Mechanical response of individual collagen fibrils in loaded tendon as measured by atomic force microscopy

A precise analysis of the mechanical response of collagen fibrils in tendon tissue is critical to understanding the ultrastructural mechanisms that underlie collagen fibril interactions (load transfer), and ultimately tendon structure–function. This study reports a novel experimental approach combining macroscopic mechanical loading of tendon with a morphometric ultrascale assessment of longitudinal and cross-sectional collagen fibril deformations. An atomic force microscope was used to characterize diameters and periodic banding (D-period) of individual type-I collagen fibrils within murine Achilles tendons that were loaded to 0%, 5%, or 10% macroscopic nominal strain, respectively. D-period banding of the collagen fibrils increased with increasing tendon strain (2.1% increase at 10% applied tendon strain, p < 0.05), while fibril diameter decreased (8% reduction, p < 0.05). No statistically significant differences between 0% and 5% applied strain were observed, indicating that the onset of fibril (D-period) straining lagged macroscopically applied tendon strains by at least 5%. This confirms previous reports of delayed onset of collagen fibril stretching and the role of collagen fibril kinematics in supporting physiological tendon loads. Fibril strains within the tissue were relatively tightly distributed in unloaded and highly strained tendons, but were more broadly distributed at 5% applied strain, indicating progressive recruitment of collagen fibrils. Using these techniques we also confirmed that collagen fibrils thin appreciably at higher levels of macroscopic tendon strain. Finally, in contrast to prevalent tendon structure–function concepts data revealed that loading of the collagen network is fairly homogenous, with no apparent predisposition for loading of collagen fibrils according to their diameter.     

Regional stiffening with aging in tibialis anterior tendons of mice occurs independent of changes in collagen fibril morphology

The incidence of tendon degeneration and rupture increases with advancing age. The mechanisms underlying this increased risk remain unknown but may arise because of age-related changes in tendon mechanical properties and structure. Our purpose was to determine the effect of aging on tendon mechanical properties and collagen fibril morphology. Regional mechanical properties and collagen fibril characteristics were determined along the length of tibialis anterior (TA) tendons from adult (8- to 12-mo-old) and old (28- to 30-mo-old) mice. Tangent modulus of all regions along the tendons increased in old age, but the increase was substantially greater in the proximal region adjacent to the muscle than in the rest of the tendon. Overall end-to-end modulus increased with old age at maximum tendon strain (799 ± 157 vs. 1,419 ± 91 MPa) and at physiologically relevant strain (377 ± 137 vs. 798 ± 104 MPa). Despite the dramatic changes in tendon mechanical properties from adulthood to old age, collagen fibril morphology and packing fraction remained relatively constant in all tendon regions examined. Since tendon properties are influenced by their external loading environment, we also examined the effect of aging on TA muscle contractile properties. Maximum isometric force did not differ between the age groups. We conclude that TA tendons stiffen in a region-dependent manner throughout the life span, but the changes in mechanical properties are not accompanied by corresponding changes in collagen fibril morphology or force-generating capacity of the TA muscle.     

Functional tissue engineering of tendon: Establishing biological success criteria for improving tendon repair

Improving tendon repair using Functional Tissue Engineering (FTE) principles has been the focus of our laboratory over the last decade. Although our primary goals were initially focused only on mechanical outcomes, we are now carefully assessing the biological properties of our tissue-engineered tendon repairs so as to link biological influences with mechanics. However, given the complexities of tendon development and healing, it remains challenging to determine which aspects of tendon biology are the most important to focus on in the context of tissue engineering. To address this problem, we have formalized a strategy to identify, prioritize, and evaluate potential biological success criteria for tendon repair. We have defined numerous biological properties of normal tendon relative to cellular phenotype, extracellular matrix and tissue ultra-structure that we would like to reproduce in our tissue-engineered repairs and prioritized these biological criteria by examining their relative importance during both normal development and natural tendon healing. Here, we propose three specific biological criteria which we believe are essential for normal tendon function: (1) scleraxis-expressing cells; (2) well-organized and axially-aligned collagen fibrils having bimodal diameter distribution; and (3) a specialized tendon-to-bone insertion site. Moving forward, these biological success criteria will be used in conjunction with our already established mechanical success criteria to evaluate the effectiveness of our tissue-engineered tendon repairs.     

