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.     

The influence of indomethacin on collagen synthesis during tendon healing in the rabbit

The influence of indomethacin on collagen synthesis in intact and healing plantaris longus tendons in the rabbit was investigated. Forty-four male New Zealand White rabbits were subjected to a standardized trauma (tenetomy + repair) on the left hindlimb. Half of the animals were subsequently treated with indomethacin, 10 mg/kg per day orally, and the other half with placebo. After 2 and 4 weeks the rabbits were injected intravenously with 3H-proline and killed 18 h later. Indomethacin affected the collagen metabolism differently depending on whether the tendons were involved in wound healing or not. In intact tendons the drug caused a small general inhibition of collagen synthesis. In the healing tendon there was a shift towards the synthesis of more insoluble collagen with little effect on the total synthesis. After 4 weeks there was also a slight but significant decrease in the amount of hydroxyproline in the most soluble collagen fraction from the tenotomized, indomethacin treated tendons.     

The influence of indomethacin on collagen synthesis during tendon healing in the rabbit

The influence of indomethacin on collagen synthesis in intact and healing plantaris longus tendons in the rabbit was investigated. Forty-four male New Zealand White rabbits were subjected to a standardized trauma (tenetomy + repair) on the left hindlimb. Half of the animals were subsequently treated with indomethacin, 10 mg/kg per day orally, and the other half with placebo. After 2 and 4 weeks the rabbits were injected intravenously with ³H-proline and killed 18 h later. Indomethacin affected the collagen metabolism differently depending on whether the tendons were involved in wound healing or not. In intact tendons the drug caused a small general inhibition of collagen synthesis. In the healing tendon there was a shift towards the synthesis of more insoluble collagen with little effect on the total synthesis. After 4 weeks there was also a slight but significant decrease in the amount of hydroxyproline in the most soluble collagen fraction from the tenotomized, indomethacin treated tendons.     

Effect of age on collagen fibril diameter in rabbit patellar tendon repair

The effect of aging on soft tissue repair is poorly understood. We examined collagen fibril diameter in repairing patellar tendons from young adult and aging rabbits. We hypothesized that repairing tendons from older (geriatric) rabbits would have similar diameter fibrils compared with the younger (young adult) rabbits. Full-length, full-thickness, central-third (2.5 to 3 mm) patellar tendon injuries were made by cutting out the center of the tendon in twelve 1-y-old and thirteen 4- to 5.5 (average, 4.25)-y-old female New Zealand White rabbits. The contralateral tendon served as an unoperated control. The rabbits were euthanized at 6, 12, and 26 wk after surgery. The collagen fibril diameter was examined by electron microscopy at the patellar end, middle, and tibial end of the patellar tendon. There was no significant decline in collagen fibril diameter at any location in the aging rabbit healing patellar tendons compared with those of the 1-y-old rabbits. This study found that collagen fibril diameter was not altered with increasing age in the healing rabbit patellar tendon.     

Acute food restriction increases collagen breakdown and phagocytosis by mature decidual cells of mice

An ultrastructural study was undertaken on antimesometrial mature decidual tissue of fed and food-restricted mice, on day 9 of pregnancy. The mean ad libitum food intake was established on mice from the 8th till the 9th day of pregnancy. Fed mice were used as controls. Experimental animals were divided into two groups: one was allowed to feed 25% of normal diet and the other 50%. Extracellular collagen fibrils were scarce in fed animals and conspicuous in food restriction. Granular electron-dense deposits and filamentous aggregates of disintegrating collagen fibrils were observed in all food-deprived mice but were rarely noted in fed animals. Intracellular vacuolar structures exhibited other typical cross-banded collagen immersed in finely granular electron-translucent material (clear vacuole) or electron-dense material containing collagen fibrils with a faint periodicity (dark vacuole). The clear and dark vacuoles were scarce in fed animals and evident in food-restricted mice, mainly in those 25% food restricted. Although collagen breakdown may be part of the normal process of decidual tissue remodelling our results suggest that it is enhanced in food-restricted animals. Thus it seems that collagen breakdown is a normal mechanism that may be regulated by the food intake of the pregnant animal.     

Ultrastructural morphometry of matrical changes induced by exercise and food restriction in the rat calcaneal tendon.

