Histological staining alters circular dichroism SHG measurements of collagen.

Circular dichroism second harmonic generation microscopy (CDSHG) is a powerful imaging technique, which allows three-dimensional visualization of collagen fibril orientation in tissues. However, recent publications have obtained contradictory results on whether CDSHG can be used to reveal the relative out-of-plane polarity of collagen fibrils. Here we compare CDSHG images of unstained tendon and tendon which has been stained with hematoxylin and eosin. We find significant differences in the CDSHG between these two conditions, which explain the recent contradictory results within the literature.

ISSLS PRIZE IN BASIC SCIENCE 2020: Beyond microstructure-circumferential specialization within the lumbar intervertebral disc annulus extends to collagen nanostructure, with counterintuitive relationships to macroscale material properties.

PURPOSE: To determine whether the annulus of lumbar intervertebral discs contains circumferential specialization in collagen nanostructure and assess whether this coincides with functional differences in macroscale material properties. METHODS: Anterior and posterior disc wall samples were prepared from 38 mature ovine lumbar segments. Regional differences in molecular thermal stability and intermolecular network integrity of the annulus' tension-bearing collagen fibres were examined with hydrothermal isometric tension (HIT) analysis, with and without preceding NaBH4 treatment to stabilize labile crosslinks. Energetics of collagen denaturation were studied by differential scanning calorimetry (DSC). Tensile mechanics of annular lamellae were studied using oblique sagittal bone-disc-bone samples loaded to rupture. Annular failure characteristics of the ruptured test segments were compared via microscopy of serial sections. RESULTS: HIT showed that tension-bearing collagen fibres of the posterior annulus were composed of collagen molecules with significantly greater thermal stability and intermolecular network integrity than those of the anterior annulus. NaBH4 treatment confirmed that labile intermolecular crosslinks did not significantly contribute to network integrity in either region. Regional differences seen in DSC were smaller than those observed in HIT, indicating structural similarities in annular collagen outside of the main fibre bundles. Mechanical testing showed that the posterior annulus was significantly weaker than the anterior annulus. For both regions, ultimate tensile strengths of annular fibres were significantly greater than those previously reported. Ruptures in both regions were predominantly due to annular failure. CONCLUSION: Specializations in collagen nanostructure exist between different circumferential regions of the annulus and coincide with significant differences in material properties. These slides can be retrieved under Electronic Supplementary Material.

Gouy phase shift measurement using interferometric second-harmonic generation.

We report on a simple way to directly measure the Gouy phase shift of a strongly focused laser beam. This is accomplished by using a recent technique, namely, interferometric second-harmonic generation. We expect that this method will be of interest in a wide range of research fields, from high-harmonic and attosecond pulse generation to femtochemistry and nonlinear microscopy.

High spatial resolution (1.1 µm and 20 nm) FTIR polarization contrast imaging reveals pre-rupture disorder in damaged tendon.

Collagen is a major constituent in many life forms; in mammals, collagen appears as a component of skin, bone, tendon and cartilage, where it performs critical functions. Vibrational spectroscopy methods are excellent for studying the structure and function of collagen-containing tissues, as they provide molecular insight into composition and organization. The latter is particularly important for collagenous materials, given that a key feature is their hierarchical, oriented structure, organized from molecular to macroscopic length scales. Here, we present the first results of high-resolution FTIR polarization contrast imaging, at 1.1 µm and 20 nm scales, on control and mechanically damaged tendon. The spectroscopic data are supported with parallel SEM and correlated AFM imaging. Our goal is to explore the changes induced in tendon after the application of damaging mechanical stress, and the consequences for the healing processes. The results and possibilities for the application of these high-spatial-resolution FTIR techniques in spectral pathology, and eventually in clinical applications, are discussed.

Single collagen fibrils isolated from high stress and low stress tendons show differing susceptibility to enzymatic degradation by the interstitial collagenase matrix metalloproteinase-1 (MMP-1).

