Biomechanics and Modeling in Mechanobiology

, Volume 12, Issue 2, pp 291–300 | Cite as

Highly sensitive single-fibril erosion assay demonstrates mechanochemical switch in native collagen fibrils

  • Brendan P. Flynn
  • Graham E. Tilburey
  • Jeffrey W. RubertiEmail author
Original Paper


It has been established that the enzyme susceptibility of collagen, the predominant load-bearing protein in vertebrates, is altered by applied tension. However, whether tensile force increases or decreases the susceptibility to enzyme is a matter of contention. It is critical to establish a definitive understanding of the direction and magnitude of the force versus catalysis rate (k C ) relationship if we are to properly interpret connective tissue development, growth, remodeling, repair, and degeneration. In this investigation, we examine collagen/enzyme mechanochemistry at the smallest scale structurally relevant to connective tissue: the native collagen fibril. A single-fibril mechanochemical erosion assay with nN force resolution was developed which permits detection of the loss of a few layers of monomer from the fibril surface. Native type I fibrils (bovine) held at three levels of tension were exposed to Clostridium histolyticum collagenase A. Fibrils held at zero-load failed rapidly and consistently (20 min) while fibrils at 1.8 pN/monomer failed more slowly (35–55 min). Strikingly, fibrils at 23.9 pN/monomer did not exhibit detectable degradation. The extracted force versus k C data were combined with previous single-molecule results to produce a “master curve” which suggests that collagen degradation is governed by an extremely sensitive mechanochemical switch.


Collagen ECM (extracellular matrix) Mechanical test Enzymatic degradation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

10237_2012_399_MOESM1_ESM.doc (126 kb)
ESM 1 (DOC 126 kb)


