Structural analysis of turkey tendon collagen upon removal of the inorganic phase

Calcified leg flexor tendons in which the inorganic phase content had been lowered by progressive demineralization were studied by small angle X-ray diffraction and thermogravimetry. The X-ray diffraction results agree very well with the data previously obtained on calcified turkey tendon indicating that the method used to decalcify tendons provides good correspondence with the process of calcification. Up to five thermal processes can be detected in the thermogravimetric scans: (1) water release; (2) collagen decomposition; (3 and 4) combustion of the residual organic components; (5) carbonate removal from the apatitic phase. The temperature of collagen decomposition decreases at lower inorganic phase content in agreement with the higher thermal stability of calcified collagen fibrils compared with uncalcified ones. The decrease of collagen thermal stability upon decalification is paralleled by a decrease of the structural order of the collagen fibrils as indicated by small angle X-ray diffraction data. Decalcification down to about 40% wt of inorganic phase does not significantly alter the inorganic blocks that are regularly arranged inside the gap zone of the collagen. Further removal of inorganic phase down to about 15% wt provokes a variation of the intensity distribution of the small angle meridional reflections that can be ascribed to a reduction of the mean height of the inorganic blocks. At inorganic phase contents below 15% wt the gap region is more free to contract upon air drying as a result of the reduction of the mean length of the inorganic blocks.     

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.     

Energy dissipation in the elastic recoil of leg tendons of Larus ridibundus

Cycles of tension and recoil were given to different tendons of black-headed gull (Larus ridibundus) in an Instron universal testing machine. It is shown that a large proportion of the energy elastically stored in straining is released upon recoil, ranging the energy lost (dissipation energy) between 8 and 13%. These results are in complete agreement with those published in the literature on mammals and birds that can be considered as the most accurate.     

Arrangement of microfibrils in collagen fibrils of tendons in the rat tail

Summary The microfibrillar arrangement in collagen fibrils of tendons in the tail of the rat was examined by electron microscopy and X-ray diffraction. Fresh and air-dried collagen fibers were examined in unstretched and stretched conditions. The results demonstrate that the microfibrils have a course parallel to the longitudinal axis of the collagen fibrils. The influence of stretching and hydration of the samples on the orientation of fibrils and microfibrils is also assessed.     

Elastic extension of leg tendons in the locomotion of horses (Equus caballus)

Several of the distal leg muscles of horses have such extremely short muscle fibres that their changes of length in locomotion must be due almost entirely to elastic extension of their tendons. Films of a horse have been analysed to determine these extensions, using data obtained by experiments on dissected legs. The tendons investigated experience peak strains of 3–6% in walking, 3–7% in trotting and 4–9% in galloping. These strains occur while the foot is on the ground.     

Analysis of the crystal arrangement in collagen fibrils of mineralizing turkey tibia tendon

Summary Mineralized pieces of tendons from the tibio-tarsus of turkeys were (i) shock-frozen, freeze-dried, embedded and cut without staining, or (ii) fixed, embedded and stained after sectioning. Micrographs were taken with an electron microscope on longitudinally cut sections. The center-to-center distances of neighboring apatitic needles within collagen fibrils were measured. For shock-frozen and freeze-dried specimens, the average of these distances is 4.7 nm and the most frequent value 4.2 nm; for fixed and stained specimens, 3.8 nm and 3.6 nm, respectively. Laser diffraction of the electron micrographs showed a dumbbell-like intensity pattern (two diffuse maxima of intensity on the equator, one on each side of the central spot), giving an average distance of about 6 nm. This value represents the upper range of the direct measurements. The measurements demonstrate that the arrangement of the collagen microfibrils is mainly preserved during mineralization. However, using laser diffraction, distances of 9–11 nm were also observed. Such large distances can also be demonstrated by X-ray diffraction on collagen fibrils stained under special conditions. This may indicate that special conditions of apatitic mineralization or staining may alter the arrangement of the microfibrils.The authors thank the Deutsche Forschungsgemeinschaft for financial support     

Growth-related changes in the mechanical properties of collagen fascicles from rabbit patellar tendons

Growth-related changes in the mechanical properties of collagen fascicles (approximately 300 microm in diameter) were studied using patellar tendons obtained from skeletally immature 1 and 2 months old and matured 6 months old rabbits. Tensile properties were determined using a specially designed micro-tensile tester. In each age group, there were no significant differences in the properties among cross-sectional locations in the tendon. Tangent modulus and tensile strength significantly increased with age; the rates of their increases between 1 and 2 months were higher than those between 2 and 6 months. The tangent modulus and tensile strength were positively correlated with the body weight of animals. However, growth-related changes in the mechanical properties were different between collagen fascicles and bulk patellar tendons, which may be attributable to such non-collagenous components as ground substances and also to mechanical interactions between collagen fascicles.     

