Striated Collagen Fibrils Research Articles

Induction of mesenchymal stem cell differentiation and cartilage formation by cross-linker-free collagen microspheres.

Because of poor self-healing ability, joint cartilage can undergo irreversible degradation in the course of various diseases or after injury. A promising approach for cartilage engineering consists of using of mesenchymal stem cells (MSC) and a differentiation factor combined with an injectable carrier biomaterial. We describe here a novel synthesis route for native collagen microspheres that does not involve the use of potentially toxic crosslinking agents. An emulsion was formed between a type I collagen solution and perfluorinated oil, stabilised by a biocompatible triblock perfluorinated copolymer surfactant. Spherical microparticles of fibrillar collagen were formed through a sol-gel transition induced by ammonia vapours. Electron microscopy observations showed that these self-cross-linked microspheres were constituted by a gel of striated collagen fibrils. Microspheres that were loaded with transforming growth factor beta (TGF-β)3 progressively released this differentiation factor over a four weeks period. Human MSC rapidly adhered to TGF-β3-loaded microspheres and, after 21d of culture, exhibited typical chondrocyte morphology and produced an uncalcified matrix made of the predominant cartilage components, aggrecan and type II collagen, but devoid of the hypertrophic marker type X collagen. Subcutaneous co-injection of MSC and TGF-β3-loaded microspheres in mice consistently led to the formation of a cartilage-like tissue, which was however hypertrophic, calcified and vascularised. In conclusion, we developed cross-linker free collagen microspheres that allowed chondrogenic differentiation of MSC in vitro and in vivo.

Influence of in vivo or vitro microenvironment on the mechanical properties and histological structure of tissue engineered cartilage

To investigate the influence of in vivo or vitro microenvironment on the mechanical properties and histological structure of tissue engineered cartilage, and to provide the appropriate parameters for cartilage construct in vitro. Human fetal articular chondrocytes were cultured and expanded in vitro, the passage 2 chondrocytes were seeded at the density of 6 x 10(7) cells/cm3 to cylindric dimensional scaffolds made by polyglycolic acid (PGA) and polylactic acid (PLA). These constructs were cultured in vitro for 4 weeks. After 4 weeks, part of samples were implanted subcutaneously into nude mice for 6 and 12 weeks, the others continued to be cultured in vitro. All specimens were harvested after 6 and 12 weeks, and evaluated by gross observation, histology, histochemistry, ultrastructure and mechanical test. All specimens in vivo and vitro eventually formed good shape hyaline cartilage. The constructs in vivo group was white color with smooth surface, and had better mechanical properties than those in vitro, by TEM we can observe the thick and striated collagen fibers in regularly arranged collagen fibril which was similar to adult articular cartilage. The constructs in vitro group was yellow color with coarse surface, the appearance and ultrastructure was similar to fetal articular cartilage. Specimens implanted for 12 weeks in vivo had better compressive modulus(38.28 +/- 3.95) MPa and collagen diameter (41.58 +/- 2.78) nm than those cultured in vitro at the same time (4.12 +/- 0.63) MPa, (15.83 +/- 1.70) nm (P < 0. 01). The structure and function of human tissue engineered cartilage became mature gradually from vitro to vivo, thick and striated collagen fibrils net similar to adult articular cartilage can be formed in constructs of vivo group,increased collagen cross-linking might be the reason that their mechanical properties been greatly improved.

Authentic fibroblast matrix in dermal equivalents normalises epidermal histogenesis and dermo-epidermal junction in organotypic co-culture

Besides medical application as composite skin grafts, in vitro constructed skin equivalents (SEs) or organotypic co-cultures represent valuable tools for cutaneous biology. Major drawbacks of conventional models, employing collagen hydrogels as dermal equivalents (DEs), are a rather poor stability and limited life span, restricting studies to early phases of skin regeneration. Here we present an improved stabilised in vitro model actually providing the basis for skin-like homeostasis. Keratinocytes were grown on dermal equivalents (DEs) reinforced by modified hyaluronic acid fibres (Hyalograft-3D) and colonised with skin fibroblasts, producing genuine dermis-type matrix. These SEs developed a superior epidermal architecture with regular differentiation and ultrastructure, which occurred also faster than in SEs based on collagen-DEs. Critical aspects of differentiation, still unbalanced in early stages, were perfectly re-normalised, most strikingly the co-expression of keratins K1/K10 and downregulation of regeneration-associated keratins such as K16. The restriction of integrin and K15 distribution as well as keratinocyte proliferation to the basal layer underlined the restored tissue polarity, while the drop of growth rates towards physiological levels implied finally accomplishment of homeostasis. This correlated to faster basement membrane (BM) formation and ultrastructurally defined dermo-epidermal junction including abundant anchoring fibrils for strong tissue connection. Whereas the fibroblasts in the scaffold initially secreted a typical provisional regenerative matrix (fibronectin, tenascin), with time collagens of mature dermis (type I and III) were accumulating giving rise to an in vivo-like matrix with regularly organised bundles of striated collagen fibrils. In contrast to the more catabolic state in conventional DEs, the de novo reconstruction of genuine dermal tissue seemed to be a key element for maintaining prolonged normal keratinocyte proliferation (followed up to 8 wks), fulfilling the criteria of tissue-homeostasis, and possibly providing a stem cell niche.

