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The microstructure and micromechanics of the tendon–bone insertion

Abstract

The exceptional mechanical properties of the load-bearing connection of tendon to bone rely on an intricate interplay of its biomolecular composition, microstructure and micromechanics. Here we identify that the Achilles tendon–bone insertion is characterized by an interface region of 500 μm with a distinct fibre organization and biomolecular composition. Within this region, we identify a heterogeneous mechanical response by micromechanical testing coupled with multiscale confocal microscopy. This leads to localized strains that can be larger than the remotely applied strain. The subset of fibres that sustain the majority of loading in the interface area changes with the angle of force application. Proteomic analysis detects enrichment of 22 proteins in the interfacial region that are predominantly involved in cartilage and skeletal development as well as proteoglycan metabolism. The presented mechanisms mark a guideline for further biomimetic strategies to rationally design hard–soft interfaces.

Figure 1: The structure of tendon fibres changes before attaching to bone.
Figure 2: Micromechanical testing of enthesis samples.
Figure 3: Fibre composition changes from collagen type I to collagen type II before attaching to bone.
Figure 4: Quantitative proteome analysis of differences in expression between Achilles tendon enthesis (E) and Achilles tendon (T).
Figure 5: Fundamental structural and molecular components of the enthesis.

References

  1. 1

    Genin, G. M. et al. Functional grading of mineral and collagen in the attachment of tendon to bone. Biophys. J. 97, 976–985 (2009).

    CAS  Google Scholar 

  2. 2

    Wren, T., Yerby, S., Beaupré, G. & Carter, D. Mechanical properties of the human Achilles tendon. Clin. Biomech. 16, 245–251 (2001).

    CAS  Google Scholar 

  3. 3

    Thomopoulos, S., Birman, V. & Genin, G. in Structural Interfaces and Attachments in Biology (eds Thomopoulos, S., Birman, V. & Genin, G.) 3–17 (Springer, 2013).

    Google Scholar 

  4. 4

    Moffat, K. L. et al. Characterization of the structure-function relationship at the ligament-to-bone interface. Proc. Natl Acad. Sci. USA 105, 7947–7952 (2008).

    CAS  Google Scholar 

  5. 5

    Buehler, M. J. & Yung, Y. C. Deformation and failure of protein materials in physiologically extreme conditions and disease. Nat. Mater. 8, 175–188 (2009).

    CAS  Google Scholar 

  6. 6

    Bogy, D. B. Edge-bonded dissimilar orthogonal elastic wedges under normal and shear loading. J. Appl. Mech. 35, 460–466 (1968).

    Google Scholar 

  7. 7

    Benjamin, M. et al. Where tendons and ligaments meet bone: attachment sites (‘entheses’) in relation to exercise and/or mechanical load. J. Anat. 208, 471–490 (2006).

    CAS  Google Scholar 

  8. 8

    Fukashiro, S., Koomi, P., Järvinen, M. & Miyashita, M. In vivo Achilles tendon loading during jumping in humans. Eur. J. Appl. Physiol. 71, 453–458 (1995).

    CAS  Google Scholar 

  9. 9

    Pedowitz, D. & Kirwan, G. Achilles tendon ruptures. Curr. Rev. Muskuloskelet. Med. 6, 285–293 (2013).

    Google Scholar 

  10. 10

    Weatherall, J. M., Mroczek, K. & Tejwani, N. Acute Achilles tendon ruptures. Orthopedics 33, 758–764 (2010).

    Google Scholar 

  11. 11

    Buehler, M. J. Nature designs tough collagen: explaining the nanostructure of collagen fibrils. Proc. Natl Acad. Sci. USA 103, 12285–12290 (2006).

    CAS  Google Scholar 

  12. 12

    Thorpe, C. T. et al. Anatomical heterogeneity of tendon: Fascicular and interfascicular tendon compartments have distinct proteomic composition. Sci. Rep. 6, 20455 (2016).

    CAS  Google Scholar 

  13. 13

    Thorpe, C. T., Birch, H. L., Clegg, P. D. & Screen, H. R. The role of the non-collagenous matrix in tendon function. Int. J. Exp. Pathol. 94, 248–259 (2013).

    CAS  Google Scholar 

  14. 14

    Lakes, R. Materials with structural hierarchy. Nature 361, 511–515 (1993).