Structural changes in human type I collagen fibrils investigated by force spectroscopy

In the field of biomechanics, collagen fibrils are believed to be robust mechanical structures characterized by a low extensibility. Until very recently, information on the mechanical properties of collagen fibrils could only be derived from ensemble measurements performed on complete tissues such as bone, skin, and tendon. Here, we measure force-elongation/relaxation profiles of single collagen fibrils using atomic force microscopy (AFM)-based force spectroscopy (FS). The elongation profiles show that in vitro-assembled human type I collagen fibrils are characterized by a large extensibility. Numerous discontinuities and a plateau in the force profile indicate major reorganization occurring within the fibrils in the 1.5- to 4.5-nN range. Our study demonstrates that newly assembled collagen fibrils are robust structures with a significant reserve of elasticity that could play a determinant role in the extracellular matrix (ECM) remodeling associated with tissue growth and morphogenesis.     

Tendon tissue microdamage and the limits of intrinsic repair

The transmission of mechanical muscle force to bone for musculoskeletal stability and movement is one of the most important functions of tendon. The load-bearing tendon core is composed of highly aligned collagen-rich fascicles interspersed with stromal cells (tenocytes). Despite being built to bear very high mechanical stresses, supra-physiological/repetitive mechanical overloading leads to tendon microdamage in fascicles, and potentially to tendon disease and rupture. To date, it is unclear to what extent intrinsic healing mechanisms of the tendon core compartment can repair microdamage. In the present study, we investigated the healing capacity of the tendon core compartment in an ex vivo tissue explant model. To do so, we isolated rat tail tendon fascicles, damaged them by applying a single stretch to various degrees of sub-rupture damage and longitudinally assessed downstream functional and structural changes over a period of several days. Functional damage was assessed by changes in the elastic modulus of the material stress-strain curves, and biological viability of the resident tenocytes. Structural damage was quantified using a fluorescent collagen hybridizing peptide (CHP) to label mechanically disrupted collagen structures. While we observed functional mechanical damage for strains above 2% of the initial fascicle length, structural collagen damage was only detectable for 6% strain and beyond. Minimally loaded/damaged fascicles (2–4% strain) progressively lost elastic modulus over the course of tissue culture, despite their collagen structures remaining intact with high degree of maintained cell viability. In contrast, more severely overloaded fascicles (6–8% strain) with damage at the molecular/collagen level showed no further loss of the elastic modulus but markedly decreased cell viability. Surprisingly, in these heavily damaged fascicles the elastic modulus partially recovered, an effect also seen in further experiments on devitalized fascicles, implying the possibility of a non-cellular but matrix-driven mechanism of molecular repair. Overall, our findings indicate that the tendon core has very little capacity for self-repair of microdamage. We conclude that stromal tenocytes likely do not play a major role in anabolic repair of tendon matrix microdamage, but rather mediate catabolic matrix breakdown and communication with extrinsic cells that are able to effect tissue repair.     

Structure–function relationships of postnatal tendon development: A parallel to healing

This review highlights recent research on structure–function relationships in tendon and comments on the parallels between development and healing. The processes of tendon development and collagen fibrillogenesis are reviewed, but due to the abundance of information in this field, this work focuses primarily on characterizing the mechanical behavior of mature and developing tendon, and how the latter parallels healing tendon. The role that extracellular matrix components, mainly collagen, proteoglycans, and collagen cross-links, play in determining the mechanical behavior of tendon will be examined in this review. Specifically, collagen fiber re-alignment and collagen fibril uncrimping relate mechanical behavior to structural alterations during development and during healing. Finally, attention is paid to a number of recent efforts to augment injured tendon and how future efforts could focus on recreating the important structure–function relationships reviewed here.     

The change of HSP47, collagen specific molecular chaperone, expression in rat skeletal muscle may regulate collagen production with gravitational conditions.

