Huey, D. J., Hu, J. C. & Athanasiou, K. A. Unlike bone, cartilage regeneration remains elusive. Science 338, 917–921 (2012).
Little, C. J., Bawolin, N. K. & Chen, X. Mechanical properties of natural cartilage and tissue-engineered constructs. Tissue Eng. B 17, 213–227 (2011).
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).
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).
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).
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).
Chen, C. et al. Cyclic equibiaxial tensile strain alters gene expression of chondrocytes via histone deacetylase 4 shuttling. PLoS ONE 11, e0154951 (2016).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Hu, J. C. & Athanasiou, K. A. A self-assembling process in articular cartilage tissue engineering. Tissue Eng. 12, 969–979 (2006).
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).
Blunk, T. et al. Differential effects of growth factors on tissue-engineered cartilage. Tissue Eng. 8, 73–84 (2002).
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).
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).
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).
Steele, J. A. et al. Combinatorial scaffold morphologies for zonal articular cartilage engineering. Acta Biomater. 10, 2065–2075 (2013).
Kevorkian, L. et al. Expression profiling of metalloproteinases and their inhibitors in cartilage. Arthritis Rheum. 50, 131–141 (2004).
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).
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).
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).
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).
Nakao, A. et al. Identification of Smad7, a TGF[β]-inducible antagonist of TGF-[β] signalling. Nature 389, 631–635 (1997).
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).
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).
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).
Liu, H. et al. Enhanced tissue regeneration potential of juvenile articular cartilage. Am. J. Sports Med. 41, 2658–2667 (2013).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Guilak, F. et al. Mechanically induced calcium waves in articular chondrocytes are inhibited by gadolinium and amiloride. J. Orthop. Res. 17, 421–429 (1999).
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).
Acerbi, I. et al. Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration. Integr. Biol. 7, 1120–1134 (2015).
Maas, S. A., Ellis, B. J., Ateshian, G. A. & Weiss, J. A. FEBio: finite elements for biomechanics. J. Biomech. Eng. 134, 011005 (2012).
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.
Author information
Authors and Affiliations
Contributions
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.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Information
Supplementary Information (PDF 1725 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmat4917
This article is cited by
-
Biomimetic ECM-Based Hybrid Scaffold for Cartilage Tissue Engineering Applications
Journal of Polymers and the Environment (2024)
-
Proteomic, mechanical, and biochemical development of tissue-engineered neocartilage
Biomaterials Research (2022)
-
Non-destructive, continuous monitoring of biochemical, mechanical, and structural maturation in engineered tissue
Scientific Reports (2022)
-
Tissue Engineering of Canine Cartilage from Surgically Debrided Osteochondritis Dissecans Fragments
Annals of Biomedical Engineering (2022)
-
Engineering large, anatomically shaped osteochondral constructs with robust interfacial shear properties
npj Regenerative Medicine (2021)