Biomedical subjects

Z Schwartz

Publications and source records attributed to Z Schwartz.

At least 199 records · Page 11Linked to original sources

PubMed1992

Stimulation of matrix vesicle enzyme activity in osteoblast-like cells by 1,25(OH)2D3 and transforming growth factor beta (TGF beta).

After demonstrating the presence of matrix vesicles in three osteosarcoma cell lines, MG-63, ROS 17/2.8 and MC-3T3-E1, we sought to determine whether two major enzymes localized to matrix vesicles, alkaline phosphatase and phospholipase A2, could be regulated by 1,25(OH)2D3 and/or TGF beta. Intravesicular calcification is probably dependent on these two enzymes. Alkaline phosphatase is essential for hydrolysis of phosphate-containing substrates and phospholipase A2 hydrolyzes diacylphosphatides in a calcium-mediated manner at lipid-aqueous interfaces leading to changes in membrane fluidity and possibly breakdown of the matrix vesicle. The 1,25(OH)2D3 induced increase of alkaline phosphatase in bone cells is localized to the matrix vesicle. TGF beta also increased alkaline phosphatase activity in two of the cell lines, MG-63 and ROS 17/2.8 but to a greater degree than 1,25(OH)2D3. Matrix vesicle alkaline phosphatase activity exhibited a greater response than that in the plasma membrane. TGF beta increased phospholipase A2 activity in both matrix vesicles and plasma membranes, therefore, no targeting was observed with respect to this enzyme. When TGF beta was combined with 1,25(OH)2D3, 1,25(OH)2D3 had no effect on phospholipase A2 and did not interfere with TGF beta stimulation of phospholipase A2 activity. When 1,25(OH)2D3 and TGF beta were combined, a tremendous synergy was observed in alkaline phosphatase specific activity in both plasma membranes and matrix vesicles with targeting to matrix vesicles. Therefore, TGF beta not only plays an important role in matrix formation and differentiation, but works in conjunction with 1,25(OH)2D3 to greatly potentiate the effects seen with 1,25(OH)2D3 alone.

Alkaline Phosphatase↗

PubMed1992

The effect of bone injury on extracellular matrix vesicle proliferation and mineral formation.

Removal of tibial bone marrow in rats is followed by primary bone formation, resorption and marrow restitution. The first week of healing is characterized by partially calcified trabeculae. After 2 weeks, a higher degree of calcification and partial resorption are observed. The third week is characterized by massive resorption of the trabeculae, which are replaced in the fourth week by new bone marrow tissue. This model was used to study primary calcification. Transmission electron micrographs of the young bone revealed osteoblasts, matrix vesicles and calcified fronts. The different vesicular types were defined as 'empty', 'amorphous', 'crystal', and 'rupture'. The vesicles were studied on days 3, 6, 8, 12, 14, 18, 21, 23 and 28 after injury. The mean diameters of most vesicles ranged between 100.3 and 121.9 nm, and their mean distance from the calcified front was less than 976.6 nm. Vesicular density, calculated as number per 10 m2, increased on the eighth day and decreased from the fourteenth day onwards. Highest diameter values were recorded on the sixth day, and decreased onward. Vesicular distance from the calcified front decreased continuously. Distribution of vesicle number, diameter, and distance in each class showed that numbers of empty and amorphous vesicles decreased and of crystal and rupture increased throughout the experiment. Distances from the calcified front and vesicular diameters varied as follows: 'rupture', 'crystal', amorphous', and 'empty', the 'rupture' type being the closest to the front and of the largest diameter. The results confirm the hypothesis that the cell is responsible for the secretion of electron lucent vesicles that accumulate Ca and Pi to form amorphous calcium phosphate complexes that convert to hydroxyapatite. Crystal growth is followed by membrane rupture.

Animals↗

PubMed1992

Regulation of matrix vesicle phospholipid metabolism is cell maturation-dependent.