The nature and function of mononuclear cells on the resorbed surfaces of bone in the reversal phase during remodeling

In a reversal phase of bone remodeling many mononuclear cells appear on the resorbed surfaces of bone with characteristic reversal lines as revealed by transmission electron microscopy (TEM). However, these mononuclear cells have been variously hypothesized or reported. The present study examined the TEM features on the resorbed surfaces of three calcified connective tissues, and aimed to clarify the nature and function of the mononuclear cells in a reversal phase. Dentine slices cultured with isolated osteoclasts, human deciduous teeth, and rat mandibles were used in this study. Specimens were fixed, decalcified, and then embedded in Epon 812, and sectioned into 0.1-microm-thick ultrathin sections. The ultrathin sections were stained with uranyl acetate and lead citrate, and then examined by TEM. Many sharply pointed collagen fibrils with striation were observed exposed on the resorbed surfaces of cultured dentine slices, but there were neither cells nor reversal lines. The same features were observed on the root dentine surfaces of human deciduous teeth. Under many mononuclear cells in a reversal phase of remodeling, reversal lines were seen on the resorbed surfaces of rat mandibles, but there were no striated collagen fibrils exposed on the bone surfaces. The alternation of the TEM features on the resorbed surfaces before and after the participation of mononuclear cells in a reversal phase of remodeling suggests the nature and function of these cells: they participate in both degrading the demineralized and disrupted matrix left on the resorbed surfaces and forming reversal lines there.

Type V collagen regulates the assembly of collagen fibrils in cultures of bovine vascular smooth muscle cells

Vascular smooth muscle cells (SMCs), the major cellular constituent of the medial layer of an artery, synthesize the majority of connective tissue proteins, including fibrillar collagen types I, III, and V/XI. Proper collagen synthesis and deposition, which are important for the integrity of the arterial wall, require the antioxidant vitamin C. Vitamin C serves as cofactor for the enzymes prolyl and lysyl hydroxylase, which are responsible for the proper hydroxylation of collagen. Here, the role of type V collagen in the assembly of collagen fibrils in the extracellular matrix (ECM) of cultured vascular SMCs was investigated. Treatment of SMCs with vitamin C resulted in a dramatic induction in the levels of the cell-layer associated pepsin-resistant type V collagen, whereas only a minor induction in the levels of types I and III collagen was detected. Of note, the deposition of type V collagen was accompanied by the formation of striated collagen fibrils in the ECM. Immunohistochemistry demonstrated that type V collagen, but not type I collagen, became masked as collagen fibrils matured. Furthermore, the relative ratio of type V to type I collagen decreased as the ECM matured as a function of days in culture, and this decrease was accompanied by an increase in the diameter of collagen fibrils. Together these results suggest that the masking of type V collagen is caused by its internalization on continuous deposition of type I collagen on the exterior of the fibril. Furthermore, they suggest that type V collagen acts as framework for the initial assembly of collagen molecules into heterotypic fibrils, regulating the diameter and architecture of these fibrils.

