Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts

The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.     

Collagen VI deficiency affects the organization of fibronectin in the extracellular matrix of cultured fibroblasts.

Fibronectin is one of the main components of the extracellular matrix and associates with a variety of other matrix molecules including collagens. We demonstrate that the absence of secreted type VI collagen in cultured primary fibroblasts affects the arrangement of fibronectin in the extracellular matrix. We observed a fine network of collagen VI filaments and fibronectin fibrils in the extracellular matrix of normal murine and human fibroblasts. The two microfibrillar systems did not colocalize, but were interconnected at some discrete sites which could be revealed by immunoelectron microscopy. Direct interaction between collagen VI and fibronectin was also demonstrated by far western assay. When primary fibroblasts from Col6a1 null mutant mice were cultured, collagen VI was not detected in the extracellular matrix and a different pattern of fibronectin organization was observed, with fibrils running parallel to the long axis of the cells. Similarly, an abnormal fibronectin deposition was observed in fibroblasts from a patient affected by Bethlem myopathy, where collagen VI secretion was drastically reduced. The same pattern was also observed in normal fibroblasts after in vivo perturbation of collagen VI-fibronectin interaction with the 3C4 anti-collagen VI monoclonal antibody. Competition experiments with soluble peptides indicated that the organization of fibronectin in the extracellular matrix was impaired by added soluble collagen VI, but not by its triple helical (pepsin-resistant) fragments. These results indicate that collagen VI mediates the three-dimensional organization of fibronectin in the extracellular matrix of cultured fibroblasts.     

Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy

Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.     

Collagen expression, ultrastructural assembly, and mineralization in cultures of chicken embryo osteoblasts

A newly defined chick calvariae osteoblast culture system that undergoes a temporal sequence of differentiation of the osteoblast phenotype with subsequent mineralization (Gerstenfeld, L. C., S. Chipman, J. Glowacki, and J. B. Lian. 1987. Dev. Biol. 122:49-60) has been examined for the regulation of collagen synthesis, ultrastructural organization of collagen fibrils, and extracellular matrix mineralization. Collagen gene expression, protein synthesis, processing, and accumulation were studied in this system over a 30-d period. Steady state mRNA levels for pro alpha 1(I) and pro alpha 2 collagen and total collagen synthesis increased 1.2- and 1.8-fold, respectively, between days 3 and 12. Thereafter, total collagen synthesis decreased 10-fold while mRNA levels decreased 2.5-fold. In contrast to the decreasing protein synthesis after day 12, total accumulated collagen in the cell layers increased sixfold from day 12 to 30. Examination of the kinetics of procollagen processing demonstrated that there was a sixfold increase in the rate of procollagen conversion to alpha chains from days 3 to 30 and the newly synthesized collagen was more efficiently incorporated into the extracellular matrix at later culture times. The macrostructural assembly of collagen and its relationship to culture mineralization were also examined. High voltage electron microscopy demonstrated that culture cell layers were three to four cells thick. Each cell layer was associated with a layer of well developed collagen fibrils orthogonally arranged with respect to adjacent layers. Fibrils had distinct 64-70-nm periodicity typical of type I collagen. Electron opaque areas found principally associated with the deepest layers of the fibrils consisted of calcium and phosphorus determined by electron probe microanalysis and were identified by electron diffraction as a very poorly crystalline hydroxyapatite mineral phase. These data demonstrate for the first time that cultured osteoblasts are capable of assembling their collagen fibrils into a bone-specific macrostructure which mineralizes in a manner similar to that characterized in vivo. Further, this matrix maturation may influence the processing kinetics of the collagen molecule.     

