Augmentation of autograft using rhBMP-2 and different carrier media in the canine spinal fusion model.

This study evaluated the use of recombinant human bone morphogenetic protein (rhBMP-2) with various types of carrier media, and the effect of rhBMP-2 as an adjunct to autogenous iliac crest bone graft in the canine spinal fusion model. BMP induces mesenchymal cells to differentiate into cartilage and bone. The recent availability of rhBMP-2 has created the opportunity to evaluate this material's properties in augmenting autogenous bone graft in spinal fusion. Currently, the most appropriate type of carrier media for rhBMP-2 is undetermined. Bilateral intertransverse spinal fusions were performed on six canine lumbar spines at L1-L2, L3-L4, and L5-L6, using autogenous posterior iliac crest bone graft at each level, creating a total of 18 segmental fusion sites. All 18 sites were then randomly assigned to one of six fusion methods: autogenous bone graft (ABG) alone, ABG + rhBMP-2, ABG + collagen (Helistat) "sandwich" + rhBMP-2, ABG + collagen (Helistat) morsels + rhBMP-2, ABG + polylactic/glycolic acid sponge (PLGA) sandwich + rhBMP-2, and ABG + open-pore polylactic acid morsels + rhBMP-2. Each material was evaluated for ease of handling and application at the index surgery. The animals underwent computed tomography (CT) scanning of the lumbar fusion sites after 8 weeks. Volumetric measurements of total fusion mass at each level were performed using two-dimensional CT scan slices and a volumetric program supplied by the Siemens Medical System. The animals were killed after imaging studies. The lumbar spine fusion sites were evaluated for integrity of the fusion mass, both visually and with manual mechanical stressing. Crossover of the fusion mass to adjoining levels was also evaluated. Histologic evaluation of all fusion sites was performed. The addition of rhBMP-2 significantly increased bone graft volume as noted on CT scan. Carrier that could be mixed with morselized bone graft offered easier handling and application and all spine segments fused. Polylactic/glycolic acid (PLGA) sites were associated with a greater incidence of voids within the fusion mass. No significant difference in carrier media for rhBMP-2 could be determined. However, PLGA was associated with a higher rate of fusion mass void formation. rhBMP-2, when added to autograft, significantly increased the volume and the maturity of the resulting fusion mass.

Comparison of diglyceride production from choline-containing phosphoglycerides in human neutrophils stimulated with N-formylmethionyl-leucylphenylalanine, ionophore A23187 or phorbol 12-myristate 13-acetate.

The turnover of choline-containing phosphoglycerides (PC) in response to agonist stimulation is well documented in human neutrophils. We have now compared the enzymic pathways of N-formylmethionyl-leucylphenylalanine (fMLP)-, A23187- and phorbol-12-myristate 13-acetate (PMA)-induced diglyceride (DG) and phosphatidic acid (PA) generation in these cells. In order to distinguish between phospholipase C- and D-mediated PC breakdown, human neutrophils were radiolabelled with 1-O-[3H]alkyl-2-acyl-glycero-3-phosphocholine and stimulated in the presence of ethanol or propranolol. The addition of 0.5% ethanol to the incubation mixture resulted in the production of phosphatidylethanol, indicative of phospholipase D activation, in response to all three stimuli. Concomitant with phosphatidylethanol formation was a partial block of PA production. The production of DG was also partially blocked by addition of ethanol. Propranolol (200 microM) was also used to assess the contributions of phospholipases C and D toward DG generation. Inhibition of PA phosphohydrolase by propranolol resulted in the complete abolition of DG generation when neutrophils were stimulated with fMLP. In contrast, propranolol only partially inhibited DG generation in response to A23187 and PMA. These results suggested that DG production in response to fMLP stimulation is mediated via the activation of phospholipase D, whereas A23187- or PMA-induced DG generation may involve more than one pathway. However, examination of the water-soluble choline metabolites produced indicated that phospholipase D was responsible for the production of PA and DG in response to all three stimuli.

Ether-linked phosphoglyceride content of human leukemia cells.

The glycerolipids of most cells are characterized by a specific proportion of ether linkages at the sn-1 position of the glycerol backbone. A number of tumors are known to have altered concentrations of ether-linked lipids compared to normal tissues. However, no through examination of the ether-lipid content of human leukemia cells has been reported despite the importance of these lipids in normal leukocyte function. In the present study samples were obtained from adults with acute myelogenous leukemia (AML), chronic granulocytic leukemia in blast crisis, and acute lymphocytic leukemia and from healthy human donors. The cellular lipids were extracted, the individual phospholipid classes were isolated, lipid phosphorus content was determined, and the lipids were converted to diglyceride benzoate derivatives for separation and quantitation of the subclasses by high performance liquid chromatography. The data indicate that all the leukemic cells analyzed have an altered phospholipid composition compared to their respective normal leukocytes. Furthermore, among the AML patients both the percentage of the choline-containing phosphoglyceride fraction (PC) which is alkyl linked and the nmoles alkyl-PC/10(6) cells differ significantly by FAB subtype. A positive correlation between the levels of alkyl-PC and the degree of cellular differentiation is observed. Although no differences are observed between chronic granulocytic leukemia in blast crisis and AML lipids, the leukemic cells contain dramatically lower levels of alkyl-linked PC than do normal polymorphonuclear leukocytes. In contrast, no differences are observed between the alkyl-PC content of normal and leukemic lymphocytes. In light of the relations among ether-lipids, protein kinase C, and cell differentiation, these data suggest the ether-linked lipids are important in myeloid cell function and differentiation.