We have developed a chondrocyte culture model for assessing the regulation of matrix vesicles at two different stages of chondrogenic maturation. These chondrocytes, resting zone (RC) and growth zone (GC), retain their phenotypic markers in culture, including production of matrix vesicles with distinctive lipid compositions and enzyme activities. Isolated matrix vesicles incubated in vitro with 1,25-(OH)2D3 (1,25) or 24,25-(OH)2D3 (24,25) respond differentially. 1,25 stimulates phospholipase A2 (PA2) in GC vesicles, but not on those from RC. 24,25 inhibits PA2 in RC vesicles, but has no effect on GC. PA2 activity is required for fatty acid turnover and is the rate-limiting step in prostaglandin production. Plasma membrane phospholipids are more susceptible to the release of arachidonic acid by PA2 than are matrix vesicle phospholipids. Matrix vesicles are distinct from the plasma membrane in terms of lipid composition and arachidonic acid incorporation. 1,25 and 24,25 stimulate arachidonic acid turnover in their target cells, but by different mechanisms. 1,25 has no effect on arachidonic acid turnover in RC; however, 24,25 inhibits turnover in RC and GC. 1,25 and 24,25 also affect isolated matrix vesicle membrane fluidity. These results suggest that vitamin D metabolites modulate PA2 activity, change the composition of membrane phospholipids by altering fatty acid composition, and affect calcium transport. The effects are mediated by altering membrane fluidity and is dependent on the stage of cell differentiation.

Animals↗

PubMed1992

Studies of matrix vesicle-induced mineralization in a gelatin gel.

Matrix vesicles isolated from fourth-passage cultures of chondrocytes were tested for their ability to induce hydroxyapatite formation in a gelatin gel in order to gain insight into the function of matrix vesicles in in situ mineralization. These matrix vesicles did not appear to be hydroxyapatite nucleators per se since the extent of mineral accumulation in the gel diffusion system was not altered by the presence of matrix vesicles alone, and in the vesicle containing gels, mineral crystals were formed whether associated with vesicles or not. In gels with these matrix vesicles and beta-glycerophosphate, despite the presence of alkaline phosphatase activity, there was no increase in mineral deposition. This suggested that in the gel system these culture-derived vesicles did not increase local phosphate concentrations. However, when known inhibitors of mineral crystal formation and growth (proteoglycan aggregates [4 mg/ml], or ATP [1 mM], or both proteoglycan and ATP) were included in the gel, more mineral was deposited in gels with the vesicles than in comparable gels without vesicles, indicating that enzymes within these vesicles were functioning to remove the inhibition. These data support the suggestion that one function of the extracellular matrix vesicles is to transport enzymes for matrix modification.

Adenosine Triphosphate↗

PubMed1992

Cell maturation-specific autocrine/paracrine regulation of matrix vesicles.

Matrix vesicles are extracellular organelles produced with distinctive phospholipid composition and enzyme activity. They are produced by cells which typically calcify their extracellular matrix and their characteristics are cell-maturation dependent. Regulation of matrix vesicle structure and function occurs at the genomic and non-genomic levels. By following alkaline phosphatase gene transcription, protein concentration, and enzyme specific activity, we have shown that steroid hormones and growth factors exhibit a regulatory influence over gene transcription, protein synthesis, and matrix vesicle activity. Matrix vesicles respond to peptide hormones, other matrix proteins, like alpha 2-HS-glycoprotein, and autocoid mediators as well. Matrix vesicle metabolism can be directly affected by vitamin D metabolites, even in the absence of cells. The results indicate that 1,25-(OH)2D3(1,25D) or 24,25-(OH)2D3(24,25D) produced by the cells in culture can modulate matrix vesicle activity, and suggest that calcifying cells can modulate events in the matrix via autocrine/paracrine stimulation or inhibition of the matrix vesicles. 1,25D and 24,25D regulate matrix vesicle phospholipase A2 activity, fatty acid turnover, arachidonic acid release, PGE2 production and membrane fluidity, which act on the matrix vesicle to alter enzyme activity. Since vitamin D metabolite production is sensitive to both hormones and growth factors, there is potential for fine tuning matrix vesicle behavior.