Actin filaments are required for fibripositor-mediated collagen fibril alignment in tendon

Cells in tendon deposit parallel arrays of collagen fibrils to form a functional tissue, but how this is achieved is unknown. The cellular mechanism is thought to involve the formation of intracellular collagen fibrils within Golgi to plasma membrane carriers. This is facilitated by the intracellular processing of procollagen to collagen by members of the tolloid and ADAMTS families of enzymes. The carriers subsequently connect to the extracellular matrix via finger-like projections of the plasma membrane, known as fibripositors. In this study we have shown, using three-dimensional electron microscopy, the alignment of fibripositors with intracellular fibrils as well as an orientated cable of actin filaments lining the cytosolic face of a fibripositor. To demonstrate a specific role for the cytoskeleton in coordinating extracellular matrix assembly, cytochalasin was used to disassemble actin filaments and nocodazole or colchicine were used to disrupt microtubules. Microtubule disruption delayed procollagen transport through the secretory pathway, but fibripositor numbers were unaffected. Actin filament disassembly resulted in rapid loss of fibripositors and a subsequent disappearance of intracellular fibrils. Procollagen secretion or processing was not affected by cytochalasin treatment, but the parallelism of extracellular collagen fibrils was altered. In this case a significant proportion of collagen fibrils were found to no longer be orientated with the long axis of the tendon. The results suggest an important role for the actin cytoskeleton in the alignment and organization of the collagenous extracellular matrix in embryonic tendon.     

The small proteoglycan biglycan is associated with thick collagen fibrils in the mouse decidua.

In mice, embryo implantation induces profound changes in the endometrium. These changes include redifferentiation of endometrial fibroblasts and extensive remodeling of extracellular matrix components. We have previously shown that, during this process, there is an impressive increase in the thickness of collagen fibrils present in decidualised areas that surround the implantation site, while collagen fibrils in non-decidualised areas and in the interimplantation site remain thin. In vitro and in vivo experiments have identified small leucine rich proteoglycans (SLRPs) as regulators of collagen fibrillogenesis. In a previous study, we demonstrated a difference between the pre-implantation and the post-implantation expression and distribution of four SLRPs types in uterine tissues. The present study, utilising immunocytochemical electron microscopy, shows that biglycan is associated with the presence of thick collagen fibrils in decidualised regions of the endometrium and that decorin is associated exclusively with thin collagen fibrils in non-decidualised endometrial areas. These results strongly indicate that biglycan plays a role in collagen fibrillogenesis and probably participates in the determination of collagen fibril thickness in the mouse decidua.     

Cellular remodelling of individual collagen fibrils visualized by time-lapse AFM

The extracellular matrix in tissues such as bone, tendon and cornea contains ordered, parallel arrays of collagen type I fibrils. Cells embedded in these matrices frequently co-align with the collagen fibrils, suggesting that ordered fibrils provide structural or signalling cues for cell polarization. To study mechanisms of matrix-induced cell alignment, we used nanoscopically defined two-dimensional matrices assembled of highly aligned collagen type I fibrils. On these matrices, different cell lines expressing integrin alpha(2)beta(1) polarized strongly in the fibril direction. In contrast, alpha(2)beta(1)-deficient cells adhered but polarized less well, suggesting a role of integrin alpha(2)beta(1) in the alignment process. Time-lapse atomic force microscopy (AFM) demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization was prevented. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix, leading to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction.     

Ultrastructure of extracellular matrix of embryonic chick limb bud cartilage

The ultrastructure of the extracellular matrix of chick limb buds at stages 24--36 was investigated with the electron microscope and with polarization microscopic evaluation of topo-optical reactions. Two types of extracellular matrix structures could be distinguished. In the primary matrix of presumptive cartilaginous areas at developmental stage 25, topo-optical reactions revealed polycarboxylated glycosaminoglycans oriented parallel to the cell surface. With the electron microscope few fine filaments and small matrix granules could be found. In the secondary cartilage matrix, from stage 26 on, topo-optical reactions demonstrated glycosaminoglycans and collagen being predominantly ordered parallel to each other. Their amount and the degree of their orientation gradually increased during the developmental period investigated. Electron microscopy demonstrated collagen filaments and matrix granules in increasing number at stages 26--36. While some parallel ordering of filaments was seen in the electron micrographs, no sign of spatial ordering of glycosaminoglycans could be found with this technique.     

