A comparison of fibronectin and laminin binding to undemineralized and demineralized tooth root surfaces.
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Animals↗
Biomedical subjects
J Sodek
Publications and source records attributed to J Sodek.
Collagenase and collagenase inhibitor activities in crevicular fluid of patients receiving treatment for localized juvenile periodontitis.
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Adolescent↗
Osteonectin is a minor component of mineralized connective tissues in rat.
Osteonectin is a major glycoprotein of porcine and bovine bones and teeth that is found associated with hydroxylapatite crystal surfaces. From the ability of osteonectin to bind calcium ions, it has been proposed as a possible nucleator of hydroxylapatite crystal formation. Analysis of hydroxylapatite-bound proteins of rat bone and dentine, however, has revealed that osteonectin represents only 2.5 +/- 1.5% of the hydroxylapatite-bound protein in long bones, 0.9 +/- 0.5% in calvariae, and less than 0.1% in incisor dentine of animals of different ages. Further, in vivo pulse-chase studies carried out in young adult rats have shown osteonectin to be synthesized at low levels in these tissues. Similarly, low levels of osteonectin were synthesized by rat calvarial cells in vitro. In contrast, fibroblastic cells from periodontal ligament and gingiva synthesized significantly greater amounts of osteonectin. These studies indicate that the low quantities of osteonectin in rat mineralized tissues are a consequence of low rates of formation rather than being due to rapid turnover. The virtual absence of osteonectin in incisor dentine correlates with the lack of peritubular dentine in rat, whereas the low osteonectin content of rat bones may reflect differences in their structure and biophysical properties compared with bones of larger mammals.
Animals↗
The influence of chemically-induced modifications of root surfaces on cell migration, attachment, and orientation.
Surface demineralization of tooth root surfaces has been shown to improve re-attachment of cells and to promote tissue reconstruction following periodontal surgery. Exposure of collagen fibers has been thought to facilitate migration, attachment, and orientation of fibroblasts on the root surface. However, using an in vitro assay, we have found that both attachment and orientation of human gingival fibroblasts on demineralized dentin surfaces are further improved following digestion of the exposed collagen with bacterial collagenase. In contrast, pronase and trypsin digestion of the surface collagen had no significant effect, whereas heat denaturation had an inhibitory effect. Dissociative extraction of the demineralized dentin slices with 4 mol/L guanidine hydrochloride (GuHCl) also improved attachment and orientation, and when undemineralized dentin was subjected to dissociative extraction, cell attachment was comparable and orientation superior to that on demineralized surfaces. These studies indicate that demineralization is not a prerequisite for facilitating attachment, and that enhanced attachment and orientation of cells are not dependent upon a collagenous substratum.
Animals↗
Enamel protein and osteonectin in developing teeth.
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Ameloblasts↗
Cell adhesion to low-Mr proteins extractable from mineralized and soft connective tissues.
Guanidinium chloride (4 M) containing proteinase inhibitors was used to extract proteins from porcine calvariae and long bones. The extracted proteins were separated on polyacrylamide slab gels and transferred electrophoretically to nitrocellulose strips. Proteins with cell-adhesion properties were identified by incubating the strips with cells and staining with Amido Black. In addition to binding to fibronectin, both bone cells and fibroblast-like cells adhered to proteins of Mr approximately 30 000 and approximately 14 000-17 000. 4 M-Guanidinium chloride extracts of porcine skin and gingiva yielded cell-binding proteins with similar Mr values. These data suggest that these low-Mr proteins may have a general cell-adhesion function in both soft and mineralized connective tissues.
Animals↗
Identification of pre-osteonectin produced by cell-free translation of fetal porcine calvarial mRNA.
The cell-free biosynthesis of the bone protein osteonectin was studied using mRNA from fetal porcine calvariae. Total RNA was extracted from the calvariae with guanidinium thiocyanate and was partially purified by precipitation with acid/ethanol. Translations were performed using the reticulocyte lysate system and were optimized with respect to mRNA concentration and K+ (70 mM) and Mg2+ (0.6 mM) concentration. Cell-free synthesized osteonectin, radiolabeled with [35S]methionine, was specifically immunoprecipitated with rabbit antiserum to porcine osteonectin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. When analyzed under reduced conditions, the translated protein migrated with an Mr 45,000 compared to an Mr 39,000 for cell-synthesized osteonectin. When translated in the presence of microsomal membranes, the immunoprecipitated osteonectin co-migrated with the cell-synthesized osteonectin, indicating that a signal sequence of about 45-50 amino acids (Mr 6,000) had been removed. Under nonreduced conditions the pre-osteonectin co-migrated with osteonectin (Mr 39,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that a highly folded structure is retained by disulfide bridges under denaturing conditions. The relationship between the immunoprecipitated pre-osteonectin from the cell-free translations and both the cell-synthesized and tissue-extracted osteonectin was confirmed by one-dimensional peptide mapping of Staphylococcus aureus V-8 protease digestions. The results indicate that porcine osteonectin is synthesized on polysomes in a pre-osteonectin form which is translocated vectorially into microsomal vesicles and cotranslationally processed by the removal of a signal peptide.