Correlation of ether lipid content of human leukemia cell lines and their susceptibility to 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine.

A number of synthetic ether-linked phospholipids are selectively cytotoxic to neoplastic cells. However, the mechanisms underlying this selective cytotoxicity are not known. We have investigated the ether-lipid content of HL-60 and K562 human leukemia cells in relation to their sensitivity to 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). HL-60 cells are much more sensitive than K562 cells to the cytotoxic effects of ET-18-OCH3 and, at the same time, they contain nearly twice as much ether lipid as the more resistant K562 cells. These observations suggested a relation between the cellular ether-lipid content and sensitivity to ET-18-OCH3. Further evidence linking these properties was obtained when the ether-lipid content of K562 cells was increased by incubating them in medium containing 1-O-hexadecyl-sn-glycerol. This supplementation not only increased the ether-lipid content of the cells but also increased their sensitivity to ET-18-OCH3. The 50% inhibitory concentration for ET-18-OCH3 decreased from 18.4 microM in the control cells to 9.83 microM in the supplemented cells.

Receptor-independent metabolism of platelet-activating factor by myelogenous cells.

Human neutrophils incorporate and metabolize platelet-activating factor (PAF). We dissociated these events from PAF binding to its receptors. Cells were pretreated with either pronase, a PAF antagonist (L652731), or excess PAF. This reduced PAF receptor numbers by 70 to almost 100% but had no comparable effect upon the neutrophil's ability to metabolize PAF. Furthermore, HL-60 cells efficiently metabolized, but did not specifically bind, PAF. Thus, PAF receptor availability did not correlate with PAF metabolic capacity and we conclude that myelogenous tissues can process this bioactive ligand by a receptor-independent pathway.

Choline-linked phosphoglycerides. A source of phosphatidic acid and diglycerides in stimulated neutrophils.

Stimulation of human polymorphonuclear leukocytes (PMN) may result in the metabolism of phospholipids other than phosphoinositides to generate second-messenger intermediary metabolites. We investigated agonist-induced breakdown of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC), which constitutes almost half the diradyl-GPC fraction in human PMN (Mueller, H. W., O'Flaherty, J. T., Green, D. G., Samuel, M. P., and Wykle, R. L. (1984) J. Lipid Res. 25: 383-388), in cells prelabeled with 1-O-[3H] alkyl-2-acyl-GPC. We also utilized normal-phase high pressure liquid chromatography to quantitate the accumulation of diradylglycerols (1-O-alkyl-2-acylglycerols and diacylglycerols) in stimulated PMN. Phorbol-12-myristate-13-acetate (PMA), 1-oleoyl-2-acetyl-sn-glycerol-, calcium ionophore A23187-, and f-methionyl-leucyl-phenylalanine (fMLP) stimulation of PMN resulted in a time- and concentration-dependent hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid (PA) and 1-O-[3H]alkyl-2-acylglycerol. In all cases formation of 1-O-[3H]alkyl-2-acyl-PA preceded that of 1-O-[3H]alkyl-2-acylglycerol. The times between addition of stimulus and appearance of 1-O-[3H] alkyl-2-acylglycerol varied for PMA (40 s at 1.6 microM), A23187 (5 min at 5 microM), and fMLP (30 sec at 1 microM). Preincubation of cells with 1 microgram/ml pertussis toxin (PT) inhibited the breakdown of 1-O-[3H]alkyl-2-acyl-GPC in cells stimulated with 1 microM fMLP, indicating a role for a PT-sensitive G protein with this stimulus. Quantitation of diglycerides as diradylglycerobenzoates in PMN stimulated with PMA (10 min), A23187 (10 min), or fMLP demonstrated marked accumulation of both 1-O-alkyl-2-acylglycerols and diacylglycerols. The highest increases over controls were observed for fMLP (33-fold for 1-O-alkyl-2-acylglycerols and 17-fold for diacylglycerols). In stimulated PMN prelabeled with 1-O-[3H]hexadecyl-2-acyl-GPC and 1-O-alkyl-2-acyl-sn-glycero-3-[32P]phosphocholine, the ratio of 3H to 32P in 1-O-alkyl-2-acyl-PA compared to 1-O-alkyl-2-acyl-GPC suggested the involvement of a phospholipase D in the hydrolysis of 1-O-[3H]-alkyl-2-acyl-GPC. Thus, stimulation of human PMN results in the hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC to yield 1-O-[3H] alkyl-2-acyl-PA and 1-O-[3H]alkyl-2-acylglycerol possibly initiated by activation of a phospholipase D.(ABSTRACT TRUNCATED AT 250 WORDS)

Reacylation of platelet activating factor with eicosapentaenoic acid in fish-oil-enriched monkey neutrophils.