Molecular structure and interaction of recombinant human type XVI collagen

Collagen XVI is a minor component of at least two different extracellular fibrillar networks of specialized regions of skin and cartilage. In skin, collagen XVI is integrated into particular fibrillin-rich microfibrils lacking an amorphous elastin core. In cartilage, collagen XVI is a component of small heterotypic D-banded fibrils, mainly occurring in the territorial matrix of chondrocytes. Here, we present the first direct evidence for the molecular structure and functional properties of these fibril-associated collagens with interrupted triple helices (FACIT). We have expressed recombinantly the full-length alpha1 chain of human collagen XVI in HEK 293 EBNA cells in large quantities using an episomal expression system. Secreted full-length recombinant collagen XVI forms stable disulfide-bonded homotrimers and is rapidly proteolytically processed to distinct fragments at specific protease sequence motifs, one resembling an aggrecanase recognition site. Limited trypsin digestion assays and thermal transition curves imply sequential thermal denaturation of individual triple helical domains of this recombinant collagen, similar to authentic collagen XVI. Molecular images of collagen XVI reveal rod-like molecules which harbor multiple sharp kinks attributing a highly flexible structure presumably introduced by non-collagenous (NC) regions. Terminally located cloverleaf-shaped nodules correspond to the large NC NC11 domain of trimeric collagen XVI. The total length of individual trimeric recombinant collagen XVI molecules constitutes about 240 nm as calculated by atomic force and negative staining electron microscopy. Recombinant collagen XVI interacts with fibrillin-1 and with fibronectin indicating multiple molecular interactions in which this ubiquitously expressed and versatile FACIT-collagen can participate. In vitro generated collagen XVI provides an indispensable tool for future determination of its function during supramolecular assembly of matrix aggregates and its role in maintenance, organization and interaction of fibrillar structures.     

The relationship of mechanical properties to morphology in patellar tendon autografts after posterior cruciate ligament replacement in sheep.

In a sheep model the posterior cruciate ligament (PCL) was replaced by a patellar tendon autograft (PTAG) using the central one-third of the ipsilateral patellar tendon (PT). The sheep were sacrificed at 16, 26, 52 and 104 weeks postoperation. The PTAG, and, as controls, the contralateral PCL and PT were harvested. These were examined using biomechanical testing as well as light and transmission electron microscopy, including immunohistological techniques. The material properties (maximum stress, elastic modulus) were compared to the morphological features. The cellular distribution, the distribution of glycosaminoglycans (GAGs), the collagen fibril diameter and the occurrence of Type III collagen were studied. Prior to transplantation, the PTAG was shown to be superior in maximum stress (57.2 +/- 5.5 MPa vs 41.3 +/- 1.9 MPa) and elastic modulus (368.8 +/- 49.3 MPa vs 172.3 +/- 14.6 MPa) to the PCL. The early decline in material properties of the PTAG (maximum stress 22% and elastic modulus 42% of the control) after free grafting paralleled a cell- and capillary-rich PTAG tissue with remnants of necrosis and a poorly organized extracellular matrix. Two years after implantation, with progressive alignment of the tissue matrix, maximum stress and elastic modulus acquired approximately 60 and 70% of the control, respectively. However, there was also an evidence of degenerative changes characterized by acellular areas, loss of the normal bundling pattern of collagen fibers and abnormal accumulation of GAGs. Ultrastructurally, there was a predominant shift to thin collagen fibrils in the PTAG compared to PCL and PT, both consisting of thick and thin collagen fibrils. Thin fibrils were demonstrated to be, in part, split thick fibrils as well as newly formed fibrils. Most of these thin fibrils revealed a positive reaction with antibodies to Type III collagen.     