Animals↗
Fibronectin-independent attachment of human gingival fibroblasts to interstitial and basement membrane collagens.
We have studied the ability of human gingival fibroblasts (HGF) to attach to different interstitial (types I, II and III) and basement membrane (types IV and V) collagens. HGF cells were plated onto collagen-coated Petri dishes under various conditions and the percentage of cells attaching to the collagen was determined. HGF were found to attach to all the different types of native collagens, but attached poorly to the corresponding denatured collagens. When plated in the presence of 15% fetal bovine serum (FBS) or fibronectin-depleted FBS, similar percentages (approximately 85%) of cells attached to both interstitial and basement membrane collagens, demonstrating an attachment mechanism that is independent of plasma fibronectin. That the attachment in the presence of serum was also independent of cellular fibronectin was shown by the inability of fibronectin antibodies to block attachment to any of the collagen types. HGF were also capable of attaching to all of the collagen types in the complete absence of serum. In previous studies, investigators using cell lines have suggested that cell attachment in the absence of serum is non-physiological. However, the serum-free attachment of HGF to collagen was found to be dependent on cellular protein synthesis indicating that this attachment mechanism has biological significance.
Basement Membrane↗
Stimulation of granulation tissue formation by platelet-derived growth factor in normal and diabetic rats.
Subcutaneous implantation of Hunt-Schilling wound chambers in rats induces a wound repair response causing the chamber first to fill with fluid and subsequently with connective tissue. The presence of a type I collagen gel encouraged a more rapid dispersion of cells throughout the chamber but had no effect on the rate of new collagen deposition. Addition of platelet-derived growth factor (PDGF; 50 ng/chamber) to the collagen-filled chambers caused an earlier influx of connective tissue cells, a marked increase in DNA synthesis, and a greater collagen deposition in the chamber during the first 2 wk after implantation. After 3 wk, however, the levels of collagen were similar in PDGF-supplemented and control chambers. Diabetic animals exhibited a decreased rate of repair which was restored to normal by addition of PDGF to the wound chamber. Combinations of PDGF and insulin caused an even more rapid increase in collagen deposition. These results suggest that the levels of various growth factors, particularly PDGF, may be limiting at wound sites and that supplementation of wounds with these factors can accelerate the rate of new tissue formation.
Animals↗
Specific immunohistochemical localization of osteonectin and collagen types I and III in fetal and adult porcine dental tissues.
Affinity-purified antibodies have been used in combination with the peroxidase-antiperoxidase technique to study the distribution of osteonectin and collagen types I and III in porcine dental tissues. Tissue sections (2 mm thick), including unerupted (fetal) or erupted (adult) teeth, were fixed in periodate-lysine-paraformaldehyde, demineralized in 12% w/v ethylenediaminetetraacetic acid, and after embedding, 6 micron sections were prepared for immunolocalization. Strong staining for osteonectin was observed in dentine of unerupted teeth and in the associated alveolar bone. Light to moderate staining was observed in the dental pulp, stratum intermedium, stellate reticulum, and the reticular elements in the endosteal spaces. In erupted teeth, osteonectin staining in dentine was concentrated around dentinal tubules and the associated alveolar bone stained with variable intensity. Cementum was poorly stained. However, the periodontal ligament and reticular material in the endosteal spaces showed moderate to strong staining. Weaker staining was apparent in the pulp and lamina propria of the gingiva. In comparison, type I collagen showed a similar distribution to osteonectin in both fetal and adult tissues, whereas type III collagen was generally restricted to the periodontal ligament, reticular elements of the endosteal spaces, and Sharpey's fibers in bone and cementum. Both odontoblast and ameloblast layers in fetal tissues stained for osteonectin and type III collagen.
Animals↗
Localization of laminin, type IV collagen, fibronectin, and heparan sulfate proteoglycan in chick retinal pigment epithelium basement membrane during embryonic development.