It is well known that unloading of skeletal muscle with spaceflight leads skeletal muscle atrophy. However, it remains unclear how the extracellular matrix within the muscle and the connective tissues such as tendon and ligament respond to reduced mechanical load including microgravity, although they have been thought to play important roles in both the transmission of force and the signal transduction between cells and tissues. Type-I collagen and type-IV collagen, both of the major components of extracellular matrix and connective tissues. We focused on change of these collagen synthesis with mechanical load. To obtain an insight into the effects of gravitational changing on the protein metabolism of collagen in skeletal muscle during mechanical unloading, reloading after unloading, we investigated changes in the amount of Heat shock protein 47 (HSP47), has been postulated to be a collagen-specific molecular chaperone localized in the ER (Nagata et al, 1992). Western blot analysis revealed that HSP47 in rat soleus muscle decreases at 5 days after hindlimb suspension (HS). On the other hand, HSP47 in rat soleus muscle increases at 5 days after hypergravity (HG) induced by the centrifugation. RT-PCR analysis showed HSP47 mRNA decreased with HS earlier, as compared with collagen type-I and type-IV mRNA. From these results, the amount of HSP47 changing by gravitational condition may effect on signal transfers in the primary stage of adaptation and the change of HSP47 expression in skeletal muscle may regulate collagen production with gravitational conditions.     

Fatigue loading of tendon results in collagen kinking and denaturation but does not change local tissue mechanics

Fatigue loading is a primary cause of tendon degeneration, which is characterized by the disruption of collagen fibers and the appearance of abnormal (e.g., cartilaginous, fatty, calcified) tissue deposits. The formation of such abnormal deposits, which further weakens the tissue, suggests that resident tendon cells acquire an aberrant phenotype in response to fatigue damage and the resulting altered mechanical microenvironment. While fatigue loading produces clear changes in collagen organization and molecular denaturation, no data exist regarding the effect of fatigue on the local tissue mechanical properties. Therefore, the objective of this study was to identify changes in the local tissue stiffness of tendons after fatigue loading. We hypothesized that fatigue damage would reduce local tissue stiffness, particularly in areas with significant structural damage (e.g., collagen denaturation). We tested this hypothesis by identifying regions of local fatigue damage (i.e., collagen fiber kinking and molecular denaturation) via histologic imaging and by measuring the local tissue modulus within these regions via atomic force microscopy (AFM). Counter to our initial hypothesis, we found no change in the local tissue modulus as a consequence of fatigue loading, despite widespread fiber kinking and collagen denaturation. These data suggest that immediate changes in topography and tissue structure – but not local tissue mechanics – initiate the early changes in tendon cell phenotype as a consequence of fatigue loading that ultimately culminate in tendon degeneration.     

Skewness angle of interfibrillar proteoglycans increases with applied load on mitral valve chordae tendineae

In highly aligned connective tissues, such as tendon, collagen fibrils are linked together by proteoglycans (PGs). Recent mechanical and theoretical studies on tendon micromechanics have implied that PGs mediate mechanical interactions between adjacent collagen fibrils. We used transmission electron microscopy to observe the collagen fibril-PG interactions in porcine mitral valve chordae under variable loading conditions and found that PGs attached to collagen fibrils perpendicularly in the load-free situation, and became skewed when the chordae were loaded. The average skewness angle of PGs increased with the applied load, and hence the strain in the chordae. The observation of PG skewing with the application of load demonstrates that, in mitral valve chordae, interfibrillar slippage occurs and that PGs play a role in fibril-to-fibril interaction and likely transfer force. The results of this study provide new insights into the mechanical role of PGs and support some recent theoretical models.     

Viscoelastic properties of collagen: synchrotron radiation investigations and structural model

Collagen type I is the most abundant structural protein in tendon, skin and bone, and largely determines the mechanical behaviour of these connective tissues. To obtain a better understanding of the relationship between structure and mechanical properties, tensile tests and synchrotron X-ray scattering have been carried out simultaneously, correlating the mechanical behaviour with changes in the microstructure. Because intermolecular cross-links are thought to have a great influence on the mechanical behaviour of collagen, we also carried out experiments using cross-link-deficient tail-tendon collagen from rats fed with beta-APN, in addition to normal controls. The load-elongation curve of tendon collagen has a characteristic shape with, initially, an increasing slope, corresponding to an increasing stiffness, followed by yielding and then fracture. Cross-link-deficient collagen produces a quite different curve with a marked plateau appearing in some cases, where the length of the tendon increases at constant stress. With the use of in situ X-ray diffraction, it was possible to measure simultaneously the elongation of the collagen fibrils inside the tendon and of the tendon as a whole. The overall strain of the tendon was always larger than the strain in the individual fibrils, which demonstrates that some deformation is taking place in the matrix between fibrils. Moreover, the ratio of fibril strain to tendon strain was dependent on the applied strain rate. When the speed of deformation was increased, this ratio increased in normal collagen but generally decreased in cross-link-deficient collagen, correlating to the appearance of a plateau in the force-elongation curve indicating creep. We proposed a simple structural model, which describes the tendon at a hierarchical level, where fibrils and interfibrillar matrix act as coupled viscoelastic systems. All qualitative features of the strain-rate dependence of both normal and cross-link-deficient collagen can be reproduced within this model. This complements earlier models that considered the next smallest level of hierarchy, describing the deformation of collagen fibrils in terms of changes in their molecular packing.     