Bovine forelimb flexor and extensor tendons serve as a model for examining high stress, energy storing and low stress, positional tendons, respectively. Previous research has shown structural differences between the collagen fibrils of these tissues. The nanoscale collagen fibrils of flexor tendons are smaller in size, more heavily crosslinked, and respond differently to mechanical loading. Meanwhile, energy storing tendons undergo less collagen turnover compared to positional tendons and are more commonly injured. These observations raise the question of whether collagen fibril structure influences the collagen degradation processes necessary for remodelling. Atomic force microscopy was used to image dry collagen fibrils before and after 5-hour exposure to matrix metalloproteinase-1 (MMP-1) to detect changes in fibril size. Collagen fibrils from three tissue types were studied: bovine superficial digital flexor tendons, matched-pair bovine lateral digital extensor tendons, and rat tail tendons. Compared to control fibrils exposed only to buffer, a significant decrease in fibril cross-sectional area (CSA) following MMP-1 exposure was observed for bovine extensor and rat tail fibrils, with larger fibrils experiencing a greater magnitude of CSA decrease in both fibril types. Fibrils from bovine flexor tendons, on the other hand, showed no decrease in CSA when exposed to MMP-1. The result did not appear to be linked to the small size of flexor fibrils, as equivalently sized extensor fibrils were readily degraded by the enzyme. Increased proteolytic resistance of collagen fibrils from high stress tendons may help to explain the longevity of collagen within these tissues in vivo.

Designed to fail: a novel mode of collagen fibril disruption and its relevance to tissue toughness.

Collagen fibrils are nanostructured biological cables essential to the structural integrity of many of our tissues. Consequently, understanding the structural basis of their robust mechanical properties is of great interest. Here we present what to our knowledge is a novel mode of collagen fibril disruption that provides new insights into both the structure and mechanics of native collagen fibrils. Using enzyme probes for denatured collagen and scanning electron microscopy, we show that mechanically overloading collagen fibrils from bovine tail tendons causes them to undergo a sequential, two-stage, selective molecular failure process. Denatured collagen molecules-meaning molecules with a reduced degree of time-averaged helicity compared to those packed in undamaged fibrils-were first created within kinks that developed at discrete, repeating locations along the length of fibrils. There, collagen denaturation within the kinks was concentrated within certain subfibrils. Additional denatured molecules were then created along the surface of some disrupted fibrils. The heterogeneity of the disruption within fibrils suggests that either mechanical load is not carried equally by a fibril's subcomponents or that the subcomponents do not possess homogenous mechanical properties. Meanwhile, the creation of denatured collagen molecules, which necessarily involves the energy intensive breaking of intramolecular hydrogen bonds, provides a physical basis for the toughness of collagen fibrils.

Effect of increasing mineralization on pre-osteoblast response to native collagen fibril scaffolds for bone tissue repair and regeneration.

With limited availability of auto- and allografts, there is increasing demand for alternative bone repair and regeneration materials. Inspired by a mimetic approach, the utility of producing engineered native protein scaffolds is being increasingly realized, demonstrating the need for continued research in this field. In previous work, we detailed a process for producing mineralized collagen scaffolds using tendon to create collagen templates of highly aligned, natively crosslinked collagen fibrils. The process produced mineral phase closely matching that of native bone, and integration of mineral with the collagen template was demonstrated to be easily controlled, allowing scaffolds to be mechanically tuned. In the current study, we have extended this work to investigate how variation in the mineralization level of these scaffolds affects the osteogenic response of pre-osteoblastic cells. Scaffolds were produced under three treatment groups, where collagen templates underwent 0, 5, or 20 mineralization cycles. Scaffolds in each treatment group were cultured with MC3T3-E1 cells for 1, 7, or 14 days. Morphologic assessment under SEM indicated decreased attachment to the mineralized scaffolds, supported by DNA results showing a significant drop between culture days 1 and 7 for mineralized scaffolds only. For adherent cells, increasing scaffold mineralization also delayed cell spreading. While mineralization presented a barrier to cell coverage of scaffolds, it increased osteogenic activity, with cells on the mineralized scaffolds showing significantly greater alkaline phosphatase activity and osteocalcin production. Understanding how increasing collagen mineralization effects pre-osteoblast function may enable design of more advanced mineralized collagen scaffolds for bone repair and regeneration.