    Google Scholar 

  15. 15

    Benjamin, M. & Ralphs, J. R. Tendons and ligaments—an overview. Histol. Histopathol. 12, 1135–1144 (1997).

    CAS  Google Scholar 

  16. 16

    Fratzl, P. Collagen—Structure and Mechanics 1–12 (Springer, 2008).

    Google Scholar 

  17. 17

    Baer, E., Hiltner, A. & Keith, H. D. Hierarchical structure in polymeric materials. Science 235, 1015–1022 (1987).

    CAS  Google Scholar 

  18. 18

    Birk, D. E. & Bruckner, P. in Collagen (eds Brinckmann, J., Notbohm, H. & Müller, P.) 185–205 (Springer, 2005).

    Google Scholar 

  19. 19

    Benjamin, M., Kumai, T., Milz, S. & Ralphs, J. R. The skeletal attachment of tendons–tendon entheses. Comp. Biochem. Physiol. Part A 133, 931–945 (2002).

    CAS  Google Scholar 

  20. 20

    Liu, Y. et al. Modelling the mechanics of partially mineralized collagen fibrils, fibres and tissue. J. R. Soc. Interface 11, 20130835 (2014).

    Google Scholar 

  21. 21

    Buehler, M. J. Molecular nanomechanics of nascent bone: fibrillar toughening by mineralization. Nanotechnology 18, 295102 (2007).

    Google Scholar 

  22. 22

    Gautieri, A., Vesentini, S., Redaelli, A. & Buehler, M. J. Hierarchical structure and nanomechanics of collagen microfibrils from the atomistic scale up. Nano Lett. 11, 757–766 (2011).

    CAS  Google Scholar 

  23. 23

    Nair, A. K., Gautieri, A., Chang, S. W. & Buehler, M. J. Molecular mechanics of mineralized collagen fibrils in bone. Nat. Commun. 4, 1724 (2013).

    Google Scholar 

  24. 24

    Fantner, G. E. et al. Sacrificial bonds and hidden length dissipate energy as mineralized fibrils separate during bone fracture. Nat. Mater. 4, 612–616 (2005).

    CAS  Google Scholar 

  25. 25

    Jager, I. & Fratzl, P. Mineralized collagen fibrils: a mechanical model with a staggered arrangement of mineral particles. Biophys. J. 79, 1737–1746 (2000).

    CAS  Google Scholar 

  26. 26

    Wopenka, B., Kent, A., Pasteris, J. D., Yoon, Y. & Thomopoulos, S. The tendon-to-bone transition of the rotator cuff: a preliminary raman spectroscopic study documenting the gradual mineralization across the insertion in rat tissue samples. Appl. Spectrosc. 62, 1285–1294 (2008).

    CAS  Google Scholar 

  27. 27

    Thomopoulos, S., Williams, G. R., Gimbel, J. A., Favata, M. & Soslowsky, L. J. Variation of biomechanical, structural, and compositional properties along the tendon to bone insertion site. J. Orthop. Res. 21, 413–419 (2003).

    Google Scholar 

  28. 28

    Thomopoulos, S., Marquez, J. P., Weinberger, B., Birman, V. & Genin, G. M. Collagen fiber orientation at the tendon to bone insertion and its influence on stress concentrations. J. Biomech. 39, 1842–1851 (2006).

    Google Scholar 

  29. 29

    Lu, H. H. & Thomopoulos, S. Functional attachment of soft tissues to bone: development, healing, and tissue engineering. Annu. Rev. Biomed. Eng. 15, 201–226 (2013).

    CAS  Google Scholar 

  30. 30

    Liu, Y. X., Thomopoulos, S., Birman, V., Li, J. S. & Genin, G. M. Bi-material attachment through a compliant interfacial system at the tendon-to-bone insertion site. Mech. Mater. 44, 83–92 (2012).

    CAS  Google Scholar 

  31. 31

    Liu, Y. X., Birman, V., Chen, C., Thomopoulos, S. & Genin, G. M. Mechanisms of bimaterial attachment at the interface of tendon to bone. J. Eng. Mater. Technol. 133, 011006 (2011).

    Google Scholar 

  32. 32

    Galatz, L. et al. Development of the supraspinatus tendon-to-bone insertion: localized expression of extracellular matrix and growth factor genes. J. Orthop. Res. 25, 1621–1628 (2007).

