Browsing by Author "Coetzee M"

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    Arachidonic acid and docosahexaenoic acid suppress osteoclast formation and activity in human CD14+ monocytes, in vitro.
    (PUBLIC LIBRARY SCIENCE, 2015) Kasonga AE; Deepak V; Kruger MC; Coetzee M
    An unbalanced diet can have adverse effects on health. Long chain polyunsaturated fatty acids (LCPUFAs) have been the focus of research owing to their necessity of inclusion in a healthy diet. However, the effects of LCPUFAs on human osteoclast formation and function have not been explored before. A human CD14+ monocyte differentiation model was used to elucidate the effects of an ω-3 LCPUFA, docosahexaenoic acid (DHA), and an ω-6 LCPUFA, arachidonic acid (AA), on osteoclast formation and activity. CD14+ monocytes were isolated from peripheral blood of healthy donors and stimulated with macrophage colony stimulating factor and receptor activator of nuclear factor kappa-B ligand to generate osteoclasts. Data from this study revealed that both the LCPUFAs decreased osteoclast formation potential of CD14+ monocytes in a dose-dependent manner when treated at an early stage of differentiation. Moreover, when exposed at a late stage of osteoclast differentiation AA and DHA impaired the bone resorptive potential of mature osteoclasts without affecting osteoclast numbers. AA and DHA abrogated vitronectin receptor expression in differentiating as well as mature osteoclasts. In contrast, the degree of inhibition for calcitonin receptor expression varied between the LCPUFAs with only AA causing inhibition during osteoclast differentiation. Furthermore, AA and DHA down regulated the expression of key osteoclast-specific genes in differentiating as well as mature osteoclasts. This study demonstrates for the first time that LCPUFAs can modulate osteoclast formation and function in a human primary osteoclast cell line.
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    Effects of omega 3 and omega 6-polyunsaturated fatty acids on RANKL-induced osteoclast differentiation of RAW264.7 cells: A comparative in vitro study
    (MDPI, 10/07/2014) Boeyens JCA; Deepak V; Chua W-H; Kruger MC; Joubert A; Coetzee M
    Polyunsaturated fatty acids (PUFAs) have been reported to have an anabolic effect on bone in vivo, but comparative studies to identify inhibitors of osteoclast formation amongst ω3- and ω6-PUFAs are still lacking. Here we assessed the effects of the ω3-PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the ω6-PUFAs, arachidonic acid (AA) and γ-linolenic acid (GLA) on a RAW264.7 osteoclast differentiation model. The effects of PUFAs on RANKL-induced osteoclast formation were evaluated by counting tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells. PUFAs significantly inhibited RANKL-induced osteoclast formation in a dose-dependent manner with AA- and DHA-mediated inhibition being the strongest. Furthermore, RANKL-induced mRNA- and protein expression of the key osteoclastogenic genes cathepsin K and TRAP were inhibited by AA and more potently by DHA. Owing to the attenuated osteoclastogenesis by DHA and AA, actin ring formation and bone resorptive activity of these cells as evaluated on bone-mimetic plates were severely compromised. Hence, of the tested PUFAs, AA and DHA were found to be the most effective in inhibiting RANKL-induced osteoclast formation with the latter providing the strongest inhibitory effects. Collectively, the data indicates that these PUFAs may play an important role in regulating bone diseases characterized by excessive osteoclast activity.
