Mineralized tissues: an overview.
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Bone Matrix↗
Biomedical subjects
W T Butler
Publications and source records attributed to W T Butler.
1,25-Dihydroxyvitamin D3 regulates the biosynthesis of osteopontin, a bone-derived cell attachment protein, in clonal osteoblast-like osteosarcoma cells.
We investigated the effects of 1,25-dihydroxyvitamin D3 on the synthesis of osteopontin, a phosphorylated cell attachment glycoprotein, in ROS 17/2.8 cells, a clonal osteoblast-like rat osteosarcoma cell line. We observed a dose dependent increase in uptake of [32PO4] into osteopontin secreted into the medium. An increased incorporation of [35S]-methionine into secreted osteopontin suggested the effect was that of increased protein biosynthesis. Using a radioimmunoassay we demonstrated a dose dependent increase in the amount of secreted osteopontin, an increase which could be blocked by Actinomycin D, in response to 1,25-dihydroxyvitamin D3. These results suggest that the hormonal form of vitamin D regulates the biosynthesis of osteopontin, possibly at the level of transcription.
Animals↗
Immunohistochemical demonstration of a 44-KD phosphoprotein in developing rat bones.
Polyclonal antibodies against a 44-KD phosphoprotein (44K BPP) from rat bone were raised in rabbits, affinity-purified, and used as probes to study the protein's distribution in various types of developing bones from newborn rats. Three immunostaining procedures were applied utilizing indirect immunofluorescence, avidin-biotin-peroxidase complex, and avidin-gold complex with silver enhancement. All methods gave essentially identical and/or complementary results. Antigenicity for anti-44K BPP was detected in endochondral and membranous bone. In the latter, it was also demonstrated in the osteoid. In the woven bone of lower jaw, immunoreactivity for anti-44K BPP antibodies was found in fibroblast-shaped cells (pre-osteoblasts) that were between the bone trabeculae but not in direct contact with bony extracellular material. In addition to these presumed osteoprogenitor cells, osteoblasts as well as osteocytes were strongly stained; the cytoplasmic staining was associated with the Golgi apparatus. Occasionally immunoreactivity was detected in osteoclasts, but in these cells immunostaining was either diffusely spread in the cytoplasm or present only at sites of bone erosion. These findings support the hypothesis that the 44K BPP is a protein made by osteoblasts and is localized predominantly in bone. Furthermore, the protein appears to be expressed early in histogenesis of the bone-forming cells.
Alkaline Phosphatase↗
Immunolocalization of Gla proteins (osteocalcin) in rat tooth germs: comparison between indirect immunofluorescence, peroxidase-antiperoxidase, avidin-biotin-peroxidase complex, and avidin-biotin-gold complex with silver enhancement.
Odontoblasts and osteoblasts synthesize gamma-carboxyglutamatic acid (Gla)-containing proteins which are partially deposited in the mineralizing tissues and partially released into the plasma. Using four immunostaining techniques, we have evaluated the question of whether dentin Gla proteins (DGP) are transported to the mineralization front through the odontoblast processes. Undecalcified sections of rat incisors and molar tooth germs were immunostained with affinity-purified antibodies to DGP using the following methods: indirect immunofluorescence; peroxidase-antiperoxidase (PAP); avidin-biotin-peroxidase complex (ABC-peroxidase); and avidin-biotin-gold complex with silver enhancement (ABC-GSS). The results obtained with these four procedures were compared with respect to the developmental appearance of DGP, staining intensity and presence in odontoblastic processes, predentin, dentin, and blood vessels. Qualitatively, similar results were obtained with the four, with respect to the distribution and developmental appearance of DGP, with two exceptions: indirect immunofluorescence never stained DGP within blood vessels, whereas the other methods occasionally did; and because of its sensitivity, only the ABC-GSS method revealed immunostaining for DGP in odontoblastic processes. All methods revealed weak immunostaining in predentin which was considerably enhanced with hyaluronidase treatment; however, hyaluronidase only moderately increased predentin immunostaining with ABC-GSS. Of these four procedures, ABC-GSS is the most sensitive; however, ABC-GSS appears to detect predominantly antigens at the surface of tissue sections. We conclude that DGP is present in odontoblastic processes but in low amounts; the weak staining was due either to rapid transport of DGP through the process or to the fact that this mode of transport is limited.