The ultrastructural morphometry of collagen fibril populations in 24 calcaneal tendons obtained from 12 Fischer 344 rats were studied to elucidate matrical changes induced by food restriction and/or endurance exercise. Rats were randomly assigned to four equal groups: ad libitum control (AC), ad libitum exercise (AE), restricted diet control (RC) and restricted diet exercise (RE) groups. Beginning from 6 weeks of age, animals in the two food restriction groups were fed 60% of the mean food consumption of ad libitum fed rats. Then, starting from 6-7 months of age, the rats in the two exercise groups performed 40-50 min of treadmill running at 1.2-1.6 miles h-1 every day for a total of 10 weeks. Endurance training did not significantly alter body weight, but food restriction with or without exercise resulted in a significant loss of body weight. In ad libitum fed controls, food restriction alone did not significantly alter the mean collagen fibril CSA, but predisposed a preponderance of small-sized collagen fibrils. Endurance training per se induced a significant (32%) increase in mean fibril CSA (P less than 0.05), but this adaptive response to exercise was prevented by food restriction, as indicated by a 33% decline in fibril CSA (P less than 0.05). These findings demonstrate that dietary restriction modifies the adaptation of tendon collagen morphometry in response to endurance training, and that weight loss is better achieved with food restriction than endurance exercise.     

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

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

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.     

Extracellular and intracellular degradation of collagen by trophoblast giant cells in acute fasted mice examined by electron microscopy

The fine structure of trophoblast giant cells and their interaction with collagen at the antimesometrial region on the 9th day of pregnancy was examined in fed and acute fasted mice. Collagen fibrils and filamentous aggregates (disintegrating collagen fibrils) were observed in the extracellular space. Three types of intracellular vacuoles containing collagen fibrils were present: vacuole type A exhibited typical cross-banded collagen immersed in finely granular electron-translucent material; and vacuoles type B and C showed electron-opaque granular material containing, respectively, faint cross-banded collagen and narrow clear stripes often with faint periodicity. In fed animals vacuoles type B were absent and the others were less evident.Only fasted animals showed extracellular acid phosphatase activity on collagen fibrils, filamentous aggregates and confined regions of the extracellular space. Intracellular acid phosphatase activity was observed in vacuoles type B and in lysosomes.The results indicate that trophoblast giant cells are capable of breaking down extracellular collagen and also of internalizing collagen for intracellular degradation. It is likely that these events are part of the process of invasion of the uterine wall. However, in fasted mice, collagen breakdown is more pronounced, and it may therefore contribute to the provision of amino acids and other nutrients for the undernourished fetus.     

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.     

The ultrastructure and possible role of the vacuolar component of hepatocyte nucleoli

A study was made of the nucleolar vacuoles in guinea pig hepatocytes that are poorly investigated for animal cells. A comparison of ultrathin sections, contrasted by heavy metals, with those treated according to Bernhard allowed to reveal the following intravacuolar structures: 1) fibrils 10-15 nm and 20-30 nm thick similar to perinucleolar chromatin fibrils; 2) RNP-granules 15-20 nm in diameter resembling the granular component of the nucleolus; 3) RNP-fibrils 15-20 nm thick with high electron density. The latter were visualized for the first time, their function still remains obscure. Upon stimulation of hepatocytes with partial hepatectomy, the vacuolar component changed. In 2.5 and 5 hours after the operation the vacuoles became smaller, the number of RNP structures of two types increased. Further, in 9 hours, the enlarging of vacuoles was accompanied by a sharp decrease in the number of these RNP-structures. The results obtained allow to suppose that the vacuoles of nucleolonemic nucleoli may be functioning elements, linking intra- and perinucleolar chromatin fibrils. They are depots for the RNP synthesized in the nucleolus; the rRNP is transported through the vacuolar system.     

Involvement of smooth muscle cells in collagen degradation in the postpartum uterus

Ultrastructural study of gravid and postpartum involuting human uteri revealed a number of cells containing collagen fibrils in their cytoplasm. In gravid uteri these cells could be identified as macrophages and fibroblasts; in the postpartum uteri smooth muscle cells (SMC) were also found, containing cytoplasmic collagenous vacuoles. The morphology of intracellular collagen in SMC was similar to that observed in macrophages: fragments of banded collagen fibrils with a diameter corresponding to that of extracellular collagen were located within structures considered to be phagosomes. Limiting membranes were always smooth, most often in apposition to the fibrils that were single or packed in small groups; some cytoplasmic vacuoles contained banded elongated profiles barely discernable as collagen. The collagen fibrils within SMC of the involuting human uterus are regarded as a morphological manifestation of heterogenic enclosure of collagen fibrils and their intracellular degradation. It seems that in the postpartum uterus, where a substantial amount of collagen needs to be removed rapidly, both macrophages and SMC are involved in the process of collagen phagocytosis and degradation. These data suggest that SMC may be involved in the cellular mechanism for collagen breakdown in remodelling SMC-containing tissues like the uterus and the vascular wall.     

The effect of age on the structure and composition of rat tendon fibrocartilage

Biochemical and morphological aspects of fibrocartilages of calcaneal and deep digital flexor tendons in rats aged 30, 180 and 730 days were analyzed. In both tendons a stronger staining with Alcian blue, indicating the presence of proteoglycans, was detected in rats of 30 and 180 days. In animals 730 days old, it was restricted to the pericellular area. Ultrastructural analysis showed a more prominent pericellular matrix in calcaneal tendon compared to the deep digital flexor tendon. The biochemical analysis showed higher levels of proteins and glycosaminoglycans in the calcaneal tendon of 30-day-old rats compared to older rats. In the deep digital flexor tendon, no significant differences were observed between ages. The small proteoglycan, fibromodulin, was detected in both tendons of all ages, but in young rats it appeared to be running as a 210 kDa component, probably due to the association with collagen chains or self-association.     

