Mechanical Strain Stabilizes Reconstituted Collagen Fibrils against Enzymatic Degradation by Mammalian Collagenase Matrix Metalloproteinase 8 (MMP-8)

Background

Collagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals. It is found in bone, ligament, tendon, cartilage, intervertebral disc, skin, blood vessel, and cornea. We have recently postulated that fibrillar collagens (and their complementary enzymes) comprise the basis of a smart structural system which appears to support the retention of molecules in fibrils which are under tensile mechanical strain. The theory suggests that the mechanisms which drive the preferential accumulation of collagen in loaded tissue operate at the molecular level and are not solely cell-driven. The concept reduces control of matrix morphology to an interaction between molecules and the most relevant, physical, and persistent signal: mechanical strain.

Methodology/Principal Findings

The investigation was carried out in an environmentally-controlled microbioreactor in which reconstituted type I collagen micronetworks were gently strained between micropipettes. The strained micronetworks were exposed to active matrix metalloproteinase 8 (MMP-8) and relative degradation rates for loaded and unloaded fibrils were tracked simultaneously using label-free differential interference contrast (DIC) imaging. It was found that applied tensile mechanical strain significantly increased degradation time of loaded fibrils compared to unloaded, paired controls. In many cases, strained fibrils were detectable long after unstrained fibrils were degraded.

Conclusions/Significance

In this investigation we demonstrate for the first time that applied mechanical strain preferentially preserves collagen fibrils in the presence of a physiologically-important mammalian enzyme: MMP-8. These results have the potential to contribute to our understanding of many collagen matrix phenomena including development, adaptation, remodeling and disease. Additionally, tissue engineering could benefit from the ability to sculpt desired structures from physiologically compatible and mutable collagen.     

Tenocyte contraction induces crimp formation in tendon-like tissue

Tendons are composed of longitudinally aligned collagen fibrils arranged in bundles with an undulating pattern, called crimp. The crimp structure is established during embryonic development and plays a vital role in the mechanical behaviour of tendon, acting as a shock-absorber during loading. However, the mechanism of crimp formation is unknown, partly because of the difficulties of studying tendon development in vivo. Here, we used a 3D cell culture system in which embryonic tendon fibroblasts synthesise a tendon-like construct comprised of collagen fibrils arranged in parallel bundles. Investigations using polarised light microscopy, scanning electron microscopy and fluorescence microscopy showed that tendon constructs contained a regular pattern of wavy collagen fibrils. Tensile testing indicated that this superstructure was a form of embryonic crimp producing a characteristic toe region in the stress–strain curves. Furthermore, contraction of tendon fibroblasts was the critical factor in the buckling of collagen fibrils during the formation of the crimp structure. Using these biological data, a finite element model was built that mimics the contraction of the tendon fibroblasts and monitors the response of the Extracellular matrix. The results show that the contraction of the fibroblasts is a sufficient mechanical impulse to build a planar wavy pattern. Furthermore, the value of crimp wavelength was determined by the mechanical properties of the collagen fibrils and inter-fibrillar matrix. Increasing fibril stiffness combined with constant matrix stiffness led to an increase in crimp wavelength. The data suggest a novel mechanism of crimp formation, and the finite element model indicates the minimum requirements to generate a crimp structure in embryonic tendon.     

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

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

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

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

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.     

Stress-strain experiments on individual collagen fibrils

Collagen, a molecule consisting of three braided protein helices, is the primary building block of many biological tissues including bone, tendon, cartilage, and skin. Staggered arrays of collagen molecules form fibrils, which arrange into higher-ordered structures such as fibers and fascicles. Because collagen plays a crucial role in determining the mechanical properties of these tissues, significant theoretical research is directed toward developing models of the stiffness, strength, and toughness of collagen molecules and fibrils. Experimental data to guide the development of these models, however, are sparse and limited to small strain response. Using a microelectromechanical systems platform to test partially hydrated collagen fibrils under uniaxial tension, we obtained quantitative, reproducible mechanical measurements of the stress-strain curve of type I collagen fibrils, with diameters ranging from 150-470 nm. The fibrils showed a small strain (epsilon < 0.09) modulus of 0.86 +/- 0.45 GPa. Fibrils tested to strains as high as 100% demonstrated strain softening (sigma(yield) = 0.22 +/- 0.14 GPa; epsilon(yield) = 0.21 +/- 0.13) and strain hardening, time-dependent recoverable residual strain, dehydration-induced embrittlement, and susceptibility to cyclic fatigue. The results suggest that the stress-strain behavior of collagen fibrils is dictated by global characteristic dimensions as well as internal structure.     

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.     

The in situ supermolecular structure of type I collagen.