  1. Adhikari AS, Chai J, Dunn AR (2011) Mechanical load induces a 100-fold increase in the rate of collagen proteolysis by MMP-1. J Am Chem Soc. doi: 10.1021/ja109972p
  2. Arokoski JP, Jurvelin JS, Vaatainen U, Helminen HJ (2000) Normal and pathological adaptations of articular cartilage to joint loading. Scand J Med Sci Sports 10(4): 186–198CrossRefGoogle Scholar
  3. Baragi VM, Qiu L, Gunja-Smith Z, Woessner JF Jr., Lesch CA, Guglietta A (1997) Role of metalloproteinases in the development and healing of acetic acid-induced gastric ulcer in rats. Scand J Gastroenterol 32(5): 419–426CrossRefGoogle Scholar
  4. Bass EC, Wistrom EV, Diederich CJ, Nau WH, Pellegrino R, Ruberti J, Lotz JC (2004) Heat-induced changes in porcine annulus fibrosus biomechanics. J biomech 37(2): 233–240CrossRefGoogle Scholar
  5. Beare AH, O’Kane S, Krane SM, Ferguson MW (2003) Severely impaired wound healing in the collagenase-resistant mouse. J Invest Dermatol 120(1): 153–163. doi: 10.1046/j.1523-1747.2003.12019.x CrossRefGoogle Scholar
  6. Bell E, Ivarsson B, Merrill C (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci USA 76(3): 1274–1278CrossRefGoogle Scholar
  7. Bhole AP, Flynn BP, Liles M, Saeidi N, Dimarzio CA, Ruberti JW (2009) Mechanical strain enhances survivability of collagen micronetworks in the presence of collagenase: implications for load-bearing matrix growth and stability. Philos Trans A Math Phys Eng Sci 367(1902): 3339–3362CrossRefGoogle Scholar
  8. Blain EJ, Gilbert SJ, Wardale RJ, Capper SJ, Mason DJ, Duance VC (2001) Up-regulation of matrix metalloproteinase expression and activation following cyclical compressive loading of articular cartilage in vitro. Arch Biochem Biophys 396(1): 49–55CrossRefGoogle Scholar
  9. Camp RJ, Liles M, Beale J, Saeidi N, Flynn BP, Moore E, Murthy SK, Ruberti JW (2011) Molecular mechanochemistry: low force switch slows enzymatic cleavage of human type I collagen monomer. J Am Chem Soc. doi: 10.1021/ja110098b
  10. Canty EG, Lu Y, Meadows RS, Shaw MK, Holmes DF, Kadler KE (2004) Coalignment of plasma membrane channels and protrusions (fibripositors) specifies the parallelism of tendon. J Cell Biol 165(4): 553–563. doi: 10.1083/jcb.200312071 CrossRefGoogle Scholar
  11. Cawston T, Billington C, Cleaver C, Elliott S, Hui W, Koshy P, Shingleton B, Rowan A (1999) The regulation of MMPs and TIMPs in cartilage turnover. Ann NY Acad Sci 878: 120–129CrossRefGoogle Scholar
  12. Chang S-W, Flynn BP, Ruberti JW, Buehler MJ (2012) Molecular mechanism of force induced stabilization of collagen against enzymatic breakdown. Biomaterials 33(15): 3852–3859CrossRefGoogle Scholar
  13. Chen SS, Wright NT, Humphrey JD (1997) Heat-induced changes in the mechanics of a collagenous tissue: isothermal free shrinkage. J Biomech Eng 119(4): 372–378CrossRefGoogle Scholar
  14. Cowin SC (2004) Tissue growth and remodeling. Annu Rev Biomed Eng 6: 77–107CrossRefGoogle Scholar
  15. Eppell SJ, Smith BN, Kahn H, Ballarini R (2006) Nano measurements with micro-devices: mechanical properties of hydrated collagen fibrils. J R Soc Interface 3(6): 117–121. doi: 10.1098/rsif.2005.0100 CrossRefGoogle Scholar
  16. Flynn BP, Bhole AP, Saeidi N, Liles M, Dimarzio CA, Ruberti JW (2010) Mechanical strain stabilizes reconstituted collagen fibrils against enzymatic degradation by mammalian collagenase matrix metalloproteinase 8 (MMP-8). PLoS One 5(8). doi: 10.1371/journal.pone.0012337
  17. Grams F, Reinemer P, Powers JC, Kleine T, Pieper M, Tschesche H, Huber R, Bode W (1995) X-ray structures of human neutrophil collagenase complexed with peptide hydroxamate and peptide thiol inhibitors. Implications for substrate binding and rational drug design. Eur J Biochem 228(3): 830–841CrossRefGoogle Scholar
  18. Grytz R, Meschke G, Jonas JB (2011) The collagen fibril architecture in the lamina cribrosa and peripapillary sclera predicted by a computational remodeling approach. Biomech Model Mechanobiol 10(3): 371–382. doi: 10.1007/s10237-010-0240-8 CrossRefGoogle Scholar
  19. Harrington DJ (1996) Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease. Infect Immun 64(6): 1885–1891Google Scholar
  20. Hormann H (1982) Fibronectin–mediator between cells and connective tissue. Klin Wochenschr 60(20): 1265–1277CrossRefGoogle Scholar
  21. Huang C, Yannas IV (1977) Mechanochemical studies of enzymatic degradation of insoluble collagen fibers. J Biomed Mater Res Symp 11(1):137–154Google Scholar
  22. Huang Y, Meek KM (1999) Swelling studies on the cornea and sclera: the effects of pH and ionic strength. Biophys J 77(3): 1655–1665. doi: 10.1016/S0006-3495(99)77013-X CrossRefGoogle Scholar
  23. Hulboy DL, Rudolph LA, Matrisian LM (1997) Matrix metalloproteinases as mediators of reproductive function. Mol Hum Reprod 3(1): 27–45CrossRefGoogle Scholar
  24. Hulmes DJ, Miller A, Parry DA, Piez KA, Woodhead-Galloway J (1973) Analysis of the primary structure of collagen for the origins of molecular packing. J Mol Biol 79(1): 137–148CrossRefGoogle Scholar
  25. Humphrey JD (2001) Stress, strain, and mechanotransduction in cells. J Biomech Eng 123(6): 638–641CrossRefGoogle Scholar
  26. Inada M, Wang Y, Byrne MH, Rahman MU, Miyaura C, Lopez-Otin C, Krane SM (2004) Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification. Proc Natl Acad Sci USA 101(49): 17192–17197. doi: 10.1073/pnas.0407788101 CrossRefGoogle Scholar
  27. Kadler K (2004) Matrix loading: assembly of extracellular matrix collagen fibrils during embryogenesis. Birth Defects Res C Embryo Today 72(1): 1–11CrossRefGoogle Scholar