Development of collagen fibril organization and collagen crimp patterns during tendon healing

Normal tendon comprises coaxially aligned bundles of crimped collagen fibres each of which possesses a fibrillar substructure. In acute traumatic injury this level of organization is disrupted and the mechanical function of the tendon impaired. During repair, a degree of recovery of the fibrillar structure takes place. In this tudy we have assessed the re-establishment of tendon organization after injury on the basis of the collagen fibril diameter distribution and the collagen crimp parameters. Crimp became undetectable following injury but one month later was present throughout the tissue. At this time the periodicity was greatly reduced by comparison with that of the normal tendon and normal values were not re-established within 14 months following injury. Collagen fibril diameters remained abnormally small over this same period of time. In particular, fibrils of diameters in excess of 100 nm, commonly found in normal and contralateral tendons, were totally absent from the observed distributions in the healing tendons. Such large diameter fibrils often account for as much as 50% of the total mass of collagen present in the uninjured tissue. Thus the mechanical properties of the healing tendon may remain significantly different from those of normal tendon for a minimum time of 14 months after injury.     

In situ cell–matrix mechanics in tendon fascicles and seeded collagen gels: implications for the multiscale design of biomaterials

Designing biomaterials to mimic and function within the complex mechanobiological conditions of connective tissues requires a detailed understanding of the micromechanical environment of the cell. The objective of our study was to measure the in situ cell–matrix strains from applied tension in both tendon fascicles and cell-seeded type I collagen scaffolds using laser scanning confocal microscopy techniques. Tendon fascicles and collagen gels were fluorescently labelled to simultaneously visualise the extracellular matrix and cell nuclei under applied tensile strains of 5%. There were significant differences observed in the micromechanics at the cell–matrix scale suggesting that the type I collagen scaffold did not replicate the pattern of native tendon strains. In particular, although the overall in situ tensile strains in the matrix were quite similar (～2.5%) between the tendon fascicles and the collagen scaffolds, there were significant differences at the cell–matrix boundary with visible shear across cell nuclei of >1 μm measured in native tendon which was not observed at all in the collagen scaffolds. Similarly, there was significant non-uniformity of intercellular strains with relative sliding observed between cell rows in tendon which again was not observed in the collagen scaffolds where the strain environment was much more uniform. If the native micromechanical environment is not replicated in biomaterial scaffolds, then the cells may receive incorrect or mixed mechanical signals which could affect their biosynthetic response to mechanical load in tissue engineering applications. This study highlights the importance of considering the microscale mechanics in the design of biomaterial scaffolds and the need to incorporate such features in computational models of connective tissues.     

Effect of preconditioning and stress relaxation on local collagen fiber re-alignment: inhomogeneous properties of rat supraspinatus tendon

Repeatedly and consistently measuring the mechanical properties of tendon is important but presents a challenge. Preconditioning can provide tendons with a consistent loading history to make comparisons between groups from mechanical testing experiments. However, the specific mechanisms occurring during preconditioning are unknown. Previous studies have suggested that microstructural changes, such as collagen fiber re-alignment, may be a result of preconditioning. Local collagen fiber re-alignment is quantified throughout tensile mechanical testing using a testing system integrated with a polarized light setup, consisting of a backlight, 90 deg-offset rotating polarizer sheets on each side of the test sample, and a digital camera, in a rat supraspinatus tendon model, and corresponding mechanical properties are measured. Local circular variance values are compared throughout the mechanical test to determine if and where collagen fiber re-alignment occurred. The inhomogeneity of the tendon is examined by comparing local circular variance values, optical moduli and optical transition strain values. Although the largest amount of collagen fiber re-alignment was found during preconditioning, significant re-alignment was also demonstrated in the toe and linear regions of the mechanical test. No significant changes in re-alignment were seen during stress relaxation. The insertion site of the supraspinatus tendon demonstrated a lower linear modulus and a more disorganized collagen fiber distribution throughout all mechanical testing points compared to the tendon midsubstance. This study identified a correlation between collagen fiber re-alignment and preconditioning and suggests that collagen fiber re-alignment may be a potential mechanism of preconditioning and merits further investigation. In particular, the conditions necessary for collagen fibers to re-orient away from the direction of loading and the dependency of collagen reorganization on its initial distribution must be examined.     

