Two forms of apatite deposited during mineralization of the hen tendon.

Old hen tendon provides a model suitable for the study of calcification in an extracellular matrix. In the present study, we observed the mineralizing substances of hen tendon by scanning electron microscopy of plasma-osmium-coated specimens and by transmission electron microscopy of those processed by a plasma-polymerization film replica method. The mineralizing front area revealed a number of elliptical particles fused to each other and forming rod-like structures oriented parallel to collagen fibrils. The area of advanced mineralization possessed non-mineralizing cavities, in which tendon cells were likely to exist. At this site, we recognized a second form of mineral structure, one in which the crystals had a scale-like morphology and were deposited onto the major first-form mineral component. This crystal form was similar to hydroxyapatite synthesized under wet reaction conditions. These findings strongly suggest that the second form of mineral formed independent of collagen fibrils existed together with the predominant, collagen-dependent form of mineral. We speculate that cell membranes and an extremely slow mineralization process may contribute to the formation of this form of mineral during the mineralization process in the hen tendon.     

The twisted collagen network of the box-fish scutes

The present article describes the three-dimensional arrangement of collagen fibrils in dermal plates of different species of Ostraciidae. These dermal plates or 'scutes' are transformed scales, which have a polygonal shape and form a rigid tiling. They are natural composites, associating a fibrous network with a mineral deposit lying at two different levels of the scute, the 'ceiling' and the 'floor', plus a set of similarly mineralized walls joining the two levels. The three-dimensional structure of the collagen network can be compared to that of 'plywood': fibrils align parallel within superposed layers of uniform thickness, and their direction changes from layer to layer. In the dermal plate, two types of plywood have been evidenced: (1) one lying between the two mineralized plates, where the orientation of fibrils rotates continuously, and (2) one under the lower plate, with thick layers of fibrils, each showing a constant orientation, but abrupt angular changes are observed at the transition from one layer to the following one. In oblique sections, both types of plywood reveal large series of arced patterns, testifying to a twisted arrangement of collagen fibrils, analogous to the arrangement of molecules or polymers in cholesteric liquid crystals. The network is reinforced by some collagen fibrils running unidirectionally and almost normally to the lamellate structure. Moreover in the overall organization of the scute, these plywood systems form a set of nested boxes. This original architecture is compared to the arrangement of the collagenous network previously described in most fish scales and in other extracellular matrices.     

Mechanical properties of mineralized collagen fibrils as influenced by demineralization

Dentin and bone derive their mechanical properties from a complex arrangement of collagen type-I fibrils reinforced with nanocrystalline apatite mineral in extra- and intrafibrillar compartments. While mechanical properties have been determined for the bulk of the mineralized tissue, information on the mechanics of the individual fibril is limited. Here, atomic force microscopy was used on individual collagen fibrils to study structural and mechanical changes during acid etching. The characteristic 67 nm periodicity of gap zones was not observed on the mineralized fibril, but became apparent and increasingly pronounced with continuous demineralization. AFM-nanoindentation showed a decrease in modulus from 1.5 GPa to 50 MPa during acid etching of individual collagen fibrils and revealed that the modulus profile followed the axial periodicity. The nanomechanical data, Raman spectroscopy and SAXS support the hypothesis that intrafibrillar mineral etches at a substantially slower rate than the extrafibrillar mineral. These findings are relevant for understanding the biomechanics and design principles of calcified tissues derived from collagen matrices.     

Changes in glycosaminoglycan binding to collagen during desmoid mineralization as revealed by different electron-microscopic staining techniques.