Animals↗
Developmental appearance of Gla proteins (osteocalcin) and alkaline phosphatase in tooth germs and bones of the rat.
The tissue distribution and developmental appearance of alkaline phosphatase and Gla proteins, relative to mineralization were examined. Undecalcified sections of tooth germs, calvaria and alveolar bone of the rat were immunostained for Gla proteins, (indirect immunofluorescence and avidin-biotin-peroxidase-complex) and then stained for alkaline phosphatase activity. In the growth regions of tooth germs, Gla protein was observed in young odontoblasts and in early predentin, at a stage prior to formation of the first mineralized dentin. Similar results were obtained for bone: young osteoblasts as well as osteoid were immunopositive for Gla protein prior to formation of the first mineralized bone. Histochemical staining revealed that differentiating odontoblasts and osteoblasts exhibited alkaline phosphatase activity at stages prior to appearance of Gla proteins and that cells adjacent to odontoblasts and osteoblasts (not directly involved in the formation of predentin/dentin and osteoid/bone) stained for alkaline phosphatase, but not for Gla proteins. We conclude, that in these bone- and dentin-forming cells, alkaline phosphatase activity is expressed before the appearance of Gla proteins, but that both appear before the onset of mineralization. We also conclude that Gla protein is a more specific marker for bone and dentin formation than histochemical alkaline phosphatase activity, since only the cells directly involved in these processes were immunostained for Gla protein.
Alkaline Phosphatase↗
Protein content and amino-acid content of consolidated carious lesions in human enamel and of experimental lesions in bovine enamel exposed to the human mouth.
Consolidation is a natural defence reaction that results in arrest of enamel caries. Experimental consolidated lesions (ECL) were compared with naturally-consolidated lesions (NCL): ECL were obtained by exposing pre-softened, bovine-enamel slabs to the oral environment in 3 subjects for 2 h, 24 h or 7 days, and NCL were sampled from extracted human teeth with white or yellow spots of arrested caries. Protein content of ECL from 2 subjects were similar to each other and to that of NCL throughout the experimental period. The ECL of the other subject showed a gradual increase in protein content with significantly higher values at day 7. The predominant amino acids in ECL were glutamic acid, proline and alanine, and in NCL, glutamic acid, glycine and alanine. The amino-acid composition of the 7-day ECL was closer to that of NCL than were that of the 2 and 24 h ECL. Intra-oral ageing caused significant reductions in proline and glycine and pronounced increases in aspartic acid, threonine, alanine and leucine. Thus the adsorbed or incorporated organic material in the ECL changed from having components dissimilar to NCL to ones similar to NCL. This intra-oral model might be useful for studies of the organic material incorporated into enamel during the process of consolidation.
Amino Acids↗
Medical education in the 1980s: increasing demands, decreasing resources.
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Education, Medical↗
The structure of a small collagenous fragment isolated from chicken hyaline cartilage.
In previous experiments, two collagenous fragments were isolated from pepsin digests of chicken hyaline cartilage and called the high molecular weight, (HMW) and low molecular weight (LMW) fractions [3]. In the present experiments, the chains of LMW were isolated after denaturation and subsequent reduction and alkylation of interchain disulfide bridges and were further fractionated by carboxymethyl-cellulose chromatography. Four peaks were resolved during chromatography and were designated LMW 1, 2A, 2B, and 3. Amino acid analyses and peptide mapping after cleavage with trypsin, V8 protease, and cyanogen bromide showed that three genetically distinct chains must be present in LMW. Fractions 2A and 2B were very similar, but not identical, in structure. LMW 1, 2A plus 2B, and 3 were consistently isolated in approximately equal proportions, suggesting that the probable chain organization of LMW is [1][2A + 2B][3]. This suggestion was supported further by experiments that attempted to fractionate LMW by carboxymethyl-cellulose chromatography after denaturation but without reduction and alkylation of interchain disulfide bridges. No fractionation of LMW was achieved, the single peak subsequently being shown to contain LMW 1, 2A plus 2B, and 3.