    Google Scholar 

  33. 33

    Hu, Y. et al. Stochastic interdigitation as a toughening mechanism at the interface between tendon and bone. Biophys. J. 108, 431–437 (2015).

    CAS  Google Scholar 

  34. 34

    de S. e Silva, J. M. et al. Three-dimensional non-destructive soft-tissue visualization with X-ray staining micro-tomography. Sci. Rep. 5, 14088 (2015).

    Google Scholar 

  35. 35

    Balijepalli, R. G., Begley, M. R., Fleck, N. A., McMeeking, R. M. & Arzt, E. Numerical simulation of the edge stress singularity and the adhesion strength for compliant mushroom fibrils adhered to rigid substrates. Int. J. Solids Struct. 85–86, 160–171 (2016).

    Google Scholar 

  36. 36

    Schwartz, A. & Thomopoulos, S. in Structural Interfaces and Attachments in Biology (eds Thomopoulos, S., Birman, V. & Genin, G.) 229–257 (Springer, 2013).

    Google Scholar 

  37. 37

    Zhang, L. et al. A coupled fiber-matrix model demonstrates highly inhomogeneous microstructural interactions in soft tissues under tensile load. J. Biomech. Eng 135, 011008 (2013).

    Google Scholar 

  38. 38

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

    CAS  Google Scholar 

  39. 39

    Mouw, J. K., Ou, G. & Weaver, V. M. Extracellular matrix assembly: a multiscale deconstruction. Nat. Rev. Mol. Cell. Biol. 15, 771–785 (2014).

    CAS  Google Scholar 

  40. 40

    Yoon, J. H. & Halper, J. Tendon proteoglycans: biochemistry and function. J. Musculoskelet. Neuronal. Interact. 5, 22–34 (2005).

    CAS  Google Scholar 

  41. 41

    Rigozzi, S., Muller, R., Stemmer, A. & Snedeker, J. G. Tendon glycosaminoglycan proteoglycan sidechains promote collagen fibril sliding-AFM observations at the nanoscale. J. Biomech. 46, 813–818 (2013).

    CAS  Google Scholar 

  42. 42

    Kuettner, K. E. & Kimura, J. H. Proteoglycans: an overview. J. Cell. Biochem. 27, 327–336 (1985).

    CAS  Google Scholar 

  43. 43

    Ritchie, R. O. The conflicts between strength and toughness. Nat. Mater. 10, 817–822 (2011).

    CAS  Google Scholar 

  44. 44

    Shaw, H. M. & Benjamin, M. Structure-function relationships of entheses in relation to mechanical load and exercise. Scand. J. Med. Sci. Sports 17, 303–315 (2007).

    CAS  Google Scholar 

  45. 45

    Deymier-Black, A. C., Pasteris, J. D., Genin, G. M. & Thomopoulos, S. Allometry of the tendon enthesis: mechanisms of load transfer between tendon and bone. J. Biomech. Eng. 137, 111005 (2015).

    Google Scholar 

  46. 46

    Han, W. M. et al. Microstructural heterogeneity directs micromechanics and mechanobiology in native and engineered fibrocartilage. Nat. Mater. 15, 477–484 (2016).

    CAS  Google Scholar 

  47. 47

    Chung, J. Y. & Chaudhury, M. K. Soft and hard adhesion. J. Adhes. 81, 1119–1145 (2007).

    Google Scholar 

  48. 48

    Lee, B. P., Messersmith, P. B., Israelachvili, J. N. & Waite, J. H. Mussel-inspired adhesives and coatings. Annu. Rev. Mater. Res. 41, 99–132 (2011).

    CAS  Google Scholar 

  49. 49

    Ho, S. P., Marshall, S. J., Ryder, M. I. & Marshall, G. W. The tooth attachment mechanism defined by structure, chemical composition and mechanical properties of collagen fibers in the periodontium. Biomaterials 28, 5238–5245 (2007).

    CAS  Google Scholar 

  50. 50

    Yuk, H., Zhang, T., Lin, S., Parada, G. A. & Zhao, X. Tough bonding of hydrogels to diverse non-porous surfaces. Nat. Mater. 15, 190–196 (2016).

