Structural characterization of the mandibular condyle in human fetuses: light and electron microscopy studies.

Mandibular condyles from 18- to 20-week-old human fetuses were examined in the light and electron microscope with particular attention to intratissue organization and extracellular matrix. In the human fetus the condyle has been divided into five layers: (1) the most superficial, articular layer, (2) chondroprogenitor cell layer, (3) condroblast cell layer, (4) nonmineralized hypertrophic cell layer, and (5) mineralized hypertrophic cell layer. The articular layer is rich in collagen fibers (mostly of the type I collagen), but the cells seldom divide. By contrast, in the chondroprogenitor cell layer and upper part of the chondroblastic cell layer mitosis gives rise to new cells. The matrix in the latter layer is composed of thick banded 'lucent' fibrils in a loose feltwork of granules representing cartilage proteoglycans. The daughter cells in the progenitor cell layer undergo differentiation which is apparently completed along the lower border of the mineralized hypertrophic cell layer--the ossification front. The matrix in the hypertrophic cell layer reveals distinct matrix vesicles that undergo mineralization and subsequently coalesce to form larger sheets of mineralized extracellular matrix. Mineralized cartilage serves as a backbone for new bone formation as marrow-derived osteoblasts and osteoclasts attach to remnants of mineralized cartilage, which enables the turning on of the remodeling cycles involved in new bone formation. It can be concluded that the process of endochondral ossification as has been reported in lower animals is recapitulated in the human fetus, thus the dynamics associated with condylar morphogenesis is maintained through phylogeny.     

Collagen formation — observations on its intracellular packaging and transport

Summary Odontoblasts, osteoblasts and fibroblasts of young rats were examined in the electron microscope after staining thin sections either with lead citrate alone or with uranyl acetate prior to lead citrate.With lead citrate alone, collagen fibrils in the extracellular matrix stand out as lucent structures against a moderately electron dense background. Within the cells, lucency is restricted to certain dilated portions of the Golgi saccules as well as to the secretory granules located nearby and in the secretory pole of the cells. The lucency present in these compartments may be attributed to fibrils that are similar to the lucent collagen fibrils in the extracellular matrix. Other cellular compartments, e.g. the rough ER, do not display lucency.When preparations are stained with uranyl acetate prior to lead citrate, lucency is observed neither in the matrix nor in the cells. In the matrix, collagen fibrils are easily identifiable by their cross banded pattern. In the odontoblasts, dilated portions of Golgi saccules between the outer and inner face contain filaments aligned in parallel that are approximately 3 000 Å in length. In saccules on the inner face filament aggregates are present, some of them exhibiting a cross banding pattern. In secretory granules, however, the contents appear rather homogeneous.It is suggested that filament aggregates of collagen can assemble in the Golgi apparatus from filamentous units. These are transported through the cell by way of secretion granules and are discharged to the extracellular matrix by exocytosis.This investigation was supported by grants of the Medical Research Council of Canada. The author wishes to express appreciation to Dr. C. P. Leblond for his guidance in the course of this work.     

Cellular stages in cartilage formation as revealed by morphometry, radioautography and type II collagen immunostaining of the mandibular condyle from weanling rats

