Encapsulation of foreign particles in vitro by separated blood cells from crayfish,Astacus leptodactylus

Summary Bone matrix consists of type-I collagen and noncollagenous proteins. The latter represent only 10% of its total protein content. Since type-I collagen is also present in various other connective tissue sites (e.g., skin) it cannot be considered as bone specific. Among the non-collagenous components osteonectin — a 32 kilodalton (KD) glycoprotein linking mineral to collagen fibrils — is thought to be bone specific due to its biochemical properties. In the present study various skeletal and non-skeletal tissues were investigated for the presence of osteonectin by means of immunocytochemical methods. Two polyclonal antibodies against human and bovine osteonectin were applied. Immunocytochemically, osteonectin could be demonstrated in active osteoblasts and osteoprogenitor cells as well as in young osteocytes, while aged, quiescent osteocytes did not contain the protein, suggesting that the protein is a marker of the osteoblastic functional differentiation of bone cells. Osteonectin was absent in all non-skeletal tissues with the exception of chondrocytes in so-called mineralizing chondroid bone.     

Matrix proteins associated with bone calcification are present in human vascular smooth muscle cells grownin vitro

Summary Atherosclerotic lesions are composed of cellular elements that have migrated from the vessel lumen and wall to form the cellular component of the developing plaque. The cellular elements are influenced by various growth-regulatory molecules, cytokines, chemoattractants, and vasoregulatory molecules that regulate the synthesis of the extracellular matrix composing the plaque. Because vascular smooth muscle cells (VSMC) constitute the major cellular elements of the atherosclerotic plaque and are thought to be responsible for the extracellular matrix that becomes calcified in mature plaques, immunostaining for collagenous and noncollagenous proteins typically associated with bone matrix was conducted on VSMC grownin vitro. VSMC obtained from human aorta were grown in chambers on glass slides and immunostained for procollagen type I, bone sialoprotein, osteonectin, osteocalcin, osteopontin, decorin, and biglycan. VSMC demonstrated an intense staining for procollagen type I, and a moderately intense staining for the noncollagenous proteins, bone sialoprotein and osteonectin, two proteins closely associated with bone mineralization. Minimal immunostaining was noted for osteocalcin, osteopontin, decorin, and biglycan. The presence in VSMC of collagenous and noncollagenous proteins associated with bone mineralization suggest that the smooth muscle cells in the developing atherosclerotic plaque play an important role in the deposition of the extracellular matrix involved in calcification of developing lesions.     

Expression of mRNAs for collagens and other matrix components in dedifferentiating and redifferentiating human chondrocytes in culture

Cell cultures were initiated from epiphyseal cartilages, diaphyseal periosteum, and muscle of 16-week human fetuses. Total RNAs isolated from these cultures were analyzed for the levels of mRNAs for major fibrillar collagens, two proteoglycan core proteins and osteonectin. In standard monolayer cultures the differentiated chondrocyte phenotype was replaced by a dedifferentiated one: the mRNA levels of cartilage-specific type II collagen decreased upon subculturing, while those of types I and III collagen, and the core proteins increased. When the cells were transferred to grow in agarose, redifferentiation (reappearance of type II collagen mRNA) occurred. Fibroblasts grown from periosteum and muscle were found to contain mRNAs for types I and III collagen and proteoglycan cores. When these cells were transferred to agarose they acquired a shape indistinguishable from chondrocytes, but no type II collagen mRNA was observed.     

Purification and partial characterization of small proteoglycans I and II, bone sialoproteins I and II, and osteonectin from the mineral compartment of developing human bone

