Effects of MTX alone or with supplementary treatment with (FO) and/or genistein (Gen) on osteogenic differentiation potential of bone marrow stromal cells isolated from treated rats.

<p>Images of a culture well showing bone marrow stromal cell-derived CFU-f colonies stained positive for alkaline phosphatase (ALP, arrows) of (<b>A</b>) a control rat and (<b>B</b>) a MTX alone treated rat on day 9 post the first MTX injection. (<b>C</b>) Treatment effects on size of osteoprogenitor cell pool in bone marrow. Mineralizing colonies stained positive by Alizarin Red (arrows) of (<b>D</b>) a control rat and (<b>E</b>) a MTX alone treated rat. (<b>F</b>) <i>Ex vivo</i> mineralization assay with bone marrow cells isolated from rats. RT-PCR relative mRNA expression of (<b>G</b>) Runx2 and of (<b>H</b>) bone matrix protein osteocalcin (OCN) assessed in bone marrow stromal cells of treated rats (relative to Cyclophilin-A). Labelled means without a common letter differ (P<0.05).</p

Effects of MTX with or without (FO) and/or genistein (Gen) supplementation on expression of anti-inflammatory cytokines IL-4 and IL-10.

<p>Levels of mRNA expression in metaphysis bones of treated rats as quantitated by real time RT-PCR: (<b>A</b>) IL-4 and (<b>B</b>) IL-10. Protein levels (pg/mL) of circulating (<b>C</b>) IL-4 and (<b>D</b>) IL-10 in plasma samples of treated rats as measured by multiplex cytokine assay. Labelled means without a common letter differ (P<0.05).</p

Effects of MTX with or without (FO) and/or genistein (Gen) supplementation on osteoclastogenesis potential.

<p>Images of TRAP-stained tibial metaphysis (arrows pointing multinucleated TRAP-positive osteoclasts) of (<b>A</b>) a control rat and (<b>B</b>) a MTX alone treated rat on day 9 post the first MTX injection showing more osteoclasts present. (<b>C</b>) Average osteoclast numbers at tibial primary and secondary spongiosa. Images of TRAP positively-stained cells formed (arrows pointing multinucleated TRAP-positive osteoclast-like cells) in an <i>ex vivo</i> osteoclastogenesis assay of (<b>D</b>) a control rat and (<b>E</b>) a MTX alone treated rat on day 9 post the first MTX injection showing more osteoclasts formed. (<b>F</b>) <i>Ex vivo</i> osteoclast formation from bone marrow cells isolated from treated rats. Labelled means without a common letter differ (P<0.05).</p

Effects of MTX with or without (FO) and/or genistein (Gen) supplementation on expression of osteoclastogenesis-regulatory or related genes.

<p>Levels of mRNA expression in metaphysis bones of treated rats as quantitated by real time RT-PCR: (<b>A</b>) RANKL/OPG ratio, (<b>B</b>) TNF-α, and (<b>C</b>) IL-6. (<b>D</b>) Levels (pg/mL) of circulating IL-6 protein in plasma samples of treated rats as measured by multiplex cytokine assay. Labelled means without a common letter differ (P<0.05).</p

List of primers used in RT-PCR.

<p>List of primers used in RT-PCR.</p

Effects of MTX with or without fish oil (FO) and/or genistein (Gen) supplementation on growth plate, primary and secondary spongiosa.

<p>Paraffin sections of the tibial metaphysis region (PS =  Primary spongiosa, SS =  Secondary spongiosa, which are separated by a dashed line) of (<b>A</b>) a normal rat, (<b>B</b>) a MTX+H₂O treated rat showing reduced height of primary spongiosa and. metaphyseal bone volume, (<b>C</b>) a MTX+FO treated rat, and (<b>D</b>) a MTX+Gen treated rat. (<b>E</b>) Growth plate total height (µm). (<b>F</b>) Primary spongiosa height (µm). (<b>G</b>) Secondary spongiosa BV/TV (%). (<b>H</b>) Secondary spongiosa osteoblast/mm² trabecular bone area. Values are means ± SEM; n = 7–8 for all groups. Labelled means without a common letter differ (P<0.05).</p