In vivo investigation of tendon responses to mechanical loading

Tendons transmit skeletal muscle forces to bone and are essential in all voluntary movement. In turn, movement appears to affect tendon properties, and in recent years considerable effort has been put into discovering how tendon tissue responds to mechanical stimuli in vivo. Months and years of mechanical loading can influence the gross morphology of tendon, seen as an increase tendon cross sectional area (CSA). Similarly, tendon stiffness appears to be affected by weeks to months of loading. Increased stiffness can relate to changes in CSA and/or tendon material properties (modulus), though the relative contribution of these parameters is largely unclear. The possible mechanisms behind alterations in tendon material properties include changes in collagen fibril morphology and levels of cross-linking between collagen molecules. Furthermore, increased levels of collagen synthesis and expression are seen as a response to acute exercise and training, and may be a central parameter in tendon adaptation to loading. There are indications that this collagen-induction relates to the auto-/paracrine action of collagen-stimulating growth factors, such as TGFβ-1 and IGF-I, which are expressed in response to mechanical stimuli.     

Tensile properties of human collagen fibrils and fascicles are insensitive to environmental salts

To carry out realistic in vitro mechanical testing on anatomical tissue, a choice has to be made regarding the buffering environment. Therefore, it is important to understand how the environment may influence the measurement to ensure the highest level of accuracy. The most physiologically relevant loading direction of tendon is along its longitudinal axis. Thus, in this study, we focus on the tensile mechanical properties of two hierarchical levels from human patellar tendon, namely: individual collagen fibrils and fascicles. Investigations on collagen fibrils and fascicles were made at pH 7.4 in solutions of phosphate-buffered saline at three different concentrations as well as two HEPES buffered solutions containing NaCl or NaCl + CaCl₂. An atomic force microscope technique was used for tensile testing of individual collagen fibrils. Only a slight increase in relative energy dissipation was observed at the highest phosphate-buffered saline concentration for both the fibrils and fascicles, indicating a stabilizing effect of ionic screening, but changes were much less than reported for radial compression. Due to the small magnitude of the effects, the tensile mechanical properties of collagen fibrils and fascicles from the patellar tendon of mature humans are essentially insensitive to environmental salt concentration and composition at physiological pH.     

Lateral force transmission between human tendon fascicles.

Whether adjacent collagen fascicles transmit force in parallel is unknown. The purpose of the present study was to examine the magnitude of lateral force transmission between adjacent collagen fascicles from the human patellar and Achilles tendon. From each sample two adjacent strands of fascicles (phi 300-530 mum) enclosed in a fascicular membrane were dissected. The specimen was deformed to approximately 3% strain in three independent load-displacement cycles in a small-scale tensile testing device. Cycle 1: the fascicles and the fascicular membrane were intact. Cycle 2: one fascicle was transversally cut while the other fascicle and the fascicular membrane were kept intact. Cycle 3: both fascicles were cut in opposite ends while the fascicular membrane was left intact. A decline in peak force of 45% and 55% from cycle 1 to cycle 2, and 93% and 92% from cycle 2 to cycle 3 was observed in the patellar and Achilles tendon fascicles, respectively. A decline in stiffness of 39% and 60% from cycle 1 to cycle 2, and of 93% and 100% from cycle 2 to cycle 3 was observed in the patellar and Achilles tendon fascicles, respectively. The present data demonstrate that lateral force transmission between adjacent collagen fascicles in human tendons is small or negligible, suggesting that tendon fascicles largely act as independent structures and that force transmission principally takes place within the individual fascicles.     

