The structure and function of normally mineralizing avian tendons

The leg tendons of certain avian species normally calcify. The gastrocnemius, or Achilles, tendon of the domestic turkey, Meleagris gallopavo, is one such example. Its structure and biomechanical properties have been studied to model the adaptive nature of this tendon to external forces, including the means by which mineral deposition occurs and the functional role mineralization may play in this tissue. Structurally, the distal rounded, thick gastrocnemius bifurcates into two smaller proximal segments that mineralize with time. Mineral deposition occurs at or near the bifurcation, proceeding in a distal-to-proximal direction along the segments toward caudal and medial muscle insertions of the bird hip. Mineral formation appears mediated first by extracellular matrix vesicles and later by type I collagen fibrils. Biomechanical analyses indicate lower tensile strength and moduli for the thick distal gastrocnemius compared to narrow, fan-shaped proximal segments. Tendon mineralization here appears to be strain-induced, the muscle forces causing matrix deformation leading conceptually to calcium binding through the exposure of charged groups on collagen, release of sequestered calcium by proteoglycans, and increased diffusion. Functionally, the mineralized tendons limit further tendon deformation, reduce tendon strain at a given stress, and provide greater load-bearing capacity to the tissue. They also serve as important and efficient elastic energy storage reservoirs, increasing the amount of stored elastic energy by preventing flexible type I collagen regions from stretching and preserving muscle energy during locomotion of the animals.     

Mechanical implications of the domain structure of fiber-forming collagens: comparison of the molecular and fibrillar flexibilities of the alpha1-chains found in types I-III collagen

Fibrillar collagens store, transmit and dissipate elastic energy during tensile deformation. Results of previous studies suggest that the collagen molecule is made up of alternating rigid and flexible domains, and extension of the flexible domains is associated with elastic energy storage. In this study, we model the flexibility of the alpha1-chains found in types I-III collagen molecules and microfibrils in order to understand the molecular basis of elastic energy storage in collagen fibers by analysing the areas under conformational plots for dipeptide sequences. Results of stereochemical modeling suggest that the collagen triple helix is made up of rigid and flexible domains that alternate with periods that are multiples of three amino acid residues. The relative flexibility of dipeptide sequences found in the flexible regions is about a factor of five higher than that found for the flexibility of the rigid regions, and the flexibility of types II and III collagen molecules appears to be higher than that found for the type I collagen molecule. The different collagen alpha1-chains were compared by correlating the flexibilities. The results suggest that the flexibilities of the alpha1-chains of types I and III collagen are more closely related than the flexibilities of the alpha1-chains in types I and II and II and III collagen. The flexible domains found in the alpha1-chains of types I-III collagen were found to be conserved in the microfibril and had periods of about 15 amino acid residues and multiples thereof. The flexibility profiles of types I and II collagen microfibrils were found to be more highly correlated than those for types I and III and II and III. These results suggest that the domain structure of the alpha1-chains found in types I-III collagen is an efficient means for storage of elastic energy during stretching while preserving the triple helical structure of the overall molecule. It is proposed that all collagens that form fibers are designed to act as storage elements for elastic energy. The function of fibers rich in type I collagen is to store and then transmit this energy while fibers rich in types II and III collagen may store and then reflect elastic energy for dissipation through viscous fibrillar slippage. Impaired elastic energy storage by extracellular matrices may lead to cellular damage and changes in signaling by mechanochemical transduction at the extracellular matrix-cell interface.     

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.     