Using nondegradative isolation procedures we purified and characterized five major noncollagenous proteins from developing human bone. Small bone proteoglycan I, Mr approximately 350,000 on sodium dodecyl sulfate (SDS), 4-20% gradient polyacrylamide gels has a different amino-terminal sequence of NH2-Asp-Glu-Glu-()-Gly-Ala-Asp-Thr and is not cross-reactive with the small bone proteoglycan II, Mr approximately 200,000 on SDS-gradient polyacrylamide gels. Bone proteoglycan II is 95% N terminally blocked and the small amount that can be sequenced has an amino-terminal sequence (NH2-Asp-Glu-Ala-()-Gly-Ile. . .) that is apparently similar but not identical to a small proteoglycan isolated by Brennan, M.J., Oldberg, A., Pierschbacher, M.D., and Ruoslahti, E. (1984) J. Biol. Chem. 259, 13742-13750 from human fetal placenta membrane. Two bone sialoproteins, each of which migrates at a Mr approximately 80,000 on SDS gels, have also been isolated. Bone sialoprotein I has an amino-terminal sequence of NH2-Ile-Pro-Val-Lys-Gln-Ala. . . which is different from that of bone sialoprotein II with an amino-terminal sequence of NH2-Phe-Ser-Met-Lys-Asn-Leu. . . The two bone sialoproteins do not cross-react on Western blot analysis. Human bone osteonectin contains a large number of cysteines, more than 90% of which appear to be in disulfide bonds. The N-terminal amino acid sequence of human bone osteonectin was nearly identical to bovine bone osteonectin and had many similarities to a protein found in mouse parietal endoderm (Mason, I.J., Taylor, A., Williams, J.G., Sage, H., and Hogan, B.L.M. (1986) EMBO J. 5, 1831-1837.     

Structure and binding properties of collagen type XIV isolated from human placenta

Collagen XIV was isolated from neutral salt extracts of human placenta and purified by several chromatographic steps including affinity binding to heparin. The same procedures also led to the purification of a tissue form of fibronectin. Collagen XIV was demonstrated by partial sequence analysis of its Col1 and Col2 domains and by electron microscopy to be a disulphide-linked molecule with a characteristic cross-shape. The individual chains had a size of approximately 210 kD, which was reduced to approximately 180 kD (domain NC3) after treatment with bacterial collagenase. Specific antibodies mainly to NC3 epitopes were obtained by affinity chromatography and used in tissue and cell analyses by immunoblotting and radioimmunoassays. Two sequences from NC3 were identified on fragments obtained after trypsin cleavage. They were identical to cDNA-derived sequences of undulin, a noncollagenous extracellular matrix protein. This suggests that collagen XIV and undulin may be different splice variants from the same gene. Heparin binding was confirmed in ligand assays with a large basement membrane heparan sulphate proteoglycan. This binding could be inhibited by heparin and heparan sulphate but not by chondroitin sulphate. In addition, collagen XIV bound to the triple helical domain of collagen VI. The interactions with heparin sulphate proteoglycan and collagen VI were not shared by the NC3 domain, or by reduced and alkylated collagen XIV. No or only low binding was observed for collagens I-V, pN- collagens I and III, and several noncollagenous matrix proteins, including laminin, recombinant nidogen, BM-40/osteonectin, plasma and tissue fibronectin, vitronectin, and von Willebrand factor. Insignificant activity was also shown in cell attachment assays with nine established cell lines.     

Bone protects proteins over thousands of years: extraction, analysis, and interpretation of extracellular matrix proteins in archeological skeletal remains

In a good state of preservation, bone conserves the entire protein pattern of extracellular bone matrix proteins over thousands of years. The quality of the profiles of matrix proteins isolated from ancient bones (ranging from the pre-Pottery Neolithic Phase to Early Modern Times from different archaeological sites in different geographical areas), separated by electrophoresis, is as good as those from recent bones. Molecules arising from collagenous proteins (e.g., collagen type I), from the noncollagenous group (e.g., osteonectin), and from the immune system (e.g., immunoglobulin G) were identified in Western blots by specific antibodies. A comparative study of the immunoglobulin G content of the bones of five prehistoric children showed the lowest immunoglobulin G content in a child who suffered from chronic scurvy. Ancient bone proteins were also separated by two-dimensional electrophoresis. This technique makes fractionation of the complex protein mixtures of extracellular bone matrix more reproducible. Bone retains a chemical memory of earlier metabolic stimuli in its configuration of collagenous and noncollagenous proteins. In combination with the results of the microscopic examination of ancient bone, it should be possible to obtain more reliable information on the history and the evolution of diseases, based on analysis of intact proteins.     

