Tendon crimps and peritendinous tissues responding to tensional forces

Tendons transmit forces generated from muscle to bone making joint movements possible. Tendon collagen has a complex supramolecular structure forming many hierarchical levels of association; its main functional unit is the collagen fibril forming fibers and fascicles. Since tendons are enclosed by loose connective sheaths in continuity with muscle sheaths, it is likely that tendon sheaths could play a role in absorbing/transmitting the forces created by muscle contraction. In this study rat Achilles tendons were passively stretched in vivo to be observed at polarized light microscope (PLM), scanning electron microscope (SEM) and transmission electron microscope (TEM). At PLM tendon collagen fibers in relaxed rat Achilles tendons ran straight and parallel, showing a periodic crimp pattern. Similarly tendon sheaths showed apparent crimps. At higher magnification SEM and TEM revealed that in each tendon crimp large and heterogeneous collagen fibrils running straight and parallel suddenly changed their direction undergoing localized and variable modifications. These fibril modifications were named fibrillar crimps. Tendon sheaths displayed small and uniform fibrils running parallel with a wavy course without any ultrastructural aspects of crimp. Since in passively stretched Achilles tendons fibrillar crimps were still observed, it is likely that during the tendon stretching, and presumably during the tendon elongation in muscle contraction, the fibrillar crimp may be the real structural component of the tendon crimp acting as shock absorber. The peritendinous sheath can be stretched as tendon, but is not actively involved in the mechanism of shock absorber as the fibrillar crimp. The different functional behaviour of tendons and sheaths may be due to the different structural and molecular arrangement of their fibrils.     

The myotendinous junction of the smooth feather muscles (mm. pennati)

Summary Smooth feather muscles (mm. pennati) consist of bundles of smooth muscle cells which are attached to the feather follicles by short elastic tendons. In addition, some muscle bundles are interrupted by elastic tendons. The elastic tendon is composed of longitudinally arranged elastic fibers which branch and wavy bundles of collagen fibrils. Smooth muscle cells of the muscle bundles are attached to each other by desmosome-like junctions and by fusion of the basal laminae. The cytoplasm of the muscle cells is characterized by conspicuous thick filaments and abundant thin and intermediate filaments. These are attached to band-like dense patches (dense bands) at the plasma membrane which are particularly broad at the tapering end of the muscle cell. The contact surface between smooth muscle cells and their elastic tendon is considerably increased (i) by deep finger-like invaginations and indentations located at the tapering muscle end, and (ii) by branching of the coarse elastic fibers into slender processes, which are attached to the richly folded surface of the muscle cell endings by peripheral microfibrils. This intimate interlocking closely resembles the myotendinous junctions in skeletal muscle. In addition to fibroblasts and fibrocytes, the myotendinous junction of the young growing chicks contains numerous so-called myofibroblasts, which are suggested to represent smooth muscle cells differentiating into fibroblasts of the developing tendon.Dedicated to Professor Dr. Helmut Leonhardt on the occasion of his 60th birthdaySupported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91/1)     

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.     

Three-dimensional ultrastructure of human tendons.

The three-dimensional ultrastructure of human tendons has been studied. Epitenon and peritenon consist of a dense network of longitudinal, oblique and transversal collagen fibrils crossing the tendon fibres. The internal structure of tendon fibres is also complex. The collagen fibrils are oriented not only longitudinally but also transversely and horizontally. The longitudinal fibrils do not run only parallel but also cross each other forming spirals (plaits). These fibril bundles are bound together by a three-dimensional collagen fibril network of endotenon. In the myotendinous junction the surface of the muscle cells form processes. A network of tendineal collagen fibrils fills the recesses between the muscle cell processes penetrating the basement membrane of these processes. This complex ultrastructure of human tendons most likely offers a good buffer system against longitudinal, transversal, horizontal as well as rotational forces during movement and activity.     

