Tendon is a connective tissue that transmits mechanical loads and forces from muscle to bone and in doing so enables locomotion and enhances joint stability. It is a living tissue and responds to mechanical forces by changing its metabolism as well as its structural and mechanical properties. Injuries to tendon, including overuse (tendinopathy), affect millions of people in occupational and athletic settings. After injury, tendon can heal itself;​ however the healed tendon typically does not reach the biochemical and mechanical properties of the tendon prior to injury. The actual healing dynamics of tendon has been reported to be more complicated than other soft tissues and does not recover to the same degree compared to before the injury.

An injured tendon usually shows scartissue in the repaired area which results in higher stiffness and tensile strength then the surrounding healthy tissue. In some cases, even people who had a tendon rupture and were treated with controlled immobilization (i.e. a plaster), had a very high incidence of re-rupture compared to people who were surgically treated. A comprehensive understanding of the pathological and biomechanical processes that underlie tendon function and pathology is essential to improved clinical diagnosis and treatment of tendon pathology. For this reason a deeper investigation of the tendon components is needed to elucidate the structure-function relationships in tendons.

Tendon structure, composed of collagen fibrils embedded within a proteoglycan (PG) rich extracellular matrix (ECM), is the key to its mechanical behavior. Collagen fibers are the major constituent of tendon and are believed to bear most of the tensile load. The PGs are thought to mediate the organization of collagen ultrastructure and facilitate force transmission along the discontinuous collagen fibrils. A considerable number of scientific investigations have already been performed to define the influence of ultrastructure on the mechanical properties of tendon. However, the large body of published experimental evidence yields conflicting conclusions with regard to the fundamental ultrastructural determinants of tendon behavior. Thus, the aim of this thesis is to elucidate these contrasting results by taking a “bottom up” approach, by which the basic intermolecular forces of binding within the ECM between the collagen fibrils and PGs are considered, in conjunction with the tensile mechanical properties of individual fibrils. This may help reconcile the conflicting and counterintuitive results that have thus far been observed in laboratory experiments, and will ultimately help inform clinical diagnosis and treatment of connective tissue disorders. Specifically, a new protocol to mechanically test Achilles tendon was developed and validated in order to quantify the mechanical contribution of the PG concentration in tensile load. Tendons were chemically digested of their PG secondary chains — the glycosaminoglycans (GAGs) — and compared with the native ones. Results showed that along the longitudinal axis the mechanical behavior was heterogeneous in tensile load, especially at the muscle and the bone insertion, highlighting the importance of performing local strain analysis with regard to tensile tendon mechanics. The contribution of PGs to tensile tendon mechanics at the macroscale was not straightforward and points to a heterogeneous and complex structure-function relationship in tendon.

Additionally, two inbred strains of mice showing similar macroscopic mechanical behavior but different elastic modulus were further investigated in their collagen fibril morphology to understand their mechanical contribution. New image-analysis tools were designed to parameterize the collagen fibril morphology. The group with higher elastic modulus, structurally exhibited a larger mean collagen fibril radius, smaller specific fibril surface (i.e. the perimeter of the fibril in contact with the non-collagenous ECM), and a lower concentration of GAGs. As in previous studies, larger collagen fibril radius appeared to be associated with a stiffer tendon, but this functional difference could also be attributed to reduced potential surface area exchange between fibrils and the surrounding PG rich matrix, in which the hydrophilic GAG side chains may promote inter-fibril sliding.

Finally, the interactions between the PGs and the collagen fibrils were studied at the ultrascale in their physiological matrix. A new experimental approach combining macroscopic mechanical loading of tendon with a morphometric ultrascale assessment of longitudinal and cross-sectional collagen fibril deformations was developed and validated. Tendons with different PG concentration were submitted to different target strains at the macroscale and chemically fixed. Collagen fibrils were characterized with atomic force microscopy (AFM). The mechanical contribution of PG at the ultrascale was quantified by comparing the difference in the collagen fibril elongation and diameter between native and GAG-depleted tendon. Tendons with a lower concentration of GAG showed greater elongation in the collagen fibril.

In conclusion, in conjunction with all the specific studies performed in this thesis, GAG concentration did not significantly influence the overall tendon mechanical response. However, at the ultrascale GAGs were responsible for changes in collagen fibril elongation, which increased at lower GAG concentration. Thus, GAGs seem not to link the collagen fibrils in order to mechanically transfer forces between them in tension, but rather hydrate the ECM, and promote collagen fibril sliding under tension, most likely to prevent damage.