Animals↗
The biosynthesis of dentin phosphophoryns by rat incisor odontoblasts in organ culture.
An in vitro system consisting of rat incisor fragments was used to study the process of dentinogenesis. In order to establish the usefulness of the organ culture, the biosynthesis and deposition of the major noncollagenous components of dentin, the phosphophoryns, were followed for specific lengths of time in culture. Three criteria were satisfied: (1) the synthesis of proteins which appeared to be chemically identical to the native proteins of dentin, (2) the accumulation of the phosphophoryns within the matrix or time, and (3) the association of the secreted proteins with the mineral phase of dentin. The synthesis of phosphophoryns was determined by using both (3H)-serine and (32P)-inorganic phosphate as precursors for synthesis of protein and post-translational modification of serine to phosphoserine. In vitro synthesized phosphophoryns were characterized by 1) their accumulation and EDTA extractability from within dentin, 2) calcium chloride precipitability, 3) elution on anion-exchange columns (DEAE cellulose and AGMP50), and 4) Mr's on SDS-PAGE and Sepharose CL-6B columns. This novel system of studying dentinogenesis provides a model with which to study the regulation of extracellular matrix protein synthesis and may be useful for revealing the effect of other agents which influence tooth development and mineralized tissue metabolism in general.
Animals↗
Immunolocalization of gamma-carboxyglutamic acid containing proteins in developing rat bones.
The localization of a gamma-carboxyglutamic acid (Gla)-containing protein, BGP (also called osteocalcin) was examined in developing calvaria, alveolar bones and long bones of newborn rats by immunostaining with the peroxidase-antiperoxidase method. In undemineralized tissues osteoblasts of intramembranous bones stained positive; osteoid was negative, whereas young mineralized bone stained weakly. After a mild demineralization all bones stained positive; no staining was found in cartilage, muscles or soft connective tissues. In addition to the osteoblasts, osteocytes were also immunoreactive. Osteoclasts, identified by a subsequent staining for acid phosphatase activity, demonstrated no immunostaining for BGP. These data support the hypothesis that BGP is synthesized by osteoblasts and osteocytes and is subsequently deposited in the mineralizing bone.
1-Carboxyglutamic Acid↗
Immunolocalization of gamma-carboxyglutamic acid-containing proteins in developing molar tooth germs of the rat.
The localization of dentin gamma-carboxyglutamic acid containing proteins (DGP's) was examined in rat molar tooth germs at various stages of development by immunostaining with the peroxidase-antiperoxidase method. Odontoblasts and their processes stained positive, whereas all other cells were negative. No staining was found in predentin. Dentin stained only after a mild demineralization. The results support the concept that DGP's are deposited by odontoblasts directly into the mineralizing dentin.
1-Carboxyglutamic Acid↗
Vitamin D and skeletal tissues.
It is now accepted that vitamin D is an integral part of a complex endocrine system, one with far-reaching implications in mineral metabolism. Reviews of the sources, functions and metabolism of vitamin D, as currently understood, are presented as a prelude to discussions of the role of vitamin D in calcium and phosphorous homeostatis and possible specific roles for vitamin D in mineralized tissues. Data describing a possible regulatory function for vitamin D in bone and bone protein metabolism are presented. Some of the controversy which presently exists regarding the biochemical mechanism of the action of this vitamin is discussed. Finally, the possible relationship of vitamin D and disorders of skeletal tissues is described.
Animals↗
Appearance of dentin gamma-carboxyglutamic acid-containing proteins in developing rat molars in vitro.