Microfibrillar composition of umbilical cord matrix: Characterization of fibrillin, collagen VI and intact collagen V

Ultrastructural studies made on human umbilical cord revealed that the striated collagen fibrils of the Wharton's jelly matrix are mixed with many microfibrillar structures. Microfibrils were found with a tubular cross-section of 10–12 nm diameter and were organized as beaded filaments characteristic of fibrillin-rich microfibrils. Beads had an average diameter of 25 nm and were spaced at about 50–80 nm. This ultrastructural observation was confirmed by indirect immunofluorescent staining of the jelly matrix using monoclonal antibody to fibrillin. Another constituent of the microfibrillar network was present as typical 100-nm periodic filaments of type VI collagen. Indirect immunofluorescent staining using antibodies to collagen VI showed for the first time that this collagen appeared to be distributed largely in the jelly matrix. In addition, other microfibrils with no specific banding pattern were observed. These microfibrils may constitute an organization of type V collagen different from the one which is generally assembled in heterotypic fibrils with collagen I. Among the latter heterotypic fibrils, type V collagen was studied using an anti-peptide antibody to the most N-terminal non-collagenous region of its α2(V) chain. This antibody recognized a filamentous mesh decorating the bundles of collagen fibrils by immunofluorescent staining. This indicates that at least this part of α2(V) chain may be accessible to the antibody at the surface of the fibrils.

Type VI collagen bound to collagen fibrils by chondroitin/dermatan sulfate glycosaminoglycan in mouse corneal stroma

We investigated the ultrastructural localization of type VI collagen in mouse corneal stroma and its relationship to striated collagen fibrils and glycosaminoglycans, using chondroitinase ABC digestion and immunoelectron microscopy with colloidal gold particles. After chondroitinase ABC digestion, the arrangement of striated collagen fibrils was disrupted, and large spaces containing widely scattered fibrils appeared. The spaces were filled by filamentous networks that were stained by anti-type VI collagen IgG, which were apparently clumps of beaded filament. Interfibrillar type VI collagen beaded filaments and immunogold particles decreased. Our results indicate that type VI collagen is bound to the striated collagen fibrils by mediation of chrondroitin/dermatan sulfate glycosaminoglycan or proteoglycan. We believe that this interaction is essential to the orderly arrangement of the striated collagen fibrils, which results in corneal transparency.

Organization of Fibrillar Collagen in the Human and Bovine Cornea

The localization and fibrillar organization of collagen types V and III in the human and bovine corneal stromas were studied. In the chicken cornea, type V co-assembles with type I collagen as heterotypic fibrils and this interaction is involved in the regulation of fibril diameter necessary for corneal transparency. To determine whether this is a regulatory mechanism common to the corneas of different species the human and bovine corneal stroma were studied. Collagen type V was found in the epithelium and Bowman's membrane in the untreated adult human and bovine cornea using immunofluorescence microscopy. In the absence of any treatment, there was no type V reactivity within the stroma. However, type V collagen was detected homogeneously throughout the corneal stroma after treatments that partially disrupt fibril structure. The reactivity was strongest in the cornea, weaker in the limbus and weakest in the sclera. Fetal corneas showed similar reactivity for type V collagen, but unlike the adult, the stroma was slightly reactive. Immunoelectron microscopy demonstrated that type V collagen was associated with disrupted, but not with intact, fibrils in both human and bovine corneal stroma. Type III collagen reactivity was not detected in the cornea, but was present subepithelially in the limbus and in the scleral stroma. These data indicate that type V collagen is a component of striated collagen fibrils throughout the human and bovine corneal stromas. The interaction of types I and V collagen as heterotypic fibrils masks the helical epitope recognized by the monoclonal antibody against type V collagen. The heterotypic interactions of collagen type V indicate a role in the regulation of fibril diameter analogous to that described in the avian cornea.

Positive Regulation of Corneal Type V Collagen mRNA: Analysis by Chicken–Human Heterokaryon Formation

Our previous studies have suggested that type V collagen is at least one factor responsible for the characteristically small, uniform diameter of striated collagen fibrils of the corneal stroma. These fibrils, which are heterotypic combinations of collagen types I and V, contain four- to fivefold more type V collagen than those of tendon and sclera. The latter are much larger and more heterodisperse. This high content of type V collagen in cornea is reflected by an equally elevated content of α1(V) chain mRNA in corneal fibroblasts. Thus, the increased production of the molecule in cornea appears to be regulated at the level of transcription and/or mRNA stability. One possible explanation for this is that corneal fibroblasts contain positive regulatory factors that specifically upregulate transcription of the type V collagen genes and/or increase their mRNA stability. To test this possibility, we have produced transient heterokaryons by fusing chicken corneal fibroblasts with two human noncorneal cell lines selected as containing little if any α1(V) mRNA. If the chicken corneal cells contain positive regulators that can act across species, these regulators should result in increased levels of the human α1(V) transcript. The results were evaluated by reverse transcript–polymerase chain reaction employing a primer pair selected for its ability specifically to amplify part of the human α1(V) mRNA. In fusions between chicken corneal fibroblasts and the human cell lines, after a lag of 10–14 h the heterokaryon-containing cultures showedde novoappearance or upregulation of human α1(V) chain mRNA, compared with that of the parental cell lines. Cultures of the mixed cell types that had not been fused showed no such upregulation, so the effect was not mediated by diffusible substances acting between the cells. Chicken tendon fibroblasts, a low producer of type V collagen, when tested in the same assay, evoked no detectible increase in the human transcript. Thus, corneal cells do contain positive regulators for α1(V) chain mRNA, and this effect is at least somewhat cell specific.