Variations in collagen fibril structure in tendons

Variation of collagen fibril structure in tendon was investigated by x-ray diffraction. Anatomically distinct tendons from single species, as well as tendons from different species, were examined to determine the variations that exist in both the axial and lateral structure of the collagen fibrils. The meridional diffraction is derived from the axial collagen fibril structure. Anatomically distinct tendons of a particular species give meridional patterns that are indistinguishable within experimental error. The meridional diffraction patterns from tendons of different mammals are similar but show small species-specific variations, most noticeably in the 14th–18th orders. Tendons of birds also give meridional patterns that are similar to each other, but the avian patterns differ considerably from the mammalian ones. Avian tendons give stronger odd and weaker even low orders, a feature consistent with a reduced gap:overlap ratio, and have a distinctive intensity pattern for the higher meridional orders. Interpretation of these differences has been approached using biochemical data, diffraction by reconsituted fibers of purified collagen, and Fourier transform analysis. From these methods, it appears that the variations observed in the lower orders (2nd–8th) and in the higher orders (29th–52nd) are probably related to differences in the primary structure of the Type I collagen found in the different species. The variations observed in the 14th–18th orders appear not to be related to features within the triple-helical domain of the molecule. Equatorial diffraction yields information on the lateral packing of collagen molecules in the fibrils, and considerable variation was seen in different tendons. Rat tail tendon gives sharp Bragg reflections, demonstrating the presence of a crystalline lateral arrangement of molecules in the fibril. For the first time, sharp lattice reflections similar to those in rat tail tendon have been observed in nontail tendons, including rat achilles tendon, rabbit leg tendon, and wing and leg tendons of quail. In the rabbit and quail tendons, one of the strong equatorial reflections characteristic of the rat tendon pattern, at 1.26 nm, was absent. The positions of the equatorial maxima, which are a measure of intermolecular spacing, varied considerably, being smallest in the specimens displaying crystalline packing. The intermolecular distance in chiken and turkey leg tendons is longer than that found in mammalian tendons, or in avian wing tendons, which supports the hypothesis that a larger intermolecular spacing is characteristic of tendons that calcify. Thus, x-ray diffraction indicates there are reproducible differences in both the axial and lateral structure of collagen fibrils among different tendons. This work on tendon, a tissue containing almost exclusively Type I collagen as its major component, should serve as a basis for analyzing the structure of other connective tissues, which contain different genetic types of collagen and larger amounts of noncollagenous components.     

Comparative morphology of muscle-skeleton attachments in the Echinodermata

Summary Muscles are generally attached to the skeleton by interconnecting tendons. Each tendon necessarily has a junction with the muscle and another with the skeleton. The ultrastructure of the skeleton is identical in all echinoderm classes. Nevertheless, we found three different types of muscle-skeleton junctions. (1) In Crinoida the muscles are attached almost directly to the calcite trabeculae. (2) Asteroida and Ophiuroida have tendons that arise from the basal laminae of the muscle bundles. They consist of unstriated microfibrils that are attached on the muscle side to electron-dense areas below the sarcolemma of the fingershaped muscle ends. On the skeleton side they embrace the outermost calcite trabeculae. (3) In Echinoida the strong muscles are joined to the skeleton by means of composite tendons. They consist of unstriated tendon cords that adhere to the muscles and of bundles of striated fibrils coiled around the calcite trabeculae. Both kinds of tendons are interconnected in the same way as the links of a chain. Composite tendons are found in junctions that are exposed to multidirectional stress. In Holothuroida there are no true muscle-skeleton junctions and the muscles are apposed to connective tissue.The muscle-tendon junctions in Echinodermata differ fundamentally from the junctions in the protostome Arthropoda or Mollusca, but they were found to be very similar in structure to the muscle-tendon junctions in Vertebrata. This coincidence may refer to a phylogenetic relationship of the two deuterostome phyla. But the tendon-skeleton junctions of the two phyla are dissimilar, for Echinodermata and Vertebrata differ fundamentally in their skeletons. Vertebrate bone consists of extracellular fibrils combined with minute crystals of hydroxylapatite. Echinoderm ossicles are intercellular in origin. They are nothing but the calcified vacuolar system of syncytial sclerocytes, and extracellular fibrils never enter the mineral phase.Abbreviations bl basal lamina - c calcite trabecula - dp distal processes of sclerocytes - el electron-dense layer - m muscle - sf striated tendon fibrils - uf unstriated tendon fibrils - tc trabecle coat     

Collagen fibril size and crimp morphology in ruptured and intact Achilles tendons.

