Collagen family of proteins

Collagen molecules are structural macro-molecules of the extracellular matrix that include in their structure one or several domains that have a characteristic triple helical conformation. They have been classified by types that define distinct sets of polypeptide chains that can form homo- and heterotrimeric assemblies. All the collagen molecules participate in supramolecular aggregates that are stabilized in part by interactions between triple helical domains. Fourteen collagen types have been defined so far. They form a wide range of structures. Most notable are 1) fibrils that are found in most connective tissues and are made by alloys of fibrillar collagens (types I, II, III, V, and XI) and 2) sheets constituting basement membranes (type IV collagen), Descemet's membrane (type VIII collagen), worm cuticle, and organic exoskeleton of sponges. Other collagens, present in smaller quantities in tissues, play the role of connecting elements between these major structures and other tissue components. The fibril-associated collagens with interrupted triple helices (FACITs) (types IX, XII, and XIV) appear to connect fibrils to other matrix elements. Type VII collagen assemble into anchoring fibrils that bind epithelial basement membranes and entrap collagen fibrils from the underlying stroma to glue the two structures together. Type VI collagen forms thin-beaded filaments that may interact with fibrils and cells.     

THE FINE STRUCTURE OF BONE CELLS

An electron microscopic study of Araldite-embedded, undecalcified human woven and chick lamellar bone is presented. The fine structure of the cells of bone in their normal milieu is described. Active osteoblasts possess abundant granular endoplasmic reticulum, numerous small vesicles, and a few secretion droplets. Their long cytoplasmic processes penetrate the osteoid. The transition of osteoblasts into osteoid osteocytes and then into osteocytes is traced and found to involve a progressive reduction of cytoplasmic organelles. Adjoining the osteocytes and their processes is a layer of amorphous material which is interposed between the cell surfaces and the bone walls of their respective cavities. Osteoclasts contain numerous non-membrane-associated ribosomes, abundant mitochondria, and little granular endoplasmic reticulum, thus differing markedly from other bone cells. The brush border is a complex of cytoplasmic processes adjacent to a resorption zone in bone. No unmineralized collagen is seen at resorption sites and it appears that collagen is removed before or at the time of mineral solution. All bone surfaces are covered by cells, some of which lack distinctive qualities and are designated endosteal lining cells. The structure of osteoid, bone, and early mineralization sites is illustrated and discussed.     

Ultrastructural aspects after cryoultramicrotomy of bone tissue and sutural cartilage in neonatal mice calvaria

Using transmission electron microscopy after cryoultramicrotomy, mineralized as well as unmineralized bone tissues and sutural cartilage were observed in neonatal mice calvaria. A good definition of osteoblasts (nucleus, membranes, organelles) and extracellular constituents (collagen fibrils, matrix vesicles, mineral substance) was obtained. The sutural zone was composed of an unmineralized cartilaginous tissue with more or less hypertrophic cells surrounded by a finely fibrillar matrix.     

Demonstration of the capacity of nacre to induce bone formation by human osteoblasts maintained in vitro.

Nacre implanted in vivo in bone is osteogenic suggesting that it may possess factor(s) which stimulate bone formation. The present study was undertaken to test the hypothesis that nacre can induce mineralization by human osteoblasts in vitro. Nacre chips were placed on a layer of first passage human osteoblasts. None of the chemical inducers generally required to obtain bone formation in vitro was added to the cultures. Osteoblasts proliferated and were clearly attracted by nacre chips to which they attached. Induction of mineralization appeared preferentially in bundles of osteoblasts surrounding the nacre chips. Three-dimensional nodules were formed by a dense osteoid matrix with cuboidal osteoblasts at the periphery and osteocytic-like cells in the center. These nodules contained foci with features of mineralized structures and bone-like structures, both radiodense to X-ray. Active osteoblasts (e.m.) with abundant rough endoplasmic reticulum, extrusion of collagen fibrils and budding of vesicles were observed. Matrix vesicles induced mineral deposition. Extracellular collagen fibrils appeared cross-banded and electrodense indicating mineralization. These results demonstrate that a complete sequence of bone formation is reproduced when human osteoblasts are cultured in the presence of nacre. This model provides a new approach to study the steps of osteoblastic differentiation and the mechanisms of induction of mineralization.     

THE FINE STRUCTURE OF DIPLOCOCCUS PNEUMONIAE

The fine structure of an unencapsulated strain of Diplococcus pneumoniae is described. A striking feature of these bacteria is an intracytoplasmic membrane system which appears to be an extension of septa of dividing bacteria. The possible function of these structures and their relationship to the plasma membrane and other types of intracytoplasmic membranes found in pneumococcus is discussed.     

