Search

STUDY DESIGN.: Mechanically induced disruption and subsequent microscopic investigation of lumbar intervertebral discs following a previously published testing protocol, but using a much higher rate of loading. OBJECTIVE.: To explore if loading rate affects the internal disruption mechanics of lumbar intervertebral discs. SUMMARY OF BACKGROUND DATA.: The failure mechanics of some bone-ligament-bone constructs vary with the rate of tensile load application. Like many ligaments, recent reports indicate that the mechanical response of the disc wall varies with strain-rate. It is possible that the internal failure mechanics of the disc wall also varies with strain-rate. METHODS.: Nuclear pressurization was used to deliver sudden pressure impulses directly to the nucleus of ovine lumbar motion segments. Pressure impulses were delivered to 12 neutrally positioned motion segments, and 15 motion segments held at 7° flexion. Aside from loading rate, testing was conducted in the same manner as 2 previously published studies that employed a gradual nuclear pressurization regime. Following testing, the internal damage resulting to each disc was analyzed using micro-CT and serial microscopy in tandem. RESULTS.: Radial tears of the medioposterior disc wall were the most frequent cause of disc failure. In most cases, radial tears involved a combination of anular and endplate disruption: Neutrally positioned discs frequently suffered tears within the superior cartilaginous endplate adjacent to the transition zone and/or inner anulus. Flexed discs frequently suffered tears adjacent to the outer anulus at the cartilaginous/vertebral endplate junction, or within the vertebral endplate. Both groups frequently suffered endplate tears adjacent to the mid anulus at the inferior cartilaginous/vertebral endplate junction. CONCLUSION.: The internal morphologies of the disc disruptions created in this study using high strain-rate impulse pressurization differed significantly from those documented previously for both neutrally positioned and flexed discs subjected to gradual low strain-rate pressurization. These morphologic differences show that the internal failure mechanics of lumbar intervertebral discs vary with the rate of internal radial load application. [ABSTRACT FROM AUTHOR]

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.

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 ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

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 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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 NaBH 4 crosslink stabilization treatment allowed us to probe the mechanisms underlying the observed changes. Untreated tendons and NaBH 4 -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 NaBH 4 -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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (© 2019 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc.)

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 NaBH 4 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. NaBH 4 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.

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., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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., (© 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

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.

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.

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.

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., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Collagen is the primary structural protein in animals. Serving as nanoscale biological ropes, collagen fibrils are responsible for providing strength to a variety of connective tissues such as tendon, skin, and bone. Understanding structure-function relationships in collagenous tissues requires the ability to conduct a variety of mechanical experiments on single collagen fibrils. Though significant advances have been made, certain tests are not possible using the techniques currently available. In this report we present a new atomic force microscopy (AFM) based method for tensile manipulation and subsequent nanoscale structural assessment of single collagen fibrils. While the method documented here cannot currently capture force data during loading, it offers the great advantage of allowing structural assessment after subrupture loading. To demonstrate the utility of this technique, we describe the results of 23 tensile experiments in which collagen fibrils were loaded to varying levels of strain and subsequently imaged in both the hydrated and dehydrated states. We show that following a dehydration-rehydration cycle (necessary for sample preparation), fibrils experience an increase in height and decrease in radial modulus in response to one loading-unloading cycle to strain <5%. This change is not altered by a second cycle to strain >5%. In fibril segments that ruptured during their second loading cycle, we show that the fibril structure is affected away from the rupture site in the form of discrete permanent deformations. By comparing the severity of select damage sites in both hydrated and dehydrated conditions, we demonstrate that dehydration masks damage features, leading to an underestimate of the degree of structural disruption. Overall, the method shows promise as a powerful tool for the investigation of structure-function relationships in nanoscale fibrous materials., Competing Interests: The authors have declared that no competing interests exist.

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.

