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H Limeback

Publications and source records attributed to H Limeback.

At least 37 records · Page 2Linked to original sources
PubMed1992

Osteogenic phase-specific co-regulation of collagen synthesis and mineralization by beta-glycerophosphate in chick periosteal cultures.

Mineralized bone formation in vitro can be induced by the alkaline phosphatase substrate beta-glycerophosphate (GP). GP may not only be essential for mineralization in vitro, but could also modulate other metabolic activities of bone cells, particularly if GP is presented to these cells during different phases of development. To assess GP modulation of bone cell metabolism, biochemical and autoradiographic analyses of chick periosteal cultures treated with GP were performed. About 50% less (p less than 0.05) Type I collagen was produced in periosteal cultures treated with GP. If the fibrous portion of the periostem was microdissected from the osteogenic layer prior to culture, GP inhibition of Type I collagen synthesis was even more marked (60%: p less than 0.05). To define organic phosphate-sensitive phases of osteogenesis, cultures were exposed to GP for various time periods. Mineralization occurred reproducibly when periosteal cultures were treated with GP from the outset of the incubation period (positive control). However, if GP was added after the third day of incubation, phosphate content was the same as in positive control cultures, whereas calcium content was significantly (20%: p less than 0.05) lower. Moreover, if GP was added on day 6, there was virtually no calcium accumulation by day 12, while massive amounts of phosphate had accumulated. Taken together, these findings indicate that organic phosphates may modulate phenotypic expression of osteogenic cells, and that osteogenic cells traverse an organic phosphate-sensitive phase, after which they may be incapable of normal mineralization.

Animals↗
PubMed1992

Correlation of structure and viscoelastic properties in the pericardia of four mammalian species.

Although the pericardium is recognized as having important contributions to ventricular function, the relationship between its functional role and structural composition remains poorly understood. Conflicting evidence from low strain rate experiments has shown that differences exist between the mechanical properties of canine, human, and bovine pericardium but with no structural explanation for these differences. This paper examines the pericardia of calves, dogs, pigs, and sheep using a structural/mechanical approach with techniques novel to the pericardial literature. High strain rate mechanical testing for stress-strain response, stress relaxation, and forced vibration response has shown the pericardium to be much more viscoelastic than previously believed under large deformations, but to be quite elastic in small vibrations. The thinner canine and porcine pericardia were found to be significantly stiffer than the thicker bovine and ovine tissues, but equivalently viscoelastic. Biochemical analysis shows these thinner tissues to have significantly higher levels of type III collagen combined with a higher degree of cross-linking. This is the first structural explanation for differences in mechanical properties between the pericardia of different species.

Animals↗
PubMed1992

The effects of hypocalcemia/hypophosphatemia on porcine bone and dental hard tissues in an inherited form of type 1 pseudo-vitamin D deficiency rickets.

The effects of Vitamin D deficiency rickets on the formation of mineralized dental tissues were studied in a breed of pigs which had moderate and marked hypocalcemia because of an inherited defect in the renal production of the biologically-active Vitamin D metabolites. Affected piglets developed classical symptoms of rickets which were fatal unless Vitamin D supplements were given. The dissected mandibles of homozygous (rickets) and heterozygous (normal) pigs were photographed and radiographed. Compared with those of normal pigs, the mandibles of homozygous pigs demonstrated slowed development/eruption of permanent teeth, under-mineralized bone, underdeveloped dentin (enlarged pulp chambers), interglobular dentin, and enamel hypoplasia. Enamel defects in rachitic pig teeth were difficult to observe radiographically, but could be detected visually and by SDS-PAGE analysis of the enamel protein components of developing and maturing enamel. There was significant retention of amelogenins in the enamel maturation zones of developing molars taken from rachitic pigs, but there was no obvious difference in the patterns of amelogenin processing.

Amelogenin↗
PubMed1992

Direct flow cytometric quantification of alkaline phosphatase activity in rat bone marrow stromal cells.