Ultrastructure of mineralized nodules formed in rat bone marrow stromal cell culture in vitro.

Rat bone marrow stromal cells were cultured in vitro. At days 14-15 of culture, dense clusters of polygonal cells were formed, and they mineralized 2-3 days later. The cells resembling osteoblasts or young osteocytes were histologically observed to be embedded in mineralized or unmineralized extracellular matrices of the nodules. Next, these mineralized nodules were electron-microscopically examined. The osteoblastic cells associated with the nodules had a well-developed rough endoplasmic reticulum, an evident Golgi apparatus and some mitochondria as their intracellular organellae. Some lysosomes and microfilaments were also visible in the cytoplasms. Moreover, some cells protruded cell processes toward the neighboring cells through the extracellular matrix. The extracellular matrix consisted of numerous collagen fibrils which were striated with 60-70 nm axial periodicity and which was similar to bone tissue collagen. A large number of matrix vesicles were scattered among the collagen fibrils in the unmineralized area of the nodules. In contrast, in the mineralized area, numerous matrix vesicles at different stages of maturation and many calcified spherules were observed. That is the mineralization in this culture system was considered to be initiated in association with the matrix vesicles and to progress along the collagen fibrils. From these findings, it was confirmed by the present study that the mineralized nodules formed in this bone marrow stromal cell culture were ultrastructurally similar to bone and that the mineralization also proceeded by going through the normal calcification process. This culture system is considered to be available to study osteogenic differentiation and calcification mechanisms.     

In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria

We investigated the capacity of a clonal osteogenic cell line MC3T3-E1, established from newborn mouse calvaria and selected on the basis of high alkaline phosphatase (ALP) activity in the confluent state, to differentiate into osteoblasts and mineralize in vitro. The cells in the growing state showed a fibroblastic morphology and grew to form multiple layers. On day 21, clusters of cells exhibiting typical osteoblastic morphology were found in osmiophilic nodular regions. Such nodules increased in number and size with incubation time and became easily identifiable with the naked eye by day 40-50. In the central part of well-developed nodules, osteocytes were embedded in heavily mineralized bone matrix. Osteoblasts were arranged at the periphery of the bone spicules and were surrounded by lysosome-rich cells and a fibroblastic cell layer. Numerous matrix vesicles were scattered around the osteoblasts and young osteocytes. Matrix vesicles and plasma membranes of osteoblasts, young osteocytes, and lysosome-rich cells showed strong reaction to cytochemical stainings for ALP activity and calcium ions. Minerals were initially localized in the matrix vesicles and then deposited on well-banded collagen fibrils. Deposited minerals consisted exclusively of calcium and phosphorus, and some of the crystals had matured into hydroxyapatite crystals. These results indicate that MC3T3-E1 cells have the capacity to differentiate into osteoblasts and osteocytes and to form calcified bone tissue in vitro.     

STUDIES ON THE BIOSYNTHESIS OF COLLAGEN : I. THE GROWTH OF FOWL OSTEOBLASTS AND THE FORMATION OF COLLAGEN IN TISSUE CULTURE

1. A tissue culture method was devised in which suspensions of osteoblasts, obtained directly from frontal bones of fowl embryos, were grown in a fluid, fibrin-free medium. 2. Maximum growth of the tissue, as measured by dry weight, with the formation of collagen protein, based on the estimation of hydroxyproline, was obtained in periods of up to 6 days. 3. Appreciable amounts of protein-bound hydroxyproline were formed during the first 24 hour growth period, but electron microscopy of portions of the same cultures failed to demonstrate the presence of any typical collagen fibrils. 4. The subsequent formation of many characteristic collagen fibrils was not associated with a significant rise in the mean hydroxyproline content of the tissue. 5. The cytoplasmic granules of the osteoblasts stained intensely with the P.A.S. technique when the collagen fibrils were being formed. 6. It is suggested that collagen-forming cells synthesise and secrete a hydroxyproline-rich precursor of protein or large peptide nature, which subsequently becomes directly transformed into typical collagen fibrils.     