Type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin were localized in the basement membrane (BM) of chick retinal pigment epithelium (RPE) during various stages of eye development. At different times over a 4-17 day period after fertilization, chick embryo eyes were dissected, fixed in periodate-lysine-paraformaldehyde, and 6 micron frozen sections through the central regions of the eye were prepared. Sections were postfixed in -20 degrees C methanol and stained immediately by indirect immunofluorescence using sheep anti-mouse laminin, sheep antimouse type IV collagen, rabbit anti-mouse heparan sulfate proteoglycan, and mouse monoclonal anti-porcine plasma fibronectin. Fluorescein-labeled F(ab')2 fragments of the appropriate immunoglobulins (IgGs) were used as secondary antibodies. Laminin could be readily demonstrated in the BM of the RPE during all stages of development. The staining for type IV collagen, fibronectin, and heparan sulfate proteoglycan HSPG) was less intense than that for laminin, but was also localized in the BM along the basal side of the RPE. In addition to staining the BM, antiserum to HSPG, gave a diffuse labeling from day 9 onward, above the RPE extending into the region of the photoreceptors. Whereas the intensity of staining generally increased between day 4 and day 17 of development, the distribution of the different BM components did not change. Hence the presence of type IV collagen, laminin, fibronectin, and HSPG in the BM of RPE in vivo during all the stages of development investigated supports the concept that these macromolecules are important basic components of this, and other, BMs. Furthermore, these results indicate that the composition of the BM of RPE cells in vivo is similar to the BM material deposited by RPE cells in vitro (Turksen K, Aubin JE, Sodek JE, Kalnins VI: Collagen Rel Res, 4:413-426, 1984) and that the in vitro cultures can therefore serve as a useful model for studying BM formation.
Animals↗
Synthesis of collagenase and collagenase inhibitors by osteoblast-like cells in culture.
A rat osteosarcoma cell clone (ROS 17/2), and osteoblast-enriched populations from rat calvaria cultured in the presence of concanavalin A, have been shown to produce latent collagenase and collagenase inhibitors. The enzymes and inhibitor activities from the ROS 17/2 cells were concentrated by ammonium sulphate precipitation and separated by gel filtration on AcA 54 resin. The size of the latent collagenase (Mr approximately equal to 58000) was reduced on conversion to active enzyme (Mr approximately equal to 48000) by p-aminophenylmercuric acetate. Latent and active forms of gelatinase activity, similar in size to the corresponding forms of collagenase, were also resolved. The collagenase inhibitor activity, which was sensitive to organomercurials, was recovered in two peaks (Mr approximately equal to 68000 and 30000). The active collagenase cleaved interstitial collagens (type I = III greater than II) producing typical 3/4 and 1/4 fragments. This activity was inhibited by the metal ion chelators ethylenediaminetetraacetic acid and o-phenanthroline. Additional specific cleavages of native collagen were also observed which, from the susceptibility of this activity to phenylmethylsulphonyl fluoride, leupeptin and antipain, suggested the presence of a second collagenolytic enzyme. This synthesis of collagenolytic enzymes by these osteoblast-like cells suggests that individual osteoblasts, like fibroblasts, are capable of both synthesizing and degrading their respective organic matrices in vivo.
Ammonium Sulfate↗
Biosynthesis of osteonectin by fetal porcine calvarial cells in vitro.
The biosynthesis of osteonectin, a major glycoprotein of bone, has been studied in vitro using bone cells from fetal porcine calvariae. The calvarial cells, which were shown to produce osteonectin by immunotransfer and immunocytochemical analysis, were pulse labeled with [35S]methionine and the radiolabeled osteonectin in the cell layers and in the chase-medium was isolated by specific immunoprecipitation. The osteonectin, representing 0.1% of the radiolabeled cell layer and 1% of the medium proteins, co-migrated with authentic bovine osteonectin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced and nonreduced conditions (Mr approximately 40,000), and was bound selectively to hydroxyapatite in the presence of 4 M guanidine HCl. The co-migration of the osteonectins indicates that the tissue-extracted osteonectin represents an intact protein and that a pro- form of osteonectin is unlikely. However, a preosteonectin form (Mr approximately 46,000) could be immunoprecipitated from cell-free translation of calvarial mRNA. The minimal secretion time for osteonectin synthesized in serum-containing medium was found to be approximately 40 min but maximal accumulation did not occur until 2 h postlabeling. In serum-free media, extensive degradation of osteonectin, characterized by the presence of two immunoprecipitable fragments (Mr approximately 36,000 and 31,000), was evident.
Animals↗
Collagenase synthesis by osteoblast-like cells.
Cultures of osteoblast-like cells from a rat sarcoma and osteoblast-enriched populations of rat calvarial cells synthesize and secrete a true collagenase and collagenase inhibitor. The enzyme, which is produced in a latent form, appears to be similar to the enzyme produced by fibroblasts.