Changes in the cross-linking of collagen from rat tail tendons due to diabetes

The acid solubility of Type I collagen from rat tail tendons decreases due to diabetes. This finding has been taken as evidence that collagen from diabetics may be more cross-linked than normal. We compared CNBr peptide maps prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for [3H] NaBH4-reduced tail tendons from streptozotocin-diabetic rats with maps from age-matched control rats. At least through 30 weeks of diabetes, the distribution of mass of both cross-linked and uncross-linked CNBr peptides was identical in diabetic and control tendons. Therefore, the number of cross-linked peptides did not increase due to diabetes. We analyzed the 3H-cross-linking compounds present on the CNBr peptides and found that the 3H content of peptides cross-linked in control tendons through the bivalent, reduced cross-links hydroxylysinonorleucine and lysinonorleucine was diminished on corresponding peptides from diabetic tendons as a function of duration of diabetes. The cross-linked peptides, however, persisted. Therefore, we conclude that a larger fraction of these bivalent cross-links is found in an unknown, non-reducible form in tendons from diabetic compared with control rats. This resembles a phenomenon normally associated with maturation and/or aging where the non-reducible form of the cross-links is acid-stable. An increase in the fraction of the cross-links that is non-reducible and acid-stable would explain, at least in part, the decrease in acid solubility of the collagen. Non-enzymatic glycation (NEG) was not very specific, since most CNBr peptides bound some glucose. However, peptides from the alpha 2-chain seemed to be preferential targets for NEG. While NEG clearly increased due to diabetes, we found no evidence that increased NEG led to an increased number of cross-links in tail tendon collagen from streptozotocin diabetic rats.     

Levels of calcium and soluble collagen in turkey egg shell membranes

1. This experiment examined the effect of weeks in egg production and type of housing confinement of turkey hens on calcium and soluble collagen levels in egg shell membranes; and discussion was given to their apparent relationship to gas exchange in turkey eggs. 2. The high level of acid-soluble collagen in inner and outer egg shell membranes of aging caged hens compared with the same aged floor-penned hens may have a relationship with the low hatchability generally recognized in caged hens. 3. The levels of calcium found in the outer shell membrane are low and appeared to decrease with the age of the hen. 4. There were no differences over time in levels of total collagen and neutral salt-soluble collagen (newly formed collagen) found in egg shell membranes of turkey hens confined in cages or floor pens. 5. It is suggested that the acid-soluble collagen levels found in inner shell membranes may have a relationship in limiting respiratory gas exchange during latter incubation time, and thus limit embryo survival.     

Effect of ionizing radiation on the structure of the collagen fibers of human tendons

In studying the effect of ionizing radiation on the properties of human Achilles tendon collagen fibres, the following parameters were analyzed: hydrothermal contraction temperature, module of elasticity, the number of cross-links, free and bound water levels, acids-soluble fraction content, and ultrastructure. With radiation doses of 2-10 Gy no changes in the collagen status were noted. An increased (from 5 to 25 Gy) radiation dose caused changes in physicochemical properties which was indicative of the formation, in the connective tissue collagen, of radiation-induced intermolecular cross-links stabilizing the biopolymer structure.     

Ageing changes in the tensile properties of tendons: influence of collagen fibril volume fraction

Connective tissues are biological composites comprising of collagen fibrils embedded in (and reinforcing) the hydrated proteoglycan-rich (PG) gel within the extracellular matrices (ECMs). Age-related changes to the mechanical properties of tissues are often associated with changes to the structure of the ECM, namely, fibril diameter. However, quantitative attempts to correlate fibril diameter to mechanical properties have yielded inconclusive evidence. Here, we described a novel approach that was based on the rule of mixtures for fiber composites to evaluate the dependence of age-related changes in tendon tensile strength (sigma) and stiffness (E) on the collagen fibril cross-sectional area fraction (rho), which is related to the fibril volume fraction. Tail tendons from C57BL6 mice from age groups 1.6-35.3 months old were stretched to failure to determine sigma and E. Parallel measurements of rho as a function of age were made using transmission electron microscopy. Mathematical models (rule of mixtures) of fibrils reinforcing a PG gel in tendons were used to investigate the influence of rho on ageing changes in sigma and E. The magnitudes of sigma, E, and rho increased rapidly from 1.6 months to 4.0 months (P-values <0.05) before reaching a constant (age independent) from 4.0 months to 29.0 months (P-values >0.05); this trend continued for E and rho (P-values >0.05) from 29.0 months to 35.3 months, but not for sigma, which decreased gradually (P-values <0.05). Linear regression analysis revealed that age-related changes in sigma and E correlated positively to rho (P-values <0.05). Collagen fibril cross-sectional area fraction rho is a significant predictor of ageing changes in sigma and E in the tail tendons of C57BL6 mice.     