The present study examined the hypothesis that collagen fibril diameter and crimp angle in ruptured human Achilles tendons differed from that of intact ones. Tissue samples were obtained from the central core (distal core) and the posterior periphery (distal superficial) at the rupture site, and the proximally intact (proximal superficial) part of the tendon in 10 subjects (38+/-8 years) with a complete tendon rupture. For comparisons corresponding tissue samples were procured from age (38+/-7 years) and gender matched intact Achilles tendons during routine forensic autopsy. The cross-sectional area density and diameter distribution of fibrils were analyzed using stereological techniques of digitized electron microscopy biopsy cross-sections, while crimp angle was measured by the changing banding pattern of collagen fibers when rotated between crossed polars. Nine of 10 persons with tendon ruptures reported that the injury did not occur during exceedingly large forces, and none experienced any symptoms in the days or months prior to the injury. Fibril diameter distribution showed no region-specific differences in either the ruptured or intact tendons for either group. However, in the distal core there were fewer fibrils in the ruptured compared to the intact tendons in 60-150 nm range, P<0.01. Similarly, in the distal superficial portion there were fewer fibrils in the ruptured compared to the intact tendons in the 90-120 nm range, 2P<0.05, while there were no differences in the proximal superficial tendons. Crimp angle did not display any region-specific differences, or any difference between the rupture and intact tendons. In conclusion, these data suggest that although crimp morphology is unchanged there appears to be a site-specific loss of larger fibrils in the core and periphery of the Achilles tendon rupture site. Moreover, the lack of symptoms prior to the rupture suggests that clinical tendinopathy is not an etiological factor in complete tendon ruptures.     

Col V siRNA engineered tenocytes for tendon tissue engineering

The presence of uniformly small collagen fibrils in tendon repair is believed to play a major role in suboptimal tendon healing. Collagen V is significantly elevated in healing tendons and plays an important role in fibrillogenesis. The objective of this study was to investigate the effect of a particular chain of collagen V on the fibrillogenesis of Sprague-Dawley rat tenocytes, as well as the efficacy of Col V siRNA engineered tenocytes for tendon tissue engineering. RNA interference gene therapy and a scaffold free tissue engineered tendon model were employed. The results showed that scaffold free tissue engineered tendon had tissue-specific tendon structure. Down regulation of collagen V α1 or α2 chains by siRNAs (Col5α1 siRNA, Col5α2 siRNA) had different effects on collagen I and decorin gene expressions. Col5α1 siRNA treated tenocytes had smaller collagen fibrils with abnormal morphology; while those Col5α2 siRNA treated tenocytes had the same morphology as normal tenocytes. Furthermore, it was found that tendons formed by coculture of Col5α1 siRNA treated tenocytes with normal tenocytes at a proper ratio had larger collagen fibrils and relative normal contour. Conclusively, it was demonstrated that Col V siRNA engineered tenocytes improved tendon tissue regeneration. And an optimal level of collagen V is vital in regulating collagen fibrillogenesis. This may provide a basis for future development of novel cellular- and molecular biology-based therapeutics for tendon diseases.     

Sacrificial bonds in polymer brushes from rat tail tendon functioning as nanoscale velcro

Polymers play an important role in many biological systems, so a fundamental understanding of their cross-links is crucial not only for the development of medicines but also for the development of biomimetic materials. The biomechanical movements of all mammals are aided by tendon fibrils. The self-organization and biomechanical functions of tendon fibrils are determined by the properties of the cross-links between their individual molecules and the interactions among the cross-links. The cross-links of collagen and proteoglycan molecules are particularly important in tendons and, perhaps, bone. To probe cross-links between tendon molecules, we used the cantilever tip of an atomic force microscope in a pulling setup. Applying higher forces to rat tail tendon molecules with the tip led to a local disruption of the highly organized shell of tendon fibrils and to the formation or an increase of a polymer brush of molecules sticking out of the surface. The cross-linking between these molecules was influenced by divalent Ca2+ ions. Furthermore, the molecules of the polymer brush seemed to bind back to the fibrils in several minutes. We propose that sacrificial bonds significantly influence the tendon fibrils' self-organization and self-healing and therefore contribute to toughness and strength.     