Elastic energy storage in an unmineralized collagen type I molecular model with explicit solvation and water infiltration

Collagen type I is a structural protein that provides tensile strength to tendons and ligaments. Type I collagen molecules form collagen fibers, which are viscoelastic and can therefore store energy elastically via molecular elongation and dissipate viscous energy through molecular rearrangement and fibrillar slippage. The ability to store elastic energy is important for the resiliency of tendons and ligaments, which must be able to deform and revert to their initial lengths with changes in load.In an earlier paper by one of the present authors, molecular modeling was used to investigate the role of mineralization upon elastic energy storage in collagen type I. Their collagen model showed a similar trend to their experimental data but with an over-estimation of elastic energy storage. Their simulations were conducted in vacuum and employed a distance-dependent dielectric function. In this study, we performed a re-evaluation of Freeman and Silver's model data incorporating the effects of explicit solvation and water infiltration, in order to determine whether the model data could be improved with a more accurate representation of the solvent and osmotic effects. We observed an average decrease in the model's elastic energy storage of 45.1%±6.9% in closer proximity to Freeman and Silver's experimental data. This suggests that although the distance-dependent dielectric implicit solvation approach was favored for its increased speed and decreased computational requirements, an explicit representation of water may be necessary to more accurately model solvent interactions in this particular system. In this paper, we discuss the collagen model described by Freeman and Silver, the present model building approach, the application of the present model to that of Freeman and Silver, and additional assumptions and limitations.     

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.     

Tensile properties of calcified and uncalcified avian tendons

The mechanical properties of turkey and heron leg tendons have been investigated in dynamic tensile tests. Heron tendons have properties similar to those found for various mammalian tendons. The Young's modulus and the density of turkey tendons increase with increasing calcification. Ultimate tensile stresses are similar to those found for uncalcified tendon, but Young's modulus may reach about 16 GPa, a value normally associated with bone. Calcification lowers the amount of strain energy that can be stored temporarily in the tendons of the legs. The contribution made by elastic strain energy storage to lowering the cost of running is reduced.     

The integrated function of muscles and tendons during locomotion

The mechanical roles of tendon and muscle contractile elements during locomotion are often considered independently, but functionally they are tightly integrated. Tendons can enhance muscle performance for a wide range of locomotor activities because muscle-tendon units shorten and lengthen at velocities that would be mechanically unfavorable for muscle fibers functioning alone. During activities that require little net mechanical power output, such as steady-speed running, tendons reduce muscular work by storing and recovering cyclic changes in the mechanical energy of the body. Tendon stretch and recoil not only reduces muscular work, but also allows muscle fibers to operate nearly isometrically, where, due to the force-velocity relation, skeletal muscle fibers develop high forces. Elastic energy storage and recovery in tendons may also provide a key mechanism to enable individual muscles to alter their mechanical function, from isometric force-producers during steady speed running to actively shortening power-producers during high-power activities like acceleration or uphill running. Evidence from studies of muscle contraction and limb dynamics in turkeys suggests that during running accelerations work is transferred directly from muscle to tendon as tendon stretch early in the step is powered by muscle shortening. The energy stored in the tendon is later released to help power the increase in energy of the body. These tendon length changes redistribute muscle power, enabling contractile elements to shorten at relatively constant velocities and power outputs, independent of the pattern of flexion/extension at a joint. Tendon elastic energy storage and recovery extends the functional range of muscles by uncoupling the pattern of muscle fiber shortening from the pattern of movement of the body.     

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.     

Self-assembly of collagen fibers. Influence of fibrillar alignment and decorin on mechanical properties.

Collagen is the primary structural element in extracellular matrices. In the form of fibers it acts to transmit forces, dissipate energy, and prevent premature mechanical failure in normal tissues. Deformation of collagen fibers involves molecular stretching and slippage, fibrillar slippage, and, ultimately, defibrillation. Our laboratory has developed a process for self-assembly of macroscopic collagen fibers that have structures and mechanical properties similar to rat tail tendon fibers. The purpose of this study is to determine the effects of subfibrillar orientation and decorin incorporation on the mechanical properties of collagen fibers. Self-assembled collagen fibers were stretched 0-50% before cross-linking and then characterized by microscopy and mechanical testing. Results of these studies indicate that fibrillar orientation, packing, and ultimate tensile strength can be increased by stretching. In addition, it is shown that decorin incorporation increases ultimate tensile strength of uncross-linked fibers. Based on the observed results it is hypothesized that decorin facilitates fibrillar slippage during deformation and thereby improves the tensile properties of collagen fibers.     

