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.     

Collagen fibril morphology and organization: implications for force transmission in ligament and tendon.

Connective tissue mechanical behavior is primarily determined by the composition and organization of collagen. In ligaments and tendons, type I collagen is the principal structural element of the extracellular matrix, which acts to transmit force between bones or bone and muscle, respectively. Therefore, characterization of collagen fibril morphology and organization in fetal and skeletally mature animals is essential to understanding how tissues develop and obtain their mechanical attributes. In this study, tendons and ligaments from fetal rat, bovine, and feline, and mature rat were examined with scanning electron microscopy. At early fetal developmental stages, collagen fibrils show fibril overlap and interweaving, apparent fibril ends, and numerous bifurcating/fusing fibrils. Late in fetal development, collagen fibril ends are still present and fibril bundles (fibers) are clearly visible. Examination of collagen fibrils from skeletally mature tissues, reveals highly organized regions but still include fibril interweaving, and regions that are more randomly organized. Fibril bifurcations/fusions are still present in mature tissues but are less numerous than in fetal tissue. To address the continuity of fibrils in mature tissues, fibrils were examined in individual micrographs and consecutive overlaid micrographs. Extensive microscopic analysis of mature tendons and ligaments detected no fibril ends. These data strongly suggest that fibrils in mature ligament and tendon are either continuous or functionally continuous. Based upon this information and published data, we conclude that force within these tissues is directly transferred through collagen fibrils and not through an interfibrillar coupling, such as a proteoglycan bridge.     

Mechanical properties of human patellar tendon at the hierarchical levels of tendon and fibril

Tendons are strong hierarchical structures, but how tensile forces are transmitted between different levels remains incompletely understood. Collagen fibrils are thought to be primary determinants of whole tendon properties, and therefore we hypothesized that the whole human patellar tendon and its distinct collagen fibrils would display similar mechanical properties. Human patellar tendons (n = 5) were mechanically tested in vivo by ultrasonography. Biopsies were obtained from each tendon, and individual collagen fibrils were dissected and tested mechanically by atomic force microscopy. The Young's modulus was 2.0 ± 0.5 GPa, and the toe region reached 3.3 ± 1.9% strain in whole patellar tendons. Based on dry cross-sectional area, the Young's modulus of isolated collagen fibrils was 2.8 ± 0.3 GPa, and the toe region reached 0.86 ± 0.08% strain. The measured fibril modulus was insufficient to account for the modulus of the tendon in vivo when fibril content in the tendon was accounted for. Thus, our original hypothesis was not supported, although the in vitro fibril modulus corresponded well with reported in vitro tendon values. This correspondence together with the fibril modulus not being greater than that of tendon supports that fibrillar rather than interfibrillar properties govern the subfailure tendon response, making the fibrillar level a meaningful target of intervention. The lower modulus found in vitro suggests a possible adverse effect of removing the tissue from its natural environment. In addition to the primary work comparing the two hierarchical levels, we also verified the existence of viscoelastic behavior in isolated human collagen fibrils.     

Characterizing local collagen fiber re-alignment and crimp behavior throughout mechanical testing in a mature mouse supraspinatus tendon model

BackgroundCollagen fiber re-alignment and uncrimping are two postulated mechanisms of tendon structural response to load. Recent studies have examined structural changes in response to mechanical testing in a postnatal development mouse supraspinatus tendon model (SST), however, those changes in the mature mouse have not been characterized. The objective of this study was to characterize collagen fiber re-alignment and crimp behavior throughout mechanical testing in a mature mouse SST.Method of approachA tensile mechanical testing set-up integrated with a polarized light system was utilized for alignment and mechanical analysis. Local collagen fiber crimp frequency was quantified immediately following the designated loading protocol using a traditional tensile set up and a flash-freezing method. The effect of number of preconditioning cycles on collagen fiber re-alignment, crimp frequency and mechanical properties in midsubstance and insertion site locations were examined.ResultsDecreases in collagen fiber crimp frequency were identified at the toe-region of the mechanical test at both locations. The insertion site re-aligned throughout the entire test, while the midsubstance re-aligned during preconditioning and the test's linear-region. The insertion site demonstrated a more disorganized collagen fiber distribution, lower mechanical properties and a higher cross-sectional area compared to the midsubstance location.ConclusionsLocal collagen fiber re-alignment, crimp behavior and mechanical properties were characterized in a mature mouse SST model. The insertion site and midsubstance respond differently to mechanical load and have different mechanisms of structural response. Additionally, results support that collagen fiber crimp is a physiologic phenomenon that may explain the mechanical test toe-region.     