Animals↗

PubMed1992

Differential regulation of prostaglandin E2 synthesis and phospholipase A2 activity by 1,25-(OH)2D3 in three osteoblast-like cell lines (MC-3T3-E1, ROS 17/2.8, and MG-63).

Both 1,25-(OH)2D3 and prostaglandin E2 (PGE2) stimulate alkaline phosphatase activity in MC-3T3-E1 cells. Previous studies, demonstrating a correlation between 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activities in matrix vesicles isolated from growth cartilage chondrocyte cultures, suggest that one mechanism of vitamin D action may be via autocrine or paracrine action of PGE2. Since most PGE2 is derived from arachidonic acid released by the action of phospholipase A2, we examined whether 1,25-(OH)2D3 stimulates phospholipase A2 activity in three osteoblastic cell lines: ROS 17/2.8 cells, MC-3T3-E1 cells, and MG-63 cells. 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activity were correlated with production of PGE2 and PGE1 in the MC-3T3-E1 cells. Alkaline phosphatase specific activity was enriched in the matrix vesicles produced by all three cell types and was stimulated by 1,25-(OH)2D3 at 10(-8) to 10(-7) M. Although phospholipase A2 specific activity was enriched in the matrix vesicles produced only by the ROS 17/2.8 cell cultures, stimulation of this enzyme activity was observed only in the MC-3T3-E1 cell cultures. The effects of 1,25-(OH)2D3 on phospholipase A2 were dose-dependent and were significant at 10(-8) to 10(-7) M. There was a significant increase in PGE2 production in the MC-3T3-E1 cell cultures only. Indomethacin reduced PGE2 production to base line values. Even at baseline, MC-3T3-E1 cells produced ten times more PGE2 than did the ROS 17/2.8 or MG-63 cell cultures. The effects of 1,25-(OH)2D3 on PGE1 were comparable to those on PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1992

Regulation of prostaglandin E2 production by vitamin D metabolites in growth zone and resting zone chondrocyte cultures is dependent on cell maturation.

The production of PGE2 by chondrocytes and its regulation by vitamin D metabolites was examined in this study as a function of cell maturation. Costochondral chondrocytes, derived from the resting zone and growth zone cartilage, were grown in culture to fourth passage. At confluence, they were exposed to 10(-8)-10(-11)M 1,25-(OH)2D3 or to 10(-7)-10(-10)M 24,25-(OH)2D3 for either five minutes or 3, 6, 12, or 24 hours. Indomethacin (10(-7)M) was added to one-half of the cultures to block the production of PGE2. The amount of PGE2 released into the media was determined by radioimmunoassay. Both growth zone and resting zone cells produced PGE2 in a time-dependent manner; PGE2 concentration was greater in the resting zone cell cultures. 1,25-(OH)2D3 stimulated PGE2 production by growth zone cells in a dose-dependent manner, significant at 10(-8)-10(-10)M. This effect was observed at 3 hours and remained elevated during the 24 hours of culture. 1,25-(OH)2D3 had no effect on PGE2 production by resting zone cells. However, 24,25-(OH)2D3 (10(-7)-10(-8)M) inhibited PGE2 production from 3-24 hours. No effect was noted when 24,25-(OH)2D3 was added to growth zone cells. Indomethacin reduced PGE2 production to baseline values in all groups examined. The results indicate that chondrocytes in culture produce PGE2. Production is regulated by vitamin D3 metabolites and is cell maturation-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

24,25-Dihydroxyvitamin D 3↗

PubMed1992

In vitro studies on the regulation of endochondral ossification by vitamin D.