Regional adaptations in three rat tendons

Although detailed histological and immunocytochemical studies have been published for the rat calcanear tendon (CT), little is known of the structure, composition and biomechanics of the deep (DFT) and superficial (SFT) flexor tendons. In this study, we examined the structural specialization of these three tendons in 90-day-old rats by applying histochemical and biochemical assays to different tendon regions (proximal, intermediate and distal regions of the DFT and SFT, and proximal and distal regions of the CT). There were regional differences in tissue structure, glycosaminoglycan type and content, swelling properties and in the amount and distribution of elastic fibers. Dermatan sulfate occurred in all regions, but chondroitin sulfate predominated in the intermediate region of the DFT and in the distal region of the CT. These two chondroitin sulfate-bearing regions showed swelling in water, while all other regions lost fluid in water. Fibrocartilaginous sites were observed on the CT, one at the insertion to the bone and another distally at the innermost area of the tendon. The intermediate region of the DFT showed round cells disposed in lacunae, while the proximal and distal regions were typically fibrous. The intermediate region of the SFT showed a wavy array of collagen bundles but neither toluidine blue staining in the matrix nor round cells. Elastic fibers were present in each region of the three tendons, but were more prominent in the intermediate zone of the SFT. These results demonstrate regional variation in the three tendons. Tendon differentiation may occur by an increase in the number of elastic fibers and by variations in the arrangement of collagen fibers, without fibrocartilage formation.     

Structure and function of tuna tail tendons

The caudal tendons in tunas and other scombrid fish link myotomal muscle directly to the caudal fin rays, and thus serve to transfer muscle power to the hydrofoil-like tail during swimming. These robust collagenous tendons have structural and mechanical similarity to tendons found in other vertebrates, notably the leg tendons of terrestrial mammals. Biochemical studies indicate that tuna tendon collagen is composed of the (alpha1)(2),alpha2 heterotrimer that is typical of vertebrate Type I collagen, while tuna skin collagen has the unusual alpha1,alpha2,alpha3 trimer previously described in the skin of some other teleost species. Tuna collagen, like that of other fish, has high solubility due to the presence of an acid-labile intermolecular cross-link. Unlike collagen in mammalian tendons, no differences related to cross-link maturation were detected among tendons in tuna ranging from 0.05 to 72 kg (approx. 0.25-6 years). Tendons excised post-mortem were subjected to load cycling to determine the modulus of elasticity and resilience (mean of 1.3 GPa and 90%, respectively). These material properties compare closely to those of leg tendons from adult mammals that can function as effective biological springs in terrestrial locomotion, but the breaking strength is substantially lower. Peak tendon forces recorded during steady swimming appear to impose strains of much less than 1% of tendon length, and no more than 1.5% during bursts. Thus, the caudal tendons in tunas do not appear to function as elastic storage elements, even at maximal swimming effort.     

Molecular assessment of the elastic properties of collagen-like homotrimer sequences

Knowledge of the mechanical behavior of collagen molecules is critical for understanding the mechanical properties of collagen fibrils that constitute the main architectural building block of a number of connective tissues. In this study, the elastic properties of four different type I collagen 30-residue long molecular sequences, were studied by performing stretching simulations using the molecular mechanics approach. The energy–molecular length relationship was achieved by means of the geometry optimization procedure for collagen molecule strains up to 10%. The energy was interpolated by a second order function, and the second order of the derivative with respect to the mean length corresponded to the molecule stiffness. According to the hypothesis of linear elastic behavior, except for one sequence, the elastic modulus was around 2.40 GPa. These values are larger than fibril values, and they confirm the hypothesis that tendon mechanical properties are deeply related to tendon hierarchical structure. A possible explanation of the lowest values obtained for one sequence (1.33–1.53 GPa) is provided and discussed.     

A new molecular model for collagen elasticity based on synchrotron X-ray scattering evidence.