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.     

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.     

Membrane-bound intracellular collagen fibrils in fibroblasts and myofibroblasts of regenerating rabbit calcaneal tendons.

The ultrastructures of 33 rabbit calcaneal tendons were studied to determine (1) whether vacuolar fibrils are present in three regions of tendons undergoing normal healing after tenotomy and repair, and (2) to stimulate collagen synthesis via functional loading, and hence determine the effect of loading on the presence of vacuolar fibrils in healing tendons. In all the loaded tendons, electron microscopy revealed membrane-bound collagen fibril equivalents in sections of neotendon obtained from the site of tenotomy, and in sections of tendon segments proximal and distal to the site of surgery. Similar vacuolar fibrils were visualized in sections of the proximal and distal segments of the non-loaded regenerating tendons, and also in sections of neotendons formed at the site of tenotomy after 12 and 15 days of healing without functional loading. No such fibrils were visualized in the non-tenotomized normal control tendons. These findings indicate that chemical agents and disease are not necessary to induce the appearance of intracytoplasmic fibrils in vivo and that functional loading augments the presence of fibril-bearing vacuoles in regenerating tendons.     

Ultrastructural features of adult human tendon

Summary A variety of human tendons have been studied at the electron microscope level. The fibers of these tendons are composed of collagen fibrils that average 1,750 Å and 600 Å in diameter. A third population that measures 100 Å in diameter may represent immature collagen or filaments that are incorporated into tendon elastic fibers. The larger collagen fibrils vary in ratio with respect to one another, and are connected by interfibrillar bridges which in some cases appear to extend through the substance of the fibril. The collagen fibrils of the paratenon are less-well organized than those of the tendon proper and average 600 Å in diameter. Tendons that exhibit the property of lateral stretch (plantaris and palmaris) were compared at the ultrastructural level with tendons that do not have this property. No differences between the two tendon types could be determined in normal or spread preparations, indicating that the differences in physical characteristics are a result of fiber rather than fibril organization.Supported by Edward G. Schlieder Foundation GrantThe authors wish to thank Mrs. Janell Buck and Mrs. Eunice Schwartz for their excellent technical and secretarial assistance, and Mr. Garbis Kerimian for his excellent photographic work     

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.     

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.     

Advanced Glycation End-Products Reduce Collagen Molecular Sliding to Affect Collagen Fibril Damage Mechanisms but Not Stiffness

Advanced glycation end-products (AGE) contribute to age-related connective tissue damage and functional deficit. The documented association between AGE formation on collagens and the correlated progressive stiffening of tissues has widely been presumed causative, despite the lack of mechanistic understanding. The present study investigates precisely how AGEs affect mechanical function of the collagen fibril – the supramolecular functional load-bearing unit within most tissues. We employed synchrotron small-angle X-ray scattering (SAXS) and carefully controlled mechanical testing after introducing AGEs in explants of rat-tail tendon using the metabolite methylglyoxal (MGO). Mass spectrometry and collagen fluorescence verified substantial formation of AGEs by the treatment. Associated mechanical changes of the tissue (increased stiffness and failure strength, decreased stress relaxation) were consistent with reports from the literature. SAXS analysis revealed clear changes in molecular deformation within MGO treated fibrils. Underlying the associated increase in tissue strength, we infer from the data that MGO modified collagen fibrils supported higher loads to failure by maintaining an intact quarter-staggered conformation to nearly twice the level of fibril strain in controls. This apparent increase in fibril failure resistance was characterized by reduced side-by-side sliding of collagen molecules within fibrils, reflecting lateral molecular interconnectivity by AGEs. Surprisingly, no change in maximum fibril modulus (2.5 GPa) accompanied the changes in fibril failure behavior, strongly contradicting the widespread assumption that tissue stiffening in ageing and diabetes is directly related to AGE increased fibril stiffness. We conclude that AGEs can alter physiologically relevant failure behavior of collagen fibrils, but that tissue level changes in stiffness likely occur at higher levels of tissue architecture.     

