Biomedical subjects

Barbara D Boyan

Publications and source records attributed to Barbara D Boyan.

25 records · Page 2Linked to original sources

PubMed2002

Transforming growth factor-beta1 regulation of growth zone chondrocytes is mediated by multiple interacting pathways.

Transforming growth factor beta 1 (TGF-beta1) affects growth plate chondrocytes through Smad-mediated mechanisms and has been shown to increase protein kinase C (PKC). This study determined if PKC mediates the physiological response of rat costochondral growth zone (GC) chondrocytes to TGF-beta1; if the physiological response occurs via type II or type III TGF-beta receptors, and, if so, which receptor mediates the increase in PKC; and the signal transduction pathways involved. Treatment of confluent GC cells with TGF-beta1 stimulated [(3)H]thymidine and [(35)S]sulfate incorporation as well as alkaline phosphatase (ALPase) and PKC specific activities. Inhibition of PKC with chelerythrine, staurosporine, or H-7 caused a dose-dependent decrease in these parameters, indicating that PKC signaling was involved. TGF-beta1-dependent PKC and the physiological response of GC cells to TGF-beta1 was reversed by anti-type II TGF-beta receptor antibody and soluble type II TGF-beta receptor, showing that TGF-beta1 mediates these effects through the type II receptor. The increase in [3H]thymidine incorporation and ALPase specific activity were also regulated by protein kinase A (PKA) signaling, since the effects of TGF-beta1 were partially blocked by the PKA inhibitor H-8. The mechanism of TGF-beta1 activation of PKC is through phospholipase A(2) (PLA(2)) and not through phospholipase C (PLC). Arachidonic acid increased PKC in control cultures and was additive with TGF-beta1. Prostanoids are required, as indomethacin blocked the effect of TGF-beta1, and Cox-1, but not Cox-2, is involved. TGF-beta1 stimulates prostaglandin E(2) (PGE(2)) production and exogenous PGE(2) stimulates PKC, but not as much as TGF-beta1, suggesting that PGE(2) is not sufficient for all of the prostaglandin effect. In contrast, TGF-beta1 was not regulated by diacylglycerol; neither dioctanoylglycerol (DOG) nor inhibition of diacylglycerol kinase with R59022 had an effect. G-proteins mediate TGF-beta1 signaling at different levels in the cascade. TGF-beta1-dependent increases in PGE(2) levels and PKC were augmented by the G protein activator GTP gamma S, whereas inhibition of G-protein activity via GDP beta S, pertussis toxin, or cholera toxin blocked stimulation of PKC by TGF-beta1, indicating that both G(i) and G(s) are involved. Inhibition of PKA with H-8 partially blocked TGF-beta1-dependent PKC, suggesting that PKA inhibition on the physiological response was via PKA regulation of PKC signaling. This indicates that multiple interacting signaling pathways are involved: TGF-beta1 stimulates PLA(2) and prostaglandin release via the action of Cox-1 on arachidonic acid. PGE(2) activates the EP2 receptor, leading to G-protein-dependent activation of PKA. PKA signaling results in increased PKC activity and PKC signaling regulates proliferation, differentiation, and matrix synthesis.

Alkaline Phosphatase↗

PubMed2002

1alpha,25(OH)2D3 regulates chondrocyte matrix vesicle protein kinase C (PKC) directly via G-protein-dependent mechanisms and indirectly via incorporation of PKC during matrix vesicle biogenesis.

Matrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1alpha,25(OH)(2)D(3). Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1alpha,25(OH)(2)D(3) in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1alpha,25(OH)(2)D(3) during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1alpha,25(OH)(2)D(3), PKCzeta in matrix vesicles was inhibited, while PKCalpha in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKCzeta in matrix vesicles was increased, while PKCalpha in plasma membranes was unchanged. The effect of 1alpha,25(OH)(2)D(3) was stereospecific and metabolite-specific. Monensin blocked the increase in matrix vesicle PKC after 24 h, suggesting the secosteroid-regulated packaging of PKC. In addition, the 1alpha,25(OH)(2)D(3) membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1alpha,25(OH)(2)D(3)-dependent changes in PKC after both long and short treatment times. In contrast, antibodies to annexin II had no effect, and there was no evidence for the presence of the nuclear VDR on Western blots. To investigate the signaling pathways involved in regulating matrix vesicle PKC activity after biosynthesis, matrix vesicles were isolated and then treated for 9 min with 1alpha,25(OH)(2)D(3) in the presence and absence of specific inhibitors. Inhibition of phosphatidylinositol-phospholipase C, phospholipase D, or G(i)/G(s) had no effect. However, inhibition of G(q) blocked the effect of 1alpha,25(OH)(2)D(3). The rapid effect of 1alpha,25(OH)(2)D(3) also involved the 1,25-mVDR. Moreover, arachidonic acid was found to stimulate PKC when added directly to isolated matrix vesicles. These results indicate that matrix vesicle PKC is regulated by 1alpha,25(OH)(2)D(3) at three levels: 1) during matrix vesicle biogenesis; 2) through direct action on the membrane; and 3) through production of other factors such as arachidonic acid.

Animals↗

PubMed2002

Evidence for distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) in osteoblasts.

1 alpha,25-(OH)(2)D(3) exerts its effects on chondrocytes and enterocytes via nuclear receptors (1,25-nVDR) and a separate membrane receptor (1,25-mVDR) that activates protein kinase C (PKC). 24R,25-(OH)(2)D(3) also stimulates PKC in chondrocytes, but through other membrane mechanisms. This study examined the hypothesis that osteoblasts possess distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) that are involved in the activation of PKC and that receptor expression varies as a function of cell maturation state. 1 alpha,25-(OH)(2)D(3) stimulated PKC in well differentiated (UMR-106, MC-3T3-E1) and moderately differentiated (ROS 17/2.8) osteoblast-like cells, and in cultures of fetal rat calvarial (FRC) cells and 2T3 cells treated with rhBMP-2 to promote differentiation. 24R,25-(OH)(2)D(3) stimulated PKC in FRC and 2T3 cultures that had not been treated to induce differentiation, and in ROS 17/2.8 cells. MG63 cells, a relatively undifferentiated osteoblast-like cell line, had no response to either metabolite. Ab99, a polyclonal antibody generated to the chick enterocyte 1,25-mVDR, but not a specific antibody to the 1,25-nVDR, inhibited response to 1 alpha,25-(OH)(2)D(3). 1 alpha,25-(OH)(2)D(3) exhibited specific binding to plasma membrane preparations from cells demonstrating a PKC response to this metabolite that is typical of positive cooperativity. Western blots of these membrane proteins reacted with Ab99, and the Ab99-positive protein had an Mr of 64 kDa. There was no cross-reaction with antibodies to the C- or N-terminus of annexin II. The effect of 24,25-(OH)(2)D(3) on PKC was stereospecific; 24S,25-(OH)(2)D(3) had no effect. These results demonstrate that response to 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) depends on osteoblast maturation state and suggest that specific and distinct membrane receptors are involved.

24,25-Dihydroxyvitamin D 3↗

PubMed2002

Membrane mediated signaling mechanisms are used differentially by metabolites of vitamin D(3) in musculoskeletal cells.