Collagen is the most abundant structural protein in vertebrates. The specific shape of its stress-strain curve is crucial for the function of a number of organs. Although the macroscopic mechanical behavior of collagen is well known, there is still no explanation of the elastic process at the supramolecular level. We have performed in situ synchrotron x-ray scattering experiments, which show that the amount of lateral molecular order increases upon stretching of collagen fibers. In strain cycling experiments the relation between strain and diffuse equatorial scattering was found to be linear in the "heel" region of the stress-strain curve. A new molecular model for collagen elasticity is proposed, which, based on the existence of thermally activated molecular kinks, reproduces this linearity and gives a simple explanation for the form of the stress-strain curve of collagen.     

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)     

A failure model for ligaments

We propose a failure model for ligament which assumes that sequential uncrimping and stretching of collagen fibers is responsible for the mechanical response of ligament. We further assume that the fibers rupture sequentially and in a brittle, strain-limited manner. The model was fit to stress strain curves obtained from medial collateral ligaments of New Zealand White rabbits from two age groups (4 and 7 months). The model indicated that collagen modulus values ranged from 300 to 680 MPa and that fiber failure strains ranged from 6 to 22%. The model provides a convenient means of describing the elastic and failure response of ligament using four structurally based parameters.     

Biomimetic approaches to tendon repair

The linear organization of collagen fibers in tendons results in optimal stiffness and strength at low strains under tensile load. However, this organization makes repairing ruptured or lacerated tendons extremely difficult. Current suturing techniques to join split ends of tendons, while providing sufficient mechanical strength to prevent gapping, are inadequate to carry normal loads. Immobilization protocols necessary to restore tendon congruity result in scar formation at the repair site and peripheral adhesions that limit excursion. These problems are reviewed to emphasize the need for novel approaches to tendon repair, one of which is the development of biomimetic tendons. The objective of the empirical work described here was to produce biologically-based, biocompatible tendon replacements with appropriate mechanical properties to enable immediate mobilization following surgical repair. Nor-dihydroguaiaretic acid (NDGA), a di-catechol from creosote bush, caused a dose dependent increase in the material properties of reconstituted collagen fibers, achieving a 100-fold increase in strength and stiffness over untreated fibers. The maximum tensile strength of the optimized NDGA treated fibers averaged 90 MPa; the elastic modulus of these fibers averaged 580 MPa. These properties were independent of strain rates ranging from 0.60 to 600 mm/min. Fatigue tests established that neither strength nor stiffness were affected after 80 k cycles at 5% strain. Treated fibers were not cytotoxic to tendon fibroblasts. Fibroblasts attached and proliferated on NDGA treated collagen normally. NDGA-fibers did not elicit a foreign body response nor did they stimulate an immune reaction during six weeks in vivo. The fibers survived 6 weeks with little evidence of fragmentation or degradation. The polymerization scheme described here produces a fiber-reinforced NDGA-polymer with mechanical properties approaching an elastic solid. The strength, stiffness and fatigue properties of the NDGA-treated fibers are comparable to those of tendon. These fibers are biocompatible with tendon fibroblasts and elicit little rejection or antigenic response in vivo. These results indicate that NDGA polymerization may provide a viable approach for producing collagenous materials that can be used to bridge gaps in ruptured or lacerated tendons. The tendon-like properties of the NDGA-fiber would allow early mobilization after surgical repair. We predict that timely loading of parted tendons joined by this novel biomaterial will enhance mechanically driven production of neo-tendon by the colonizing fibroblasts and result in superior repair and rapid return to normal properties.     

Passive force generation and titin isoforms in mammalian skeletal muscle.