The role played by cell addition, cell enlargement, and matrix deposition in the endochondral growth of the condyle was assessed in weanling rats by four approaches making use of the light microscope: morphometry, 3H-thymidine radioautography, 3H-proline radioautography, and immunostaining for the cartilage-specific type II collagen. From the articular surface down, the condyle may be divided into five layers made up of cells embedded in a matrix: 1) the articular layer composed of static cells in a matrix rich in fibers presumed to be of type I collagen, 2) the polymorphic cell layer including the progenitor cells from which arise the cells undergoing endochondral changes, 3) the flattened cell layer in which cells produce a precartilagenous matrix devoid of type II collagen while undergoing differentiation in two stages: a "chondroblast" stage and a short "flattened chondrocyte" stage when intracellular type II collagen elaboration begins, 4) the upper hypertrophic cell layer, in which cells are "typical chondrocytes" that enlarge at a rapid rate, actively produce type II collagen, and deposit it into a cartilagenous matrix, and 5) the lower hypertrophic cell layer, composed of chondrocytes at a stage of terminal enlargement while the cartilagenous matrix is adapting for mineralization. 3H-thymidine radioautographic results indicate that the turnover time of progenitor cells in the polymorphic cell layer is about 2.9 days. The time spent by cells at each stage of development is estimated to be 1.4 days as chondroblasts, 0.5 days as flattened chondrocytes, 2.3 days as the chondrocytes of the upper hypertrophic cell layer, and 1.1 days as those of the lower hypertrophic cell layer. Calculations referring to a 1 x 1-mm square-sided column extending from the articular surface to the zone of vascular invasion provide the daily rate of cell addition (0.0077 mm3), extracellular matrix deposition (0.0127 mm3), and cell enlargement (0.0302 mm3). Hence the respective contribution of the three factors to condyle growth is in a ratio of about 1:1.6:4. This result emphasizes the role played by cell enlargement in the overall growth of the condyle.     

Immunohistochemical localization of type II and type I collagens in articular cartilage of the femoral head of dexamethasone-treated rats

The immunohistochemical localization of type II and type I collagens was examined in the articular cartilage of the femoral head of growing rats injected systemically with 5 mg kg−1 dexamethasone for 2 weeks every other day. The intensities of immunostaining for type II collagen, measured by microphotometry, was highest in the flattened cell layer and high in the hypertrophic cell layer, moderate in the proliferative cell and transitional cell layers and low in the superficial layer. After dexamethasone administration, the intensities decreased markedly in the flattened cell layer and slightly in the hypertrophic cell layer, although the decreases in other layers were negligible. The staining intensities for type I collagen were highest in the flattened cell layer, low in the superficial and transitional cell layers and very low in the proliferative and hypertrophic cell layers. After dexamethasone administration, the intensities increased markedly in the flattened cell layer and slightly in the superficial and proliferative cell layers, but did not change in the transitional and hypertrophic cell layers. Thus, dexamethasone administration caused a decrease in type II collagen and an increase in type I collagen in the matrix of the surface portion of articular cartilage. The composition of isoforms of collagen in the matrix changed after the steroid administration. The results strongly suggest that the shift in collagen composition from type II to type I predominance is a cause of the degeneration of the articular cartilage after glucocorticoid administration. This revised version was published online in November 2006 with corrections to the Cover Date.     

Defects in the cartilaginous growth plates of brachymorphic mice

Homozygous brachymorphic (bm/bm) mice are characterized by disproportionately short stature. Newborn bm/bm epiphyseal cartilages are shorter than normal although the cells in the different zones of growth are relatively well organized. The extracellular matrix reacts poorly with stains specific for sulfated glycosaminoglycans. The ultrastructural appearance of the cartilage matrix indicates normal collagen fibrils; however, proteoglycan aggregate granules are smaller than normal and are present in reduced numbers, particularly in the columnar and hypertrophic zones of the growth plate. In addition, a prominent network of fine filaments, which are extractable in 4 M guanidine hydrochloride, are present in the bm/bm cartilage matrix. These findings suggest that a defect affecting the proteoglycan component of cartilage occurs in bm/bm mice.     

Inhibition of limb chondrogenesis in vitro by vitamin A: alterations in cell surface characteristics

Mesenchyme cells derived from embryonic mouse limb buds were cultured at high cell density. During the first 24 h in culture, groups of mesenchyme cells condensed and formed cell contacts and specialized junctions. These condensations were the nodule primordia which gave rise to cartilage nodules. The cell contacts were lost as the mesenchyme cells in the primordia developed into cartilage nodules. The mature nodules contained chondrocytes isolated from one another by an extensive extracellular matrix consisting of cartilage type collagen fibrils and proteoglycan granules. The differentiation of the mesenchyme cells to chondrocytes was also characterized by the loss of a 240,000-MW cell surface glycoprotein and the appearance of an 80,000-MW surface protein. The addition of vitamin A to the medium on Day 1 inhibited chondrogenesis. The cells were closely packed together, and the limited extracellular space contained thick, banded collagen fibrils with no proteoglycan granules. The cells exhibited extensive areas of close membrane contact and specialized junctions. Vitamin A-treated cultures also retained the 240,000-MW surface glycoprotein and retarded the appearance of the 80,000-MW cell surface protein. The results of this study suggest that cell surface features normally present on mesenchyme cells are maintained and exaggerated by vitamin A.     