Mineralization at collagen fibrils is regulated by glycosaminoglycans (GAG). Alterations in proteoglycan composition during mineralization as well as inhibition of mineralization by GAGs are well documented. Collagen-GAG interactions during desmoid osteogenesis in fetal rat calvariae were investigated ultrastructurally by means of different fixation techniques. Mineralization was restricted to the collagen of the osteoid at the ectocranial side. Beyond the osteoid, one layer containing degenerated cells was found, followed by sheets of healthy osteoblasts with nonmineralized collagen fibrils. These fibrils were ordered in bundles, but were irregularly arranged in the mineralized osteoid. After fixation in glutaraldehyde-ruthenium red (GA-RR), small RR-positive granules were periodically attached to the fibrils of the nonmineralized collagen. These granules were absent at collagen in the mineralized osteoid. Periodically bound granules (periodicity of 62 nm) could clearly be demonstrated along collagen fibrils by pretreatment with the positively charged protamine sulfate and subsequent fixation in GA-RR in the nonmineralized collagen. In the mineralized osteoid, however, these granules were present, but periodic binding was missing. Heparin pretreatment followed by fixation in GA-RR revealed periodically bound fine strands between collagen fibrils running parallel in the nonmineralized collagen; these threads were absent in the mineralizing osteoid. Restriction of mineralization to osteoid at the mineralization border may be reflected by the observed changes in GAG binding to collagen fibrils within the osteoid of developing fetal calvariae in contrast to binding to collagen in nonmineralized areas.     

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.     

Neutron diffraction studies of collagen in fully mineralized bone

Neutron diffraction measurements have been made of the equatorial and meridional spacings of collagen in fully mineralized mature bovine bone and demineralized bone collagen, in both wet and dry conditions. The collagen equatorial spacing in wet mineralized bovine bone is 1.24 nm, substantially lower than the 1.53 nm value observed in wet demineralized bovine bone collagen. Corresponding spacings for dry bone and demineralized bone collagen are 1.16 nm and 1.12 nm, respectively. The collagen meridional long spacing in mineralized bovine bone is 63.6 nm wet and 63.4 nm dry. These data indicate that collagen in fully mineralized bovine bone is considerably more closely packed than had been assumed previously, with a packing density similar to that of the relatively crystalline collagens such as wet rat tail tendon. The data also suggest that less space is available for mineral within the collagen fibrils in bovine bone than had previously been assumed, and that the major portion of the mineral in this bone must be located outside the fibrils.     

Ultrastructural data on the scales of the dipnoan Protoptems annectens (Sarcopterygii, Osteichthyes)

The structure and the mineralization of the scales of the living dipnoan (lungfish) Protoptems annectens have been investigated by transmission electron microscopy (TEM). The thin and imbricated scales are composed of two layers: the squamulae and the basal plate. At the outer surface, the squamulae form isolated plates superficially ornamented with spines and concretions and made up of acellular bone. After demineralization, the squamulae show a heterogeneous organic matrix composed of thin randomly oriented collagen fibrils forming a loose network within which the concretions appear as electronlucent circular areas. Abundant and aggregated concretions are located within the spines. The crystallites are oriented by the collagen fibrils except in the concretions. Anchoring bundles composed of parallel collagen fibrils arise from the squamulae and connect the scales to the overlying dermis.
The basal plate, the most developed part of the scale, is made up of isopedine. Its main component consists of thick, closely packed collagen fibrils organized in a 'double twisted plywood-like structure'. Fibroblasts are present in the basal plate. Mineralization occurs only in few plies located beneath the squamulae. Mandl's corpuscles are found in front of the mineralization front. The mineral deposit is oriented by the collagen fibrils.
The scales of Protoptems annectens differ from the typical elasmoid scales of the teleosts by the peculiar structure of the squamulae, nevertheless they show enough structural characteristics to support the hypothesis that they can be considered as scales of the elasmoid grade, which have retained some plesiomorphic characteristics.     

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.     

Embryonic avian cornea contains layers of collagen with greater than average stability

A unique morphological feature of the embryonic avian cornea is the uniformity of its complement of striated collagen fibrils, each of which has a diameter of 25 nm. We have asked whether this apparent morphological uniformity also reflects an inherent uniformity of the structural and physical properties of these fibrils. For this we have examined the in situ thermal stability of the type I collagen within these fibrils. Corneal tissue sections were reacted at progressively higher temperatures with conformation-dependent monoclonal antibodies directed against the triple-helical domain of the type I collagen molecule. These studies show that the cornea contains layers of collagen fibrils with greater than average stability. The two most prominent of these extend uninterrupted across the entire width of the cornea, and then appear to insert into thick bundles of scleral collagen, which in turn appear to insert into the scleral ossicles, a ring of bony plates which circumscribe the sclera of the avian eye. Once formed, the bands may act to stabilize the shape of the cornea or, conversely, to alter it during accommodation.     