The influence of torsion on disc herniation when combined with flexion.

The role of torsion in the mechanical derangement of intervertebral discs remains largely undefined. The current study sought to investigate if torsion, when applied in combination with flexion, affects the internal failure mechanics of the disc wall when exposed to high nuclear pressure. Thirty ovine lumbar motion segments were each positioned in 2 degrees axial rotation plus 7 degrees flexion. Whilst maintained in this posture, the nucleus of each segment was gradually injected with a viscous radio-opaque gel, via an injection screw placed longitudinally within the inferior vertebra, until failure occurred. Segments were then inspected using micro-CT and optical microscopy in tandem. Five motion segments failed to pressurize correctly. Of the remaining 25 successfully tested motion segments, 17 suffered vertebral endplate rupture and 8 suffered disc failure. Disc failure occurred in mature motion segments significantly more often than immature segments. The most common mode of disc failure was a central posterior radial tear involving a systematic annulus-endplate-annulus failure pattern. The endplate portion of these radial tears often propagated contralateral to the direction of applied axial rotation, and, at the lateral margin, only those fibres inclined in the direction of the applied torque were affected. Apart from the 2 degrees of applied axial rotation, the methods employed in this study replicated those used in a previously published study. Consequently, the different outcome obtained in this study can be directly attributed to the applied axial rotation. These inter-study differences show that when combined with flexion, torsion markedly reduces the nuclear pressure required to form clinically relevant radial tears that involve cartilaginous endplate failure. Conversely, torsion appears to increase the disc wall's resistance to radial tears that do not involve cartilaginous endplate failure, effectively halving the disc wall's overall risk of rupture.

Effect of testing temperature on the nanostructural response of tendon to tensile mechanical overload.

Despite many in vitro mechanical experiments of tendon being conducted at room temperature, few assessments have been made to determine how the structural response of tendon to mechanical overload may vary with ambient temperature. We explored whether damage to the collagen nanostructure of tendon resulting from tensile rupture varies with temperature. Use of bovine tail tendons in combination with NaBH4 crosslink stabilization treatment allowed us to probe the mechanisms underlying the observed changes. Untreated tendons and NaBH4-stabilized tendons were pulled to rupture at temperatures of 24, 37, and 55 °C. Of nine mechanical parameters measured from the resulting stress-strain curves, only yield stress differed between the tendons tested at 37 and 24 °C. When tested at 55 °C, untreated tendons showed large reductions in ultimate strength and toughness, while NaBH4-stabilized tendons showed smaller reductions. Differential scanning calorimetry was used to assess damage to the collagen fibril nanostructure of tendons resulting from rupture, with samples from the ruptured tendons compared to samples from the same tendons removed prior to loading. While there was indication that overload-induced molecular packing disruption to collagen fibrils may be heightened at 37 °C, statistical increases in damage compared to that occurring at 24 °C were only seen when testing was conducted at 55 °C. The results show that the temperature sensitivity of tendon to ramp loading depends on crosslinking within the tissue. In poorly crosslinked tissues, collagen may be more susceptible to mechanical damage when tested at physiologic temperature compared to room temperature. For tendons with a high density of thermally stable crosslinks, such as the human Achilles or patellar tendons, testing at room temperature should produce comparable results to testing at physiologic temperature.

Use of tendon to produce decellularized sheets of mineralized collagen fibrils for bone tissue repair and regeneration.

