K. L. Goh 1 Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 639798, Singapore e-mail: gohkl@ntu.edu.sg D. F. Holmes H.-Y. Lu S. Richardson K. E. Kadler School of Biological Science, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK P. P. Purslow Department of Food Science, University of Guelph, Guelph, ON, N1G 2W1, Canada T. J. Wess Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff University, Redwood Building, Cardiff, CF10 3NB, UK Ageing Changes in the Tensile Properties of Tendons: Influence of Collagen Fibril Volume Fraction Connective tissues are biological composites comprising of collagen fibrils embedded in (and reinforcing) the hydrated proteoglycan-rich (PG) gel within the extracellular ma- trices (ECMs). Age-related changes to the mechanical properties of tissues are often associated with changes to the structure of the ECM, namely, fibril diameter. However, quantitative attempts to correlate fibril diameter to mechanical properties have yielded inconclusive evidence. Here, we described a novel approach that was based on the rule of mixtures for fiber composites to evaluate the dependence of age-related changes in tendon tensile strength () and stiffness (E) on the collagen fibril cross-sectional area fraction (), which is related to the fibril volume fraction. Tail tendons from C57BL6 mice from age groups 1.6–35.3 months old were stretched to failure to determine  and E. Parallel measurements of  as a function of age were made using transmission electron microscopy. Mathematical models (rule of mixtures) of fibrils reinforcing a PG gel in tendons were used to investigate the influence of  on ageing changes in  and E. The magnitudes of , E, and  increased rapidly from 1.6 months to 4.0 months (P-values 0.05) before reaching a constant (age independent) from 4.0 months to 29.0 months (P-values 0.05); this trend continued for E and  (P-values 0.05) from 29.0 months to 35.3 months, but not for , which decreased gradually (P-values 0.05). Linear regression analysis revealed that age-related changes in  and E correlated positively to  (P-values 0.05). Collagen fibril cross-sectional area fraction  is a significant predictor of ageing changes in  and E in the tail tendons of C57BL6 mice. DOI: 10.1115/1.2898732 Keywords: stiffness, strength, area fraction, collagen, rule of mixtures 1 Introduction Ageing changes in the mechanical properties of connective tis- sues 1–14 are often associated with changes to the structure of the extracellular matrix ECM, namely, fibril diameter 6–8,15,16. Current attempts to explain the relationship between the structure of collagen fibrils within the tissue and the mechani- cal property of the tissue were derived from qualitative analyses 15–19. Large fibril diameters, which are observed in tendon with increasing age, may be associated with an increased density of intrafibrillar covalent cross-links and may influence the ability to resist tensile load 15,20. On the other hand, smaller diameters may influence the ability to withstand creep because the reduced diameter could increase the surface area per unit mass of the fibrils and, in turn, increase the density of noncovalent cross-links between the fibrils and the components in the ECM 15,20. These explanations are motivated by concepts established for engineer- ing fiber composite materials; here, we have theories for micro- mechanics of fibers, which describe how fibers provide reinforce- ment to a material under loading regimes corresponding to elastic and plastic stress transfer as explained by shear-lag and shear- sliding models, respectively 21,22. However, there is no clear evidence demonstrating the correla- tion of the age-related changes in the mechanical properties of tissues to fibril diameter 9,23,24. In many tissues, histograms of fibril diameter distribution reveal a spread of values that were qualitatively described as either unimodal or bimodal depending on the age and tissue type 15,17,20,21,25. Apart from the nar- row spread of fibril diameters observed in foetal tissues, the range of diameters increases thereafter from young to adulthood 15,20. The irregularity of fibril cross sections also increased with age, and this has been attributed to fibril-fibril fusion 26,27; in this respect, the diameter parameter could only provide an approxi- mate description of the structure. Thus, the nature of the fibril cross section means that the correlation between the tensile prop- erties and fibril diameters is complex and cannot be reliably pre- dicted using a statistical analysis 9,18,23,28. There are evidences that suggest that the collagen fibril cross- sectional area fraction  is significantly correlated to the tensile properties of tissues, such as tendons 9 and the cervix 29. Underlying this relationship is a concept based on the rule of mixtures for fiber composites. The rule of mixtures addresses a two-phase system in which one phase is regarded as a reinforcing phase for the other when the system is subjected to an external load 30,31. Connective tissues are biological composites com- prising of collagen fibrils embedded in and reinforcing the hy- drated proteoglycan-rich PG gel within the ECMs 28,32. Ac- cording to the rule, the structural property of the ECM, such as collagen fibril volume fraction , following the notation given in Ref. 33, can be related to the tensile stiffness E and strength  18,28,32. It then follows that  is analogous to  9. While the correlation has been demonstrated on tendons from the tails of 3 w old 0.7 month and 8 wk old 1.9 month mice 9, it is not 1 Corresponding author. Contributed by the Bioengineering Division of ASME for publication in the JOUR- NAL OF BIOMECHANICAL ENGINEERING. Manuscript received May 31, 2006; final manu- script received September 11, 2007; published online March 31, 2008. Review con- ducted by Jeffrey A. Weiss. Journal of Biomechanical Engineering APRIL 2008, Vol. 130 / 021011-1 Copyright © 2008 by ASME Downloaded 24 Jun 2008 to 131.104.117.36. Redistribution subject to ASME license or copyright; see http://www.asme.org/terms/Terms_Use.cfm