A model for the ultrastructure of bone based on electron microscopy of ion-milled sections

The relationship between the mineral component of bone and associated collagen has been a matter of continued dispute. We use transmission electron microscopy (TEM) of cryogenically ion milled sections of fully-mineralized cortical bone to study the spatial and topological relationship between mineral and collagen. We observe that hydroxyapatite (HA) occurs largely as elongated plate-like structures which are external to and oriented parallel to the collagen fibrils. Dark field images suggest that the structures ("mineral structures") are polycrystalline. They are approximately 5 nm thick, 70 nm wide and several hundred nm long. Using energy-dispersive X-ray analysis we show that approximately 70% of the HA occurs as mineral structures external to the fibrils. The remainder is found constrained to the gap zones. Comparative studies of other species suggest that this structural motif is ubiquitous in all vertebrates.     

The structure of the osteoderms in the Gekko: Tarentola mauritanica

Histological and cytological analysis reveals that the osteoderms of Tarentola mauritanica are composed of an outer part superimposed on a basal region. The structure of both parts can be related to that of the surrounding dermis. The basal part of the osteoderms, inserted in the dense dermis, is made up of abundant closely packed collagen fibrils that orient the mineral deposit. The outer part, located in the superficial loose dermis, is crossed by few bundles of mineralized collagen fibrils arising from the basal part. These bundles connect the osteoderm to the overlying loose dermis. The outer superficial part is characterized by the presence of mineralized globules surrounding the mineralized collagen bundles. In these globules, the crystals are deposited on a microfibrillar matrix rich in acidic mucosubstances and composed of radially oriented, tangled microfilaments that lie among the collagen bundles. The two different mineralizing systems in the osteoderms of Tarentola mauritanica may reflect two different organic matrices. The mineral is deposited in a preexisting dermal tissue, as a "metaplastic ossification," and is another expression of the potential retained by the reptilian dermis to form mineralized structures.     

Structure and function of bone collagen fibrils

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

Microstructural Features of Non-Union of Human Humeral Shaft Fracture

Microstructures of non-unions of human humeral shaft fractures were investigated by using scanning electron microscopy, transmission electron microscopy, and X-ray microdiffraction. The non-union has a trabeculae structural framework similar to woven bone. Among the trabeculae are cavities that are subdivided into small chambers by thin plates of collagen fibrils. Some chambers are filled with variously shaped mineralized particles several micrometers in size. The collagen fibrils in both the trabeculae and the thin plates were only slightly mineralized by hydroxyapatite. Vesicles loaded with noncrystalline calcium phosphate (NCP) were observed in most mineralized particles, and brushite crystals with special morphology were seen to be embedded in some particles in irregular shapes. X-ray microdiffraction results indicated that the mineral phases in the non-unions were mainly NCP in addition to small amounts of hydroxyapatite and brushite. NCP deposition and insufficient mineralization of the collagen fibrils may be two important microstructural features of the non-unions of human humeral shaft fractures different from normally repaired bone callus.     

Energy dispersive X-ray diffraction by collagen fibrils in costal cartilage using synchrotron radiation

Low-angle X-ray diffraction patterns from the isotropic distribution of collagen fibrils, which occur in low concentrations in costal cartilage, were recorded using synchrotron radiation. An energy dispersive technique was used to exploit the properties of synchrotron radiation to the full. The third, fourth, fifth and sixth diffraction orders from the axial periodicity of the fibrils were recorded and used to calculate a value for this periodicity of 67 ± 1 nm. This result is in good agreement with measurements made on amianthoid areas as well as from fibrils in tendon, which consist of a chemically distinct form of collagen.     

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.     

Early mineral deposition in calcifying tendon characterized by high voltage electron microscopy and three-dimensional graphic imaging.

