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.     

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.     

Mechanical properties of human patellar tendon at the hierarchical levels of tendon and fibril

Tendons are strong hierarchical structures, but how tensile forces are transmitted between different levels remains incompletely understood. Collagen fibrils are thought to be primary determinants of whole tendon properties, and therefore we hypothesized that the whole human patellar tendon and its distinct collagen fibrils would display similar mechanical properties. Human patellar tendons (n = 5) were mechanically tested in vivo by ultrasonography. Biopsies were obtained from each tendon, and individual collagen fibrils were dissected and tested mechanically by atomic force microscopy. The Young's modulus was 2.0 ± 0.5 GPa, and the toe region reached 3.3 ± 1.9% strain in whole patellar tendons. Based on dry cross-sectional area, the Young's modulus of isolated collagen fibrils was 2.8 ± 0.3 GPa, and the toe region reached 0.86 ± 0.08% strain. The measured fibril modulus was insufficient to account for the modulus of the tendon in vivo when fibril content in the tendon was accounted for. Thus, our original hypothesis was not supported, although the in vitro fibril modulus corresponded well with reported in vitro tendon values. This correspondence together with the fibril modulus not being greater than that of tendon supports that fibrillar rather than interfibrillar properties govern the subfailure tendon response, making the fibrillar level a meaningful target of intervention. The lower modulus found in vitro suggests a possible adverse effect of removing the tissue from its natural environment. In addition to the primary work comparing the two hierarchical levels, we also verified the existence of viscoelastic behavior in isolated human collagen fibrils.     

Frog tendon structure and its relationship with locomotor modes

Tendon collagen fibrils are the basic force‐transmitting units of the tendon. Yet, surprisingly little is known about the diversity in tendon anatomy and ultrastructure, and the possible relationships between this diversity and locomotor modes utilized. Our main objectives were to investigate: (a) the ultra‐structural anatomy of the tendons in the digits of frogs; (b) the diversity of collagen fibril diameters across frogs with different locomotor modes; (c) the relationship between morphology, as expressed by the morphology of collagen fibrils and tendons, and locomotor modes. To assess the relationship between morphology and the locomotor modes of the sampled taxa we performed a principal component analysis considering body length, fibrillar cross sectional area (CSA) and tendon CSA. A MANOVA showed that differences between species with different locomotor modes were significant with collagen fibril diameter being the discriminating factor. Overall, our data related the greatest collagen fibril diameter to the most demanding locomotor modes, conversely, the smallest collagen fibril CSA and the highest tendon CSA were observed in animals showing a hopping locomotion requiring likely little absorption of landing forces given the short jump distances.     

Development of collagen fibril organization and collagen crimp patterns during tendon healing

Normal tendon comprises coaxially aligned bundles of crimped collagen fibres each of which possesses a fibrillar substructure. In acute traumatic injury this level of organization is disrupted and the mechanical function of the tendon impaired. During repair, a degree of recovery of the fibrillar structure takes place. In this tudy we have assessed the re-establishment of tendon organization after injury on the basis of the collagen fibril diameter distribution and the collagen crimp parameters. Crimp became undetectable following injury but one month later was present throughout the tissue. At this time the periodicity was greatly reduced by comparison with that of the normal tendon and normal values were not re-established within 14 months following injury. Collagen fibril diameters remained abnormally small over this same period of time. In particular, fibrils of diameters in excess of 100 nm, commonly found in normal and contralateral tendons, were totally absent from the observed distributions in the healing tendons. Such large diameter fibrils often account for as much as 50% of the total mass of collagen present in the uninjured tissue. Thus the mechanical properties of the healing tendon may remain significantly different from those of normal tendon for a minimum time of 14 months after injury.     

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.     

Quantitative electron microscope observations of the collagen fibrils in rat-tail tendon

An electron microscope study of collagen fibrils from fixed tail tendons of rats has revealed that from some time shortly after birth until maturity, the fibril diameters have a bimodal distribution. The “two” types of fibril are indistinguishable in both transverse and longitudinal section. Unfixed specimens of eight-week-old-tail tendon showed a similar bimodal distribution of diameters though the positions of the peak values compared to fixed specimens of an eight-week-old-tail tendon were shifted upwards by about 30%. It has also been shown quantitatively that the polar collagen fibrils are directed randomly “up” and “down” with respect to their neighbors. Whilst it has been suggested by others that anastomosis is a feature of collagen structure, the results presented here do not support this hypothesis. Fibrillar units ～ 140 Å in diameter have been observed and the possibilities that these are elastic fibers or the breakdown products of collagen fibrils have been considered.     

Evidence of a discrete axial structure in unimodal collagen fibrils

The collagen fibrils of cornea, blood vessel walls, skin, gut, interstitial tissues, the sheath of tendons and nerves, and other connective tissues are known to be made of helically wound subfibrils winding at a constant angle to the fibril axis. A critical aspect of this model is that it requires the axial microfibrils to warp in an implausible way. This architecture lends itself quite naturally to an epitaxial layout where collagen microfibrils envelop a central core of a different nature. Here we demonstrate an axial domain in collagen fibrils from rabbit nerve sheath and tendon sheath by means of transmission electron microscopy after a histochemical reaction designed to evidence all polysaccharides and by tapping-mode atomic force microscopy. This axial domain was consistently found in fibrils with helical microfibrils but was not observed in tendon, whose microfibrils run longitudinal and parallel.     