With demand for alternatives to autograft and allograft materials continuing to rise, development of new scaffolds for bone tissue repair and regeneration remains of significant interest. Engineered collagen-calcium phosphate (CaP) constructs can offer desirable attributes, including absence of foreign body response and possession of inherent osteogenic potential. Despite their promise, current collagen-CaP constructs are limited to nonload-bearing applications. In this article, we describe a process for creating decellularized sheets of highly aligned, natively cross-linked, and mineralized collagen fibrils, which may be useful for developing multilaminate collagen-CaP constructs with improved mechanical properties. Decellularized bovine tendons were cryosectioned to produce thin sheets of aligned collagen fibrils. Mineralization of the sheets was then performed using an alternate soaking method incorporating a polymer-induced liquid precursor (PILP) process to promote intrafibrillar mineralization, along with incorporation of physiologically relevant amounts of citrate, Mg, and carbonate. Characteristics of the produced scaffolds were assessed using energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Scaffolds were also compared with both native bovine cortical bone and pure hydroxyapatite using X-ray powder diffraction (XRD), and Fourier transform infrared spectroscopy attenuated total reflection (FTIR-ATR). Structural and chemical analyses show that the scaffold preparation process that we described is successful in creating mineralized collagen sheets, possessing a mineral phase similar to that found in bone as well as a close association between collagen fibrils and mineral plates.

Alternate soaking enables easy control of mineralized collagen scaffold mechanics from nano- to macro-scale.

The mechanical properties of biologic scaffolds are critical to cellular interactions and hence functional response within the body. In the case of scaffolds for bone tissue regeneration, engineered scaffolds created by combining collagen with inorganic mineral are increasingly being explored, due to their favourable structural and chemical characteristics. Development of a method for controlling the mechanics of these scaffolds could lead to significant additional advantages by harnessing the intrinsic mechnotransduction pathways of stem cells via appropriate control of scaffold mechanical properties. Here we present a method for controlling the macroscale flexural modulus of mineralized collagen sheets, and the radial indentation modulus of the sheets' constituent collagen fibrils. Scaffolds were created starting with sheets of highly aligned, natively structured collagen fibrils, prepared via cryosectioning of decellularized tendon. Sheets underwent an alternate soaking mineralization procedure, with sequential exposure to citrate-doped calcium and carbonate-containing phosphate solutions, both of which included poly aspartic acid. The extent of scaffold mineralization was controlled via number of repeated mineralization cycles: 0 (unmineralized), 5, 10, and 20 cycles were trialed. Following scaffold preparation, ultrastructure, macroscale flexural modulus, and nanoscale indentation modulus were assessed. Surface architecture studied by SEM, and inspection of individual extracted fibrils by TEM and AFM confirmed that fibrils became increasingly laden with mineral as the number of mineralization cycles increased. Measurements of collagen fibril nanomechanics using AFM showed that the radial modulus of collagen fibrils increased linearly with mineralization cycles completed, from 215 ± 125 MPa for fibrils from unmineralized (0 cycle) scaffolds to 778 ± 302 MPa for fibrils from the 20 mineralization cycle scaffolds. Measurements of scaffold macromechanics via flexural testing also showed a linear increase in flexural modulus with increasing number of mineralization cycles completed, from 18 ± 7 MPa for the 5 cycle scaffolds to 156 ± 50 MPa for the 20 cycle scaffolds. The process detailed herein provides a way to create mineralized collagen scaffolds with easily controllable mechanical properties.

A new longitudinal variation in the structure of collagen fibrils and its relationship to locations of mechanical damage susceptibility.

The hierarchical architecture of the collagen fibril is well understood, involving non-integer staggering of collagen molecules which results in a 67 nm periodic molecular density variation termed D-banding. Other than this variation, collagen fibrils are considered to be homogeneous at the micro-scale and beyond. Interestingly, serial kink structures have been shown to form at discrete locations along the length of collagen fibrils from some mechanically overloaded tendons. The formation of these kinks at discrete locations along the length of fibrils (discrete plasticity) may indicate pre-existing structural variations at a length scale greater than that of the D-banding. Using a high velocity nanomechanical mapping technique, 25 tendon collagen fibrils, were mechanically and structurally mapped along 10 µm of their length in dehydrated and hydrated states with resolutions of 20 nm and 8 nm respectively. Analysis of the variation in hydrated indentation modulus along individual collagen fibrils revealed a micro-scale structural variation not observed in the hydrated or dehydrated structural maps. The spacing distribution of this variation was similar to that observed for inter-kink distances seen in SEM images of discrete plasticity type damage. We propose that longitudinal variation in collagen fibril structure leads to localized mechanical susceptibility to damage under overload. Furthermore, we suggest that this variation has its origins in heterogeneous crosslink density along the length of collagen fibrils. The presence of pre-existing sites of mechanical vulnerability along the length of collagen fibrils may be important to biological remodeling of tendon, with mechanically-activated sites having distinct protein binding capabilities and enzyme susceptibility.