Immunoelectron microscopy of type X collagen: supramolecular forms within embryonic chick cartilage

To determine the supramolecular forms in which avian type X collagen molecules assemble within the matrix of hypertrophic cartilage, we performed immunoelectron microscopy with colloidal gold-labeled monoclonal antibodies. In addition double-labeled analyses were performed for the molecule and type II collagen, employing two monoclonal antibodies attached to different size gold particles. Both in situ limb cartilages and the extracellular matrix of chondrocyte cultures were examined. We observed in both systems that the type X collagen is present in two forms. One is as fine filaments (less than 5 nm in diameter) within mats which are found predominantly in the pericellular matrix of the hypertrophic chondrocytes. The second form is in association with the fibrils (10-20 nm in diameter) which also react with the antibody for type II collagen. It seems that the filamentous mats represent a form in which the type X collagen is initially secreted from the cell. The type X associated with the striated fibrils most likely represents a secondary association of the molecule with preexisting type II/IX/XI fibrils. The data are consistent with our previously proposed hypothesis that type X collagen is involved in, and perhaps even "targets," certain matrix components for degradation and removal.     

Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system

Conditions were defined for promoting cell growth, hypertrophy, and extracellular matrix mineralization of a culture system derived from embryonic chick vertebral chondrocytes. Ascorbic acid supplementation by itself led to the hypertrophic phenotype as assessed by respective 10- and 15-fold increases in alkaline phosphatase enzyme activity and type X synthesis. Maximal extracellular matrix mineralization was obtained, however, when cultures were grown in a nutrient-enriched medium supplemented with both ascorbic acid and 20 mM beta-glycerophosphate. Temporal studies over a 3-wk period showed a 3-4-fold increase in DNA accompanied by a nearly constant DNA to protein ratio. In this period, total collagen increased from 3 to 20% of the cell layer protein; total calcium and phosphorus contents increased 15-20-fold. Proteoglycan synthesis was maximal until day 12 but thereafter showed a fourfold decrease. In contrast, total collagen synthesis showed a greater than 10-fold increase until day 18, a result suggesting that collagen synthesis was replacing proteoglycan synthesis during cellular hypertrophy. Separate analysis of individual collagen types demonstrated a low level of type I collagen synthesis throughout the 21-d time course. Collagen types II and X synthesis increased during the first 2 wk of culture; thereafter, collagen type II synthesis decreased while collagen type X synthesis continued to rise. Type IX synthesis remained at undetectable levels throughout the time course. The levels of collagen types I, II, IX, and X mRNA and the large proteoglycan core protein mRNA paralleled their levels of synthesis, data indicating pretranslational control of synthesis. Ultrastructural examination revealed cellular and extracellular morphology similar to that for a developing hypertrophic phenotype in vivo. Chondrocytes in lacunae were surrounded by a well-formed extracellular matrix of randomly distributed collagen type II fibrils (approximately 20-nm diam) and extensive proteoglycan. Numerous vesicular structures could be detected. Cultures mineralized reproducibly and crystals were located in extracellular matrices, principally associated with collagen fibrils. There was no clear evidence of mineral association with extracellular vesicles. The mineral was composed of calcium and phosphorus on electron probe microanalysis and was identified as a very poorly crystalline hydroxyapatite on electron diffraction. In summary, these data suggest that this culture system consists of chondrocytes which undergo differentiation in vitro as assessed by their elevated levels of alkaline phosphatase and type X collagen and their ultrastructural appearance.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural visualization of complex carbohydrates in epiphyseal cartilage with the tannic acid-metal salt methods