The research described in this article has focused on the complex autocrine, paracrine, and endocrine regulation of endochondral ossification using vitamin D metabolites and TGF-beta as models. By comparing results from a number of laboratories utilizing a diverse array of in vivo and in vitro systems, a coherent picture is beginning to emerge. Vitamin D metabolites influence cell differentiation and maturation and have direct effects on cell function. Differentiation of the mesenchymal cells into chondroblasts is regulated by both 1,25-(OH)2D3 and 24,25-(OH)2D3, as well as by TGF-beta. The resting zone chondrocytes respond primarily to 24,25-(OH)2D3 in terms of matrix synthesis and matrix vesicle biochemistry. They synthesize both metabolites and other factors that stabilize matrix vesicle enzymes like AHSG. In addition to the paracrine role these factors may play in regulating the matrix, it is possible that they may influence the cells in the growth plate itself. Growth zone chondrocytes also synthesize both metabolites, but respond primarily to 1,25-(OH)2D3 for the parameters measured in the studies described. These cells also synthesize TGF-beta which further increases alkaline phosphatase activity, perhaps via an autocrine stimulation of the cell. While cells from the calcified zone have not yet been studied directly in culture, it is likely that they respond to paracrine signals from the avascular cartilage as well as to serum-derived factors. How the signals are transferred among the cells is unknown. Certainly one can postulate information flow in both upward and downward directions. The signal transduction mechanisms for the factors at the cellular level are complex. While it is known that 1,25-(OH)2D3 stimulates gene transcription and stabilization of mRNA for proteins like alkaline phosphatase, its nongenomic effects are only beginning to emerge. Membrane effects of this metabolite have been shown in intestine and kidney in conjunction with studies on Ca flux. It is becoming increasingly evident that other steroid hormones may operate in similar ways. Studies with the rat costochondral chondrocytes are the first to show that there are specific membrane effects for at least two vitamin D metabolites and that membrane enzymes, including those involved in phospholipid metabolism, can be differentially regulated by them. Furthermore, these experiments have provided for the first time a clear hypothesis for how cells can regulate events in the extracellular matrix after the matrix vesicles are produced and incorporated into the matrix.

Animals↗

PubMed1992

Production of 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 by growth zone and resting zone chondrocytes is dependent on cell maturation and is regulated by hormones and growth factors.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 have been shown to promote chondrocyte proliferation and differentiation; resting zone chondrocytes respond primarily to 24,25-(OH)2D3, whereas growth zone chondrocytes respond primarily to 1,25-(OH)2D3. This study determined whether resting zone and growth zone cells produce 24,25-(OH)2D3 or 1,25-(OH)2D3; whether this production is regulated by 1,25-(OH)2D3 (10(-8) M), 24,25-(OH)2D3 (10(-7) M), dexamethasone (10(-7) M), or recombinant human transforming growth factor-beta 1 (11 ng/ml); and whether the metabolites produced are biologically active. Confluent fourth passage rat costochondral growth zone or resting zone chondrocytes were cultured in Dulbecco's Modified Eagle's Medium containing [3H]25-hydroxyvitamin D3 ([3H]25OHD3), 2% fetal bovine serum, and antibiotics. Metabolism of [3H]25OHD3 was measured by analyzing the lipid extracts of the conditioned medium and the cell layer for [3H]1,25OHD3, [3H]1,25-(OH)2D3, and [3H]24,25-(OH)2D3 using flow-through scintillation spectroscopy of HPLC eluates. Chemically synthesized radioinert vitamin D3 metabolites were used as standards, and their migration was determined by absorbance at 254 nm. To ensure that the radioactive peaks were 1,25-(OH)2D3 and 24,25-(OH)2D3, the fractions were rechromatographed into three other HPLC solvent systems. Biological activity was confirmed; the addition of HPLC-purified 1,25-(OH)2D3 produced by growth zone chondrocytes elicited a dose-dependent stimulation of alkaline phosphatase specific activity in growth zone cell cultures, but had no effect on the resting zone cells. There was a time-dependent increase in both [3H]1,25-(OH)2D3 and [3H]24,25-(OH)2D3 in the conditioned medium of both types of cultures. At 24 h, the percent conversion of [3H]25OHD3 to [3H]1,25-(OH)2D3 was 5.3 +/- 1.2, and the percent conversion to [3H]24,25-(OH)2D3 was 1.8 +/- 0.4 in growth zone chondrocyte cultures. No such effect was found in cultures freeze-thawed five times or without cells. When resting zone cells were cultured with [3H]25OHD3, the percent conversion to 1,25-(OH)2D3 and 24,25-(OH)2D3 was 4.5 +/- 1.0 and 1.7 +/- 0.4, respectively. The addition of dexamethasone significantly increased the percent production of 1,25-(OH)2D3 at 6 and 24 h and at 6 h by resting zone and growth zone cells, respectively, compared to the control values. Recombinant human transforming growth factor-beta 1 increased the percent production of 1,25-(OH)2D3 after 1 h in resting zone cells and, after 24 h, the production of 24,25-(OH)2D3 in growth zone cells. Radiolabeled 1,25-(OH)2D3 and 24,25-(OH)2D3 were not detected in the cell layer.(ABSTRACT TRUNCATED AT 400 WORDS)