Temporal and spatial gene expression of major bone extracellular matrix molecules during embryonic mandibular osteogenesis in rats

It is not known how gene expression of bone extracellular matrix molecules is controlled temporally and spatially, or how it is related with morphological differentiation of osteoblasts during embryonic osteogenesis in vivo. The present study was designed to examine gene expressions of type I collagen, osteonectin, bone sialoprotein, osteopontin, and osteocalcin during mandibular osteogenesis using in situ hybridization. Wistar rat embryos 13–20 days post coitum were used. The condensation of mesenchymal cells was formed in 14-day rat embryonic mandibles and expressed genes of pro-(I) collagen, osteonectin, bone sialoprotein and osteopontin. Cuboidal osteoblasts surrounding the uncalcified bone matrix were seen as early as in 15-day embryonic mandibles, while flat osteoblasts lining the surface of the calcified bone were seen from 16-day embryonic mandibles. Cuboidal osteoblasts expressed pro-1(I) collagen, osteonectin and bone sialoprotein intensely but osteopontin very weakly. In contrast, flat osteoblasts expressed osteopontin very strongly. Osteocytes expressed the extracellular matrix molecules actively, in particular, osteopontin. The present study demonstrated the distinct gene expression pattern of type I collagen, osteonectin, bone sialoprotein, osteopontin and osteocalcin during embryonic mandibular osteogenesis in vivo.     

Confocal imaging and timing of secretion of matrix proteins by osteoblasts derived from avian long bone

Primary osteoblasts derived from avian long bone have been evaluated in terms of spatial and temporal expression of known osteoblastic marker proteins during the early phases of cell culture. Confocal imaging of matrix proteins revealed that osteocalcin, bone sialoprotein, osteopontin, and osteonectin were restricted to the cell interior at day 4 of culture; secretion and deposition into the extra-cellular matrix of bone sialoprotein and osteopontin was evident at 8 and 12 days of culture. Osteocalcin and osteonectin were not deposited in the matrix within the timeframe of the study. Total collagen levels produced and alkaline phosphatase activity were substantial by day 4 of culture, and increased from that point 4.0- and 5.5-fold, respectively, by culture day 12. The expression of type I collagen, PTHrP receptor, osteopontin, bone sialoprotein and osteocalcin was followed by Northern blot analysis. Type I collagen and osteopontin mRNA were expressed at constant levels throughout the culture period. Over the 12 days of culture both PTH/PTHrP receptor and bone sialoprotein mRNA expression were found to increase by 2.3- and 2.5-fold, respectively. In contrast, the expression of osteocalcin message decreased by 2.5-fold by day 8 of culture.     

Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

Isolated mouse osteoblasts that retain their osteogenic activity in culture were incubated with [35S] sulfate. Two radiolabeled proteins, in addition to proteoglycans, were extracted from the calcified matrix of osteoblast cultures. All the sulfate label in both proteins was in the form of tyrosine sulfate as assessed by amino acid analysis and thin layer chromatography following alkaline hydrolysis. The elution behavior on DEAE-Sephacel of the major sulfated protein and the apparent Mr on sodium dodecyl sulfate gels were characteristic of bone sialoprotein II extracted from rat. This protein was shown to cross-react with an antiserum raised against bovine bone sialoprotein II, indicating that bone sialoprotein II synthesized by cultured mouse osteoblasts is a tyrosine-sulfated protein. The minor sulfated protein was tentatively identified as bone sialoprotein I or osteopontin based on its elution properties on DEAE-Sephacel and anomalous behavior on sodium dodecyl sulfate gels similar to those reported for rat bone sialoprotein I.     

Immunocytochemical demonstration of extracellular matrix proteins in isolated osteocytes

 Cultures of isolated osteocytes may offer an appropriate system to study osteocyte function, since isolated osteocytes in culture behave very much like osteocytes in vivo. In this paper we studied the capacity of osteocytes to change their surrounding extracellular matrix by production of matrix proteins. With an immunocytochemical method we determined the presence of collagen type I, fibronectin, osteocalcin, osteopontin and osteonectin in cultures of isolated chicken osteocytes, osteoblasts and periosteal fibroblasts. In osteoblast and periosteal fibroblast cultures, large extracellular networks of collagen type I and fibronectin were formed, but in osteocyte populations, extracellular threads of collagen or fibronectin were only rarely found. The percentage of cells positive for osteocalcin, osteonectin and osteopontin in the Golgi apparatus, on the other hand, was highest in the osteocyte population. These results show that osteocytes have the ability to alter the composition of their surrounding extracellular matrix by producing matrix proteins. We suggest this property is of importance for the regulation of the calcification of the bone matrix immediately surrounding the cells. More importantly, as osteocytes depend for their role as mechanosensor cells on their interaction with matrix proteins, the adaptation of the surrounding matrix offers a way to regulate their response to mechanical loading. Accepted: 9 July 1996     