Cell preparations in cytochemistry are conventionally analyzed with transmitted light after fixation and reaction with agents such as azo-coupling dyes. With cell suspensions stained with fluorescent cytochemical dyes, cells can also be analyzed and sorted by flow cytometry. We have exploited the intense red fluorescence of Fast Red Violet LB generated in cytochemical reactions to perform flow cytometric analyses of alkaline phosphatase (AP) expression in rat bone marrow stromal cells. By modifying staining protocols of single-cell suspensions, we demonstrate that in comparison to staining with Fast Red TR, the method is specific, can distinguish among various levels of enzyme expression within the whole population, and permits enzyme kinetic studies of heterogeneous cell populations. The method was applied to study the effect of the glucocorticoid dexamethasone (Dx) on cell proliferation and AP expression. In low AP-expressing cells, Dx treatment at 10(-8) M increased the [3H]-thymidine labeling index from 3.85% to 5.24% (p less than 0.01). In contrast, high AP-expressing cells were unlabeled by [3H]-thymidine. The staining and analytical methods reported here facilitate the detection, isolation, and quantification of subpopulations of bone marrow stromal cells that express alkaline phosphatase activity. These experiments demonstrate the value of flow cytometry as an adjunct to conventional cytochemical methods.

Alkaline Phosphatase↗
PubMed1991

The extent and distribution of intratubular collagen fibrils in human dentine.

Dentine of 27 permanent human teeth was examined by scanning electron microscopy. The teeth were incisors, canines, premolars and molars, ranging in age from 18 to 54 yr. Intratubular collagen was found in 65% of the dental tubules in inner dentine (closest to the pulp) with 16% of the tubules containing large collagen bundles occupying more than one-fifth of the lumen. In middle dentine the corresponding figures were 42 and 7%, and for outer dentine, 12 and 0% This pattern of distribution was the same for all tooth families examined and appeared to be unrelated to age.

Adolescent↗
PubMed1991

Molecular mechanisms in dental hard tissue mineralization.

During the period covered by this issue, over 500 papers were published in the area of biology of dental hard tissues. This review is limited to publications that focus on the organic matrix components believed to be important in the formation of dentin, cementum, and enamel. The advances made in this area for the collagen-based dental hard tissues (dentin and cementum) have been primarily in the isolation and partial characterization of the noncollagenous proteins such as proteoglycans, phosphoproteins, and proteins normally found in bone. These proteins are being studied because of their potential role for directing hydroxyapatite nucleation or crystal growth. The progress made in the enamel field has been primarily in the area of molecular biology. Enamel is quite different physically from dentin and cementum because it is formed from a noncollagenous matrix (mostly amelogenin), which is almost completely removed and replaced with hydroxyapatite. Serum proteins have now been found in all dental hard tissues including enamel. Opinions on the clinical significance of these advances are provided.

Amelogenesis↗
PubMed1990

Biochemical characterization of stable high molecular-weight aggregates of amelogenins formed during porcine enamel development.

Analysis of the enamel matrix during porcine tooth formation has revealed a number of high molecular-weight (Mr) enamel proteins (greater than 30 kDa), which are related to the major amelogenins (20-26 kDa). To examine the nature of these proteins, amelogenins were extracted and separated by conventional gel filtration and reverse phase HPLC. Many of the proteins in the high Mr fraction reacted with a polyclonal antibody, affinity-purified against a mixture of 20-26 kDa amelogenins. Another antibody, affinity-purified against a fraction containing the LRAP, reacted with amelogenins 30-36 kDa in size but not with amelogenins 40 kDa or larger, indicating that the high Mr amelogenins were a heterogeneous group of enamel proteins. Analysis of amino acid composition and N-terminal amino acid sequence, as well as PASGE of electrophoretically eluted proteins, indicated that the high Mr amelogenins were aggregates of various major amelogenins. Three amelogenin aggregates (43, 40 and 32 kDa) isolated by electrophoretic elution were less stable at 100 degrees C in SDS-containing buffer than at 60 degrees C. In contrast to the major amelogenins, which are found in constant proportions throughout enamel development, the high Mr amelogenins appeared to increase in maturing enamel relative to the total matrix protein. Thus, at least in the pig high Mr amelogenins appear to be naturally occurring, stable aggregates of major amelogenins. It is proposed that amelogenin aggregation occurs as a consequence of the diminishing spaces between growing crystals in maturing enamel.