The present study has ultrastructurally applied the tannic acid-ferric chloride (TA-Fe) and the TA-uranyl acetate (TA-UA) methods to thin sections of glutaraldehyde-fixed, unosmicated embedded epiphyseal cartilage from rat tibiae to demonstrate complex carbohydrates. The strongest TA-Fe and TA-UA staining was observed after fixation of the specimens in glutaraldehyde containing TA. TA-Fe (pH 1.5) strongly stained matrix granules presumed to be proteoglycan monomers and chondrocyte secretory granules at various maturational stages but did not stain collagen fibrils and glycogen. TA-UA (pH 4.2) strongly stained matrix granules, intracellular glycogen, and chondrocyte secretory granules, and moderately stained collagen fibrils in the cartilage matrix. Ribosomes and nuclei were not stained above background staining with UA alone. In alpha-amylase-digested specimens, all TA-UA-reactive cytoplasmic glycogen was selectively removed. Testicular hyaluronidase digestion of specimens selectively removed TA-UA staining in matrix granules and all TA-Fe staining. When the pH of the UA solution was reduced to 1.5, TA-UA staining of glycogen and collagen was markedly decreased or absent, whereas staining of anionic sites was unaltered and significantly greater than with UA staining alone. Thus the TA-metal salt methods are pH dependent and allow differential intracellular and extracellular localization of complex carbohydrates in cartilage tissues at the electron microscope level.     

Collagen fibril assembly by corneal fibroblasts in three-dimensional collagen gel cultures: small-diameter heterotypic fibrils are deposited in the absence of keratan sulfate proteoglycan.

Extracellular matrix assembly is a multistep process and the various steps in collagen fibrillogenesis are thought to be influenced by a number of factors, including other noncollagenous matrix molecules. The synthesis and deposition of extracellular matrix by corneal fibroblasts grown within three-dimensional collagen gel cultures were examined to elucidate the factors important in the establishment of tissue-specific matrix architecture. Corneal fibroblasts in collagen gel cultures form layers and deposit small-diameter collagen fibrils (approximately 25 nm) typical of the mature corneal stroma. The matrix synthesized contains type VI collagen in a filamentous network and type I and type V collagen assembled as heterotypic fibrils. The amount of type V collagen synthesized is relatively high and comparable to that seen in the corneal stroma. This matrix is deposited between cell layers in a manner reminiscent of the secondary corneal stroma, but is not deposited as densely or as organized as would be found in situ. No keratan sulfate proteoglycan, a proteoglycan found only in the corneal stroma, was synthesized by the fibroblasts in the collagen gel cultures. The assembly and deposition of small-diameter fibrils with a collagen composition and structure identical to that seen in the corneal stroma in the absence of proteoglycans typical of the secondary corneal stroma imply that although proteoglycan-collagen interactions may function in the establishment of interfibrillar spacing and lamellar organization, collagen-collagen interactions are the major parameter in the regulation of fibril diameter.     

Ultrastructure of elastic cartilage in the rat external ear

Summary The structure of elastic cartilage in the external ear of the rat was investigated by transmission and scanning electron microscopy.The narrow subperichondrial, boundary zone contains predominantly ovoid cells rich in cell organelles: mitochondria, Golgi complex, granular endoplasmic reticulum and small (40–100 nm) vesicles. Scarce glycogen granules and bundles of 6–7 nm cytoplasmic filaments are also present. Deeper in the boundary zone, one or more cytoplasmic lipid droplets appear and cytofilaments become more abundant.Fully differentiated chondrocytes in the central zone of the cartilage plate resemble white adipose cells. They are globular and contain a single, large cytoplasmic lipid droplet. The cytoplasm is reduced to a thin peripheral rim; it contains a flattened nucleus, few cytoplasmic organelles and abundant, densely packed, cytoplasmic filaments.The intercellular matrix is very sparse. The pericellular ring consists of collagen fibrils about 20 nm in diameter and a proteoglycan cartilage matrix in the form of a stellate reticulum. The complex of these two structures appears in the scanning electron micrographs as a network of randomly oriented, ca 100 nm thick fibrils. Spaces between pericellular rings of matrix also contain thick elastic fibers or plates, apparently devoid of microfibrils. In scanning electron micrographs elastic fibers could be detected only in a few areas, in which they were not obscured by other constituents of the matrix. Immature forms of elastic fibers, oxytalan (pre-elastic) and elaunin fibers, were found in the perichondrial and boundary zones.     