Type VI collagen in mouse masseter tendon, from osseous attachment to myotendinous junction

The association of masseter tendon type VI collagen with other extracellular matrix (ECM) components was examined from osseous attachment to myotendinous junction by immunohistochemistry and transmission electron microscopy with ATP treatment and enzyme digestion. In the tendon proper, fibrocytes extended their processes among bundles of striated collagen fibrils and associated with adjacent cells through amorphous materials, thus forming a three-dimensional network. The amorphous or filamentous material was observed around the fibrocyte cell body and along the cell processes, where the localization of type VI collagen was confirmed by immunohistochemistry using anti-type VI collagen antibody. After treatment with 20 mM adenosine 5'-triphosphate (ATP), 100 nm periodic fibrils, an aggregated form of type VI collagen, were formed in the place where amorphous or filamentous material was present before the treatment. In myotendinous junction, the ATP-aggregated periodic fibrils were observed to associate with the external lamina of the muscle cells as well as among junctional tendon collagen fibrils. In the tendon-bone boundary, ATP-aggregated periodic fibrils were observed around fibrocartilage-like cells in the uncalcifying area but not in the calcification front. Prolonged ATP treatment or hyaluronidase predigestion caused the formation of type VI collagen periodic fibrils in the area near the calcified matrix. The distribution of type VI collagen in mouse masseter tendon is different in different anatomical position. This may reflect the different functional demand for this collagen.

Glycosaminoglycan and collagen fibrillar interactions in the mouse corneal stroma.

When sections of mouse corneal stroma were treated with 20 mM adenosine 5'-triphosphate (ATP) in phosphate buffered saline, pH 4.0, at 37 degrees C and observed by electron microscopy, numerous periodic fibrils with about 100-nm periodicity appeared which were the aggregated form of type VI collagen (type VI collagen fibrils). They occurred in close association with D-periodic fibrillar collagens (striated collagen fibrils). However, when the tissue was digested with chondroitinase ABC or testicular hyaluronidase prior to the ATP treatment, type IV collagen fibrils were segregated from striated collagen fibrils, even though the type VI collagen fibrils themselves aggregated to form the 100 nm-periodic structures. Keratanase or Streptomyces hyaluronidase had no such effect. One possible suggestion is that the ATP-aggregated type VI collagen fibrils are connected with striated collagen fibrils through chondroitin/dermatan sulfate glycosaminoglycans.

An Ultrastructural Investigation of an Early Manifestation of the Posterior Polymorphous Dystrophy of the Cornea

Posterior polymorphous dystrophy of the cornea is an uncommon, but well-recognized, congenital disease affecting Descemet's membrane and endothelium. Visual impairment usually is minimal and slowly progressive. Edema develops in a small number of patients, necessitating a corneal graft in middle age. An early manifestation of posterior polymorphous dystrophy with corneal edema present at birth or soon thereafter is extremely rare; only a few ultrastructural reports are available. The corneal button of a 5-month-old baby with posterior polymorphous dystrophy was examined by conventional light and electron microscopy. The anterior banded zone of Descemet's membrane was thinned (2 microns) in the axial part of the cornea, contained defects in the paraxial part, and was absent at the periphery. Where present, the anterior banded zone was lined posteriorly by a fibrocellular layer (containing fibroblast-like spindle cells and striated collagen fibrils) and a fibrillar layer forming an abnormal posterior collagenous layer. This layer was lined posteriorly by degenerate overlapping endothelial cells, which also were in apposition to the stroma in regions with an absent anterior banded zone. The defects in Descemet's membrane were used by the abnormal keratocytes to migrate between the anterior banded zone and the posterior collagenous layer. At the periphery, a multilayer of spindle cells was present behind the stroma. Failure to produce a continuous anterior banded zone indicates an onset of the disease before the twelfth week of gestation. It appears that, in posterior polymorphous dystrophy, there is a mosaic of better preserved and dystrophic endothelial cells and that the presence or absence of the normal components of Descemet's membrane is determined by the proportion of dystrophic cells in the endothelial cell population.