In situ fibril stretch and sliding is location-dependent in mouse supraspinatus tendons

Tendons are able to transmit high loads efficiently due to their finely optimized hierarchical collagen structure. Two mechanisms by which tendons respond to load are collagen fibril sliding and deformation (stretch). Although many studies have demonstrated that regional variations in tendon structure, composition, and organization contribute to the full tendon׳s mechanical response, the location-dependent response to loading at the fibril level has not been investigated. In addition, the instantaneous response of fibrils to loading, which is clinically relevant for repetitive stretch or fatigue injuries, has also not been studied. Therefore, the purpose of this study was to quantify the instantaneous response of collagen fibrils throughout a mechanical loading protocol, both in the insertion site and in the midsubstance of the mouse supraspinatus tendon. Utilizing a novel atomic force microscopy-based imaging technique, tendons at various strain levels were directly visualized and analyzed for changes in fibril d-period with increasing tendon strain. At the insertion site, d-period significantly increased from 0% to 1% tendon strain, increased again from 3% to 5% strain, and decreased after 5% strain. At the midsubstance, d-period increased from 0% to 1% strain and then decreased after 7% strain. In addition, fibril d-period heterogeneity (fibril sliding) was present, primarily at 3% strain with a large majority occurring in the tendon midsubstance. This study builds upon previous work by adding information on the instantaneous and regional-dependent fibrillar response to mechanical loading and presents data proposing that collagen fibril sliding and stretch are directly related to tissue organization and function.     

Matrix metabolism rate differs in functionally distinct tendons.

Tendon matrix integrity is vital to ensure adequate mechanical properties for efficient function. Although historically tendon was considered to be relatively inert, recent studies have shown that tendon matrix turnover is active. During normal physiological activities some tendons are subjected to stress and strains much closer to their failure properties than others. Tendons with low safety margins are those which function as energy stores such as the equine superficial digital flexor tendon (SDFT) and human Achilles tendon (AT). We postulate therefore that energy storing tendons suffer a higher degree of micro-damage and thus have a higher rate of matrix turnover than positional tendons. The hypothesis was tested using tissue from the equine SDFT and common digital extensor tendon (CDET). Matrix turnover was assessed indirectly by a combination of measurements for matrix age, markers of degradation, potential for degradation and protein expression. Results show that despite higher cellularity, the SDFT has lower relative levels of mRNA for collagen types I and III. Non-collagenous proteins, although expressed at different levels per cell, do not appear to differ between tendon types. Relative levels of mRNA for MMP1, MMP13 and both pro-MMP3 and MMP13 protein activity were significantly higher in the CDET. Correspondingly levels of cross-linked carboxyterminal telopeptide of type I collagen (ICTP) were higher in the CDET and tissue fluorescence lower suggesting more rapid turnover of the collagenous component. Reduced or inhibited collagen turnover in the SDFT may account for the high level of degeneration and subsequent injury compared to the CDET.     

The dynamics of collagen uncrimping and lateral contraction in tendon and the effect of ionic concentration

Under tensile loading, tendon undergoes a number of unique structural changes that govern its mechanical response. For example, stretching a tendon is known to induce both the progressive “uncrimping” of wavy collagen fibrils and extensive lateral contraction mediated by fluid flow out of the tissue. However, it is not known whether these processes are interdependent. Moreover, the rate-dependence of collagen uncrimping and its contribution to tendon's viscoelastic mechanical properties are unknown. Therefore, the objective of this study was to (a) develop a methodology allowing for simultaneous measurement of crimp, stress, axial strain and lateral contraction in tendon under dynamic loading; (b) determine the interdependence of collagen uncrimping and lateral contraction by testing tendons in different swelling conditions; and (c) assess how the process of collagen uncrimping depends on loading rate. Murine flexor carpi ulnaris (FCU) tendons in varying ionic environments were dynamically stretched to a set strain level and imaged through a plane polariscope with the polarizer and analyzer at a fixed angle. Analysis of the resulting images allowed for direct measurement of the crimp frequency and indirect measurement of the tendon thickness. Our findings demonstrate that collagen uncrimping and lateral contraction can occur independently and interstitial fluid impacts tendon mechanics directly. Furthermore, tensile stress, transverse contraction and degree of collagen uncrimping were all rate-dependent, suggesting that collagen uncrimping plays a role in tendon's dynamic mechanical response. This study is the first to characterize the time-dependence of collagen uncrimping in tendon, and establishes structure–function relationships for healthy tendons that can be used to better understand and assess changes in tendon mechanics after disease or injury.