Collagen self-assembly and the development of tendon mechanical properties

The development of the musculoskeleton and the ability to locomote requires controlled cell division as well as spatial control over deposition of extracellular matrix. Self-assembly of procollagen and its final processing into collagen fibrils occurs extracellularly. The formation of crosslinked collagen fibers results in the conversion of weak liquid-like embryonic tissues to tough elastic solids that can store energy and do work. Collagen fibers in the form of fascicles are the major structural units found in tendon. The purpose of this paper is to review the literature on collagen self-assembly and tendon development and to relate this information to the development of elastic energy storage in non-mineralizing and mineralizing tendons. Of particular interest is the mechanism by which energy is stored in tendons during locomotion. In vivo, collagen self-assembly occurs by the deposition of thin fibrils in recesses within the cell membrane. These thin fibrils later grow in length and width by lateral fusion of intermediates. In vitro, collagen self-assembly occurs by both linear and lateral growth steps with parallel events seen in vivo; however, in the absence of cellular control and enzymatic cleavage of the propeptides, the growth mechanism is altered, and the fibrils are irregular in cross section. Results of mechanical studies suggest that prior to locomotion the mechanical response of tendon to loading is dominated by the viscous sliding of collagen fibrils. In contrast, after birth when locomotion begins, the mechanical response is dominated by elastic stretching of crosslinked collagen molecules.     

Influence of decorin on the mechanical, compositional, and structural properties of the mouse patellar tendon

The interactions of small leucine-rich proteoglycans (SLRPs) with collagen fibrils, their association with water, and their role in fibrillogenesis suggests that SLRPs may play an important role in tendon mechanics. Some studies have assessed the role of SLRPs in the mechanical response of the tendon, but the relationships between sophisticated mechanics, assembly of collagen, and SLRPs have not been well characterized. Decorin content was varied in a dose dependent manner using decorin null, decorin heterozygote, and wild type mice. Quantitative measures of mechanical (tension and compression), compositional, and structural changes of the mouse patellar tendon were evaluated. Viscoelastic, tensile dynamic modulus was increased in the decorin heterozygous tendons compared to wild type. These tendons also had a significant decrease in total collagen and no structural changes compared to wild type. Decorin null tendons did not have any mechanical changes; however, a significant decrease in the average fibril diameter was found. No differences were seen between genotypes in elastic or compressive properties, and all tendons demonstrated viscoelastic mechanical dependence on strain rate and frequency. These results suggest that decorin, a member of the SLRP family, plays a role in tendon viscoelasticity that cannot be completely explained by its role in collagen fibrillogenesis. In addition, reductions in decorin do not cause large changes in indentation compressive properties, suggesting that other factors contribute to these properties. Understanding these relationships may ultimately help guide development of tissue engineered constructs or treatment modalities.     

Murine patellar tendon biomechanical properties and regional strain patterns during natural tendon-to-bone healing after acute injury

Tendon-to-bone healing following acute injury is generally poor and often fails to restore normal tendon biomechanical properties. In recent years, the murine patellar tendon (PT) has become an important model system for studying tendon healing and repair due to its genetic tractability and accessible location within the knee. However, the mechanical properties of native murine PT, specifically the regional differences in tissue strains during loading, and the biomechanical outcomes of natural PT-to-bone healing have not been well characterized. Thus, in this study, we analyzed the global biomechanical properties and regional strain patterns of both normal and naturally healing murine PT at three time points (2, 5, and 8 weeks) following acute surgical rupture of the tibial enthesis. Normal murine PT exhibited distinct regional variations in tissue strain, with the insertion region experiencing approximately 2.5 times greater strain than the midsubstance at failure (10.80±2.52% vs. 4.11±1.40%; mean±SEM). Injured tendons showed reduced structural (ultimate load and linear stiffness) and material (ultimate stress and linear modulus) properties compared to both normal and contralateral sham-operated tendons at all healing time points. Injured tendons also displayed increased local strain in the insertion region compared to contralateral shams at both physiologic and failure load levels. 93.3% of injured tendons failed at the tibial insertion, compared to only 60% and 66.7% of normal and sham tendons, respectively. These results indicate that 8 weeks of natural tendon-to-bone healing does not restore normal biomechanical function to the murine PT following injury.     