Extracellular matrix organization and the spatial relationship between collagen fibrils, vesicular structures, and the first deposits of mineral in the calcifying leg tendon from the domestic turkey, Meleagris gallopavo, have been investigated by high voltage electron microscopy and three-dimensional computer graphic imaging of serial thick tissue sections. The work demonstrates that the tendon extracellular matrix is a complex assembly of somewhat flexible, highly aligned collagen fibrils with different diameters and occasionally opposite directionality. Smaller collagen fibrils appear to branch from larger fibrils or to aggregate to form those of greater size. While the matrices are dominated by fibrils, space exists between adjacent packed fibrils. The three-dimensional perspective indicates that approximately 60% of the total tendon volume is extrafibrillar over the regions examined. The first observable mineral in this tissue is extrafibrillar and appears to derive from vesicles. This view of three-dimensional matrix-mineral spatial relations supports earlier two-dimensional results that mineral is initially associated with membrane-invested vesicles and is deposited between collagen fibrils, but it is distinct in showing the mineral at different depths in the matrix rather than at a single depth as deduced from two-dimensional conventional electron microscopy. These results are important in the onset and development of tendon calcification in that they suggest, first, that collagen fibrils appear to be aligned three-dimensionally such that their hole zones are in contiguous arrangement. This situation may create channels or grooves within the collagen volume to accommodate extensive mineral deposition in association with the fibrils. Second, the results indicate that there are widely dispersed sites of vesicle-mediated mineralization in the tendon matrix, that the bulk of mineralization in this tissue is collagen-mediated, and that, while vesicles may possibly exert some local influence temporally on mineralization of neighboring collagen, vesicle- and collagen-mediated mineralization arise at spatially and structurally distinct sites by independent nucleation phenomena. Such concepts are fundamental in considerations of possible mechanisms of mineralization of tendon and potentially of other normally calcifying vertebrate tissues in general.     

The nanometer-scale structure of amyloid-Β visualized by atomic force microscopy

Amyloid-Β (AΒ) is the major protein component of neuritic plaques found in Alzheimer's disease. Evidence suggests that the physical aggregation state of AΒ directly influences neurotoxicity and specific cellular biochemical events. Atomic force microscopy (AFM) is used to investigate the three-dimensional structure of aggregated AΒ and characterize aggregate/fibril size, structure, and distribution. Aggregates are characterized by fibril length and packing densities. The packing densities correspond to the differential thickness of fiber aggregates along az axis (fiber height above thex-y imaging surface). Densely packed aggregates (≥100 nm thick) were observed. At the edges of these densely packed regions and in dispersed regions, three types of AΒ fibrils were observed. These were classified by fibril thickness into three size ranges: 2–3 nm thick, 4–6 nm thick, and 8–12 nm thick. Some of the two thicker classes of fibrils exhibited pronounced axial periodicity. Substructural features observed included fibril branching or annealing and a height periodicity which varied with fibril thickness. When identical samples were visualized with AFM and electron microscopy (EM) the thicker fibrils (4–6 nm and 8–12 nm thick) had similar morphology. In comparison, the densely packed regions of ～≥100 nm thickness observed by AFM were difficult to resolve by EM. The small, 2- to 3-nm-thick, fibrils were not observed by EM even though they were routinely imaged by AFM. These studies demonstrate that AFM imaging of AΒ fibrils can, for the first time, resolve nanometer-scale,z-axis, surface-height (thickness) fibril features. Concurrentx-y surface scans of fibrils reveal the surface submicrometer structure and organization of aggregated AΒ. Thus, when AFM imaging of AΒ is combined with, and correlated to, careful studies of cellular AΒ toxicity it may be possible to relate certain AΒ structural features to cellular neurotoxicity.     