Animals↗
Changes in the distribution of laminin, fibronectin, type IV collagen and heparan sulfate proteoglycan during colony formation by chick retinal pigment epithelial cells in vitro.
Retinal pigment epithelial cells isolated by non-enzymatic means from 8 day old chick embryos were grown as explants on glass coverslips in culture. Retinal pigment epithelial cells grown in this way form colonies in which three zones, each containing cells with distinctly different morphology, pigmentation, adhesion pattern and cytoskeletal organization, can be distinguished (Turksen et al., 1983). Using specific antisera against laminin, fibronectin, type IV collagen, and heparan sulfate proteoglycan, we have found differences in the distribution of these basement membrane components in the three zones of each colony. When cells were stained with laminin, type IV collagen and fibronectin antibodies, extensive filamentous arrays were observed on the substratum side of the cells. In contrast to type IV collagen which was deposited to a similar degree in all three zones of each colony, laminin and fibronectin were most prominent in the central zone in which the packed cuboidal differentiated cells are located. In contrast to the other components, the heparan sulfate proteoglycan appeared to be associated primarily with the cell surface. Our results support the general view that basement membrane components could influence cell shape through an effect on the cytoskeleton, and play a role in the maintenance and expression of the differentiated state. Thus retinal pigment epithelial cells might provide a very useful model system for studying the interactions between the cytoskeleton and extracellular matrices and the biosynthesis of the BM components in epithelial cells.
Animals↗
Change in phenotype in long-term cultures of a clonal rat bone cell line: switch to the synthesis of alpha 1(I)-trimer collagen.
A number of bone cell clones isolated from rat calvaria have been maintained in culture for more than 3 years. Several of these clones have undergone dramatic changes in phenotype. One of these clones, RCB 2.2, was observed originally to have a fibroblastic morphology in culture and to respond to parathyroid hormone (PTH), but not prostaglandin E2 (PGE2), with an increase in intracellular cAMP. Throughout several passages in early subcultures, these cells synthesized mostly type I collagen, with small amounts of type III and type V collagens. Whereas PTH had no detectable effect on collagen synthesis, PGE2 decreased the amount of total cell layer collagen, with the greatest effect on type III collagen, while increasing the proportion of type V collagen. Subsequent studies on these cells during 3 years in culture have indicated changes in their phenotype including a progressive change in morphology to a more cuboidal shape and a change in collagen synthesis, the cells producing large amounts of the "embryonic" collagen, alpha 1(I) trimer. The reason(s) for the change in collagen expression is unknown, but may be the result of a change in which gene(s) is being expressed.
Animals↗
An osteonectinlike protein in porcine periodontal ligament and its synthesis by periodontal ligament fibroblasts.
Periodontal ligament, a soft connective tissue that lies between cementum and alveolar bone in the periodontium, has been shown to contain an osteonectinlike protein. The similarity between porcine ligament osteonectin and bovine bone osteonectin was evident from immunochemical studies, from migration characteristics on sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and from binding studies on hydroxyapatite. Using immunotransfer and immunodot analyses, ligament osteonectin was found to be extractable from tissues with 4 M guanidine-HCl (GuHCl) and 4 M GuHCl - 0.5 M EDTA and to comigrate with authentic bovine osteonectin on SDS-PAGE with a relative mass approximately 38 000. Furthermore, osteonectin from guanidine extracts of ligament was bound to hydroxyapatite in the presence of 4 M GuHCl. Immunofluorescence studies showed the osteonectin to be distributed throughout the extracellular matrix of the ligament and to be present within the ligament fibroblasts in a perinuclear, punctate distribution. Biosynthesis of osteonectin by ligament fibroblasts was studied following pulse-chase labelling with [35S]methionine and immunoprecipitation. The labelled osteonectin in the chased culture medium represented approximately 0.5% of the total labelled proteins secreted. It comigrated on SDS-PAGE with the corresponding labelled protein from pulsed cells and with the protein extracted from the tissue.
Animals↗
Polypeptide transforming growth factors isolated from bovine sources and used for wound healing in vivo.
Transforming growth factors, which are polypeptides that induce the transformed phenotype in nonneoplastic cells, have been isolated in bulk amounts from bovine salivary gland and kidney. In experiments in which wound healing chambers were implanted subcutaneously in the backs of rats, these bovine transforming growth factors accelerated the accumulation of total protein, collagen, and DNA in treated chambers. These studies thus show an effect of an isolated transforming growth factor in vivo.
Animals↗