24,25-Dihydroxyvitamin D 3↗

PubMed1992

Effects of 1 alpha(OH)-vitamin D3 and 24,25(OH)2-vitamin D3 on long bones of glucocorticoid-treated rats.

Glucocorticoids may induce osteopenia in experimental animals and in man. In order to study the possible effects of vitamin D metabolites in the prevention of glucocorticoid-induced osteopenia in rats, we administered 1 alpha(OH)-vitamin D3, 24,25(OH)2-vitamin D3 or a combination of both metabolites, by intragastric intubation, to rats treated daily by intramuscular injections of 10 mg/kg cortisone acetate. Treatment with the vitamin D metabolites started after 1 month of glucocorticoid therapy, at the time osteopenia was already present. Cortisone acetate decreased the gain weight, increased alkaline phosphatase (AP) and decreased Ca serum levels. It also decreased tibial wet and ash weight and tibial Ca content. Computerized histomorphometry of sections from the upper tibia showed decreased epiphyseal bone volume and increased bone marrow volume; decreased height of hypertrophic cartilage in the growth plate and decreased amount of persisting cartilage in the metaphyseal bone trabeculae were also observed. Administration of 24,25(OH)2D3 alone did not reduce these glucocorticoid-induced bone changes and sometimes even worsened them. 1 alpha(OH)D3 reversed many of the deleterious effects of cortisone acetate. It reduced serum AP levels, increased serum Ca levels, increased bone ash weight, epiphyseal and metaphyseal bone volume, with a concomitant reduction in epiphyseal and metaphyseal bone marrow volume. The best results were obtained by a combination of 1 alpha(OH)D3 and 24,25(OH)2D3. It is presumed that both metabolites are needed to reduce the impact of glucocorticoids on bone. 1 alpha(OH)2D3 acts on the gut, increasing Ca absorption (which was decreased by glucocorticoids), and 24,25(OH)2D3 directly acts on bone to enhance bone formation and mineralization.

24,25-Dihydroxyvitamin D 3↗

PubMed1992

Epithelial cell lines that induce bone formation in vivo produce alkaline phosphatase-enriched matrix vesicles in culture.

Hypertrophic chondrocytes and osteoblasts produce alkaline phosphatase (ALPase)-enriched matrix vesicles in vivo and in vitro and, along with certain epithelial cell lines and osteoblast precursors, induce bone when implanted in mesenchymal tissues. This study examined whether ALPase-enriched matrix vesicle production in vitro was a general property of cells that induce bone in vivo. Epithelial cell lines FL, WISH, and OK 16; connective tissue cell lines HEPM 1 and HEPM 2; neonatal rat muscle cells; rat costochondral chondrocytes; and human fibroblasts were implanted intramuscularly into nude mice. The FL and WISH cells produced tumors and induced large islands of bone with focal areas of cartilage immediately adjacent to the tumors. The chondrocytes formed cartilage nodules but did not induce bone, indicating that the ability of the cells to form a solid mass was not an a priori requirement for bone formation. No other cell type produced tumors or nodules or induced bone formation, although connective tissue cells have been shown to induce chondrogenesis in vitro and osteogenesis in vivo. Only matrix vesicles from normal chondrocytes, FL, WISH, and OK16 cultures exhibited enriched ALPase-specific activity. Matrix vesicles from FL and WISH cultures exhibited ALPase specific activities similar to those isolated from osteoblast or chondrocyte cultures. These data suggest that the ability to produce ALPase-enriched matrix vesicles in culture may be associated with the ability of cells to induce bone or cartilage in vivo.