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    Inhibisie van osteoklastvorming en beenresorpsie deur poli-onversadigde vetsure in RAW 264.7 muismonosiete
    (AOSIS Ltd, 2012) Boeyens JCA; Chua WH; Kruger M; Joubert AM; Coetzee M
    Inhibition of osteoclast differentiation and bone resorption by polyunsaturated fatty acids in RAW 264.7 murine macrophages. This study investigated the effects of polyunsaturated fatty acids on osteoclast formation and bone resorption in RAW 264.7 murine pre-osteoclasts. Data obtained suggests an inhibitory effect of these compounds on osteoclastogenesis and bone resorption in the cell line tested. Beenhermodellering in volwassenes is ‘n fisiologiese proses wat die sintese van beenmatriks deur osteoblaste en die resorpsie (afbraak) van been deur osteoklaste behels. Osteoklaste ontstaan deur die samesmelting van hematopoïetiese selle van monosiet-makrofaagafkoms en speel ‘n deurslaggewende rol in beenhermodellering. Osteoklast-ooraktiwiteit kan die afbraak van been in verskeie patologiese toestande tot gevolg hê. Kliniese- en dierestudies het aangedui dat sommige poli-onversadigde vetsure ‘n voordelige effek op been kan hê. Die doel van hierdie proefstudie was om te bepaal of omega-3 en omega-6 poli-onversadigde vetsure osteoklastvorming vanaf RAW 264.7 monosiete moduleer en daardeur die aantal volwasse resorberende osteoklaste kan beïnvloed. Monosiet/makrofaag-muisselle (RAW 264.7 pre-osteoklaste) is teen 1.5x104 selle/putjie in steriele 24-put plaatjies in die aanwesigheid van 15 ng/ml muisreseptor-aktiveerder van NFκBligand (RANKL) gesaai. RANKL is noodsaaklik vir osteoklastvorming vanaf voorgangerselle. Etanol (oplosmiddelkontrole), die omega-6 poli-onversadigde vetsure aragidoonsuur en gammalinoleensuur asook die omega-3 poli-onversadigde vetsure eikosapentaënöesuur en dokosaheksaënöesuur is by die selkulture teen konsentrasies van 5–20 μg/ml gevoeg. Palmitiensuur, ‘n versadigde vetsuur, teen ‘n konsentrasie van 20 μg/ml is as positiewe kontrole vir inhibisie van osteoklastvorming gebruik. Na vyf dae inkubasie is osteoklastvorming geëvalueer deur van tartraat-weerstandigesuurfosfatase (TRAP)-kleuring gebruik te maak. TRAP-positiewe selle met vyf of meer kerne word as veelkernige osteoklaste beskou. Soortgelyke eksperimente is uitgevoer op plaatjies wat bedek is met ‘n sintetiese anorganiese beenoppervlak. Na sewe dae inkubasie is die selle afgewas en resorpsie van die oppervlak met behulp van mikroskoopfoto’s waargeneem. Die persentasie resorpsie-oppervlak is daarna met behulp van toepaslike rekenaarsagteware bepaal. Verder is RAW 264.7 selle op beenskyfies gesaai om die effek van poli-onversadigde vetsure op die degradering van organiese en anorganiese beenkomponente te evalueer. Na nege dae is die gekondisioneerde media afgetrek en die hoeveelheid Ca2+ en kollageenfragmente, beendegraderingsprodukte onderskeidelik afkomstig vanaf die anorganiese en organiese komponente van beenafbraak, deur gepaste kolorimetriese metodes bepaal. Resultate van hierdie studie het getoon dat die vorming van veelkernige osteoklaste deur die blootstelling aan poli-onversadigde vetsure by konsentrasies van 5–20 μg/ml geïnhibeer word. Blootstelling aan al die vetsure het tot inhibering van osteoklastvorming gelei met die grootste effek by die hoogste vetsuurkonsentrasies. Dokosaheksaënöesuur (omega-3) het die mees betekenisvolle inhiberende effek oor al die konsentrasies getoon en eikosapentaënöesuur die minste. Resorpsieholtes op die gesimuleerde beenplaatjies was kleiner waar die selle aan aragidoonsuur en dokosaheksaënöesuur blootgestel was in vergelyking met dié van die oplosmiddelkontrole, wat moontlik aan die laer voorkoms van volwasse veelkernige osteoklaste toegeskryf kan word. ‘n Afname in die beendegraderingsprodukte is ook waargeneem waar selle blootgestel is aan aragidoonsuur en dokosaheksaënöesuur in vergelyking met die oplosmiddelkontrole. Die resultate ondersteun die vermoede dat poli-onversadigde vetsure die vorming van volwasse osteoklaste inhibeer en daardeur ‘n beenbeskermende effek tot gevolg mag hê. Verdere navorsing is nodig om duidelikheid oor die meganismes wat hier betrokke is, te verkry. Hierdie navorsing is deur die Mediese Navorsingsraad en die Navorsings-ontwikkelingsprogram (Universiteit van Pretoria) befonds.