When relaxed striated muscle cells are stretched, a resting tension is produced which is thought to arise from stretching long, elastic filaments composed of titin (also called connectin). Here, I show that single skinned rabbit soleus muscle fibers produce resting tension that is several-fold lower than that found in rabbit psoas fibers. At sarcomere lengths where the slope of the resting tension-sarcomere length relation is low, electron microscopy of skinned fibers indicates that thick filaments move from the center to the side of the sarcomere during prolonged activation. As sarcomeres are stretched and the resting tension sarcomere length relation becomes steeper, this movement is decreased. The sarcomere length range over which thick filament movement decreases is higher in soleus than in psoas fibers, paralleling the different lengths at which the slope of the resting tension-sarcomere length relations increase. These results indicate that the large differences in resting tension between single psoas and soleus fibers are due to different tensions exerted by the elastic elements linking the end of each thick filament to the nearest Z-disc, i.e., the titin filaments. Quantitative gel electrophoresis of proteins from single muscle fibers excludes the possibility that resting tension is less in soleus than in psoas fibers simply because they have fewer titin filaments. A small difference in the electrophoretic mobility of titin between psoas and soleus fibers suggests the alternate possibility that mammalian muscle cells use at least two titin isoforms with differing elastic properties to produce variations in resting tension.     

Glycation-induced matrix stability in the rabbit achilles tendon

Connective tissue susceptibility to nonenzymatic glycation was examined following 0, 2, 4, 6, 8, and 10 weeks of incubating the rabbit Achilles tendon in phosphate-buffered saline containing ribose (glycated). The biomechanical integrity of the glycated tendons was then compared to control tendons incubated in phosphate-buffered saline (non-glycated) at each time interval, while the biochemical stability of both groups of tendons was determined by examining collagen extractability and the formation of pentosidine at 8 weeks. Whereas there were no significant biomechanical differences between control and glycated tendons at 0- and 2-week intervals (P > 0.05), moderately significant increases in maximum load, energy to yield, and toughness of glycated tendons were observed at 4 weeks. Beyond 4 weeks of incubation, the differences between glycated and non-glycated tendons became highly significant, as glycated tendons withstood more load and tensile stress (P < 0.01 for each variable), attained significantly higher modulus of elasticity (P < 0.01), absorbed more energy (P < 0.01), and became tougher (P < 0.01) than controls. These differences in the biomechanical indices of the effects of glycation were stable between the 6th and 10th week of glycation. The maximum increases in the biomechanical measurements as a result of glycation were 29% for maximum load, 125% for stress, 19% for strain, 106% for Young's modulus of elasticity, 14% for energy to yield, and 57% for toughness. Biochemical analysis showed a 61% reduction in the extractability of neutral salt-soluble collagen, a 48% decrease in acid-soluble collagen, and a 29% decline in pepsin-soluble collagen in glycated tendons (P < 0.01). In contrast, there was a 28% increase in the amount of insoluble collagen and significantly higher amounts of pentosidine (P < 0.01) in glycated tendons. Collectively, these biomechanical and biochemical results suggest that nonenzymatic glycation may explain the altered stability of connective tissue matrix induced by the processes of diabetes and aging.     

Entropic elasticity controls nanomechanics of single tropocollagen molecules

We report molecular modeling of stretching single molecules of tropocollagen, the building block of collagen fibrils and fibers that provide mechanical support in connective tissues. For small deformation, we observe a dominance of entropic elasticity. At larger deformation, we find a transition to energetic elasticity, which is characterized by first stretching and breaking of hydrogen bonds, followed by deformation of covalent bonds in the protein backbone, eventually leading to molecular fracture. Our force-displacement curves at small forces show excellent quantitative agreement with optical tweezer experiments. Our model predicts a persistence length xi(p) approximately 16 nm, confirming experimental results suggesting that tropocollagen molecules are very flexible elastic entities. We demonstrate that assembly of single tropocollagen molecules into fibrils significantly decreases their bending flexibility, leading to decreased contributions of entropic effects during deformation. The molecular simulation results are used to develop a simple continuum model capable of describing an entire deformation range of tropocollagen molecules. Our molecular model is capable of describing different regimes of elastic and permanent deformation, without relying on empirical parameters, including a transition from entropic to energetic elasticity.