At its essence, biomechanical injury to soft tissues or tissue products means damage to collagen fibrils. To restore function, damaged collagen must be identified, then repaired or replaced. It is unclear at present what the kernel features of fibrillar damage are, how phagocytic or synthetic cells identify that damage, and how they respond. We recently identified a nanostructural motif characteristic of overloaded collagen fibrils that we have termed discrete plasticity. In this study, we have demonstrated that U937 macrophage-like cells respond specifically to overload-damaged collagen fibrils. Tendons from steer tails were bisected, one half undergoing 15 cycles of subrupture mechanical overload and the other serving as an unloaded control. Both halves were decellularized, producing sterile collagen scaffolds that contained either undamaged collagen fibrils, or fibrils with discrete plasticity damage. Matched-pairs were cultured with U937 cells differentiated to a macrophage-like form directly on the substrate. Morphological responses of the U937 cells to the two substrates-and evidence of collagenolysis by the cells-were assessed using scanning electron microscopy. Enzyme release into medium was quantified for prototypic matrix metalloproteinase-1 (MMP-1) collagenase, and MMP-9 gelatinase. When adherent to damaged collagen fibrils, the cells clustered less, showed ruffled membranes, and frequently spread: increasing their contact area with the damaged substrate. There was clear structural evidence of pericellular enzymolysis of damaged collagen-but not of control collagen. Cells on damaged collagen also released significantly less MMP-9. These results show that U937 macrophage-like cells recognize strain-induced discrete plasticity damage in collagen fibrils: an ability that may be important to their removal or repair., (© 2014 Wiley Periodicals, Inc.)

Due to the high occurrence rate of overextension injuries to tendons and ligaments, it is important to understand the fundamental mechanisms of damage to these tissues' primary load-bearing elements: collagen fibrils and their constituent molecules. Based on our recent observations of a new subrupture, overload-induced mode of fibril disruption that we call discrete plasticity, we have sought in the current study to re-explore whether the tensile overload of collagen fibrils can alter the helical conformation of collagen molecules. In order to accomplish this, we have analyzed the conformation of collagen molecules within repeatedly overloaded tendons in relation to their undamaged matched-pair controls using both differential scanning calorimetry and variable temperature trypsin digestion susceptibility. We find that tensile overload reduces the specific enthalpy of denaturation of tendons, and increases their susceptibility to trypsin digestion, even when the digestion is carried out at temperatures as low as 4 °C. Our results indicate that the tensile overload of collagen fibrils can uncoil the helix of collagen molecules, placing them in a stable, denatured state., (© 2013 Elsevier B.V. All rights reserved.)

We investigated whether immature allysine-derived cross-links provide mechanically labile linkages by exploring the effects of immature cross-link stabilization at three levels of collagen hierarchy: damaged fibril morphology, whole tendon mechanics, and molecular stability. Tendons from the tails of young adult steers were either treated with sodium borohydride (NaBH₄) to stabilize labile cross-links, exposed only to the buffer used during stabilization treatment, or maintained as untreated controls. One-half of each tendon was then subjected to five cycles of subrupture overload. Morphologic changes to collagen fibrils resulting from overload were investigated using scanning electron microscopy, and changes in the hydrothermal stability of collagen molecules were assessed using hydrothermal isometric tension testing. NaBH4 cross-link stabilization did not affect the response of tendon collagen to tensile overload at any of the three levels of hierarchy studied. Cross-link stabilization did not prevent the characteristic overload-induced mode of fibril damage that we term discrete plasticity. Similarly, stabilization did not alter the mechanical response of whole tendons to overload, and did not prevent an overload-induced thermal destabilization of collagen molecules. Our results indicate that hydrothermally labile cross-links may not be as mechanically labile as was previously thought., (© 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

A critical feature of tendons and ligaments is their ability to resist rupture when overloaded, resulting in strains or sprains instead of ruptures. To treat these injuries more effectively, it is necessary to understand how overload affects the primary load-bearing elements of these tissues: collagen fibrils. We have investigated how repeated subrupture overload alters the collagen of tendons at the nanoscale. Using scanning electron microscopy to examine fibril morphology and hydrothermal isometric tension testing to look at molecular stability, we demonstrated that tendon collagen undergoes a progressive cascade of discrete plasticity damage when repeatedly overloaded. With successive overload cycles, fibrils develop an increasing number of kinks along their length. These kinks-discrete zones of plastic deformation known to contain denatured collagen molecules-are accompanied by a progressive and eventual total loss of D-banding along the surface of fibrils, indicating a loss of native molecular packing and further molecular denaturation. Thermal analysis of molecular stability showed that the destabilization of collagen molecules within fibrils is strongly related to the amount of strain energy dissipated by the tendon after yielding during tensile overload. These novel findings raise new questions about load transmission within tendons and their fibrils and about the interplay between crosslinking, strain-energy dissipation ability, and molecular denaturation within these structures., (Copyright © 2012 Orthopaedic Research Society.)

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., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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.