Advanced glycation end-product cross-linking inhibits biomechanical plasticity and characteristic failure morphology of native tendon.

Advanced glycation end-products (AGEs) are formed in vivo from the nonenzymatic reaction between sugars and proteins. AGEs accumulate in long-lived tissues like tendons, cross-linking neighboring collagen molecules, and are in part complicit in connective tissue pathologies experienced in aging and with diabetes. We have previously described discrete plasticity: a characteristic form of nanoscale collagen fibril damage consisting of serial fibril kinking and collagen denaturation that occurs in some mechanically overloaded tendons. We suspect that this failure mechanism may be an adaptive trait of collagen fibrils and have published evidence that inflammatory cells may be able to recognize and digest the denatured collagen produced by overload. In this study, we treated bovine tail tendons with ribose to simulate long-term AGE cross-linking in vitro. We hypothesized that a high degree of cross-linking would inhibit the intermolecular sliding thought to be necessary for discrete plasticity to occur. Tendons were mechanically overloaded, and properties were investigated by differential scanning calorimetry and scanning election microscopy. Ribose cross-linking treatment altered the mechanical response of tendons after the yield point, significantly decreasing postyield extensibility and strain energy capacity before rupture. Coincident with altered mechanics, ribose cross-linking completely inhibited the discrete plasticity failure mechanism of tendon. Our results suggest that discrete plasticity, which may be an important physiological mechanism, becomes pathologically disabled by the formation of AGE cross-links in aging and diabetes. NEW & NOTEWORTHY We have previously shown that mechanically overloaded collagen fibrils in mammalian tendons accrue nanoscaled damage. This includes development of a characteristic kinking morphology within a shell of denatured collagen: discrete plasticity. Here, using a ribose-incubation model, we show that advanced glycation end-product cross-linking associated with aging and diabetes completely inhibits this mechanism. Since discrete plasticity appears to cue cellular remodeling, this result has important implications for diabetic tendinopathy.

Ultrastructural response of tendon to excessive level or duration of tensile load supports that collagen fibrils are mechanically continuous.

The basic collagen fibril structure of tendons continues to be debated in the literature. Some studies have proposed that collagen fibrils are longitudinally discontinuous, with the load-bearing ability of tendon dependent on interfibrillar shear strength. Other evidence indicates that collagen fibrils are probably structurally continuous, running uninterrupted from osteotendinous to myotendinous junction. In this study we explored the question of collagen fibril continuity in tendon by examining fibril response to tendon loading. Tendons were subjected to high stress and/or long duration tensile loading routines, after which we examined the ultrastructure of the tendons using differential scanning calorimetry and scanning electron microscopy, comparing the results from the loaded tendons to control samples taken from the same tendons prior to loading. Our results show that under ramp loading, collagen fibril damage begins near the end of the linear region in the stress-strain response (i.e., near the yield point). When tendons are allowed to gradually elongate under static load, tendon rupture is caused by failure of collagen fibrils, not uncontrolled slippage between fibrils. Our findings indicate that the collagen fibrils of tendon are at least sufficiently long to be mechanically continuous, meaning that tensile failure of tendon does not occur as the result of uncontrolled slippage between fibrils, but by failure of the fibrils themselves.

Development of overuse tendinopathy: A new descriptive model for the initiation of tendon damage during cyclic loading.