Morphological evidence of the formation of intracellular collagen fibrils in the embryonic mouse molar odontoblasts induced by colchicine administration

The effects of colchicine on collagen formation were examined ultrastructurally using secretory odontoblasts in mouse molar tooth germs isografted to the spleen for 1 week. Colchicine in concentrations of 0.025 or 0.05 mg/0.1 ml was injected intravenously 12-24 h prior to harvesting. Colchicine induced the disruption of the Golgi apparatus and caused the accumulation of various types of Golgi-associated vacuoles containing collagenous fibrillar structures. Many vacuoles containing fine particles, nonstriated parallel filaments, banding patterns with a periodicity of approximately 63-nm intervals, and occasionally segment-long-spacing-like assemblies were aggregated in the cytoplasm during the experimental period. These morphological changes in vacuole contents may reflect the initial steps for polymerization of the intracellular collagen fibrils. The majority of the aggregated vacuoles were degraded by fusion with lysosomes but banded filamentous material in some vacuoles appeared to polymerize into the collagen fibrils with native structures. These results suggested that in unsecreted vacuoles accumulated in the odontoblasts as a result of colchicine administration the polymerization of collagen fibrils with native structures can occur.     

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.     

Glucocorticoid-induced alterations in collagen of neonatal mouse condylar cartilage

Neonatal mice were treated with a single dose of triamcinolone hexacetonide, a long-acting synthetic analogue of cortisol, and their mandibular condyles were studied ultrastructurally ten days thereafter. A pronounced decrease in the number and size of matrix granules (proteoglycans) was found in the cartilaginous matrix of triamcinolone-treated condyles. In contrast, a marked increase concomitant with significant structural changes was noted in collagen fibrils. An obvious enhancement of collagen fibrillogenesis was noticed in the pre-mineralizing extracellular matrix. Atypical, wider than normal, banded collagen fibrils were found to form dense meshworks which appeared to lack any specific orientation or organization. It is proposed that glucocorticoid hormones, given systemically to neonatal mice, interfere with regulatory mechanisms involved with the biosynthesis of cartilaginous matrical macromolecules, i.e., proteoglycans and collagen and thereby promote certain aging processes within active growth centers.     

Ultrastructural observations on the intraodontoblastic collagen fibrils of the mouse tooth germs

We examined electron-microscopically and histochemically the ultrastructural features of the intraodontoblastic collagen fibrils of the mouse. These collagen fibrils were most common in secreting odontoblasts (pre-odontoblasts) of the maturating stages. In such cells they were most numerous at the peripheral zone of the Golgi apparatus, and were sometimes seen in odontoblastic processes. Intraodontoblastic collagen fibrils also had morphological variations including a banded structure enclosed by limiting membranes of vacuoles, fusion with primary lysosomes, and an electron-dense material covering with a structure that was not banded. Study of acid phosphatase activity showed that these structural changes were caused by the degradation of intraodontoblastic collagen fibrils by lysosomes. The results of studies of the permeation of lanthanum nitrate and the alkaline phosphatase reaction showed that these collagen fibrils were separate from the extracellular matrix and that there was no phagocytosis of the odontoblasts.     

Ultrastructural features of adult human tendon

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

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.     

Designed to fail: a novel mode of collagen fibril disruption and its relevance to tissue toughness

Collagen fibrils are nanostructured biological cables essential to the structural integrity of many of our tissues. Consequently, understanding the structural basis of their robust mechanical properties is of great interest. Here we present what to our knowledge is a novel mode of collagen fibril disruption that provides new insights into both the structure and mechanics of native collagen fibrils. Using enzyme probes for denatured collagen and scanning electron microscopy, we show that mechanically overloading collagen fibrils from bovine tail tendons causes them to undergo a sequential, two-stage, selective molecular failure process. Denatured collagen molecules-meaning molecules with a reduced degree of time-averaged helicity compared to those packed in undamaged fibrils-were first created within kinks that developed at discrete, repeating locations along the length of fibrils. There, collagen denaturation within the kinks was concentrated within certain subfibrils. Additional denatured molecules were then created along the surface of some disrupted fibrils. The heterogeneity of the disruption within fibrils suggests that either mechanical load is not carried equally by a fibril's subcomponents or that the subcomponents do not possess homogenous mechanical properties. Meanwhile, the creation of denatured collagen molecules, which necessarily involves the energy intensive breaking of intramolecular hydrogen bonds, provides a physical basis for the toughness of collagen fibrils.