Effects of cis-4-hydroxy-L-proline, and inhibitor of Schwann cell differentiation, on the secretion of collagenous and noncollagenous proteins by Schwann cells

The proline analog cis-4-hydroxy-L-proline (CHP) was previously shown to inhibit both Schwann cell (SC) differentiation and extracellular matrix (ECM) formation in cultures of rat SCs and dorsal root ganglion neurons. We confirmed that CHP inhibits basal lamina formation by immunofluorescence with antibodies to laminin, type IV collagen, and heparan sulfate proteoglycan. In order to test the hypothesis that CHP inhibits SC differentiation by specifically inhibiting the secretion of collagen, cultures grown in the presence or absence of CHP were metabolically labeled with [3H]leucine and the media were analyzed for relative amounts of (a) collagenous and noncollagenous proteins by assay with bacterial collagenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or (b) triple-helical collagen by pepsin digestion followed by SDS-PAGE. The results indicate that although CHP inhibited the accumulation of secreted collagen in the culture medium and disrupted collagen triple-helix formation, it also significantly inhibited the accumulation of secreted noncollagenous proteins in the medium. CHP had no significant effect on either total protein synthesis (medium plus cell layer) or cell number. We conclude that CHP does not act as a specific inhibitor of collagen secretion in this system, and thus data from these experiments cannot be used to relate SC collagen production to other aspects of SC differentiation. We discuss the evidence for and against specificity of CHP action in other systems.     

Noncollagenous bone proteins in experimental rickets in the rat

Rats were raised in the absence of vitamin D in utero and throughout post-fetal life and neither 1,25-dihydroxyvitamin D3 nor related metabolites were detected in serums. No changes were observed in the relative amount of extractable noncollagenous bone proteins (NCP) in rachitic compared to vitamin-D-repleted animals. As expected, the relative levels of the mineral-bound, serum-derived albumin and 2-HS glycoprotein were unaffected in bones of rachitic animals. Interestingly, the vitamin D deficiency also did not have dramatic effects on several bone cell-derived noncollagenous proteins including: bone proteoglycans I & 11, bone sialoprotein li osteonectin, and osteocalcin. In contrast to the proteoglycans, the bone sialoprotein II and osteonectin were found in the nonmineral compartment of the rachitic animals, presumably bound to the wide osteoid seam.     

Cells isolated from the endosteal bone surface of adult rats express differentiated osteoblastic characteristics in vitro

Endosteal bone surface cells were previously shown to be involved in the regulation of bone formation in humans. In this study, we have characterized the cells isolated from the endosteal bone surface in adult rats. Fragments of periosteum-free tibia were obtained from 4-, 6- and 9-month-old rats by collagenase digestion, and the phenotypic characteristics of the osteoblastic cells migrating from the endosteal bone surface were evaluated in culture. Endosteal bone surface cells present a strong alkaline phophatase (ALP) activity as shown by cytochemistry and measured biochemically. The cells synthesize high levels of osteocalcin as measured by radioimmunoassay. Osteocalcin production was increased after stimulation with 10 nM 1,25 dihydroxyvitamin D (1,25(OH)₂ D) and the response to 1,25(OH)2 D was similar at all ages. Endosteal cells from young adult rats (4 months old) but not from older rats (6 and 9 months old) showed increased cAMP production in response to 10 nM parathyroid hormone (PTH), suggesting an agerelated decrease in the PTH-responsiveness of the bone surface cells. Immunocytochemistry using specific antibodies showed that preconfluent endosteal bone cells from adult rats expressed collagen and noncollagenous bone proteins in culture in the absence of inducers. The cells synthesized mostly type-I collagen which remained localized intracellularly. Type-III collagen was only expressed at low levels. The bone surface cells also expressed osteocalcin and bone sialoprotein, two markers of differentiated osteoblasts, as well as osteonectin. Endosteal cells plated at high density and cultured for 21 days with 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate formed multiple calcified nodules, as evidenced by von Kossa staining. This study shows that cells isolated from the endosteal bone surface of adult rats express in vitro characteristics of differentiated osteoblasts. These cell cultures can be used to study the dysfunctions of endosteal bone cells in relation to disorders of bone formation in adult rats.     