    CAS  Google Scholar 

  51. 51

    Freed, L. E., Engelmayr, J., G. C., Borenstein, J. T., Moutos, F. T. & Guilak, F. Advanced material strategies for tissue engineering scaffolds. Adv. Mater. 21, 3410–3418 (2009).

    CAS  Google Scholar 

  52. 52

    Screen, H. R. et al. The influence of swelling and matrix degradation on the microstructural integrity of tendon. Acta Biomater. 2, 505–513 (2006).

    Google Scholar 

  53. 53

    Screen, H. R. C., Seto, J., Krauss, S., Boesecke, P. & Gupta, H. S. Extrafibrillar diffusion and intrafibrillar swelling at the nanoscale are associated with stress relaxation in the soft collagenous matrix tissue of tendons. Soft Matter 7, 11243–11251 (2011).

    CAS  Google Scholar 

  54. 54

    Preibisch, S., Saalfeld, S. & Tomancak, P. Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25, 1463–1465 (2009).

    CAS  Google Scholar 

  55. 55

    Arampatzis, A., Peper, A., Bierbaum, S. & Albracht, K. Plasticity of human Achilles tendon mechanical and morphological properties in response to cyclic strain. J. Biomech. 43, 3073–3079 (2010).

    Google Scholar 

  56. 56

    Tseng, Q. et al. Spatial organization of the extracellular matrix regulates cell-cell junction positioning. Proc. Natl Acad. Sci. USA 109, 1506–1511 (2012).

    CAS  Google Scholar 

  57. 57

    Buckley, M. R., Gleghorn, J. P., Bonassar, L. J. & Cohen, I. Mapping the depth dependence of shear properties in articular cartilage. J. Biomech. 41, 2430–2437 (2008).

    Google Scholar 

  58. 58

    Raffel, M., Willert, C. E. & Kompenhans, J. Particle Image Velocimetry—A Pratical Guide (Springer, 2007).

    Google Scholar 

  59. 59

    Upton, M. L., Gilchrist, C. L., Guilak, F. & Setton, L. A. Transfer of macroscale tissue strain to microscale cell regions in the deformed meniscus. Biophys. J. 95, 2116–2124 (2008).

    CAS  Google Scholar 

  60. 60

    Cheng, V. W. T. & Screen, H. R. C. The micro-structural strain response of tendon. J. Mater. Sci. 42, 8957–8965 (2007).

    CAS  Google Scholar 

  61. 61

    Michalek, A. J., Buckley, M. R., Bonassar, L. J., Cohen, I. & Iatridis, J. C. Measurement of local strains in intervertebral disc anulus fibrosus tissue under dynamic shear: contributions of matrix fiber orientation and elastin content. J. Biomech. 42, 2279–2285 (2009).

    Google Scholar 

  62. 62

    Boyle, J. J. et al. Simple and accurate methods for quantifying deformation, disruption, and development in biological tissues. J. R. Soc. Interface 11, 20140685 (2014).

    Google Scholar 

  63. 63

    Boersema, P. J., Raijmakers, R., Lemeer, S., Mohammed, S. & Heck, A. J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 4, 484–494 (2009).

    CAS  Google Scholar 

  64. 64

    Mi, H., Muruganujan, A., Casagrande, J. T. & Thomas, P. D. Large-scale gene function analysis with the PANTHER classification system. Nat. Protoc. 8, 1551–1566 (2013).

    Google Scholar 

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Acknowledgements

Research was supported by the International Graduate School of Science and Engineering (L.R. and L.A.K.). The authors acknowledge the continuous support of the DFG via the Nanosystems Initiative Munich (NIM).

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L.R. and L.A.K. contributed equally to this work. L.R., L.A.K., H.G., J.S.-S., F.P., S.A.S., R.B. and A.R.B. designed the research. L.A.K. and J.S. performed microcomputed tomography. L.R. and L.A.K. carried out micromechanical analysis, confocal microscopy and investigated fibre composition. L.A.K. and E.K. performed proteomics experiments. L.R., L.A.K., K.W.M., R.B. and A.R.B. analysed the data and wrote the paper. All authors reviewed and revised the manuscript.

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Correspondence to R. Burgkart or A. R. Bausch.

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Rossetti, L., Kuntz, L., Kunold, E. et al. The microstructure and micromechanics of the tendon–bone insertion. Nature Mater 16, 664–670 (2017). https://doi.org/10.1038/nmat4863

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