Pepsin-generated type VI collagen is a degradation product of GP140

A major extracellular matrix glycoprotein, GP140 , synthesized by WI-38 human lung fibroblasts has previously been shown to be collagen-like. A form of GP140 that is related to extracellular matrix GP140 both antigenically and in apparent molecular mass was isolated from human placenta. Types I-VI collagen were isolated from human tissues by limited pepsin digestion, selective salt precipitation, and chromatography. Immunoblot analysis of the collagens and GP140 utilizing affinity-purified polyclonal antiserum directed against extracellular matrix GP140 demonstrated cross-reactivity of antibodies with type VI collagen. Both type VI collagen and matrix GP140 could be digested with bacterial collagenase following reduction with dithiothreitol but were collagenase insensitive under nonreducing conditions, unlike types I-V collagen. Placental and matrix GP140 and type VI collagen were shown to have receptors for 125I-labeled Lens culinaris lectin. Pepsin digestion of WI-38 extracellular matrix GP140 yielded a 64,000-dalton band which co-migrated with subunits of reduced type VI collagen on Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, reacted with anti- GP140 antiserum and 125I-labeled L. culinaris lectin, and was collagenase-sensitive only under reducing conditions. CNBr fragmentation of extracellular matrix GP140 , the 64,000-dalton pepsin-resistant peptide of GP140 and type VI collagen followed by immunoblot analysis using anti- GP140 revealed similarities in peptide maps of GP140 and type VI collagen. Our data strongly suggest that GP140 and type VI collagen share characteristics that differ from those of other collagen types and that intermolecular disulfide bonding appears to stabilize these molecules in their native unreduced form, thus conferring collagenase resistance. Finally, the SC1 and SC2 subunits of type VI collagen appear to be generated by pepsin digestion of GP140 .     

Growth of Collagen Fibril Seeds from Embryonic Tendon: Fractured Fibril Ends Nucleate New Tip Growth

Collagen fibrils are the principal tensile element of vertebrate tissues where they occur in the extracellular matrix as spatially organised arrays. A major challenge is to understand how the mechanisms of nucleation, growth and remodelling yield fibrils of tissue-specific diameter and length. Here we have developed a seeding system whereby collagen fibrils were isolated from avian embryonic tendon and added to purified collagen solution, in order to characterise fibril surface nucleation and growth mechanisms. Fragmentation of tendon in liquid nitrogen followed by Dounce homogenisation generated fibril length fragments. Most (> 94%) of the fractured ends of fibrils, which show an abrupt square profile, were found to act as nucleation sites for further growth by molecular accretion. The mechanism of this nucleation and growth process was investigated by transmission electron microscopy, atomic force microscopy and scanning transmission electron microscopy mass mapping. Typically, a single growth spur occurred on the N-terminal end of seed fibrils whilst twin spurs frequently formed on the C-terminal end before merging into a single tip projection. The surface nucleation and growth process generated a smoothly tapered tip that achieved maximum diameter when the axial extension reached ∼ 13 μm. Lateral growth also occurred along the entire length of all seed fibrils that contained tip projections. The data support a model of collagen fibril growth in which the broken ends of fibrils are nucleation sites for propagation in opposite axial directions. The observed fibril growth behaviour has direct relevance to tendon matrix remodelling and repair processes that might involve rupture of collagen fibrils.     

Trabecular meshwork's collagen network formation is inhibited by non-pigmented ciliary epithelium-derived extracellular vesicles

The present research aims to determine whether the application of non-pigmented ciliary epithelium cells derived extracellular vesicles to human trabecular meshwork cells affects the formation and secretion of collagen type I to the extracellular matrix formation. Following the extraction of non-pigmented ciliary epithelium derived extracellular vesicles by a precipitation method, their size and concentration were determined using tunable resistive pulse sensing technology. Extracellular vesicles were incubated with trabecular meshwork cells for 3 days. Morphological changes of collagen type I in the extracellular matrix of trabecular meshwork cells were visualized using confocal microscopy and scanning electron microscopy. A Sirius Red assay was used to determine the total amount of collagen. Finally, collagen type I expression levels in the extracellular matrix of trabecular meshwork cells were quantified by cell western analysis. We found that non-pigmented ciliary epithelium extracellular vesicles were very effective at preventing collagen fibres formation by the trabecular meshwork cells, and their secretion to the extracellular matrix was significantly reduced (P < .001). Morphological changes in the extracellular matrix of trabecular meshwork cells were observed. Our study indicates that non-pigmented ciliary epithelium extracellular vesicles can be used to control collagen type I fibrillogenesis in trabecular meshwork cells. These fibrils net-like structure is responsible for remodelling the extracellular matrix. Moreover, we suggest that targeting collagen type I fibril assembly may be a viable treatment for primary open-angle glaucoma abnormal matrix deposition of the extracellular matrix.     