Biochemical comparison of fibroblast populations from different periodontal tissues: characterization of matrix protein and collagenolytic enzyme synthesis

To compare the expression of extracellular matrix components by fibroblasts from different periodontal tissues, rat molar periodontal ligament fibroblasts (RPL) and rat gingival fibroblasts (RGF) were isolated and cultured from individual animals. Pulse-chase experiments using [35S]methionine as a precursor revealed that confluent populations of early passage cells of both cell types synthesized similar amounts of collagen, fibronectin, and SPARC/osteonectin. Qualitative and quantitative differences were apparent in the relative proportions of type III collagen, in the rates of procollagen processing, and in the synthesis of a small number of unidentified proteins observed by sodium dodecyl sulphate--polyacrylamide gel electrophoresis. Collagen constituted 24-26% of the radiolabelled proteins secreted by both cell types, type I being the predominant collagen, with lower amounts of type III (3-8% RGF, 8-18% RPL) and type V (approximately 1%) collagens. Procollagen processing in the culture medium of RPL cells was more rapid than for RGF cells, but was increased in multilayered cultures of both RPL and RGF. In multilayered cultures, collagen TCA fragments, indicative of tissue collagenase activity, were also identified. Active and latent tissue collagenases and a latent form of a novel collagenolytic enzyme (matrix metalloendoproteinase-V) that cleaves native TCA fragments were demonstrated in these cultures. Addition of either concanavalin A (10(-6) M) or retinoic acid (10(-5) M) to the culture medium stimulated the secretion of the latent collagenolytic enzymes. Collagenase inhibitor was also synthesized by both RGF and RPL cells. SPARC/osteonectin, a 40-kilodalton glycoprotein, represented 0.5-1.0% of the secreted radiolabelled proteins of both cell types.     

Formation of chondrous and osseous tissues in micromass cultures of rat frontonasal and mandibular ectomesenchyme

Rat frontonasal and mandibular mesenchyme was isolated from day-12 1/2 (stage-22) rat embryos and cultured at high density for up to 12 days. The stage chosen was based on the observation that mandibular mesenchyme at this stage became independent of its epithelium with respect to the production of both cartilage and bone. Frontonasal cultures developed aggregates of anastomosing columns of cells within 2 days. These grew as the cells enlarged, laying down an Alcian-blue-positive matrix by day 3 of culture. Significant mineral was detected by von Kossa staining by day 5 at which time the aggregates covered a large portion of the culture, eventually covering the entire micromass by day 10-12. Mandibular cultures developed centrally located nodular aggregates by 3 days of culture. These nodules increased in number, spreading outwards as the cells enlarged, laying down an Alcian-blue-positive matrix by day 4 and mineral by days 6-7. At this time the nodules began to elongate and coalesce, but never covered the entire culture over the 12-day period. Antibody staining revealed that in both cultures the cells were initially positive for type I collagen. Subsequently, the aggregates began expressing type II collagen, followed by type X, which coincided with the onset of mineralization. At this time some cells were negative for these cartilage markers, but positive for osteoblast markers, bone sialoprotein II, osteocalcin and type I collagen. In addition osteonectin and alkaline phosphatase were demonstrable in all of the aggregate cells late in the culture period. This provided clear evidence that chondroblast and osteoblast differentiation was proceeding within these cultures. The culture of rat facial mesenchyme should prove very useful, not only for the analysis of bone and cartilage induction and lineage relationships, but also in furthering our knowledge of craniofacial differentiation, growth and pattern formation by extending our analysis to a mammalian system.     

Collagen-binding proteins in collagenase-released matrix vesicles from cartilage. Interaction between matrix vesicle proteins and different types of collagen.