Platelet activating factor (PAF) is rapidly metabolized via a deacetylation: reacylation pathway which shows striking specificity for arachidonate at the sn-2 position of the 1-O-alkyl-2-acyl-GPC thus formed. We have now examined the effects of a diet enriched in fish oils on the metabolism of PAF and specificity for arachidonate in the reacylation reaction. [3H]PAF was incubated for various lengths of time with neutrophils from monkeys fed a control diet or one enriched in fish oils. The [3H]PAF added to the cell suspension was rapidly converted to 1-O-alkyl-2-acyl-GPC. Reverse-phase HPLC analysis of the acyl chains added at the sn-2 position revealed that arachidonate was the major fatty acid incorporated into the 1-O-alkyl-2-acyl-GPC formed by neutrophils from monkeys on the control diet. In contrast, both 1-O-alkyl-2-arachidonoyl-GPC and 1-O-alkyl-2-eicosapentaenoyl-GPC were formed by the fish-oil-enriched neutrophils. We also report on the fatty acid composition of neutrophil phospholipids during such a diet.

Cyclic adenosine-3',5'-monophosphate alters hydrolysis of phospholipids by mouse embryo palate mesenchyme cells.

The purpose of this study was to determine whether inhibition of release of arachidonic acid from mouse embryo palate mesenchyme (MEPM) cells in response to cAMP is due to a selected or generalized inhibition of hydrolysis of esterified pools of arachidonic acid. The calcium ionophore A23187 proved to be a useful probe of phospholipid hydrolases in MEPM cells, since it stimulated release of radiolabeled fatty acids from phospholipids of prelabeled MEPM cells as a function of the length of exposure, concentration, and concentration of Ca2+ in the medium. Elevation of intracellular levels of cAMP by treatment with (-) isoproterenol resulted in the inhibition of release of radiolabeled arachidonic acid in response to A23187. Analysis by quantitative gas-liquid chromatography revealed the source of the arachidonic acid released in response to the ionophore to be 1,2-diradyl-sn-glycero-3-phosphoethanolamine; elevation of intracellular levels of cAMP inhibited hydrolysis of this substrate, but may have stimulated hydrolysis of 1,2-diradyl-sn-glycero-3-phosphocholine. These findings permit the conclusions that 1) the ionophore stimulates activities of selected phospholipases A in MEPM cells and 2) cAMP modulates certain phospholipases A in MEPM cells in a specific manner.

Metabolism of prostaglandin E2 by mouse embryo palate mesenchyme cells in vitro.

Mouse embryo palate mesenchyme cells synthesize a number of prostaglandins, particularly prostaglandin E2 (PGE2). However, the ability of such cells to metabolize prostaglandins was unknown. By use of radiolabeled PGE2 we determined that palate mesenchyme cells have little ability to degrade that prostaglandin in vitro but are able to metabolize products formed from its spontaneous degradation.

Cyclic AMP inhibits the release of prostaglandins and arachidonic acid from cultures of mouse embryo palate mesenchyme cells.

Mouse embryo palate mesenchyme (MEPM) cells are able to synthesize and respond to prostaglandins. However, mechanisms that regulate their synthesis in these cells are not known. Cyclic adenosine 3',5' monophosphate (cAMP) has been implicated as being involved in differentiation of the palate, accumulates in MEPM cells in response to stimulation with selected prostaglandins, and has been found to modulate synthesis of prostaglandins by other cells and tissues. Therefore, we have investigated whether cAMP modulates synthesis of prostaglandins by MEPM mesenchyme cells and partially characterized the metabolic site at which such modulation occurs. We found that treatment of MEPM cells with various agents to stimulate a seven- to 100-fold increase in intracellular levels of cAMP inhibited release of various prostaglandins by at least 50%. Similarly, elevation of intracellular levels of cAMP inhibited release of radiolabeled arachidonic acid from membrane phospholipids by as much as 27%. The inhibitory effects of cAMP on release of prostaglandins from MEPM cells could be almost completely overcome by the addition of arachidonic acid to the culture medium. We interpret these data to mean that there is a regulatory cycle in MEPM cells in which intracellular levels of cAMP regulates synthesis of prostaglandins and prostaglandins regulate accumulation of cAMP and regulation of synthesis of prostaglandins by cAMP is predominantly through inhibition of a phospholipase.