Identification of two new collagen alpha-chains in extracts of lathyritic chick embryo tendons

Two new collagen polypeptide chains have been identified in extracts of lathyritic embryonic chick tendons. The electrophoretic migration of these polypeptides in sodium dodecyl sulfate-polyacrylamide gels indicates that they have about 20% greater apparent molecular weights than α₁ and α₂ chains of Type I collagen. These chains are not held by disulfide bonds since reduction does not affect their electrophoretic behavior. Further, they do not represent incompletely cleaved procollagen since their apparent molecular size remains greater than that of Type I collagen polypeptides after limited proteolytic digestion. Because the ratio of these polypeptides in the purified extracts is not 2:1 it appears that they are components of two separate tropocollagen molecules.     

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

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

Changes in bones of the foot and development of inserting tendons of the lower leg musculature in tibial aplasia

Skeleton, muscles and tendons were investigated in 3 lower legs with aplasia of the tibia. The tibiofibular joint appears to be rather a syndesmosis than a diarthrosis. In all 3 cases, talus and calcaneus are connected by a synostosis. Also in the other parts of the skeleton of the feet, synarthroses with different extensions are found. The muscles, normally originating from the tibia, have shifted their origin to the fibula. The proximal part of the inserting tendons under the retinacula is quite normal. Also, their distal part in the region of the metatarsalia and of the digits exhibits no alterations. In the region of the tarsus in which already alterations in bone formation are found, the tendons of the flexor muscles of the toes exhibit some variations. Often the muscles show a new, strong insertion into the bones of the tarsus. The normal connection between the proximal and distal parts of the tendons is sometimes still indicated by a thin bundle of collagenous fibers. In all 3 cases, the tibialis anterior muscle inserts into the distal part of the tendons of the flexor muscles of the toes, indicating that there are also connections between tendons of muscles from different blastemas.     

On the elastic properties of mineralized turkey leg tendon tissue: multiscale model and experiment

The key parameters influencing the elastic properties of the mineralized turkey leg tendon (MTLT) were investigated. Two structurally different tissue types appearing in the MTLT were considered: circumferential and interstitial tissue. These differ in their amount of micropores and their average diameter of the mineralized collagen fibril bundles. A multiscale model representing the apparent elastic stiffness tensor of MTLT tissue was developed using the Mori–Tanaka and the self-consistent homogenization schemes. The volume fraction of mineral (hydroxyapatite) in the fibril bundle, \(\hbox {vf}_{{\text {ha}}}^{{\text {MCFB}}}\) , and the tissue microporosity are the variables of the model. The MTLT model was analyzed performing a global sensitivity analysis (Elementary Effects method) and a parametric study. The stiffnesses parallel (axial) and perpendicular (transverse) to the MTLT long axis were the only significantly sensitive components of the apparent stiffness tensor of MTLT tissue. The most important parameters influencing these apparent stiffnesses are \(\hbox {vf}_{{\text {ha}}}^{{\text {MCFB}}}\) , tissue microporosity, as well as shape and distribution of the minerals in the fibril bundle (intra- vs. interfibrillar). The predicted apparent stiffness was converted to acoustic impedance for model validation. From measurements on embedded MTLT samples, including 50- and 200-MHz scanning acoustic microscopy as well as synchrotron radiation micro-computed tomography, we obtained site-matched acoustic impedance and \(\hbox {vf}_{{\text {ha}}}^{{\text {MCFB}}}\) data of circumferential and interstitial tissue. The experimental and the model data compare very well for both tissue types (relative error 6–8 %).     

Differential expression of lumican and fibromodulin regulate collagen fibrillogenesis in developing mouse tendons

Collagen fibrillogenesis is finely regulated during development of tissue-specific extracellular matrices. The role(s) of a leucine-rich repeat protein subfamily in the regulation of fibrillogenesis during tendon development were defined. Lumican-, fibromodulin-, and double-deficient mice demonstrated disruptions in fibrillogenesis. With development, the amount of lumican decreases to barely detectable levels while fibromodulin increases significantly, and these changing patterns may regulate this process. Electron microscopic analysis demonstrated structural abnormalities in the fibrils and alterations in the progression through different assembly steps. In lumican-deficient tendons, alterations were observed early and the mature tendon was nearly normal. Fibromodulin-deficient tendons were comparable with the lumican-null in early developmental periods and acquired a severe phenotype by maturation. The double-deficient mice had a phenotype that was additive early and comparable with the fibromodulin-deficient mice at maturation. Therefore, lumican and fibromodulin both influence initial assembly of intermediates and the entry into fibril growth, while fibromodulin facilitates the progression through growth steps leading to mature fibrils. The observed increased ratio of fibromodulin to lumican and a competition for the same binding site could mediate these transitions. These studies indicate that lumican and fibromodulin have different developmental stage and leucine-rich repeat protein specific functions in the regulation of fibrillogenesis.