Tendinopathic tissue has long been characterized by changes to collagen microstructure. However, initial tendon damage from excessive mechanical loading-a hallmark of tendinopathy development-could occur at the nanoscale level of collagen fibrils. Indeed, it is on this scale that tenocytes interact directly with tendon matrix, and excessive collagen fibril damage not visible at the microscale could trigger a degenerative cascade. In this study, we explored whether initiation of tendon damage during cyclic loading occurs via a longitudinal compression-induced buckling mechanism of collagen fibrils leading to nanoscale kinkband development. Two groups of tendons were cyclically loaded to equivalent peak stresses. In each loading cycle, tendons in one group were unloaded to the zero displacement mark, while those in the other group were unloaded to a nominal level of tension, minimizing the potential for fibril buckling. Tendons that were unloaded to the zero displacement mark ruptured significantly sooner during cyclic loading (1,446 ± 737 vs. 4,069 ± 1,129 cycles), indicating that significant fatigue damage is accrued in the low stress, toe region of the load-deformation response. Ultrastructural analysis using scanning electron microscopy of tendons stopped after 1,000 cycles showed that maintaining a nominal tension slowed the accumulation of kinkbands, supporting a longitudinal compression-induced buckling mechanism as the basis for kinkband development. Based on our results, we present a new descriptive model for the initiation of tendon damage during cyclic loading. The so-called Compression of Unrecovered Elongation or CUE Model may provide useful insight into the development of tendinopathy. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:467-476, 2018.

Ultrastructure of tendon rupture depends on strain rate and tendon type.

Previous research has shown that both the mechanics and elongation mechanisms of tendon and ligament vary with strain rate during tensile loading. In this study, we sought to determine if the ultrastructural damage created during tendon rupture also varies with strain rate. A bovine forelimb model was used, allowing two anatomically proximate but physiologically distinct tendons to be studies: the positional common digital extensor tendon, and the energy storing superficial digital flexor tendon. Samples from the two tendon types were ruptured at rates of either 1%/s or 10%/s. Relative to unruptured control samples, changes to collagen fibril structure were assessed using scanning electron microscopy (SEM), and changes to collagen molecule packing were studied using differential scanning calorimetry (DSC). Rupture at 1%/s caused discrete plasticity damage that extended along the length of collagen fibrils in both the extensor and flexor tendons. Consistent with this, DSC showed molecular packing disruption relative to control samples. Both SEM and DSC showed that extensor tendon fibrils sustained more severe damage than the more highly crosslinked flexor tendon fibrils. Increasing strain rate during rupture decreased the level of longitudinal disruption experienced by the collagen fibrils of both tendon types. Disruption to D-banding was no longer seen in the extensor tendon fibrils, and discrete plasticity damage was completely eliminated in the flexor tendon fibrils, indicating a transition to localized point failure. Ultrastructural damage resulting from tendon rupture depends on both strain rate and tendon type. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2842-2850, 2018.

Combining tensile testing and structural analysis at the single collagen fibril level.

Tensile testing to failure followed by imaging is a simple way of studying the structure-function relationship of connective tissues such as skin, tendon, and ligament. However, interpretation of these datasets is complex due to the hierarchical structures of the tissues spanning six or more orders of magnitude in length scale. Here we present a dataset obtained through the same scheme at the single collagen fibril level, the fundamental tensile element of load-bearing tissues. Tensile testing was performed on fibrils extracted from two types of bovine tendons, adsorbed on a glass surface and glued at both ends. An atomic force microscope (AFM) was used to pull fibrils to failure in bowstring geometry. The broken fibrils were then imaged by AFM for morphological characterization, by second harmonic generation microscopy to assess changes to molecular packing, and by fluorescence microscopy after incubation with a peptide probe that binds specifically to denatured collagen molecules. This dataset linking stress-strain curves to post-failure molecular changes is useful for researchers modelling or designing functional protein materials.

In tendons, differing physiological requirements lead to functionally distinct nanostructures.