In situ atomic force microscopy of partially demineralized human dentin collagen fibrils

Dentin collagen fibrils were studied in situ by atomic force microscopy (AFM). New data on size distribution and the axial repeat distance of hydrated and dehydrated collagen type I fibrils are presented. Polished dentin disks from third molars were partially demineralized with citric acid, leaving proteins and the collagen matrix. At this stage collagen fibrils were not resolved by AFM, but after exposure to NaOCl(aq) for 100-240 s, and presumably due to the removal of noncollagenous proteins, individual collagen fibrils and the fibril network of dentin connected to the mineralized substrate were revealed. High-aspect-ratio silicon tips in tapping mode were used to image the soft fibril network. Hydrated fibrils showed three distinct groups of diameters: 100, 91, and 83 nm and a narrow distribution of the axial repeat distance at 67 nm. Dehydration resulted in a broad distribution of the fibril diameters between 75 and 105 nm and a division of the axial repeat distance into three groups at 67, 62, and 57 nm. Subfibrillar features (4 nm) were observed on hydrated and dehydrated fibrils. The gap depth between the thick and thin repeating segments of the fibrils varied from 3 to 7 nm. Phase mode revealed mineral particles on the transition from the gap to the overlap zone of the fibrils. This method appears to be a powerful tool for the analysis of fibrillar collagen structures in calcified tissues and may aid in understanding the differences in collagen affected by chemical treatments or by diseases.     

Structural peculiarities of the tubercles in the skin of the turbot,Scophthalmus maximus (L., 1758) (Osteichthyes,Pleuronectiformes, Scophthalmidae)

The structure of the bony tubercles of the turbot, Scophthalmus maximus (L., 1758), was examined using ground sections, microradiography, SEM, and TEM. The tubercles are small, isolated, mineralized conical plates randomly distributed in the eyed side of the body. They are composed of three layers: the outer limiting layer, the external layer, and the basal plate, which make up the thin and flat elasmoid scales of Teleostei. The main difference between regular elasmoid scales and bony tubercles lies in the organization and the growth of the basal plate. Indeed, the conical shape of the tubercle is the result of a prominent thickening of the central part of the basal plate where the collagen matrix is organized in a complicated three-dimensional network. Densely packed thick collagen fibrils form superimposed plies organized in a plywood-like structure that resembles that of the elasmoid scales but it is criss-crossed by numerous vertical sheets of thin collagen fibrils. The tubercles originate from thin and flat plates located in the skin of larvae and juveniles, whose structure is that of regular-developing elasmoid scales. Thus, the tubercles of Scophthalmus maximus could be considered as modified elasmoid scales rather than bony structures. They might be the result of specific arrangements related to the general trend of reduction of the dermal skeleton in the teleostean lineage.     

Type VI collagen in extracellular, 100-nm periodic filaments and fibrils: identification by immunoelectron microscopy

Filaments and fibrils that exhibit a 100-nm axial periodicity and occur in the medium and in the deposited extracellular matrix of chicken embryo and human fibroblast cultures have been tentatively identified with type VI collagen on the basis of their similar structural characteristics (Bruns, R. R., 1984, J. Ultrastruct. Res., 89:136-145). Using indirect immunoelectron microscopy and specific monoclonal and polyclonal antibodies, we now report their positive identification with collagen VI and their distribution in fibroblast cultures and in tendon. Primary human foreskin fibroblast cultures, labeled with anti-type VI antibody and studied by fluorescence microscopy, showed a progressive increase in labeling and changes in distribution with time up to 8 d in culture. With immunoelectron microscopy and monoclonal antibodies to human type VI collagen followed by goat anti-mouse IgG coupled to colloidal gold, they showed in thin sections specific 100-nm periodic labeling on extracellular filaments and fibrils: one monoclonal antibody (3C4) attached to the band region and another (4B10) to the interband region of the filaments and fibrils. Rabbit antiserum to type VI collagen also localized on the band region, but the staining was less well defined. Control experiments with antibodies to fibronectin and to procollagen types I and III labeled other filaments and fibrils, but not those with a 100-nm period. Heavy metal-stained fibrils with the same periodic and structural characteristics also have been found in both adult rat tail tendon and embryonic chicken tendon subjected to prolonged incubation in culture medium or treatment with adenosine 5'-triphosphate at pH 4.6. We conclude that the 100-nm periodic filaments and fibrils represent the native aggregate form of type VI collagen. It is likely that banded fibrils of the same periodicity and appearance, reported by many observers over the years in a wide range of normal and pathological tissues, are at least in part, type VI collagen.