Amelogenesis↗
PubMed1990

Clonal distribution of osteoprogenitor cells in cultured chick periostea: functional relationship to bone formation.

Folded explants of periosteum from embryonic chick calvaria form bone-like tissue when grown in the presence of ascorbic acid, organic phosphate, and dexamethasone. All osteoblast-like cells in these cultures arise de novo by differentiation of osteoprogenitor cells present in the periosteum. To study the spatial and functional relationships between bone formation and osteoprogenitor cells, cultures were continuously labeled with [3H]thymidine for periods of 1-5 days. Radioautographs of serial 2-microns plastic sections stained for alkaline phosphatase (AP) showed maximal labeling of 30% of fibroblastic (AP-negative) cells by 3 days while osteogenic cells (AP-positive) exhibited over 95% labeling by 5 days. No differential shifts in labeling indices, grain count histograms of fibroblastic and osteogenic cells or numbers of AP-positive cells were observed, indicating no significant recruitment of cells from the fibroblastic to the osteogenic compartment. Despite the continuous presence of [3H]thymidine, less than 35% of both osteoblasts and osteocytes were labeled at 5 days, indicating that only one-third of the osteoprogenitor cells had cycled prior to differentiation. Spatial clustering of [3H]thymidine-labeled cells was measured by computer-assisted morphometry and application of the Poisson distribution to assess contagion. Cluster size and number of labeled cells per cluster did not vary between 1-3 days, but the number of clusters increased 20-fold between Day 1 and Day 3. Clusters were predominantly AP-positive and located close to bone. Three-dimensional reconstruction from serial sections showed that clusters formed long, tubular arrays of osteogenic cells up to eight cells in length and located within 2-3 cell layers from the bone surface. Selective killing of S-phase cells with two pulse labels of high specific activity [3H]thymidine at 1 and 2 days of culture completely blocked bone formation. These data indicate that a very small population of cycling osteoprogenitor cells is essential for bone formation in vitro and give rise to relatively small numbers of clonally distributed progenitors with limited proliferative capacity. The progeny of these clusters undergo restricted migration and differentiate into osteoblasts.

Alkaline Phosphatase↗
PubMed1989

Serum albumin and its acid hydrolysis peptides dominate preparations of mineral-bound enamel proteins.

Serum albumin is a major noncollagenous protein component of bone, dentine, and, according to our results, enamel. Preparations of mineral-bound proteins from porcine developing enamel contain a single 67 kD protein at neutral pH or three proteins (67, 63, and 53 kD) at low pH that were assumed to be enamelins, a minor class of enamel proteins. A more complete analysis of these proteins in this study showed that they were derived from porcine serum albumin (PSA). This was demonstrated by amino acid analysis, by N-terminal sequence analysis, by immunoblot studies using an anti-PSA antibody, and by SDS-PAGE analysis of the acid hydrolysis, cyanogen bromide, and tryptic peptides. Examination of enamel at different developmental stages showed that PSA deposited in enamel from the enamel organ and from the dentine during development, not during the dissection process. These results indicate that true enamelins must represent a very small fraction of the total mineral-bound protein matrix in porcine developing teeth, and this has important implications on the role of mineral-bound proteins during mineralization of enamel.