Alkaline Phosphatase↗

PubMed1992

Initial effects of partially purified bone morphogenetic protein on the expression of glycosaminoglycan, collagen, and alkaline phosphatase in nonunion cell cultures.

Bone morphogenetic protein (BMP) stimulates mesenchymal cells to differentiate, resulting in de novo endochondral ossification in vivo. The response of fibrocartilage and periosteal cells from human and canine nonunion tissues to partially purified BMP was examined in culture. Cells derived from neonatal rat muscle explants were used for comparison. Alkaline phosphatase activity and expression of alkaline phosphatase and Types I and II collagen mRNAs were compared to that of rat chondrocytes. Synthesis of Type II collagen by the muscle cells was verified by enzyme-linked immunosorbent assay (ELISA). Addition of BMP to the muscle cell and nonunion cell cultures resulted in a dose-dependent decrease in cell number. There was a decrease in matrix vesicle and plasma membrane alkaline phosphatase activity concomitant with an increase in mRNA levels for alkaline phosphatase and collagen genes. Synthesis of immunoreactive Type II collagen increased. These data indicate that neonatal rat muscle cells and nonunion cells may respond in a similar fashion to BMP. Bone morphogenetic protein stimulated hyaluronic acid synthesis at three days, but chondroitin sulfate synthesis did not increase until ten days exposure to BMP. These data, together with those summarized above, suggest that more than three days may be required for complete expression of the chondrocyte phenotype typical of endochondral ossification.

Alkaline Phosphatase↗

PubMed1991

Inhibition of 1,25-(OH)2D3- and 24,25-(OH)2D3-dependent stimulation of alkaline phosphatase activity by A23187 suggests a role for calcium in the mechanism of vitamin D regulation of chondrocyte cultures.

This study used the ionophore, A23187, to examine the hypothesis that the regulation of alkaline phosphatase and phospholipase A2 activity by vitamin D3 metabolites in cartilage cells is mediated by changes in calcium influx. Confluent, fourth-passage cultures of growth zone and resting zone chondrocytes from the costochondral cartilage of 125 g rats were incubated with 0.01-10 microM A23187. Specific activities of alkaline phosphatase and phospholipase A2 were measured in the cell layer and in isolated plasma membranes and matrix vesicles. There was an inhibition of alkaline phosphatase specific activity at 0.1 microM A23187 in resting zone cells and at 0.1 and 1 microM in growth zone chondrocytes. At these concentrations of ionophore, the 45Ca content of the chondrocytes was shown to increase. Both the plasma membrane and matrix vesicle enzyme activities were inhibited. There was no effect of ionophore on matrix vesicle or plasma membrane phospholipase A2 in either cell type. In contrast, alkaline phosphatase activity is stimulated when growth zone chondrocytes are incubated with 1,25-(OH)2D3 and in resting zone cells incubated with 24,25-(OH)2D3. Phospholipase A2 activity is differentially affected depending on the metabolite used and the cell examined. Addition of ionophore to cultures preincubated with 1,25-(OH)2D3 or 24,25-(OH)2D3 blocked the stimulation of alkaline phosphatase by the vitamin D3 metabolites in a dose-dependent manner. The effects of ionophore were not due to a direct effect on the membrane enzymes since enzyme activity is isolated membranes incubated with A23187 in vitro was unaffected. These results suggest a role for calcium in the action of vitamin D metabolites on chondrocyte membrane enzyme activity but indicate that mechanisms other than merely Ca2+ influx per se are involved.

24,25-Dihydroxyvitamin D 3↗

PubMed1991

Effect of glass ceramic and titanium implants on primary calcification during rat tibial bone healing.