An in vitro model of mineralization was devised in order to study the developmental appearance of dentin gamma-carboxyglutamic acid-containing proteins (DGPs) in relation to the onset of mineralization. Maxillary third molars from 11-day-old rats were cultured with or without fetal calf serum (FCS) as modified from Navia et al. (1984). Molars were incubated without radiolabel, or with either 45CaCl2 (5 microCi/ml) for 24 hr at various stages of a ten-day culture period or [3H]-leucine (10 microCi/ml) for 24 hr at the eighth day of culture. Molars were lyophilized and extracted with 10% formic acid overnight at 4 degrees C. DGPs in extracts were detected by immunologic and chromatographic techniques; DGPs in molar sections were detected by immunolocalization using indirect immunofluorescence. Molar development was evaluated histologically using the Von Kossa staining technique. Molars cultured with FCS showed histologic evidence for mineralized dentin and enamel and a significant increase in 45Ca uptake after the sixth day in vitro. Eleven-day-old molars in vivo and molars cultured without FCS showed no evidence of the presence of mineralized tissues. [3H]-Leucine-labeled DGPs were isolated and identified by affinity and reversed-phase high-performance liquid chromatography and by gel electrophoresis from both mineralized and unmineralized molars. DGP antigens were localized immunohistochemically using rabbit anti-rat antibodies raised against a highly purified DGP preparation. In the unmineralized molar, antigenicity was seen in odontoblasts but not in predentin matrix, preodontoblasts, or in any other cell type. Antigens in the mineralized molar were localized to odontoblasts and dentin.(ABSTRACT TRUNCATED AT 250 WORDS)
1-Carboxyglutamic Acid↗
Acidic glycoproteins from bovine compact bone.
Several acidic glycoproteins with apparent molecular weights of 85,000 (bone sialoprotein, BSP), 80,000, 67,000, 60,000, 40,000 (osteonectin) and 30,000 were isolated from adult bovine compact bone. About 20-25% of the 67 K preparation was serum albumin. This contaminant was removed by passing the protein through Blue Sepharose CL-6B; the material that emerged in the void volume of this affinity column contained the bone 67 K glycoprotein free of serum albumin. All six of the proteins had high amounts of aspartic and glutamic acid. The amino acid compositions of 80 K, 67 K, 40 K and 30 K glycoproteins were very similar. The 60 K glycoprotein had an amino acid content that differed from the others with notably higher proline, alanine and valine values. All six protein samples contained mannose, galactose, glucosamine, galactosamine and sialic acid, but no fucose. The carbohydrate contents varied from about 10% for osteonectin to 38% for BSP. Antibodies were raised against electrophoretically purified osteonectin and used in enzyme-linked immunosorbent assays (ELISA). In addition to reactivity with osteonectin, the antibodies displayed strong reactivity to 80 K and 30 K glycoprotein preparations, but did not react with 67 K or 60 K bone glycoproteins or bovine serum albumin. None of the acidic glycoproteins cross-reacted with antibodies to bovine serum albumin, bovine serum proteins or human alpha 2-HS-glycoprotein.
Amino Acids↗
Proteoglycans of adult bovine compact bone.