Staining of proteoglycans in mouse lung alveoli. I. Ultrastructural localization of anionic sites

Summary In order to contrast anionic sites, in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules.After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl₂ concentration used during staining. At 0.4m MgCl₂, the length was mostly within the range 100–180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.     

Different patterns of collagen-proteoglycan interaction: a scanning electron microscopy and atomic force microscopy study

The extracellular matrix of unfixed, unstained rat corneal stroma, visualized with high-resolution scanning electron microscopy and atomic force microscopy after minimal preliminary treatment, appears composed of straight, parallel, uniform collagen fibrils regularly spaced by a three-dimensional, irregular network of thin, delicate proteoglycan filaments. Rat tail tendon, observed under identical conditions, appears instead made of heterogeneous, closely packed fibrils interwoven with orthogonal proteoglycan filaments. Pre-treatment with cupromeronic blue just thickens the filaments without affecting their spatial layout. Digestion with chondroitinase ABC rids the tendon matrix of all its interconnecting filaments while the corneal stroma architecture remains virtually unaffected, its fibrils always being separated by an evident interfibrillar spacing which is never observed in tendon. Our observations indicate that matrix proteoglycans are responsible for both the highly regular interfibrillar spacing which is distinctive of corneal stroma, and the strong interfibrillar binding observed in tendon. These opposite interaction patterns appear to be distinctive of different proteoglycan species. The molecular details of proteoglycan interactions are still incompletely understood and are the subject of ongoing research.     

Connective tissue of the livers of newborn and adult marmosets (Callithrix jacchus)

Immunofluorescence microscopic and electron microscopic investigations revealed components of the matrix and of the basal lamina (collagen type I, III, IV and V, BL-heparan sulfate and fibronectin) in the sinus wall (Disse's space) of the livers of newborn and adult marmosets (Callithrix jacchus). Collagen type I was missing in both the two age groups. Small amounts of laminin were present in the livers of newborn and absent in those of adult animals, whereas collagen type III occurred in the form of delicate fibres. Light microscopic inspection showed a continuous distribution of all other components in the sinus wall. The amount of collagen type III and V increased depending on the age. Electron microscopic investigations revealed single or bundled fibrils (20-30 nm) and filaments (10-12 nm). After addition of tannic acid, plaques of a fine-filamentous network and incorporated granules were observed. After addition of resting Ruthenium Red, electron-dense granules (20-60 nm) were irregularly distributed in the structureless space, resting on collagenous fibrils and cell membranes. The fibrils were allocated to collagen type III, the filaments to collagen type V. The plaques were supposed to contain heparan sulfate, collagen type IV and fibronectin. The absence of a Lamina densa of the basal lamina was attributed to the absence of laminin which probably plays an important role in the formation of this layer. Differences in the distribution pattern of the matrix components and thus a functional mosaic of the permeability of Disse's space were assumed. The complete absence of collagen type I and laminin in the lobules makes the adult marmoset liver especially suited for studies on the importance of this collagen type under pathological conditions, since both components are expressed in this way.     