Decorin regulates assembly of collagen fibrils and acquisition of biomechanical properties during tendon development

Tendon function involves the development of an organized hierarchy of collagen fibrils. Small leucine-rich proteoglycans have been implicated in the regulation of fibrillogenesis and decorin is the prototypic member of this family. Decorin-deficient mice demonstrate altered fibril structure and mechanical function in mature skin and tail tendons. However, the developmental role(s) of decorin needs to be elucidated. To define these role(s) during tendon development, tendons (flexor digitorum longus) were analyzed ultrastructurally from postnatal day 10 to 90. Decorin-deficient tendons developed abnormal, irregularly contoured fibrils. Finite mixture modeling estimated that the mature tendon was a three-subpopulation mixture of fibrils with characteristic diameter ranges. During development, in each subpopulation the mean diameter was consistently larger in mutant mice. Also, diameter distributions and the percentage of fibrils in each subpopulation were altered. Biomechanical analyses demonstrated that mature decorin-deficient tendons had significantly reduced strength and stiffness; however, there was no reduction in immature tendons. Expression of decorin and biglycan, a closely related family member, was analyzed during development. Decorin increased with development while biglycan decreased. Spatially, both had a comparable localization throughout the tendon. Biglycan expression increased substantially in decorin-deficient tendons suggesting a potential functional compensation. The accumulation of structural defects during fibril growth, a period associated with decorin expression and low biglycan expression, may be the cause of compromised mechanical function in the absence of decorin. Our findings indicate that decorin is a key regulatory molecule and that the temporal switch from biglycan to decorin is an important event in the coordinate regulation of fibrillogenesis and tendon development.     

Possible role of decorin glycosaminoglycans in fibril to fibril force transfer in relative mature tendons--a computational study from molecular to microstructural level

Experimental studies on immature tendons have shown that the collagen fibril net is discontinuous. Manifold evidences, despite not being conclusive, indicate that mature tissue is discontinuous as well. According to composite theory, there is no requirement that the fibrils should extend from one end of the tissue to the other; indeed, an interfibrillar matrix with a low elastic modulus would be sufficient to guarantee the mechanical properties of the tendon. Possible mechanisms for the stress-transfer involve the interfibrillar proteoglycans and can be related to the matrix shear stress and to electrostatic non-covalent forces. Recent studies have shown that the glycosaminoglycans (GAGs) bound to decorin act like bridges between contiguous fibrils connecting adjacent fibril every 64-68 nm; this architecture would suggest their possible role in providing the mechanical integrity of the tendon structure. The present paper investigates the ability of decorin GAGs to transfer forces between adjacent fibrils. In order to test this hypothesis the stiffness of chondroitin-6-sulphate, a typical GAG associated to decorin, has been evaluated through the molecular mechanics approach. The obtained GAG stiffness is piecewise linear with an initial plateau at low strains (<800%) and a high stiffness region (3.1 x 10(-11)N/nm) afterwards. By introducing the calculated GAG stiffness in a multi-fibril model, miming the relative mature tendon architecture, the stress-strain behaviour of the collagen fibre was determined. The fibre incremental elastic modulus obtained ranges between 100 and 475 MPa for strains between 2% and 6%. The elastic modulus value depends directly on the fibril length, diameter and inversely on the interfibrillar distance. In particular, according to the obtained results, the length of the fibril is likely to play the major role in determining stiffness in mature tendons.     

Collagen fibril diameter distribution does not reflect changes in the mechanical properties of in vitro stress-deprived tendons

The purpose of this study was to determine if an association exists between the tensile properties and the collagen fibril diameter distribution in in vitro stress-deprived rat tail tendons. Rat tail tendons were paired into two groups of 21 day stress-deprived and 0 time controls and compared using transmission electron microscopy (n = 6) to measure collagen fibril diameter distribution and density, and mechanical testing (n =6) to determine ultimate stress and tensile modulus. There was a statistically significant decrease in both ultimate tensile strength (control: 17.95+/-3.99 MPa, stress-deprived: 6.79+/-3.91 MPa) and tensile modulus (control: 312.8+/-89.5 MPa, stress-deprived: 176.0+/-52.7 MPa) in the in vitro stress-deprived tendons compared to controls. However, there was no significant difference between control and stress-deprived tendons in the number of fibrils per tendon counted, mean fibril diameter, mean fibril density, or fibril size distribution. The results of this study demonstrate that the decrease in mechanical properties observed in in vitro stress-deprived rat tail tendons is not correlated with the collagen fibril diameter distribution and, therefore, the collagen fibril diameter distribution does not, by itself, dictate the decrease in mechanical properties observed in in vitro stress-deprived rat tail tendons.     