BACKGROUND: The proteins belonging to the collagen family are ubiquitous throughout the animal kingdom. The most abundant collagen, type I, readily forms fibrils that convey the principal mechanical support and structural organization in the extracellular matrix of connective tissues such as bone, skin, tendon, and vasculature. An understanding of the molecular arrangement of collagen in fibrils is essential since it relates molecular interactions to the mechanical strength of fibrous tissues and may reveal the underlying molecular pathology of numerous connective tissue diseases. RESULTS: Using synchrotron radiation, we have conducted a study of the native fibril structure at anisotropic resolution (5.4 A axial and 10 A lateral). The intensities of the tendon X-ray diffraction pattern that arise from the lateral packing (three-dimensional arrangement) of collagen molecules were measured by using a method analogous to Rietveld methods in powder crystallography and to the separation of closely spaced peaks in Laue diffraction patterns. These were then used to determine the packing structure of collagen by MIR. CONCLUSIONS: Our electron density map is the first obtained from a natural fiber using these techniques (more commonly applied to single crystal crystallography). It reveals the three-dimensional molecular packing arrangement of type I collagen and conclusively proves that the molecules are arranged on a quasihexagonal lattice. The molecular segments that contain the telopeptides (central to the function of collagen fibrils in health and disease) have been identified, revealing that they form a corrugated arrangement of crosslinked molecules that strengthen and stabilize the native fibril.     

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.     

A constitutive model for the mechanical behaviour of soft connective tissues

A model of the mechanical behaviour of soft connective tissue has been developed by considering the role of the collagen and glycosaminoglycan (GAG) components within the tissue in order to examine the mechanism by which a variation in the GAG components may exert a control over the mechanical properties of the tissue. It is proposed that the strain energy stored within the collagen fibrils of the loaded tissue can be transferred into a potential field created by the charged GAG components and their electrostatic interaction with the collagen fibrils. A fundamental mechanical unit is described to simulate this energy transfer and a combination of such units is used to represent the tissue. The computer implementation of the proposed tissue model shows it to reproduce many features which have been recognised in the rate dependent mechanical behaviour of soft tissues. These include the characteristic non-linearity of the force-deformation behaviour and the approximate invariance of the stress relaxation behaviour with deformation. The model is also consistent with earlier constitutive representations of tissue behaviour.     

A constitutive model for vascular tissue that integrates fibril, fiber and continuum levels with application to the isotropic and passive properties of the infrarenal aorta

A fundamental understanding of the mechanical properties of the extracellular matrix (ECM) is critically important to quantify the amount of macroscopic stress and/or strain transmitted to the cellular level of vascular tissue. Structural constitutive models integrate histological and mechanical information, and hence, allocate stress and strain to the different microstructural components of the vascular wall. The present work proposes a novel multi-scale structural constitutive model of passive vascular tissue, where collagen fibers are assembled by proteoglycan (PG) cross-linked collagen fibrils and reinforce an otherwise isotropic matrix material. Multiplicative kinematics account for the straightening and stretching of collagen fibrils, and an orientation density function captures the spatial organization of collagen fibers in the tissue. Mechanical and structural assumptions at the collagen fibril level define a piece-wise analytical stress-stretch response of collagen fibers, which in turn is integrated over the unit sphere to constitute the tissue's macroscopic mechanical properties. The proposed model displays the salient macroscopic features of vascular tissue, and employs the material and structural parameters of clear physical meaning. Likewise, the constitutive concept renders a highly efficient multi-scale structural approach that allows for the numerical analysis at the organ level. Model parameters were estimated from isotropic mean-population data of the normal and aneurysmatic aortic wall and used to predict in-vivo stress states of patient-specific vascular geometries, thought to demonstrate the robustness of the particular Finite Element (FE) implementation. The collagen fibril level of the multi-scale constitutive formulation provided an interface to integrate vascular wall biology and to account for collagen turnover.     

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.     

Structural dynamic of native tendon collagen

The dynamic behaviour of collagen fibrils is revealed by time-resolved X-ray investigations of native rat tail tendon fibres in tensile tests.     

Fracture Mechanics of Collagen Fibrils: Influence of Natural Cross-Links

Tendons are important load-bearing structures, which are frequently injured in both sports and work. Type I collagen fibrils are the primary components of tendons and carry most of the mechanical loads experienced by the tissue, however, knowledge of how load is transmitted between and within fibrils is limited. The presence of covalent enzymatic cross-links between collagen molecules is an important factor that has been shown to influence mechanical behavior of the tendons. To improve our understanding of how molecular bonds translate into tendon mechanics, we used an atomic force microscopy technique to measure the mechanical behavior of individual collagen fibrils loaded to failure. Fibrils from human patellar tendons, rat-tail tendons (RTTs), NaBH₄ reduced RTTs, and tail tendons of Zucker diabetic fat rats were tested. We found a characteristic three-phase stress-strain behavior in the human collagen fibrils. There was an initial rise in modulus followed by a plateau with reduced modulus, which was finally followed by an even greater increase in stress and modulus before failure. The RTTs also displayed the initial increase and plateau phase, but the third region was virtually absent and the plateau continued until failure. The importance of cross-link lability was investigated by NaBH₄ reduction of the rat-tail fibrils, which did not alter their behavior. These findings shed light on the function of cross-links at the fibril level, but further studies will be required to establish the underlying mechanisms.     