Structure and function of bone collagen fibrils

The intermolecular volume of fully hydrated collagen fibrils from a number of mineralized and non-mineralized tissues of adult rats has been determined both by an exclusion technique and by a method which involves the monitoring of specific X-ray diffraction parameters. The intermolecular volume of either bone or dentinal fibrils is approximately twice that of either tail or achilles tendon, and the most frequent intermolecular distance in bone or dentine fibrils is approximately 3 Å larger than of the tendons.A number of fibrillar structures are most compatible with the intermolecular volume of rat tail tendon. These include hexagonal molecular packing and orthogonal arrays of microfibrils comprising seven parallel molecular strands. The intermolecular volume of bone or dentinal collagen fibrils, on the other hand, appears to arise from structures having a disordered or pseudo-hexagonal molecular packing, in which the most frequent intermolecular distance is about 19 Å.The space associated with collagen fibrils in adult bone is such that 70 to 80% of the mineral is located within the intermolecular space of the fibrils—approximately equal amounts of mineral being in spaces having lateral dimensions of 25 to 75 Å and 6 to 12 Å, respectively. Particles located in the latter kind of intermolecular space probably constitute, to a large extent, the non-crystalline mineral phase of adult bone.The stereo-chemical constraints on the transport of mineral ions into and within collagen fibrils of bone and tendon support the postulate that bone collagen is an in vivo catalyst for mineral deposition and further suggests that its catalytic activity may be partially regulated through its molecular packing.     

Biochemical and biomechanical properties of tendons in two commercial types of chickens

The quality of the attachment of meat to bone is often reported to be insufficient by more and more poultry's consumers. This is particularly true for thigh meat in broilers. The aim of this study was to compare muscle to bone attachment (namely, tendons) from a biomechanical and a biochemical point of view in 50 standard (S) and 50 Label Rouge (LR) chickens. Carcasses weighted around 1.7 kg in the two groups. Two tendons were harvested and proceeded for passive stretch tests, prior to cooking or not, to determine main mechanical characteristics (maximum load, stiffness and longitudinal strain). Biochemical parameters such as dry matter percentage, total collagen content, collagen solubility and sulphated glycosaminoglycans (sGAGs) content were also determined. Results showed that biomechanical values differ largely between the two studied tendons. For a given tendon, the values were also different between the two groups of chickens mainly after cooking. The results clearly showed that, mainly after cooking, the mechanical resistance of tendon to stretch was better in LR than in S chickens. LR chickens were reported to have tendons with higher collagen and sGAGs contents associated with a lower collagen solubility. These differences may explain biomechanical differences observed for the two types of tendons and could be due to increased age and/or higher physical activity of LR chickens.     

Decorin expression is important for age-related changes in tendon structure and mechanical properties

The aging population is at an increased risk of tendon injury and tendinopathy. Elucidating the molecular basis of tendon aging is crucial to understanding the age-related changes in structure and function in this vulnerable tissue. In this study, the structural and functional features of tendon aging are investigated. In addition, the roles of decorin and biglycan in the aging process were analyzed using transgenic mice at both mature and aged time points. Our hypothesis is that the increase in tendon injuries in the aging population is the result of altered structural properties that reduce the biomechanical function of the tendon and consequently increase susceptibility to injury. Decorin and biglycan are important regulators of tendon structure and therefore, we further hypothesized that decreased function in aged tendons is partly the result of altered decorin and biglycan expression. Biomechanical analyses of mature (day 150) and aged (day 570) patellar tendons revealed deteriorating viscoelastic properties with age. Histology and polarized light microscopy demonstrated decreased cellularity, alterations in tenocyte shape, and reduced collagen fiber alignment in the aged tendons. Ultrastructural analysis of fibril diameter distributions indicated an altered distribution in aged tendons with an increase of large diameter fibrils. Aged wild type tendons maintained expression of decorin which was associated with the structural and functional changes seen in aged tendons. Aged patellar tendons exhibited altered and generally inferior properties across multiple assays. However, decorin-null tendons exhibited significantly decreased effects of aging compared to the other genotypes. The amelioration of the functional deficits seen in the absence of decorin in aged tendons was associated with altered tendon fibril structure. Fibril diameter distributions in the decorin-null aged tendons were comparable to those observed in the mature wild type tendon with the absence of the subpopulation containing large diameter fibrils. Collectively, our findings provide evidence for age-dependent alterations in tendon architecture and functional activity, and further show that lack of stromal decorin attenuates these changes.     