In vitro characterization of lactoferrin aggregation and amyloid formation

Lactoferrin has previously been identified in amyloid deposits in the cornea, seminal vesicles, and brain. We report in this paper a highly amyloidogenic region of lactoferrin (sequence of NAGDVAFV). This region was initially identified by sequence comparison with medin, a 5.5 kDa amyloidogenic fragment derived from lactadherin. Subsequent characterization revealed that this peptide forms amyloid fibrils at pH 7.4 when incubated at 37 degrees C. Furthermore, although full-length lactoferrin does not by itself form amyloid fibrils, the protein does bind to the peptide fibrils as revealed by an increase in thioflavin T fluorescence and the presence of enlarged fibrils by transmission electron microscopy and polarized light microscopy. The binding of lactoferrin is a selective interaction with the NAGDVAFV fibrils. Lactoferrin does not bind to insulin or lysozyme fibrils, and the NAGDVAFV fibrils do not bind to soluble insulin or lysozyme. The lactoferrin appears to coat the peptide fibril surface to form mixed peptide/protein fibrils, but again there is no evidence for the formation of lactoferrin-only fibrils. This interaction, therefore, seems to involve selective binding rather than conventional seeding of fibril formation. We suggest that such a process could be generally important in the formation of amyloid fibrils in vivo since the identification of both full-length protein and protein fragments is common in ex vivo amyloid deposits.     

Characterization of opticin and evidence of stable dimerization in solution

Opticin is a class III member of the extracellular matrix small leucine-rich repeat protein (SLRP) family that was initially identified in the eye in association with the collagen fibrils of the vitreous humor. Recombinant and tissue-extracted forms of bovine opticin were subjected to biochemical and biophysical characterization. Following SDS-PAGE the predominant component produced by both forms was a broad band between 45-52 kDa. There was evidence for two-stage processing and, additionally, a proteolytic cleavage product of approximately 25 kDa. Deconvolution of circular dichroism spectra revealed beta-sheet (41%), beta-turn (21%), and alpha-helix (10%), and thermal denaturation experiments showed a transition with a midpoint of 47 degrees C. Weight-averaged molecular mass measurements using both light scattering and analytical ultracentrifugation demonstrated that opticin exists in solution as a stable dimer of approximately 90 kDa, which can be dissociated into a monomer by denaturation with 2.5 m guanidine hydrochloride or during SDS-polyacrylamide electrophoresis. Opticin remains a dimer after removal of the amino-terminal region by O-sialoglycoprotein endopeptidase digestion, suggesting that dimer formation is mediated by the leucine-rich repeats. Dimerization could have a number of functional consequences, including divalent ligand interactions.     

Localization of the Extracellular Matrix Protein SC1 to Synapses in the Adult Rat Brain

Extracellular matrix molecules play important roles in neural developmental processes such as axon guidance and synaptogenesis. When development is complete, many of these molecules are down-regulated, however the molecules that remain highly expressed are often involved in modulation of synaptic function. SC1 is an example of an extracellular matrix protein whose expression remains high in the adult rat brain. Confocal microscopy revealed that SC1 demonstrates a punctate pattern in synaptic enriched regions of the cerebral cortex and cerebellum. Higher resolution analysis using electron microscopy indicated that SC1 localizes to synapses, particularly the postsynaptic terminal. SC1 was also detected in perisynaptic glial processes that envelop synapses. This work was supported by the National Science and Engineering Research Council of Canada.     