1 alpha,25(OH)(2)D(3) and 24R,25(OH)(2)D(3) mediate their effects on chondrocytes and osteoblasts in part through increased activity of protein kinase C (PKC). For both cell types, 1 alpha,25(OH)(2)D(3) exerts its effects primarily on more mature cells within the lineage, whereas 24R,25(OH)(2)D(3) exerts its effects primarily on relatively immature cells. Studies using the rat costochondral cartilage growth plate as a model indicate that the two metabolites increase PKC activity by different mechanisms. In growth zone cells (prehypertrophic/upper hypertrophic cell zones), 1 alpha,25(OH)(2)D(3) causes a rapid increase in PKC that does not involve new gene expression. 1 alpha,25(OH)(2)D(3) binds its membrane receptor (1,25-mVDR), resulting in activation of phospholipase A(2) and the rapid release of arachidonic acid, as well as activation of phosphatidylinositol-specific phospholipase C, resulting in formation of diacylglycerol and inositol-1,4,5-tris phosphate (IP(3)). IP(3) leads to release of intracellular Ca(2+) from the rough endoplasmic reticulum, and together with diacylglycerol, the increased Ca(2+) activates PKC. PKC is then translocated to the plasma membrane, where it initiates a phosphorylation cascade, ultimately phosphorylating the extracellular signal-regulated kinase-1 and -2 (ERK1/2) family of MAP kinases (MAPK). PKC increases are maximal at 9 min, and MAPK increases are maximal at 90 min in these cells. By contrast, 24R,25(OH)(2)D(3) increases PKC through activation of phospholipase D in resting zone cells. Peak production of diacylglycerol via phospholipase D2 is at 90 min, as are peak increases in PKC. Some of the effect is direct on existing plasma membrane PKC, but most is due to new PKC expression; translocation is not involved. Arachidonic acid and its metabolites also play differential roles in the mechanisms, stimulating PKC in growth zone cells and inhibiting PKC in resting zone cells. 24R,25(OH)(2)D(3) decreases phospholipase A(2) activity and prostaglandin production, thereby overcoming this potential inhibitory component, which may account for the delay in the PKC response. Ultimately, ERK1/2 is phosphorylated. PKC-dependent MAPK activity transduces some, but not all, of the physiological responses of each cell type to its respective vitamin D metabolite, suggesting that the membrane receptor(s) and nuclear receptor(s) may function interdependently to regulate proliferation and differentiation of musculoskeletal cells, but different pathways are involved at different stages of phenotypic maturation.

24,25-Dihydroxyvitamin D 3↗

PubMed2002

Ceramic and PMMA particles differentially affect osteoblast phenotype.

There is increasing evidence that wear debris particles present in periprosthetic tissues have direct effects on osteoblasts. The nature of the cell response varies with the chemistry of the particle and the number of particles. Most studies have used Ti, Ti-6Al-4V, and ultrahigh molecular weight polyethylene (UHMWPE) particles since these materials are most frequently used in implants and as a result, these particles predominate in peri-prosthetic tissues. Ceramics have also been used successfully as load-bearing surfaces in implants for years, although it is unknown how wear debris from these surfaces may contribute to aseptic bone loss. Further, particles resulting from polymethylmethacrylate (PMMA) cements used for fixation may also be involved in aseptic loosening of implants, but how these particles may affect bone formation is unknown. In the present study, we examined whether aluminum oxide (Al2O3), zirconium oxide (ZrO2), and PMMA particles exert effects on osteoblast proliferation, phenotypic expression, and local factor production, and if so, whether the effects were specific to the particle type. ZrO2 particles were produced in a custom-made axial mixer in which ZrO2 containers were filled with ZrO2 bars and 95% ethanol and then rotated continuously at room temperature. PMMA particles were prepared in a ZrO2 roller mill. Al2O3 was produced and provided by Aesculap AG. Particles were endotoxin-free with equivalent circle diameters <3 microm; Al2O3 particles were significantly smaller than ZrO2 or PMMA particles. Particle suspensions were added to confluent cultures of MG63 osteoblast-like cells after diluting them 1:100, 1:10, and 1:1 with culture medium. Cells were incubated with the particles for 24 h. Transmission electron microscopy showed that MG63 cells phagocytosed Al2O3 particles and exhibited ultrastructural changes consistent with cytotoxicity. This was supported by biochemical changes as well. Proliferation, alkaline phosphatase activity, and TGF-beta1 levels were decreased. ZrO2 and PMMA particles increased proliferation and alkaline phosphatase specific activity. The effect of ZrO2 on alkaline phosphatase was targeted to matrix vesicles, the effect of PMMA was greater on the cells. All particles increased prostaglandin E2 production. These results show that Al2O3, ZrO2, and PMMA particles elicit direct effects on osteoblasts and that cell response depends on the particle type. None of the particles tested had the same effect as noted previously for UHMWPE: increased proliferation and decreased alkaline phosphatase. These results may indicate that the response of peri-prosthetic tissues to wear particles may be modulated by the relative contributions of the various particle types present.