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 immunolocalization of cartilage oligomeric matrix protein (COMP) in relation to collagen fibrils in the equine tendon.

The structure and organisation of the extracellular matrix, and in particular the axial alignment of type I collagen fibrils, are essential for the tensile strength of tendons. The resident tenocytes synthesize and maintain the composition of the extracellular matrix, which changes with age and maturation. Other components of the extracellular matrix include less abundant collagen types II, III, V, VI, XII, proteoglycans and glycoproteins. Cartilage oligomeric matrix protein (COMP) is an abundant non-collagenous pentameric glycoprotein in the tendon, which can bind to collagen types I and II. The function of COMP in the tendon is not clear, but it may act as a catalyst in fibrillogenesis. Its concentration changes with age, maturation and load. The present study delineates the ultrastructural distribution of COMP and its correlation to collagen fibril thickness in different compartments in two flexor tendons from horses of different ages (foetus, 8 months, 3 years, 12 years). The immunolabeling for COMP was higher in the superficial digital flexor tendon compared with the deep digital flexor tendon and it increased with the age of the animal, with the highest concentration in the 3-year-olds. Fibril diameter differed between age groups and a more homogenous fibril population was found in the fetal tendons. A positive correlation between high COMP immunolabeling and the percentage of small fibrils (<60 nm) were present in the SDFT. COMP immunolabeling was enriched at the gap region of the collagen fibril. In situ hybridization revealed the strongest expression in tendons from the 3-year-old horses whereas there was no expression in foetal tendon.     

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 role of tenascin-C in adaptation of tendons to compressive loading

Although most tendon regions are subjected primarily to high tensile loads, selected regions, primarily those that directly contact bones that change the direction of the tendon, must withstand high compressive loads as well. Compressed tendon regions differ from regions subjected to primarily tensile loads: they have a fibrocartilaginous structure with spherical cells surrounded by a matrix containing aggrecan and collagen types I and II, in contrast regions not exposed to compression have a fibrous structure with spindle shaped fibroblasts surrounded by a matrix of dense, longitudinally oriented type I collagen fibrils. The spherical shape of cells in fibrocartilagenous regions indicates these cells are more loosely attached to the matrix than their spindle-shaped counterparts in fibrous regions, a feature that may help to minimize cell deformation during tendon compression. We hypothesized that expression of tenascin-C, an anti-adhesive protein, is part of the adaptation of tendon cells to compression that helps establish and maintain fibrocartilaginous regions. To test this hypothesis we compared tenascin-C content and expression in compressed (distal) versus uncompressed (proximal) segments of bovine flexor tendons. Immunohistochemistry and immunoblot analyses showed that tenascin-C content was increased in the distal tendon where it co-distributed with type II collagen and aggrecan. Tendon cells from the distal segments expressed more tenascin-C than did cells from the proximal segments for up to four days in cell culture, indicating that increased tenascin-C expression is a relatively stable feature of the distal cells. These observations support the hypothesis that tenascin-C expression is a cellular adaptation to compression that helps establish and maintain fibrocartilagenous regions of tendons.     

Proteoglycans in chicken gastrocnemius tendons change with exercise

Growth, loading, and mobilization lead to changes in tendon structure. Recent studies have shown that proteoglycans (PGs) regulate the organization of collagen fibrils, the main structural components of tendons. We hypothesized that moderate exercise alters PG synthesis in the avian gastrocnemius tendon. To test our hypothesis we compared the PG content in gastrocnemius tendons from control 6.5-week-old chickens with that in tendons from 6.5-week-old chickens that underwent exercise. Our results show high levels and a wide variety of glycosaminoglycans (GAGs) in 6.5-week-old tendons. Chondroitin-4-sulfate disaccharide was the major GAG disaccharide in control and exercised 6.5-week-old gastrocnemius tendons. Exercise led to an increase in the size of the tendons, the content of hyaluronic acid, and the level of decorin. High levels of keratan sulfate (KS) were found in the lower halves of gastrocnemius tendons, although the amount of KS decreased with exercise. This corresponded well with lower content of aggrecan in the lower halves of exercised tendons. In conclusion, our data support the hypothesis that exercise alters the content of PGs in chicken tendons.     

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.