The effect of bone bonding (KG Cera, Mina 13, and titanium) and nonbone bonding (KGy-213, M 8/1) implants on primary calcification in endosteal bone was examined by comparing changes in the morphometry of matrix vesicles to those occurring during normal bone healing following ablation of rat tibial marrow. The concentration of matrix vesicles, their diameter, and their distance from the calcification front were determined using computerized cytomorphometry at the transmission electron microscopic level. The results demonstrated that bone bonding materials supported an increase in matrix vesicle concentration when compared with control bone at 6 and 14 days postimplantation. At 14 days, there were fewer matrix vesicles in the bone adjacent to the nonbonding implants. Though matrix vesicle diameter decreased in the control bone between 6 and 14 days, it increased in all of the experimental samples. Diameters were significantly greater in the bone bonding samples at 14 days and significantly lower in the nonbonding samples at 6 days. Distance from the calcification front decreased between 6 and 14 days in all groups except in bone adjacent to the KGy-213 implants. In bone adjacent to the bone bonding implants, distance from the calcification front was comparable to or further than that of control bone; in the nonbonding samples it was closer to the calcification front. These results demonstrate that production and maturation of matrix vesicles is influenced in a differential manner by the presence of implant materials.

Animals↗

PubMed1991

Alpha 2-HS-glycoprotein: expression in chondrocytes and augmentation of alkaline phosphatase and phospholipase A2 activity.

The alpha 2-HS-glycoprotein is a plasma protein synthesized in liver and enriched in bone. The concentration of alpha 2-HS-glycoprotein dynamically changes in various physiological conditions and is highest in bone during growth, suggesting that it is involved in regulation of endochondral ossification. Northern blot analysis demonstrated that mRNA transcripts from growth zone and resting zone costochondral chondrocyte cultures hybridized with alpha 2-HS-glycoprotein cDNA. However, a difference of mRNA transcript size was observed, with chondrocyte mRNA transcripts being 2.2 kb, while mRNA isolated from liver was 1.6 kb. Presence of alpha 2-HS-glycoprotein in cartilage cells was found by immunohistochemical staining of human fetal epiphyses using anti-human alpha 2-HS-glycoprotein antibody. To understand the role of alpha 2-HS-glycoprotein in cartilage growth, the effects of exogenous alpha 2-HS-glycoprotein were correlated with alkaline phosphatase (ALPase) and phospholipase A2 (PA2) activity in the chondrocyte cultures. Alkaline phosphatase specific activity was stimulated by alpha 2-HS-glycoprotein at concentrations between 0.25 and 1.25 micrograms/mL in the growth zone and resting zone cultures 2.7 and 2.0-fold, respectively. Matrix vesicle PA2 activity was increased only in the growth zone chondrocyte cultures. These results suggested that alpha 2-HS-glycoprotein may contribute to the regulation of the expression of the chondrocyte phenotype. Steady state mRNA levels of ALPase were analyzed in chondrocytes after additions of alpha 2-HS-glycoprotein. The ALPase mRNA levels remained stationary during the stimulation of enzymatic activity, indicating that the effect of alpha 2-HS-glycoprotein upon alkaline phosphatase activity is not at the transcriptional level.

Alkaline Phosphatase↗

PubMed1991

Direct and sex-specific enhancement of bone formation and calcification by sex steroids in fetal mice long bone in vitro (biochemical and morphometric study.

The study was carried out to examine the direct effect of the sex hormones 17 beta-estradiol (E2) and testosterone on the modeling of cultured fetal mouse long bones separated according to their sex. The culture system used allowed for the simultaneous assessment of bone growth, mineralization, and resorption on each bone. Bones from 16-day-old male and female mouse fetuses were cultured in BGJ medium, supplemented with either 10% fetal calf serum or 4 mg/ml BSA (serum-free medium) for 48 h. The bones were harvested, and their length; the length of their diaphyses; their hydroxyproline, calcium, and phosphorus contents; and their 45Ca release were measured. Histomorphometric analyses on midlongitudinal sections of bones from parallel experiments were also performed. The results indicate that in medium supplemented with 10% fetal calf serum, E2 had a dose-dependent stimulatory effect on bone formation and mineralization at 10(-7) and 10(-9) M, with no effect on bone resorption. This effect was specific to bones from female mice and to E2, since 17-alpha-estradiol had no effect. Testosterone had similar effects specific to bones from male mice, resulting in the stimulation of bone formation and mineralization at 10(-7)- and 10(-9)-M concentrations. These effects were absent when serum-free medium was used. E2 and testosterone had an anabolic effect on endochondral and periosteal bone formation and mineralization, but no effect on bone resorption. This effect is dependent on the presence of a serum factor(s).