Deposition and ultrastructural organization of collagen and proteoglycans in the extracellular matrix of gel-cultured fibroblasts

Human skin fibroblasts were cultivated within the three-dimensional space of polymerized alginate and collagen, respectively. The in vitro synthesis of collagens and proteoglycans was measured during the first 3 days of culture, and the deposition as well as the ultrastructural organization of newly synthesized extracellular matrix components were examined by electron microscopy. The amount of collagens and proteoglycans synthesized by fibroblasts, embedded in calcium alginate gels as well as in collagen lattices, was lowered as compared to monolayer cultures. Furthermore, it was found that collagen synthesis was reduced to a greater extent in alginate gels than in collagen lattices. On the contrary, total proteoglycan biosynthesis was similarly reduced either in alginate gels or in collagen lattices. At the end of a 3-day-culture period, filamentous material as well as cross-striated banded structures were found extracellularly in the alginate gel. According to their periodicity, their banding pattern, their association with polyanionic matrix components and their sensitivity towards glycosaminoglycan-degrading enzymes we could distinguish (1) sheets of amorphous non-banded material consisting of irregularly arranged filaments and containing dermatan sulfate-rich proteoglycans (type I structures), (2) sheets of long-spacing fibrils consisting of parallel orientated filaments and containing chondroitin sulfate-rich proteoglycans (= zebra bodies; type II structures), and (3) fibrillar structures with a complex banding pattern different from that of native collagen fibrils (type III structures). In fibroblasts cultured in collagen lattices, we only sporadically found depositions which are identified as type I structures. Using indirect immunoelectron microscopy and monospecific polyclonal antibodies, we localized type VI collagen in type I structures and type II structures. Type III structures can be identified as type I collagen derived as becomes obvious by comparison with segment long spacing crystallites of type I collagen.     

Two type XII-like collagens localize to the surface of banded collagen fibrils

Two recently identified collagen molecules, termed twelve-like A and twelve-like B (TL-A and TL-B) have properties similar to type XII collagen. These molecules have been localized in human and calf tissues by immunoelectron microscopy. The observations strongly suggest that both molecules are located along the surface of banded collagen fibers. The epitopes recognized by the antibodies are contained in large, nontriple-helical domains at one end of the collagen helix. The epitopes are visualized at a distance from the surface of the banded fibers roughly equal to the length of the nonhelical domains, suggesting that the nonhelical domains extend from the fibril, while the triple-helical domains are likely to bind directly to the fibril surface. Occasionally, both TL-A and TL-B demonstrate periodic distribution along the fibril surface. The period corresponds to the primary interband distance of the banded fibrils. Not all fibrils in a fiber bundle are labeled, nor is the labeling continuous along the length of labeled fibrils. Simultaneous labeling of TL-A and type VI collagen only rarely shows colocalization, suggesting that TL-A and TL-B do not mediate interactions between the type VI collagen beaded filaments and banded collagen fibrils. Also, interfibrillar distances are approximately equivalent in the presence and absence of these type XII-like molecules. While the results do not directly indicate a specific function for these molecules, the localization at the fibril surface suggests that they mediate interactions between the fibrils and other matrix macromolecules or with cells.     

Morphology of the pericellular capsule in articular cartilage revealed by hyaluronidase digestion

To allow a more valid comparison between our previous ultrastructural data and the immunolocalization of type IX and other minor collagen species in cryosectioned cartilage, we examined both normal and testicular hyaluronidase-digested canine tibial cartilage by electron microscopy. Removal of matrix proteoglycans caused the pericellular capsule to collapse against the cell surface, suggesting that its normal anatomical position is mediated by pericellular matrix hydration. Detailed examination of the pericellular capsule and pericellular channel revealed fine, faintly banded fibrils and an amorphous component somewhat similar in structure to basement membrane collagens. Matrix vesicles and the electron-dense material of the interterritorial matrix were only partially digested by hyaluronidase. We propose that the pericellular capsule is composed of a "felt-like" network of minor collagen species which act synergistically to maintain both the composition of the pericellular matrix and the integrity of the chondrocyte/pericellular matrix complex during compressive loading.     