  28. Kang JD, Georgescu HI, McIntyre-Larkin L, Stefanovic-Racic M, Donaldson WF III, Evans CH (1996) Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2. Spine (Phila Pa 1976) 21(3):271–277Google Scholar
  29. Kilian O, Pfeil U, Wenisch S, Heiss C, Kraus R, Schnettler R (2007) Enhanced alpha 1(I) mRNA expression in frozen shoulder and dupuytren tissue. Eur J Med Res 12(12): 585–590Google Scholar
  30. Koskinen SO, Heinemeier KM, Olesen JL, Langberg H, Kjaer M (2004) Physical exercise can influence local levels of matrix metalloproteinases and their inhibitors in tendon-related connective tissue. J Appl Physiol 96(3): 861–864CrossRefGoogle Scholar
  31. Leikina E, Mertts MV, Kuznetsova N, Leikin S (2002) Type I collagen is thermally unstable at body temperature. Proc Natl Acad Sci USA 99(3): 1314–1318. doi: 10.1073/pnas.032307099 CrossRefGoogle Scholar
  32. Lyubimov AV, Vasiliev JM (1982) Distribution of fibronectin-containing structures on the surface of lamelloplasm and endoplasm of fibroblasts; hypothesis of receptor-mediated assembly of fibronectin structures. Cell Biol Int Rep 6(2): 105–112CrossRefGoogle Scholar
  33. Mallya SK, Mookhtiar KA, Van Wart HE (1992) Kinetics of hydrolysis of type I, II, and III collagens by the class I and II Clostridium histolyticum collagenases. J Protein Chem 11(1): 99–107CrossRefGoogle Scholar
  34. McCawley LJ, Matrisian LM (2000) Matrix metalloproteinases: multifunctional contributors to tumor progression. Mol Med Today 6(4): 149–156. doi: S1357-4310(00)01686-5 CrossRefGoogle Scholar
  35. Meek KM, Leonard DW (1993) Ultrastructure of the corneal stroma: a comparative study. Biophys J 64: 273–280CrossRefGoogle Scholar
  36. Michna H, Hartmann G (1989) Adaptation of tendon collagen to exercise. Int Orthop 13(3): 161–165CrossRefGoogle Scholar
  37. Miles CA, Ghelashvili M (1999) Polymer-in-a-box mechanism for the thermal stabilization of collagen molecules in fibers. Biophys J 76(6): 3243–3252. doi: 10.1016/S0006-3495(99)77476-X CrossRefGoogle Scholar
  38. Minond D, Lauer-Fields JL, Nagase H, Fields GB (2004) Matrix metalloproteinase triple-helical peptidase activities are differentially regulated by substrate stability. Biochemistry 43(36): 11474–11481. doi: 10.1021/bi048938i CrossRefGoogle Scholar
  39. Nabeshima Y, Grood ES, Sakurai A, Herman JH (1996) Uniaxial tension inhibits tendon collagen degradation by collagenase in vitro. J Orthop Res 14: 123–130CrossRefGoogle Scholar
  40. Nemetschek T, Jonak R, Nemetschek-Gansler H, Riedl H, Rosenbaum G (1978) [On the determination of changes in the large periodic structure of collagen (author’s transl)]. Z Naturforsch C 33(11-12): 928–936Google Scholar
  41. Nerenberg PS, Salsas-Escat R, Stultz CM (2008) Do collagenases unwind triple-helical collagen before peptide bond hydrolysis? Reinterpreting experimental observations with mathematical models. Proteins 70(4): 1154–1161. doi: 10.1002/prot.21687 CrossRefGoogle Scholar
  42. Okada T, Hayashi T, Ikada Y (1992) Degradation of collagen suture in vitro and in vivo. Biomaterials 13: 448–454CrossRefGoogle Scholar
  43. Perumal S, Antipova O, Orgel JP (2008) Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis. Proc Natl Acad Sci USA 105(8): 2824–2829. doi: 10.1073/pnas.0710588105 CrossRefGoogle Scholar
  44. Prajapati RT, Chavally-Mis B, Herbage D, Eastwood M, Brown RA (2000) Mechanical loading regulates protease production by fibroblasts in three-dimensional collagen substrates. Wound Repair Regen 8(3): 226–237CrossRefGoogle Scholar
  45. Ruberti JW, Hallab NJ (2005) Strain-controlled enzymatic cleavage of collagen in loaded matrix. Biochem Biophys Res Commun 336(2): 483–489. doi: 10.1016/j.bbrc.2005.08.128 CrossRefGoogle Scholar
  46. Sander EA, Barocas VH, Tranquillo RT (2011) Initial fiber alignment pattern alters extracellular matrix synthesis in fibroblast-populated fibrin gel cruciforms and correlates with predicted tension. Ann Biomed Eng 39(2): 714–729. doi: 10.1007/s10439-010-0192-2 CrossRefGoogle Scholar
  47. Stultz CM (2002) Localized unfolding of collagen explains collagenase cleavage near imino-poor sites. J Mol Biol 319(5): 997–1003. doi: 10.1016/S0022-2836(02)00421-7 CrossRefGoogle Scholar
  48. Tzafriri AR, Bercovier M, Parnas H (2002) Reaction diffusion model of the enzymatic erosion of insoluble fibrillar matrices. Biophys J 83: 776–793CrossRefGoogle Scholar
  49. Van der Rijt JA, Van der Werf KO, Bennink ML, Dijkstra PJ, Feijen J (2006) Micromechanical testing of individual collagen fibrils. Macromol Biosci 6(9): 697–702. doi: 10.1002/mabi.200600063 CrossRefGoogle Scholar
  50. Welgus HG, Jeffrey JJ, Stricklin GP, Roswit WT, Eisen AZ (1980) Characteristics of the action of human skin fibroblast collagenase on fibrillar collagen. J Biolo Chem 255: 6808–6813Google Scholar
  51. Wyatt KE, Bourne JW, Torzilli PA (2009) Deformation-dependent enzyme mechanokinetic cleavage of type I collagen. J Biomech Eng 131(5): 051004CrossRefGoogle Scholar
  52. Zareian R, Church KP, Saeidi N, Flynn BP, Beale JW, Ruberti JW (2010) Probing collagen/enzyme mechanochemistry in native tissue with dynamic, enzyme-induced creep. Langmuir 26(12): 9917–9926. doi: 10.1021/la100384e CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Brendan P. Flynn
    • 1
  • Graham E. Tilburey
    • 1
  • Jeffrey W. Ruberti
    • 1
    Email author
  1. 1.Department of Mechanical and Industrial EngineeringNortheastern UniversityBostonUSA

Personalised recommendations