An ultrastructural study of extracellular fibrillar components of developing mouse mandibular condyle with special reference to type VI collagen

The localization of type VI collagen was examined from birth to 8 weeks of age. Immunohistochemical staining with anti-type VI collagen antibody was strongly positive in the hypertrophic zone and moderately positive in the fibrous zone and the outer periphery of the proliferative zone, but negative in the inner area of the proliferative zone and mature zone. After ATP treatment, type VI collagen periodic fibrils with about 80-nm intervals were frequently observed but only in the fibrous zone. They occurred mainly in the superficial area of this zone, where striated collagen fibrils were sparse, while a few were noted in the inner area, where bundles of collagen fibrils were abundant. From these distributional differences of both components, a subzonation of the fibrous zone into superficial and inner areas is suggested. Moreover, with ATP treatment there were fewer type VI collagen periodic fibrils formed with increasing age (8 weeks). Testicular hyaluronidase digestion before ATP treatment facilitated the formation of periodic fibrils, in all the ages examined, in the intercellular space and around the fibroblastic cells. The interaction of type VI collagen with other components such as collagen fibrils, glycosaminoglycans or proteoglycans may play a part in maintaining the structural integrity of extracellular matrix in the mouse mandibular condyle.

Collagens in the aged human macula

Immunogold cytochemistry was used to investigate the fine structural distribution of collagen types I-VI in Bruch's membrane and choroid of the aged human macula. Macular tissue was obtained from ten eyes, and processed for cryoultramicrotomy and London Resin white embedding. Striated collagen fibrils within the inner and outer collagenous layers were found to contain collagen types I, III and V. In addition, type V collagen was also present in the basement membrane of the choriocapillaris. Gross thickening of the choriocapillaris basement membrane was attributed to the deposition of type IV collagen. However, type IV collagen appeared to be absent from the basement membrane of the retinal pigment epithelium. The interesting location of type VI collagen on the choroidal side of the choriocapillaris suggested that its function is to anchor the choriocapillaris onto the choroid. The collagens studied were absent from fibrous banded material, long-spacing collagen, the elastic layer and amorphous granular material. It was concluded that, of the collagen types studied, only the deposition of type IV collagen contributes to the age-related thickening of Bruch's membrane.

Development and fine structure of the bony scutes in Corydoras arcuatus (Siluriformes, callichthyidae).

The development and the structure of the bony scutes have been studied in a growth series of the armored catfish Corydoras arcuatus using light and electron microscopy. Fibroblast-like cell condensations appear in the dermis, in the posterior region of the caudal peduncle, and these will constitute the scute papillae. Collagen bundles of the preexisting dermis colonized by the papilla cells are remodeled and incorporated in the papilla to form, in addition to newly synthesized woven-fibered bony material, the initium of the scute. This process of formation differs from that described for the dermal papilla of an elasmoid scale. During growth, the osteoblasts surrounding the scute constitute the scute sac in which the scute grows. Parallel-fibered bone is deposited on both sides of the initium, and osteoblasts are incorporated within the scute matrix. The remodeling and incorporation of collagen bundles of the preexisting dermis is maintained during growth only in the deep, anterior region of the scute. The posterior region and the upper surface of the scute are close to the epidermal-dermal boundary. When growth slows down in the upper part of the scute, a characteristic, well-mineralized tissue, composed of thin vertical fibrils and granules and devoid of typical striated collagen fibrils, is deposited on the scute surface. A new term, hyaloine, is introduced for this nonosseous, highly mineralized layer constituting the upper part of the scute. Hyaloine shows thin electron-dense lines, which probably correspond to periodic growth arrests. The structure and localization of the hyaloine are compared to other well-mineralized, similar tissues found on the surface of the dermal skeleton in lower vertebrates. © 1993 Wiley-Liss, Inc.

Two cell lines from bone marrow that differ in terms of collagen synthesis, osteogenic characteristics, and matrix mineralization.