Cyclic tensile strain upregulates collagen synthesis in isolated tendon fascicles

Mechanical stimulation has been implicated as an important regulatory factor in tendon homeostasis. In this study, a custom-designed tensile loading system was used to apply controlled mechanical stimulation to isolated tendon fascicles, in order to examine the effects of 5% cyclic tensile strain at 1 Hz on cell proliferation and matrix synthesis. Sample viability and gross structural composition were maintained over a 24 h loading period. Data demonstrated no statistically significant differences in cell proliferation or glycosaminoglycan production, however, collagen synthesis was upregulated with the application of cyclic tensile strain over the 24 h period. Moreover, a greater proportion of the newly synthesised matrix was retained within the sample after loading. These data provide evidence of altered anabolic activity within tendon in response to mechanical stimuli, and suggest the importance of cyclic tensile loading for the maintenance of the collagen hierarchy within tendon.     

Assembly of type I collagen: fusion of fibril subunits and the influence of fibril diameter on mechanical properties.

Structural stability of the extracellular matrix is primarily a consequence of fibrillar collagen and the extent of cross-linking. The relationship between collagen self-assembly, consequent fibrillar shape and mechanical properties remains unclear. Our laboratory developed a model system for the preparation of self-assembled type I collagen fibers with fibrillar substructure mimicking the hierarchical structures of tendon. The present study evaluates the effects of pH and temperature during self-assembly on fibrillar structure, and relates the structural effects of these treatments on the uniaxial tensile mechanical properties of self-assembled collagen fibers. Results of the analysis of fibril diameter distributions and mechanical properties of the fibers formed under the different incubation conditions indicate that fibril diameters grow via the lateral fusion of discrete approximately 4 nm subunits, and that fibril diameter correlates positively with the low strain modulus. Fibril diameter did not correlate with either the ultimate tensile strength or the high strain elastic modulus, which suggests that lateral aggregation and consequently fibril diameter influences mechanical properties during small strain mechanical deformation. We hypothesize that self-assembly is mediated by the formation of fibrillar subunits that laterally and linearly fuse resulting in fibrillar growth. Lateral fusion appears important in generating resistance to deformation at low strain, while linear fusion leading to longer fibrils appears important in the ultimate mechanical properties at high strain.     

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

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

Tendon tissue microdamage and the limits of intrinsic repair

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

The relation between collagen fibril kinematics and mechanical properties in the mitral valve anterior leaflet

We have recently demonstrated that the mitral valve anterior leaflet (MVAL) exhibited minimal hysteresis, no strain rate sensitivity, stress relaxation but not creep (Grashow et al., 2006, Ann Biomed Eng., 34(2), pp. 315-325; Grashow et al., 2006, Ann Biomed. Eng., 34(10), pp. 1509-1518). However, the underlying structural basis for this unique quasi-elastic mechanical behavior is presently unknown. As collagen is the major structural component of the MVAL, we investigated the relation between collagen fibril kinematics (rotation and stretch) and tissue-level mechanical properties in the MVAL under biaxial loading using small angle X-ray scattering. A novel device was developed and utilized to perform simultaneous measurements of tissue level forces and strain under a planar biaxial loading state. Collagen fibril D-period strain (epsilonD) and the fibrillar angular distribution were measured under equibiaxial tension, creep, and stress relaxation to a peak tension of 90 N/m. Results indicated that, under equibiaxial tension, collagen fibril straining did not initiate until the end of the nonlinear region of the tissue-level stress-strain curve. At higher tissue tension levels, epsilonD increased linearly with increasing tension. Changes in the angular distribution of the collagen fibrils mainly occurred in the tissue toe region. Using epsilonD, the tangent modulus of collagen fibrils was estimated to be 95.5+/-25.5 MPa, which was approximately 27 times higher than the tissue tensile tangent modulus of 3.58+/-1.83 MPa. In creep tests performed at 90 N/m equibiaxial tension for 60 min, both tissue strain and epsilonD remained constant with no observable changes over the test length. In contrast, in stress relaxation tests performed for 90 min epsilonD was found to rapidly decrease in the first 10 min followed by a slower decay rate for the remainder of the test. Using a single exponential model, the time constant for the reduction in collagen fibril strain was 8.3 min, which was smaller than the tissue-level stress relaxation time constants of 22.0 and 16.9 min in the circumferential and radial directions, respectively. Moreover, there was no change in the fibril angular distribution under both creep and stress relaxation over the test period. Our results suggest that (1) the MVAL collagen fibrils do not exhibit intrinsic viscoelastic behavior, (2) tissue relaxation results from the removal of stress from the fibrils, possibly by a slipping mechanism modulated by noncollagenous components (e.g. proteoglycans), and (3) the lack of creep but the occurrence of stress relaxation suggests a "load-locking" behavior under maintained loading conditions. These unique mechanical characteristics are likely necessary for normal valvular function.     

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.