Local strain measurement reveals a varied regional dependence of tensile tendon mechanics on glycosaminoglycan content

Proteoglycans (PG) and their associated glycosaminoglycan (GAG) side chains are known to play a key role in the bearing of compressive loads in cartilage and other skeletal connective tissues. In tendons and connective tissues that are primarily loaded in tension, the influence of proteoglycans on mechanical behavior is debated due to conflicting experimental evidence that alternately supports or controverts a functional role of proteoglycans in bearing tensile load. In this study we sought to better reconcile these conflicting data by investigating the possibility that GAG content is differentially related to tensile tendon mechanics depending upon the anatomical subregion one considers. To test this hypothesis, we quantified the mechanical consequences of proteoglycan disruption within specific tendon anatomical subregions using an optical–mechanical measurement approach.Achilles tendons from adult mice were treated with chondroitinase ABC to obtain two groups consisting of native tendons and GAG-depleted tendons. All the tendons were mechanically tested and imaged with high-resolution digital video in order to optically quantify tendon strains. Tendon surface strains were locally analyzed in three main subregions: the central midsubstance, and the proximal and distal midsubstance near the muscle and bone insertions, respectively. Upon GAG digestion, the tendon midsubstance softened appreciably near the bone insertion, while elastic modulus in the central and proximal thirds was unchanged. Thus the contribution of PGs to tensile tendon mechanics is not straightforward and points to a heterogeneous and complex structure–function relationship in tendon. This study further highlights the importance of performing local strain analysis with regard to tensile tendon mechanics.     

Evidence that collagen fibrils in tendons are inhomogeneously structured in a tubelike manner

The standard model for the structure of collagen in tendon is an ascending hierarchy of bundling. Collagen triple helices bundle into microfibrils, microfibrils bundle into subfibrils, and subfibrils bundle into fibrils, the basic structural unit of tendon. This model, developed primarily on the basis of x-ray diffraction results, is necessarily vague about the cross-sectional organization of fibrils and has led to the widespread assumption of laterally homogeneous closepacking. This assumption is inconsistent with data presented here. Using atomic force microscopy and micromanipulation, we observe how collagen fibrils from tendons behave mechanically as tubes. We conclude that the collagen fibril is an inhomogeneous structure composed of a relatively hard shell and a softer, less dense core.     

Conformation and sequence evidence for two-fold symmetry in left-handed beta-helix fold

The glycosaminoglycan (GAG) side-chains of small leucine-rich proteoglycans have been postulated to mechanically cross-link adjacent collagen fibrils and contribute to tendon mechanics. Enzymatic depletion of tendon GAGs (chondroitin and dermatan sulfate) has emerged as a preferred method to experimentally assess this role. However, GAG removal is typically incomplete and the possibility remains that extant GAGs may remain mechanically functional. The current study specifically investigated the potential mechanical effect of the remaining GAGs after partial enzymatic digestion.A three-dimensional finite element model of tendon was created based upon the concept of proteoglycan mediated inter-fibril load sharing. Approximately 250 interacting, discontinuous collagen fibrils were modeled as having a length of 400 μm, being composed of rod elements of length 67 nm and E-modulus 1 GPa connected in series. Spatial distribution and diameters of these idealized fibrils were derived from a representative cross-sectional electron micrograph of tendon. Rod element lengths corresponded to the collagen fibril D-Period, widely accepted to act as a binding site for decorin and biglycan, the most abundant proteoglycans in tendon. Each element node was connected to nodes of any neighboring fibrils within a radius of 100 nm, the slack length of unstretched chondroitin sulfate. These GAG cross-links were the sole mechanism for lateral load sharing among the discontinuous fibrils, and were modeled as bilinear spring elements. Simulation of tensile testing of tendon with complete cross-linking closely reproduced corresponding experiments on rat tail tendons. Random reduction of 80% of GAG cross-links (matched to a conservative estimate of enzymatic depletion efficacy) predicted a drop of 14% in tendon modulus. Corresponding mechanical properties derived from experiments on rat tail tendons treated in buffer with and without chondroitinase ABC were apparently unaffected, regardless of GAG depletion. Further tests for equivalence, conservatively based on effect size limits predicted by the model, confirmed equivalent stiffness between enzymatically depleted tendons and their native controls.Although the model predicts that relatively small quantities of GAGs acting as primary collagen cross-linking elements could provide mechanical integrity to the tendon, partial enzymatic depletion of GAGs should result in mechanical changes that are not reflected in analogous experimental testing. We thus conclude that GAG side chains of small leucine-rich proteoglycans are not a primary determinant of tensile mechanical behavior in mature rat tail tendons.