The collagen-based tissues of animals are hierarchical structures: even tendon, the simplest collagenous tissue, has seven to eight levels of hierarchy. Tailoring tissue structure to match physiological function can occur at many different levels. We wanted to know if the control of tissue architecture to achieve function extends down to the nanoscale level of the individual, cable-like collagen fibrils. Using tendons from young adult bovine forelimbs, we performed stress-strain experiments on single collagen fibrils extracted from tendons with positional function, and tendons with energy storing function. Collagen fibrils from the two tendon types, which have known differences in intermolecular crosslinking, showed numerous differences in their responses to elongation. Unlike those from positional tendons, fibrils from energy storing tendons showed high strain stiffening and resistance to disruption in both molecular packing and conformation, helping to explain how these high stress tissues withstand millions of loading cycles with little reparative remodeling. Functional differences in load-bearing tissues are accompanied by important differences in nanoscale collagen fibril structure.

Quantitative phase measurements of tendon collagen fibres.

Collagen is the main component of structural mammalian tissues. In tendons, collagen is arranged into fibrils with diameters ranging from 30 nm to 500 nm. These fibrils are further assembled into fibres several micrometers in diameter. Upon excessive thermal or mechanical stress, damage may occur in tendons at all levels of the structural hierarchy. At the fibril level, reported damage includes swelling and the appearance of discrete sites of plastic deformation that are best observed at the nanometer-scale using, for example, scanning electron microscopy. In this paper, digital in-line holographic microscopy is used for quantitative phase imaging to measure both the refractive index and diameter of collagen fibres in a water suspension in the native state, after thermal treatments, and after mechanical overload. Fibres extracted from tendons and subsequently exposed to 70 °C for 5, 15, or 30 minutes show a significant decrease in refractive index and an increase in diameter. A significant increase in refractive index is also observed for fibres extracted from tendons that were subjected to five tensile overload cycles.

Collagen fibrils in functionally distinct tendons have differing structural responses to tendon rupture and fatigue loading.

UNLABELLED: In this study we investigate relationships between the nanoscale structure of collagen fibrils and the macroscale functional response of collagenous tissues. To do so, we study two functionally distinct classes of tendons, positional tendons and energy storing tendons, using a bovine forelimb model. Molecular-level assessment using differential scanning calorimetry (DSC), functional crosslink assessment using hydrothermal isometric tension (HIT) analysis, and ultrastructural assessment using scanning electron microscopy (SEM) were used to study undamaged, ruptured, and cyclically loaded samples from the two tendon types. HIT indicated differences in both crosslink type and crosslink density, with flexor tendons having more thermally stable crosslinks than the extensor tendons (higher TFmax of >90 vs. 75.1±2.7°C), and greater total crosslink density than the extensor tendons (higher t1/2 of 11.5±1.9 vs. 3.5±1.0h after NaBH4 treatment). Despite having a lower crosslink density than flexor tendons, extensor tendons were significantly stronger (37.6±8.1 vs. 23.1±7.7MPa) and tougher (14.3±3.6 vs. 6.8±3.4MJ/m(3)). SEM showed that collagen fibrils in the tougher, stronger extensor tendons were able to undergo remarkable levels of plastic deformation in the form of discrete plasticity, while those in the flexor tendons were not able to plastically deform. When cyclically loaded, collagen fibrils in extensor tendons accumulated fatigue damage rapidly in the form of kink bands, while those in flexor tendons did not accumulate significant fatigue damage. The results demonstrate that collagen fibrils in functionally distinct tendons respond differently to mechanical loading, and suggests that fibrillar collagens may be subject to a strength vs. fatigue resistance tradeoff. STATEMENT OF SIGNIFICANCE: Collagen fibrils-nanoscale biological cables-are the fundamental load-bearing elements of all structural human tissues. While all collagen fibrils share common features, such as being composed of a precise quarter-staggered polymeric arrangement of triple-helical collagen molecules, their structure can vary significantly between tissue types, and even between different anatomical structures of the same tissue type. To understand normal function, homeostasis, and disease of collagenous tissues requires detailed knowledge of collagen fibril structure-function. Using anatomically proximate but structurally distinct tendons, we show that collagen fibrils in functionally distinct tendons have differing susceptibilities to damage under both tensile overload and cyclic fatigue loading. Our results suggest that the structure of collagen fibrils may lead to a strength versus fatigue resistance tradeoff, where high strength is gained at the expense of fatigue resistance, and vice versa.