Animals↗

PubMed1990

Regulation of arachidonic acid turnover by 1,25-(OH)2D3 and 24,25-(OH)2D3 in growth zone and resting zone chondrocyte cultures.

Previous studies have shown that phospholipase A2 activity in rat costochondral chondrocyte cultures is differentially regulated by 1,25-(OH)2D3 and 24,25-(OH)2D3. 1,25-(OH)2D3 stimulates enzyme activity in growth zone chondrocytes but has no effect on the resting zone chondrocyte enzyme activity. 24,25-(OH)2D3 inhibits the resting zone enzyme but has no effect on the growth zone chondrocyte phospholipase A2. This study examined whether the metabolites affect arachidonic acid turnover in their target cell populations. Incorporation and release of [14C]arachidonate was measured at various times following addition of hormone to the cultures. Acylation and reacylation were measured independently by incubating half of the [14C]arachidonate-labeled cultures with p-chloromercuribenzoate. The results demonstrated that the distribution of [14C]arachidonate in membrane phospholipids differed between growth zone and resting zone chondrocytes and between the plasma membranes and matrix vesicles isolated from the growth zone chondrocyte cultures. Plasma membrane phospholipids were more susceptible to the release of [14C]arachidonic acid by exogenous phospholipases than were matrix vesicle phospholipids. The effect of 1,25-(OH)2D3 on growth zone chondrocytes was observed within 5 min. Incorporation was greatest after 60 min; release was greatest after 30 min. 24,25-(OH)2D3 stimulated consistently elevated incorporation throughout the incubation period, peaking at 15 min. Peak release was at 60 min. The results confirm that resting zone chondrocytes and growth zone chondrocytes retain a differential phenotype in culture and demonstrate that matrix vesicles are distinct from the plasma membrane in terms of lipid composition and arachidonic acid incorporation. 1,25-(OH)2D3 and 24,25-(OH)2D3 appear to stimulate arachidonic acid turnover in their target cells by different mechanisms. Changes in fatty acid acylation and reacylation may be one mode of vitamin D-3 action in cartilage.

24,25-Dihydroxyvitamin D 3↗

PubMed1990

The effect of glass-ceramic implants on matrix vesicle calcification after two weeks of rat tibial bone healing.

Type, size and distribution of extracellular matrix vesicles (MV), known mediators of primary calcification, were studied around bone-bonding and metal-oxide containing, nonbonding, glass-ceramic implants. This was performed in order to further understand the different effects of implants on bone healing. At 14 days after implantation in adult rat tibial bone the effects of different implants on MV were studied by transmission electron microscopy and computerized morphometry. A total number of 4607 MV in 245 electron micrographs were counted and grouped according to diameter, distance from the calcifying front, and classified as four types: "empty," "amorphous," "crystal," and "rupture." The sequence of types according to diameter and distance was recorded as follows around both implants tested: "rupture" MV were the closest to the front with the largest diameter, followed by "crystal," "amorphous," and "empty," MV with the largest distance from the front and the smallest diameter. Most vesicles were concentrated in a distance of less than 2.4 microns from the front and between diameters of 0.06 microns and 0.22 microns. The noncalcified extracellular matrix around bone-bonding implants contained more MV than the matrix around the nonbonding type (26.24 MV/10 microns2 and 18.76 MV/10 microns2). MV distribution according to types showed that around bonding implants there was a higher percentage of "crystal" and a lower percentage of "rupture" when compared to the nonbonding type. These results indicate that bonding implants affect osteoblastic function by increasing the vesicular number and retardation of intravesicular crystal formation. It might be suggested that bonding implants induce an increase in the process of primary calcification and a decreased rate of crystal formation resulting with the highest organization of the healing bone.

Animals↗