Effect of forskolin on collagen production in clonal osteoblastic MC3T3-E1 cells

The effect of forskolin on collagen production in osteoblasts was investigated by using clonal osteoblastic MC3T3-E1 cells cultured in a-minimum essential medium containing 0.1% bovine serum albumin. Forskolin increased the adenylate cyclase activity in membranes pelleted from homogenates of the cell line in a dose-dependent manner. The drug caused a 13-fold stimulation at 10(-4) M, indicating that the compound directly acts on adenylate cyclase, leading to an increase in the intracellular cAMP content of the cells. Collagen accumulation in the cultures was elevated by one-day treatment with 5 X 10(-5) M forskolin to about twice that in the controls. The stimulation was mainly due to an elevation in collagen synthesis but not to an inhibition of intracellular collagen degradation because forskolin dose-dependently increased collagen synthesis; it also significantly increased the amount of low-molecular-weight hydroxyproline found in the cultures. Cells treated with forskolin produced mainly type I collagen, as found in bone matrix in situ, with only small amounts of other types of collagen. Furthermore, forskolin time-dependently inhibited DNA synthesis in the cells, indicating that the increase in type I collagen synthesis by forskolin was not due to stimulated cell proliferation. These results suggest that cAMP is closely linked to the differentiation of osteoblasts in vitro.     

Cartilage induction in vitro : Ultrastructural studies

When 19-day fetal rat triceps muscle was cultured for 7 to 14 days upon decalcified, sequentially extracted adult rat bone, cartilage formed within clefts and vascular spaces of the decalcified bone. The bone substrata were prepared by extracting tibias and femurs of Sprague-Dawley rats with 1:1 chloroform:methanol, 0.6 N HCl, 2 M CaCl₂, 0.6 M EDTA, 8 M LiCl, and H₂O at 56°C. The culture medium used was CMRL 1066 with 15% newborn calf serum. During cultivation, fibroblastic mesenchymal cells migrated out of muscle and into bone crevices where they secreted a cartilaginous matrix composed of thin, randomly dispersed collagen fibrils and proteoglycan granules. The latter are characteristic for cartilage matrix. Extracted bone matrix contained mature collagen fibrils, some of which retained their typical 640-Å banding. Other collagen fibrils were partially disaggregated and expanded to reveal component 50-Å-thick, beaded micro fibrils. Such an expansion of collagen fibrils is known to result from exposure to proteoglycan solvents such as 2 M CaCl₂. The decalcified bone matrix contained many residual devitalized cells and cell fragments which often were seen in close proximity to chondrifying mesenchymal cells. This finding indicates the possibility that residual cellular material could play a role in stimulating cartilage development.     

The osteochondral ligament: a fibrous attachment between bone and articular cartilage in Rana catesbeiana

The anuran epiphyseal cartilage shows a lateral expansion that covers the external surface of the bone, besides other features that distinguish it from the corresponding avian and mammalian structures. The fibrous structure that attaches the lateral cartilage to the bone was characterized in this work. It was designated osteochondral ligament (OCL) and presented two main areas. There was an inner area that was closer to the periosteal bone and contained a layer of osteoblasts and elongated cells aligned to and interspersed with thin collagen fibers. The thin processes of the cells in this area showed strong alkaline phosphatase activity. The outer area, which was closer to the cartilage, was rich in blood vessels and contained a few cells amongst thick collagen fibers. TRITC-phaloidin staining showed the cells of the inner area to be rich in F-actin, and were observed to form a net around the cell nucleus and to fill the cell processes which extended between the collagen fibers. Cells of the outer area were poor in actin cytoskeleton, while those associated with the blood vessels showed intense staining. Tubulin-staining was weak, regardless of the OCL region. The main fibers of the extracellular matrix in the OCL extended obliquely upwards from the cartilage to the bone. The collagen fibers inserted into the bone matrix as Sharpey's fibers and became progressively thicker as they made their way through the outer area to the cartilage. Immunocytochemistry showed the presence of type I and type III collagen. Microfibrils were found around the cells and amongst the collagen fibrils. These microfibrils were composed of either type VI collagen or fibrilin, as shown by immunocytochemistry. The results presented in this paper show that the osteochondral ligament of Rana catesbeiana is a complex and specialized fibrous attachment which guarantees a strong and flexible anchorage of the lateral articular cartilage to the periosteal bone shaft, besides playing a role in bone growth.     

Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method

Summary The ultrastructural localization of alkaline phosphatase (AlP) activity has been demonstrated in epiphyseal growth cartilage and metaphyseal bone of rats. Epiphyso-metaphyseal specimens were decalcified with EDTA and treated with MgCl₂ to regenerate the enzymatic activity before incubation in a medium containing beta-glycerophosphate, MgCl₂ and CeCl₃. AlP activity was present on the outer surface of the plasmamembrane of maturing and hypertrophic chondrocytes and of osteoblasts. Moreover, the reaction product was present in chondrocyte lacunae, in matrix vesicles, and in cartilage matrix, as well as among uncalcified collagen fibrils of osteoid tissue in bone. The intensity of reaction was the lowest, or completely lacking, where the degree of matrix calcification was the highest. These results suggest that alkaline phosphatase is transported from the cells into the cartilage and bone matrix by its association with matrix vesicles and plasmamembrane components, and that its activity in cartilage and bone matrix is inhibited as it is incorporated in the mineral substance.     

Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture

A method is presented for isolating osteoblasts from newborn mouse calvaria without the use of digestive enzymes. The procedure is based on the ability of osteoblasts to migrate from bone onto small glass fragments (Jones, S.J., and A. Boyde, 1977, Cell Tissue Res., 184:179- 193). The isolated cells were cultured for up to 14 d in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 micrograms/ml of ascorbic acid. 7-d cultures were incubated for 24 h with [3H]proline. High levels of collagen synthesis relative to total protein were found, as measured by collagenase digestion of medium and cell layer proteins. Analysis of pepsin-digested proteins from the same cultures by SDS PAGE showed that type I collagen was predominantly produced with small amounts of type III and V (alpha 1 chains) collagens. Osteoblasts grown in the presence of beta-glycerophosphate were able to initiate mineral deposition in culture. Electron microscopic analysis of the cultures revealed the presence of needle- shaped apatite-like crystals associated with collagen fibrils and vesicles in the extracellular space. Mouse skin fibroblasts cultured under identical conditions failed to initiate mineralization. Electron histochemical studies revealed the presence of alkaline phosphatase activity, associated with osteoblast membranes, matrix vesicles and on or near collagen fibrils. Thus these isolated osteoblasts retained in culture their unique property of initiating mineralization and therefore represent a model of value for studying the mineralization process in vitro.     

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.     

THE ULTRASTRUCTURE OF MOUSE LUNG : SOME REMARKS REGARDING THE FINE STRUCTURE OF THE ALVEOLAR BASEMENT MEMBRANE

The fine structure of the alveolar basement membrane of mouse lung was discussed on the basis of three electron micrographs. The basement membrane, i.e., the intercellular layer between endothelium and alveolar epithelium, was found to be of variable width. In its thin parts it appeared rather homogeneous, and did not reveal well defined layers of fibrils. In its thicker portions, some of which may be due to oblique sectioning, cell fragments could be seen lying inside the basement membrane layer. Their exact nature was not determined. In other thickened portions of the membrane bundles of slender (about 23 to 25 mµ) fibrils were found and were tentatively interpreted as collagen fibrils, in spite of the fact that a periodicity could not be observed.     

Interactions of cells in zones of bone resorption under microgravity and hypokinesia

With the use of the methods of electron microscopy and autoradiography employing 3H-glycine the study was made of some morpho-functional cells-cells interactions (osteoblasts, osteocytes, macrophages, fibroblasts) in zones of adaptive remodeling of bone structures of the metaepiphyseal femoral bones of white rats which were during 28 days under experimental hypokinesia conditions, as well as of rats, flown on SLS-2 during 2 weeks. It is established that in zones of an increase of mineral matrix resorption some osteoblasts and osteocytes undergo destruction; a part of osteoblasts remains intact. The osteoclasts don't take part in destruction of osteoblasts and osteocytes. The utilization of the osteogenic cells detritus is accomplished by macrophages, coming to these zones. The resorption loci are filled not with the differentiating osteoblastic cells, as it is the case in the norm, but with fibroblasts and the bundles of collagen fibrils (fibrotic tissue) which do not undergo mineralization. Such changes are considered as one of the mechanisms of bone tissue response to a reduction of the supporting load.     

The migration of osteoblasts

Summary The endocranial matrix surfaces of parietal bones of 2-week old Albino Wistar rats were partly denuded of osteoblasts and then cultured for various periods up to 24 h, in control or PTE-enriched medium. They were examined by scanning electron microscopy and evidence for cell locomotion was found. Osteoblasts traversed the denuded bone surface and cut edges of bone in either medium, and cells also migrated out from vascular channels.Glass spicules were placed on the otherwise undisturbed osteoblast layer in similar organ cultures for 2, 3 or 5 days. Osteoblasts migrated from the bone to populate the glass, negotiating any angle. The cells in PTE-enriched media were always aligned parallel to one another and elongated, tended to align with the edges of the glass and, in time, formed a substrate of aligned fibrils whose axes were parallel to those of the cells. Osteoblasts in control medium on glass showed variable degrees of alignment and elongation and were less influenced by the edges of the glass. Non-locomotory, nearly equidiametrical cells on glass in 5d control cultures had formed a substrate of randomly oriented fibrils.Migrating osteoblasts on bone matrix did not have leading edge ruffles; isolated, migrating ones on glass did.We thank Elaine Bailey for expert assistance; Dr. Martin Evans for the facilities of his laboratory; Dr. Nicholas Maroudas for his erudite interest in our work; and the M.R.C. for financial support.     