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.     

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     

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.     

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.     

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.     

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

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

Cross-linkage sites in type I collagen fibrils studied by neutron diffraction

Cross-links in tendon collagen are essential for the biomechanical strength of healthy tissue. The nature and position of these cross-links has long been a subject for conjecture. We have approached this problem in a non-destructive manner, by studying neutron diffraction from collagen fibrils that have been specifically deuterated by reduction at keto-amine and Schiff base groups with sodium borodeuteride (NaB2H4). The intensities of the first 23 meridional reflections were recorded for both native and reduced tendons. These data were used to calculate the neutron-scattering density profile of the 67 nm (D) repeat of type I collagen fibrils in rat tail tendon. This approach not only succeeds in determining the location of the cross-linkage sites with respect to the fibril structure, as projected onto the fibre axis, but also presents a novel form of the isomorphous derivative solution to the phase problem.     

Increased C-telopeptide Cross-linking of Tendon Type I Collagen in Fibromodulin-deficient Mice

The controlled assembly of collagen monomers into fibrils, with accompanying intermolecular cross-linking by lysyl oxidase-mediated bonds, is vital to the structural and mechanical integrity of connective tissues. This process is influenced by collagen-associated proteins, including small leucine-rich proteins (SLRPs), but the regulatory mechanisms are not well understood. Deficiency in fibromodulin, an SLRP, causes abnormal collagen fibril ultrastructure and decreased mechanical strength in mouse tendons. In this study, fibromodulin deficiency rendered tendon collagen more resistant to nonproteolytic extraction. The collagen had an increased and altered cross-linking pattern at an early stage of fibril formation. Collagen extracts contained a higher proportion of stably cross-linked α1(I) chains as a result of their C-telopeptide lysines being more completely oxidized to aldehydes. The findings suggest that fibromodulin selectively affects the extent and pattern of lysyl oxidase-mediated collagen cross-linking by sterically hindering access of the enzyme to telopeptides, presumably through binding to the collagen. Such activity implies a broader role for SLRP family members in regulating collagen cross-linking placement and quantity.     

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.     

Dysfunctional tendon collagen fibrillogenesis in collagen VI null mice

Tendons are composed of fibroblasts and collagen fibrils. The fibrils are organized uniaxially and grouped together into fibers. Collagen VI is a non-fibrillar collagen expressed in developing and adult tendons. Human collagen VI mutations result in muscular dystrophy, joint hyperlaxity and contractures. The purpose of this study is to determine the functional roles of collagen VI in tendon matrix assembly. During tendon development, collagen VI was expressed throughout the extracellular matrix, but enriched around fibroblasts and their processes. To analyze the functional roles of collagen VI a mouse model with a targeted inactivation of Col6a1 gene was utilized. Ultrastructural analysis of Col6a1−/− versus wild type tendons demonstrated disorganized extracellular micro-domains and associated collagen fibers in the Col6a1−/− tendon. In Col6a1−/− tendons, fibril structure and diameter distribution were abnormal compared to wild type controls. The diameter distributions were shifted significantly toward the smaller diameters in Col6a1−/− tendons compared to controls. An analysis of fibril density (number/μm²) demonstrated a ~ 2.5 fold increase in the Col6a1−/− versus wild type tendons. In addition, the fibril arrangement and structure were aberrant in the peri-cellular regions of Col6a1−/− tendons with frequent very large fibrils and twisted fibrils observed restricted to this region. The biomechanical properties were analyzed in mature tendons. A significant decrease in cross-sectional area was observed. The percent relaxation, maximum load, maximum stress, stiffness and modulus were analyzed and Col6a1−/− tendons demonstrated a significant reduction in maximum load and stiffness compared to wild type tendons. An increase in matrix metalloproteinase activity was suggested in the absence of collagen VI. This suggests alterations in tenocyte expression due to disruption of cell-matrix interactions. The changes in expression may result in alterations in the peri-cellular environment. In addition, the absence of collagen VI may alter the sequestering of regulatory molecules such as leucine rich proteoglycans. These changes would result in dysfunctional regulation of tendon fibrillogenesis indirectly mediated by collagen VI.     

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.     

Three-dimensional organization of collagen fibres in tendon

Electron microscopic observations are presented on thin sections of excised chicken breast tendon following the introduction and diffusion of aqueous solutions of heavy metal salts. The dark banded regions of the collagen fibrils are seen to be in near-perfect register throughout the diameter of each fibril and, in many cases, to be continuous across the intervening ground substance. Clusters of uranyl ions form well-defined chains extending across the interfibrillar space between neighbouring fibrils, a distance of several hundred nanometres. It is suggested that the high degree of organization characteristic of collagen fibrils in tissue may perhaps be a property not only of the protein but also of the ground substance in which it is embedded, the fibres merely rendering visible a lattice pattern of their surroundings to which they have conformed.