Recent evidence indicates that matrix vesicles (MV) interact with cartilage-specific collagens and other matrix proteins. Both type II and X collagens bind to and cosediment with MV. Our companion study shows that MV also are tightly coupled to proteoglycan link proteins (LP) and hyaluronic acid-binding region (HABR) in cartilage matrix. Here we sought to identify proteins responsible for the nexus between MV and matrix collagens using affinity chromatography with types I, II, and X collagen-Sepharose columns. Elution with NaCl step-gradients in the presence of nonionic detergent was used to assess the affinity between the MV proteins and the covalently attached collagens. Several MV proteins were found to bind to native type I, II, and X collagens but none bound to denatured type I collagen. Alkaline phosphatase, proteoglycan LP and HABR, and the 33- and 67-kDa annexins, bound with varying affinities to the native type I, II and X columns. In particular, LP and HABR, the 67-kDa annexin, and alkaline phosphatase bound with high affinity to the cartilage-specific collagens, although LP, HABR, and a 37-kDa protein also bound less tightly to native type I collagen. Thus, several MV proteins bind specifically to native type II and X collagens and should promote interaction between MV and the extracellular matrix. Such interactions may be important in MV formation, or in MV-mediated mineralization.     

Isolation and characterization of native adult osteonectin

Noncollagenous bovine bone proteins were obtained from EDTA-solubilized extracts of adult bovine bone in the absence of denaturants. Native osteonectin was isolated from the noncollagenous bone proteins by ion-exchange chromatography using DEAE-Sephadex A-50 and DEAE-Sephadex A-25, followed by gel filtration on Sephadex G-100. Comparison of the physical and chemical properties (i.e. electrophoric mobility, amino acid composition and pI) of this protein with those reported by Termine et al. (Termine, J.D., Belcourt, A.B., Conn, K.M., and Kleinman, H.K. (1981) J. Biol. Chem. 256, 10403-10408) indicate that this protein is osteonectin. Sedimentation equilibrium analyses in the presence of 6 M guandinium chloride, 10 mM Ca2+, 10 mM EDTA, or 0.15 M NaCl all yielded a molecular weight of 29,100 +/- 900. 125I-Osteonectin underwent saturable and exchangeable binding to hydroxyapatite and calf skin collagen. Eleven mg of 125I-osteonectin bound to 1.0 g of hydroxyapatite with a Kd of 8 X 10(-8) M. The intrinsic fluorescence of bovine osteonectin was partially quenched when micromolar Ca2+ was added, indicating a high affinity Ca2+ interaction. Native osteonectin was found to reduce the rate of hydroxyapatite crystal seeded growth by 50% (1 IU) at a concentration of 1.6 X 10(-7) M at pH 7.4, 37 degrees C in 0.15 M NaCl. This makes osteonectin one of the most potent inhibitors of hydroxyapatite formation presently known and more than 5 times as effective as bone Gla protein (1 IU = 8 X 10(-7) M).     

WHY DOES BONE MATRIX CONTAIN NON-COLLAGENOUS PROTEINS? THE POSSIBLE ROLES OF OSTEOCALCIN,OSTEONECTIN, OSTEOPONTIN AND BONE SIALOPROTEIN IN BONE MINERALISATION AND RESORPTION

Four major non-collagenous bone proteins were localised by single and double immuno-histochemistry during de novo mineralisation and bone resorption. Both osteopontin and bone sialoprotein were localised ahead of the mineralisation front, suggesting that both proteins are necessary for the initiation of bone mineralisation. This supports previous suggestions that bone sialoprotein acts as a crystal nucleator. The role of osteopontin is less certain, but might be related to ensuring that only the right type of crystal is formed. Osteocalcin and osteonectin were not present in areas of first crystal formation, but were present in the fully mineralised matrix. Their role may be to control the size and speed of crystal formation. Osteopontin, bone sialoproteins and osteocalcin (but not osteonectin) were also present at reversal lines. Interpreting this localisation together with information from the literature, the following functions are suggested during resorption: Osteocalcin may act as a chemoattractant for osteoclasts, while both osteopontin and bone sialoprotein may facilitate the binding of osteoclasts via the arg-gly-asp motif.