Proteoglycans of bovine compact bone were purified by chromatography of the formic acid precipitate of an EDTA extract. The sequential chromatographic steps consisted of gel filtration on Sepharose CL-6B in 4-M guanidine HCl, ion-exchange chromatography on DEAE-Sephacel in 4-M urea and rechromatography on Sepharose CL-6B in 4-M guanidine HCl. The preparation consisted of a relatively small proteoglycan (Kav = 0.4 on Sepharose CL-6B) containing about 40% protein, 21% hexuronic acid, 23% galactosamine and lesser amounts of other monosaccharides. The core protein was shown by gradient NaDodSO4 gel electrophoresis, electrotransfer and immunodetection to be monodispersed with an Mr = 45,000. Analysis of glycopeptides obtained after papain digestion of the proteoglycan and separation from glycosaminoglycan chains by gel chromatography, indicated that both N-linked and O-linked oligosaccharides were present. The glycosaminoglycan chains liberated by papain digestion eluted from Sepharose CL-6B as a broad peak with Kav = 0.50, slightly ahead of the position of elution of bovine nasal cartilage glycosaminoglycans (Kav = 0.52); the bone glycosaminoglycans are thus slightly larger than those from cartilage and smaller than the ones attached to fetal bone proteoglycans. These chains were totally susceptible to chondroitinase AC II, a procedure that yielded unsaturated disaccharides corresponding predominantly to chondroitin-4-sulfate, and to a lesser extent chondroitin-6-sulfate. Antisera raised against adult bone proteoglycans cross-reacted with core protein of bone proteoglycan (obtained after chondroitinase digestion) but not with papain digested proteoglycan. In addition, they cross-reacted with core protein and trypsin-liberated, chondroitin sulfate rich region (AlTAl) derived from cartilage proteoglycans and, to a lesser extent, rat bone proteoglycans. No cross-reactivity could be detected to Smith-degraded cartilage proteoglycans, bone acidic glycoproteins or serum proteins.
Amino Acids↗
Incorporation of [35S]sulphate into glycosaminoglycans by mineralized tissues in vivo.
To investigate the metabolism of proteoglycans in young growing rats, calvaria, incisors, femoral diaphysis and metaphysis were labelled in vivo for 0.5-72 h with [35S]sulphate. At each time point the specific radioactivity, expressed as c.p.m. of [35S]sulphate/micrograms of uronic acid, of papain-resistant macromolecules in each tissue was determined. The identity of the glycosaminoglycans was established by the use of specific enzymic and chemical methods of degradation. Incorporation of the label into each tissue was maximal at 12 h; it then declined to 50-75% of that value by 72 h. Chondroitin sulphate was the predominant glycosaminoglycan in each tissue, representing 80-96% of the total; heparan sulphate comprised 2-14% of the total; in general, radioactive material sensitive to keratanase comprised less than 1% of the total. The relative amount of labelled chondroitin sulphate increased, whereas that of heparan sulphate decreased, with increasing time of incorporation. These data show that 25-50% of the newly synthesized glycosaminoglycans are lost from mineralizing tissues, during the time in which the newly secreted organic matrix becomes mineralized.
Animals↗
Isolation and partial characterization of proteoglycans from rat incisors.
Newly synthesized proteoglycans of rat incisors were labelled in vivo for 6h with [35S]-sulphate in order to facilitate their detection during purification and characterization. Proteoglycans were extracted from non-mineralized portions (predentine) of rat incisors with 4M-guanidinium chloride and subsequently from dentine by demineralization with a 0.4M-EDTA solution containing 4M-guanidinium chloride. Both extractions were performed at 4 degrees C in the presence of proteinase inhibitors. Purification of proteoglycans was achieved with a procedure involving gel-filtration chromatography, selective precipitation of phosphoproteins, affinity chromatography and ion-exchange chromatography. Two proteoglycan populations were found in the initial extract (Pd-PG I and Pd-PG II), whereas only one fraction (D-PG) was obtained after demineralization. The minor proteoglycan fraction from the first extract, Pd-PG I, although not totally characterized, differed sharply from the other proteoglycans in that it had a larger molecular size with larger glycosaminoglycan chains composed of chondroitin 4- and 6-sulphate isomers. In contrast, the major proteoglycans Pd-PG II and D-PG had smaller hydrodynamic sizes with smaller glycosaminoglycan chains (but larger than those from bovine nasal cartilage proteoglycans) composed exclusively of chondroitin 4-sulphate. The major proteoglycans were incapable of interacting with hyaluronic acid. In general, the amino acid compositions of the major proteoglycans of rat incisors resembled that of bovine nasal cartilage proteoglycans, but the former had lower proline, valine, isoleucine, leucine, and higher aspartic acid, contents.
Amino Acids↗