Interaction between amino propeptides of type XI procollagen alpha1 chains

Type XI collagen is a quantitatively minor yet essential constituent of the cartilage extracellular matrix. The amino propeptide of the alpha1 chain remains attached to the rest of the molecule for a longer period of time after synthesis than the other amino propeptides of type XI collagen and has been localized to the surface of thin collagen fibrils. Yeast two-hybrid system was used to demonstrate that a homodimer of alpha1(XI) amino propeptide (alpha1(XI)Npp) could form in vivo. Interaction was also confirmed using multi-angle laser light scattering, detecting an absolute weight average molar mass ranging from the size of a monomer to the size of a dimer (25,000-50,000 g/mol), respectively. Binding was shown to be saturable by ELISA. An interaction between recombinant alpha1(XI)Npp and the endogenous alpha1(XI)Npp was observed, and specificity for alpha1(XI)Npp but not alpha2(XI)Npp was demonstrated by co-precipitation. The interaction between the recombinant form of alpha1(XI)Npp and the endogenous alpha1(XI)Npp resulted in a stable association during the regeneration of cartilage extracellular matrix by fetal bovine chondrocytes maintained in pellet culture, generating a protein that migrated with an apparent molecular mass of 50-60 kDa on an SDS-polyacrylamide gel.     

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)     

Development of the basement membrane and formation of collagen fibrils below the placodes in the head of anuran larvae

The development of the basement membrane and collagen fibrils below placodes, including the corneal region of the ectoderm, lens epithelium, nasal plate, and auditory vesicle in anuran larvae was observed by transmission electron microscopy and compared with that in nonplacodal regions such as the epidermis, neural tube, and optic vesicle. In the corneal region the lamina densa becomes thick concomitantly with the development of the connecting apparatuses such as hemidesmosomes and anchoring fibrils. The collagen fibrils increase in number and form a multilayered structure, showing similar morphology to the connective tissues below the epidermis. These two areas, i.e., the corneal region and epidermis, possess much collagenous connective tissue below them. On the other hand, the neural tube and ophthalmic vesicle that originated from the neural tube each have a thin lamina densa and a small number of underlying collagen fibrils. The lamina densa does not thicken and the number of collagen fibrils do not significantly increase during development. These two areas possess little extracellular matrix. The nasal plate and auditory vesicle show intermediate characteristics between the epidermis-type and the neural tube-type areas. In these areas, the lamina densa becomes thick and hemidesmosomes and anchoring fibrils develop. The number of collagen fibrils increases during development, but does not show an orderly arrangement; rather, they are randomly distributed. It is thought that the difference in the arrangement of collagen fibrils in different tissues is due to differences in the extracellular matrix around the collagen fibrils. Placodal epithelia have the same origin as epidermis, but during development their morphological characteristics differ and they are not associated with the pattern of extracellular matrix with characteristics of epidermal and corneal multilayered collagen fibril areas.     

Collagen deposition on a preformed grid

Appearance of collagen fibrils in the cuticle was seen by electron microscopy to be preceded by fonnation of a finely filamentous matrix material. At first, the fine filaments of the matrix are unorganized. However, signs of orthogonal ordering soon appear in the most superficial portion of the cuticle, and subsequently appear more basally and closer to the underlying epidermis. Meanwhile, fibrils of different staining properties and identifiable as collagen begin to be deposited in the superficial portion of the cuticle, the same region which first showed organized fine filaments. Then, like the fine filaments before them, the collagen fibrils polymerize more basally. Collagen appears to polymerize on the preformed skeleton of fine filaments as though the fine filaments caused the collagen to assemble. Neither the polymerization nor ordering of collagen fibrils seems to require direct cellular intervention but occur first in that portion of the cuticle which is furthest away from the underlying epidermis. The fine filaments may be self ordering, extracellular macromolecules which in turn determine the polymerization of collagen fibrils.