Bone sialoprotein mRNA expression and ultrastructural localization in fetal porcine calvarial bone: comparisons with osteopontin

Summary Bone sialoprotein (BSP) and osteopontin (OPN) are two major non-collagenous proteins in bone that have similar biochemical properties and can mediate cell attachment through an RGD (Arg-Gly-Asp) motif that recognizes the vitronectin receptor. To facilitate evaluations of the biological functions of BSP and OPN in bone formation, affinity-purified rabbit polyclonal antibodies against porcine BSP and OPN were used, together with a high-resolution protein A-gold immunocytochemical technique to reveal the ultrastructural localization of these proteins in undermineralized sections of 50-day fetal porcine calvarial bone. In addition,³⁵S-labelled antisense riboprobes were prepared to demonstrate the cellular expression of BSP and OPN in the same tissues usingin situ hybridization. Immunolocalization for both BSP and OPN revealed the highest density of gold particles associated with electron-dense organic material found at the mineralization front and in ‘cement lines’. Labelling was also observed in the mineralized matrix over electron-dense material between collagen fibrils. In the osteoid of newly-formed bone, immunogold labelling for BSP and OPN was associated with loci of mineralization, which were often characterized by feathery clusters of fine needle-like crystals. Results ofin situ hybridization on the same tissues demonstrated that BSP mRNA expression was restricted to differentiated osteoblasts with particularly strong signals evident at sites ofde novo bone formation. More moderate expression of BSP was observed in ‘older’ osteoblasts and in some of the newly-entrapped osteocytes. Although expression of OPN mRNA was also observed in osteoblasts and osteocytes, the level of hybridization was similar for most bone cells and not markedly stronger than the signal observed in some stromal cells. While it is evident from these and other studies that both BSP and OPN are associated with bone formation, the differences observed in cellular expression indicate distinct roles for these proteins in bone formation.     

Ultrastructural pathology of the compound eye and optic neuropiles of the retinal degeneration mutant (w rdgB KS222) Drosophila melanogaster

Summary Lanthanum, applied to the outside of the fixed sciatic nerve of Rana pipiens, did not enter the endoneurium, but was halted by functionally tight junctions at the inner layers of the perineurium. This component of the bloodnerve barrier consists of several concentric layers of cells interspersed with an extracellular matrix of amorphous ground substance, collagen fibrils, and fine filaments. Numerous vesicular profiles are closely associated with the surface membranes of all the cells. The application of lanthanum to fixed tissue revealed that these profiles are attached to the cell surface by narrow necks, and open to the extracellular space. The attenuated cells are filled by the vesicular structures, which often appear to overlap. Stereoscopic electron microscopy showed that these vesicles did not fuse with each other or with the apposing cell surface to form transcellular channels. Channel formation does not appear to contribute significantly to the permeability of any of the perineurial layers.     

Mesodermal and neural crest derived ovine tibial and mandibular osteoblasts display distinct molecular differences

Mandibular osteoblasts originate from the neural crest and deposit bone intramembranously, mesoderm derived tibial osteoblasts by endochondral mechanisms. Bone synthesized by both cell types is identical in structure, yet functional differences between the two cell types may exist. Thus, both matched juvenile and adult mandibular and tibial osteoblasts were studied regarding their proliferative capacity, their osteogenic potential and the expression of osteogenic and origin related marker genes. Juvenile tibial cells proliferated at the highest rate while juvenile mandibular cells exhibited higher ALP activity depositing more mineralized matrix. Expression of Hoxa4 in tibial cells verified their mesodermal origin, whereas very low levels in mandibular cells confirmed their ectodermal descent. Distinct differences in the expression pattern of bone development related genes (collagen type I, osteonectin, osteocalcin, Runx2, MSX1/2, TGF-β₁, BAMBI, TWIST1, β-catenin) were found between the different cell types. The distinct dissimilarities in proliferation, alkaline phosphatase activity, the expression of characteristic genes, and mineralization may aid to explain the differences in bone healing time observed in mandibular bone when compared to long bones of the extremities.