Study Design: Hydrostatically induced disruption of flexed lumbar intervertebral discs followed by microstructural investigation., Objective: To investigate how flexion affects the anulus' ability to resist rupture during hydrostatic loading, and determine how the characteristics of the resulting disc failures compare with those observed clinically., Summary of Background Data: While compression of neutrally positioned motion segments consistently causes vertebral failure, compression of flexed segments can induce herniation. Why flexion has this effect remains unclear. A vast range of herniation characteristics have been documented clinically; whether flexion-related herniations are likely to possess a subset of these is unknown., Methods: Forty-two ovine lumbar motion segments, dissected from the same 3 levels of 14 spines, were each flexed 7 degrees or 10 degrees from the neutral position. While maintained at one of these angles, 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. Each segment was then inspected using microcomputed tomography and oblique illumination microscopy in tandem. RESULTS.: Eighteen segments suffered disc failure; 14 of these were caused by direct radial rupture of the anular wall. All radial ruptures were located in the central posterior anulus. Nine radial ruptures contained nuclear material, which had breached the posterior longitudinal ligament in 1 disc, and reached it in 5 others forming transligamentous and subligamentous nuclear extrusions, respectively. The most common radial rupture route, occurring in 10 discs, involved a systematic anulus-endplate-anulus failure pattern., Conclusion: Flexion places the anulus at risk by facilitating nuclear flow, limiting circumferential disruption while promoting radial rupture, and rendering the endplate/vertebra junction vulnerable to failure. Flexion may play a developmental role in those herniations possessing a central posterior radial rupture that incorporates a short span of endplate disruption along the apex of the vertebral rim.

Hydrothermal isometric tension (HIT) testing and high-performance liquid chromatography were used to assess the molecular stability and cross-link population of collagen in the four valves of the adult bovine heart. Untreated and NaBH(4)-treated tissues under isometric tension were heated in a water bath to a 90 degrees C isotherm that was sustained for 5 h. The denaturation temperature (T(d)), associated with hydrogen bond rupture and molecular stability, and the half-time of load decay (t(1/2)), associated with peptide bond hydrolysis and intermolecular cross-linking, were calculated from acquired load/temperature/time data. An unpaired group of samples of the same population was biochemically assayed for the types and quantities of enzymatic cross-links present. Tissues known to endure higher in vivo transvalvular pressures had lower T(d) values, suggesting that molecular stability is inversely related to in vivo loading. The treated inflow valves (mitral and tricuspid) had significantly lower t(1/2) values than did treated outflow valves (aortic and pulmonary), suggesting lower overall cross-linking in the inflow valves. Inflow valves were also found to fail during HIT testing significantly more often than outflow valves, also suggestive of a decreased cross-link population. Inflow valves may be remodeling at a faster rate and may be at an earlier state of molecular "maturity" than outflow valves. At the molecular level, the thermal stability of collagen is associated with in vivo loading and may be influenced by the mature, aldimine-derived cross-link, histidinohydroxylysinonorleucine. We conclude that the valves of the heart utilize differing, location-specific strategies to resist biomechanical fatigue loading.

Study Design: Mechanically induced annular disruption of lumbar intervertebral discs followed by microstructural investigation., Objective: To investigate the role that elevated nuclear pressures play in disrupting the lumbar intervertebral disc's annulus fibrosus., Summary of Background Data: Compound mechanical loadings have been used to recreate clinically relevant annular disruptions in vitro. However, the role that individual loading parameters play in disrupting the lumbar disc's annulus remains unclear., Methods: The nuclei of ovine lumbar intervertebral discs were gradually pressurized by injecting a viscous radio-opaque gel via their inferior vertebrae. Pressurization was conducted until catastrophic failure of the disc occurred. Investigation of the resulting annular disruption was carried out using microcomputed tomography and differential interference contrast microscopy., Results: Gel extrusion from the posterior annulus was the most common mode of disc failure. Unlike other aspects of the annular wall, the posterior region was unable to distribute hydrostatic pressures circumferentially. In each extrusion case, severe disruption of the posterior annulus occurred. Although intralamellar disruption occurred in the mid annulus, interlamellar disruption occurred in the outer posterior annulus. Radial ruptures between lamellae always occurred in the mid-axial plane., Conclusion: With respect to the annular wall, the posterior region is most susceptible to failure in the presence of high nuclear pressure, even when loaded in the neutral position. Weak interlamellar cohesion of the outer posterior lamellae may explain why the majority of herniations remain contained as protrusions within the outer annular wall.