Two cloned cell lines were isolated from cultures of mouse bone-marrow cells. One of the lines, D1, exhibited osteogenic properties and synthesized type-I collagen (alpha 1)2 alpha 2. The second cell line, D2, was not osteogenic and produced a collagen homotrimer (alpha 1)3. Whereas the extracellular matrix of the D1 cell cultures contained striated collagen fibrils, presumably composed of type-I collagen, the homotrimer-producing D2 cells did not demonstrate striated collagen fibrils. Instead, they had thin filaments without detectable striations. Sodium ascorbate stimulated collagen synthesis at the transcriptional level in both the D1 and the D2 cells. The bone-producing characteristics of D1 in vitro included high levels of alkaline phosphatase, increased cyclic adenosine monophosphate on treatment with parathyroid hormone, and expression of osteocalcin mRNA. The D1 cells, unlike the D2 cells, produced a mineralized matrix in vitro. Mineralization in the cultures of the D1 cells occurred in nodules of increased cell density, which also contained the cells with the highest concentrations of collagen mRNA, as shown by in situ hybridization. When the D1 cells were implanted in a diffusion chamber in vivo, a mixture of both osteogenic and adipogenic tissues was formed. This indicates that the D1 cell line is derived from an early marrow stromal precursor that is multipotential.

Collagens in the aged human macular sclera.

Scleral tissue from the region of the human macula was studied by the immunogold labeling technique (cryoultramicrotomy and LR white resin embedding) in an attempt to identify the fine structural distribution of collagen types I-VI. Labeling of the striated collagen fibrils suggested colocalisation of collagen types I, III and V with type V occurring at the fibril surface. Both types V and VI collagen were localised to filamentous strands in the interfibrillar matrix. Collagen types II and IV were absent from the scleral stroma.

New concepts of corneal swelling biophysics

When sections of mouse corneal stroma were treated with 20 mm adenosine 5′-triphosphate (ATP) in phosphate buffered saline, pH 4.0, at 37°C and observed by electron microscopy, numerous periodic fibrils with about 100-nm periodicity appeared which were the aggregated form of type VI collagen (type VI collagen fibrils). They occurred in close association with D-periodic fibrillar collagens (striated collagen fibrils). However, when the tissue was digested with chondroitinase ABC on testicular hyaluronidase prior to the ATP treatment, type VI collagen fibrils were segregated from striated collagen fibrils, even though the type VI collagen fibrils themselves aggregated to form the 100 nm-periodic structures. Keratanase or Streptomyces hyaluronidase had no such effect. One possible suggestion is that that ATP-aggregated type VI collagen fibrils are connected with striated collagen fibrils through chondroitin/dermatan sulfate glycosaminoglycans.

Immunogold Ultrastructural Localization of Collagens in the Aged Human Outflow System

AbstractImmunogold labeling was applied at the ultrastructural level to the identification of collagen types I, II, III, V, and VI in the human trabecular meshwork obtained from 11 surgically enucleated globes. Both London resin white embedding and cryoultramicrotomy were used, and for types V and VI, the latter provided more sensitive localization. Type II collagen was not identified. Types I and III were localized to the striated collagen fibrils of the trabecular core, the basement membrane of the trabecular beams, and loose aggregates in the juxtacanalicular tissue. Types V and VI formed a fine network around the striated fibrils in the trabecular cores and linkage strands to the basement membranes. The outer wall of Schlemm's canal contained collagens I, III, V, and VI. Long-spacing collagen and elastin-like material did not label with any of the antibodies used in this study.

Immunogold fine structural localization of extracellular matrix components in aged human cornea II. Collagen types V and VI

Using immunogold immunocytochemical techniques we studied the distribution of collagen types V and VI in corneal tissue from seven enucleated human eyes (age range, 63-78 years). Results obtained by cryoultramicrotomy were marginally more intense than those obtained using London Resin white (LR white) embedding. Type V collagen was present in the striated collagen fibrils in Bowman's layer, in the stroma and in a thin, non-banded anterior zone of Descemet's membrane. Our results suggest that types I, III and V collagen co-distribute in striated collagen fibrils. By contrast, type VI collagen was located in fine filaments in the interfibrillar matrix of the stroma, in Bowman's layer and in the anchoring plaques of the sub-epithelial basement-membrane complex. This implies an importance in epithelial adhesion which was previously unsuspected. Keratocyte bodies were electron-dense, amorphous extracellular deposits of matrix-like material, and these were labelled with types III, V and VI collagen antibodies. Long-spacing collagen was observed in the corneal stroma, and this deposit did not contain any of the collagen types studied.