Immunohistochemical studies on the tissue localization of collagen types I, III, IV, V and VI in schwannomas. Correlation with ultrastructural features of the extracellular matrix

The distinctive tissue localization of collagen types in typical schwannomas with Antoni type A and B areas was demonstrated immunohistochemically using affinity-purified antibodies against types I, III, IV, V and VI collagen and comparative ultrastructural studies were made on the extracellular matrix components. Antoni type A tissue, which was composed of tightly packed spindle cells with long cytoplasmic processes surrounded by a continuous basement membrane and a few fibrillar components of the extracellular matrix, was almost exclusively immunoreactive for type IV collagen, presumably representing the basement membrane. Verocay bodies, which are organoid structures of Antoni type A tissue, had a variety of more abundant extracellular fibrous components, such as banded collagen fibrils, fibrous long-spacing fibrils and microfibrils. These were positive for type I and III, as well as type IV collagen. In Antoni type B areas, where two types to tumor cells designated Schwann cell-like and fibroblast-like were scattered in large amounts of amorphous extracellular matrix containing microfibrils and thick banded collagen fibrils, type VI collagen as well as types I, III and IV collagen were consistently detected. Type V collagen was localized in dense fibrous tissue areas and around blood vessels. These findings indicate that the differently organized cellular patterns of schwannomas, identified as Antoni types A and B, are characterized not only by the ultrastructural features of the extracellular matrix, but also by the distinctive collagen types produced by neoplastic Schwann cells.     

Ultrastructure of the submandibular gland in the rabbit

The secretory endpieces of the rabbit submandibular gland are unusual in that they consist of seromucous acini (not demilunes) that empty into serous tubules that in turn drain into intercalated ducts. Seromucous granules consist of a moderately dense spherule in a fibrillogranular matrix. Serous granules contain a feltwork of filaments, which are liberated as a tangled skein during exocytosis. Peculiar granulated cells that have secretory granules of complex morphology are present at each end of the serous tubules. Intercalated ducts are, cytologically speaking, relatively simple, but the duct cells may contain a few oblong secretory granules. Striated ducts are typical in structure, although postfixation with ferrocyanide-reduced osmium reveals significant amounts of glycogen in the basal processes. Modified mitochondria are present in striated duct cells, but their frequency varies from rabbit to rabbit. Such mitochondria contain either an array of parallel, rigid cristae linked by intermembranous bridges, or a bundle of helical filaments within an expanded crista. Interspersed with the striated duct cells, especially near the duct origin, are some highly vacuolated cells with sparse mitochondria. Excretory ducts consisting of stratified columnar (sometimes pseudostratified) epithelium often show bleb formation of the luminal surface of the tall cells.     

Interaction of collagen with hydrophobic protein granules in the egg capsule of the dogfish scyliorhinus canicula

The egg capsule of the dogfish is a composite material containing collagenous fibrils and 2 mum spherical hydrophobic protein granules. The latter appear to owe much of their hydrophobicity to an exceptionally high tyrosine content (approximately 20% of total amino acid residues). The hydrophobic component appears to form as an emulsion in the secretory granules of the D and E zone gland cells of the nidamental gland. Droplets of the hydrophobic material appear to become coated with remarkably regular layers of radially-arranged collagen molecules which form a series of concentric, evenly spaced layers around each hydrophobic granule. Numerous disclinations were seen where the layers around adjacent granules interfered with one another. The layers are thought to represent a lamellar liquid crystalline phase previously described for this collagen (Knight et al., 1993). The fine structural appearance of the concentric layers and evidence for radial arrangement of collagen molecules within them is compatible with the suggestion that the layers are built from a dumbbell-shaped unit approximately 35 nm long with hydrophobic groups concentrated at the ends. This unit may represent a dumbbell-shaped molecule or an oligomer of two or more molecules lying parallel with one another in a head-to-tail arrangement. Such a unit can be readily incorporated into models for the micellar, hexagonal columnar and final fibrillar phases previously described for this collagen (Knight et al., 1993). Evidence from the TEM study of stretched egg capsule wall suggests that there is a mechanical interaction between the hydrophobic granules and the collagen fibrils in the fully formed material. We suggest that the radial, concentric layered arrangement of collagen molecules is established by hydrophobic interactions within the liquid crystalline material and locked into place by oxidative covalent cross-linking to give a 3-dimensional cross-linked meshwork of collagen fibrils and hydrophobic granules. The latter arrangement helps to account for the high tensilestrength and toughness of this material.