Alkaline Phosphatase↗

PubMed2002

The palatal advanced flap: a pedicle flap for primary coverage of immediately placed implants.

The placement of implants immediately after tooth extraction has become a relatively common event. This approach has gained popularity because it reduces the time between tooth extraction and placement of the final prosthetic restoration. One problem that remains unresolved with this procedure, however, is that there is usually space left in the area surrounding the coronal portion of the implant. As none of the techniques published to date is completely satisfactory, we developed an approach that is a modification of the palatal pedicle flaps used for closure of oro-antral fistulas. This technique allows for precise rotational advancement of the palatal tissue in a coronal direction, thus facilitating complete coverage of the extraction site. The procedure was used on 38 patients treated for primary coverage of postextraction placed implants. Thirty-two of the patients required single tooth replacement, three patients received two implants each, and three patients received three implants each. In 27 of the cases, the procedure was performed in conjunction with barrier membranes; of the 38 cases, 25 patients required the use of DFDBA to fill the defect created by the extraction procedure. The average defect width was 4.70 +/- 0.2 mm. Complete primary coverage was obtained in all cases without any changes in the size or anatomy of the buccal vestibule; complete coverage was maintained over the entire healing period of 6-8 months (mean +/- SEM, 6.49 +/- 0.10 months). Even with guided tissue regeneration cases, there was no exposure of the barrier membrane or cover screw of the implant during healing. After a period of 1-5 years (mean +/- SEM, 39.35 +/- 2.69 months), none of the implants was lost. We conclude that the palatal advanced flap procedure is useful, fast, and easy to perform in cases of immediate implant placement after tooth extraction.

Adult↗

PubMed2002

Nitric oxide and prostaglandin E2 production in response to ultra-high molecular weight polyethylene particles depends on osteoblast maturation state.

BACKGROUND: Recent studies have shown that osteoblast-like cells respond directly to ultra-high molecular weight polyethylene particles in culture, suggesting that they may be involved in aseptic loosening of endoprostheses. We tested the hypothesis that the state of cell maturation plays a role in the response of osteogenic cells to ultra-high molecular weight polyethylene particles. METHODS: MG63 cells (immature osteoblast-like cells), OCT-1 cells (mature secretory osteoblast-like cells), and MLO-Y4 cells (osteocyte-like cells) were treated for twenty-four hours with commercial ultra-high molecular weight polyethylene particles with an average diameter of 1 mm. The effect of particle treatment on cell proliferation was assessed by measuring the number of cells, whereas the effects on differentiation and local factor production were assessed by measuring the production of osteocalcin, prostaglandin E2, and nitric oxide. The effect of particles on apoptosis was also evaluated. RESULTS: The addition of ultra-high molecular weight polyethylene particles increased the number of MG63 cells, did not affect the number of OCT-1 cells, and led to a decrease in the number of MLO-Y4 cells. The observed changes in cell number were not due to programmed cell death, as no more than 3% of the cells in cultures treated with the highest concentration of particles were undergoing apoptosis. Osteocalcin production was not affected by the addition of particles. Prostaglandin E2 production was increased in all three types of cultures, but the effect was greatest in OCT-1 cell cultures, as was the absolute amount of prostaglandin E2 produced. Nitric oxide production was unaffected in MG63 cell cultures, but it was stimulated in OCT-1 and MLO-Y4 cell cultures. CONCLUSIONS: The results of the present study support the hypothesis that osteoblast cell maturation state plays an important role in the response to ultra-high molecular weight polyethylene particles and that the terminally differentiated osteocyte may be involved in the bone response to wear debris in vivo.

Alprostadil↗