Amino Acid Sequence↗
PubMed1988

Identification and characterization of enamel proteinases isolated from developing enamel. Amelogeninolytic serine proteinases are associated with enamel maturation in pig.

During tooth formation nearly all of the protein matrix of enamel is removed before final mineralization. To study this process, enamel proteins and proteinases were extracted from pig enamel at different stages of tooth development. In the enamel maturation zones, the major enamel matrix proteins, the amelogenins, were rapidly processed and removed. Possibly associated with this process in vivo are two groups of proteinases which were identified in the enamel extracts by enzymography using amelogenin-substrate and gelatin-substrate polyacrylamide gels and by the degradation in vitro of guanidinium chloride-extracted amelogenins. One group of proteinases with gelatinolytic activity consisted of several neutral metalloendoproteinases having Mr values from 62,000 to 130,000. These proteinases were inactive against amelogenins, casein and albumin, and were present in approximately equal proportions in enamel at all developmental stages. In the other group, two serine proteinases, with apparent non-reduced Mr of 31,000 and 36,000 exhibited amelogeninolytic activity. The substrate preference of the enamel serine proteinases was indicated by their limited degradation of casein and their inability to degrade gelatin and albumin. Contrasting with the distribution of the metalloendoproteinase enzymes, the serine proteinases were found only in the enamel scrapings taken from late-maturing enamel. The amelogenin degradation patterns in vivo, observed in the enamel scrapings, were similar to those produced in assays in vitro using partially purified fractions of enamel proteinases and amelogenin substrate. Together, these data strongly indicate an important role for the serine proteinases, and possibly the gelatinolytic proteinases, in the organized processing of the enamel protein matrix during enamel formation.

Amelogenin↗
PubMed1987

Isolation and characterization of pig enamelins.

Enamel proteins were extracted from pig developing enamel by sequential extraction procedures. Two proteins identified as enamelins by slab-gel electrophoresis (Mr 67,000 and 63,000) were separated from amelogenins by gel sieving and ion-exchange chromatography. Their enamelin characteristic was confirmed by hydroxyapatite-binding studies and amino acid analysis. Degradation of extracted enamel proteins was also studied in vitro. The larger of the two enamelins appeared to be resistant to degradation by endogenous enamel proteinases. Hydroxyapatite showed strong binding with the enamelins, but did not prevent the degradation of the Mr-63,000 enamelin. These results indicate that at least one high-Mr enamelin in pig developing enamel is a source of enamelin breakdown products.

Amino Acids↗
PubMed1987

Enamel protein and collagen production by cells subcultured from porcine tooth bud explants.

Fibroblast (F) and epithelial (E) cells were obtained as primary outgrowths from explants of fetal porcine maxillary molars and subcultured up to four passages in monolayers enriched with either cell type. Histology of a tooth bud after 1 day in culture showed intact odontogenic E cell layers which were the probable source of the E cell outgrowths. After 2 months in culture, the fourth passage E cells demonstrated morphological differentiation by an alteration in cell packing and the formation of domes and nodules, when E and F cells were cocultured. Occasionally the nodules grew to considerable size, indicating the potential of these cells to aggregate and reorganize into odontogenic tissues even on culture dishes. The cells were characterized in monolayer culture by immunocytochemical staining. Laminin and type IV collagen staining was distributed diffusely throughout the culture, whereas type I collagen and osteonectin staining was predominantly localized in the F cells. Radiolabelled proteins from both E and F cell media produced similar collagen patterns (95% type I, 4% type V, 1% other), except that the F cells appeared to produce active collagenase. In addition, the E cells produced two radiolabelled proteins (relative masses of 50,000 and 53,000) that reacted with an affinity-purified antibody directed against porcine amelogenin. These experiments show that cells subcultured from tooth buds and grown in monolayer cultures can be used to study tooth organogenesis in vitro, as well as enamel protein biosynthesis.

Amelogenin↗
PubMed1984

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↗
PubMed1984

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↗