22 research outputs found

    Osteogenic induction: Alizarin Red S staining and Osteogenic Biomarker expression.

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    <p>Representative photomicrographs of 24-well plate wells stained with Alizarin Red S after (A) two weeks or (B) one week of treatment with osteogenic medium (OM) based on Fetal Bovine Serum (FBS) or Platelet Lysate (PL). Parallel samples cultured in maintenance medium (MM) lacking osteogenic factors served as controls for spontaneous differentiation. Gene upregulation of 12 osteogenic biomarker genes in cGMP-hBM-MSC populations derived from six donors was measured after treatment with OM-FBS (hatched columns) or OM-PL (plain columns) for (C) two weeks or (D) one week. Measurements from triplicate determinations were all statistically significant (p < 0.05).</p

    Inter-donor heterogeneity for osteogenic biomarker expression.

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    <p>The level of named gene upregulation in response to osteogenic medium treatment for (A) two weeks (2W-OM) or (B) one week (1W-OM) determined from triplicate measurements was tested to ensure statistical significance (*p < 0.05), error bars indicating standard error of the mean.</p

    Biomarker characterisation of ALZ<sup>+</sup>, VK<sup>+</sup>, <i>MKI67</i><sup>+</sup>, BF<sup>+</sup> and BF<sup>-</sup> cell populations.

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    <p>Histograms of the average extent of osteogenic biomarker gene upregulation in cGMP-hBM-MSC derived from (A) donors #1, #2, #3, #4 with cells positive for Alizarin red S (ALZ<sup>+</sup>) when treated with OM-PL for two weeks (red column) or one week (light column). (B) donors #1, #2, #3 with cells positive for Von Kossa staining (VK<sup>+</sup>) when treated with OM-PL for two weeks (black column) or one week (light column). (C) donors #1, #3, #4 with significant <i>MKI67</i><sup>+</sup> upregulation when cells were treated with OM-PL for two weeks (grey column) or one week (light column). (D) donors #1, #2, #3, #6 with good bone formation (BF<sup>+</sup>) when cells were treated with OM-PL for two weeks (pink column) or one week (light column). (E) Venn diagrams show the relation between osteogenic function and significantly upregulated biomarkers after (left hand side) OM-PL treatment for two weeks (2W) or (right hand side) OM-PL treatment for one week. (1W). (F) donors #4, #5, incapable of good bone formation (BF<sup>-</sup>) when cells were treated with OM-PL for two weeks (purple column) or one week (light column). Error bars indicate S.D. of means. (*) Constituent mean values were statistically significant (p<0.05).</p

    Inter-donor heterogeneity for bone formation <i>in-vivo</i>.

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    <p>Photomicrographs of H&E stained sections of decalcified paraffin-embedded Xenografts under bright field illumination. The Xenografts consisted of hydroxyapatite ß-tricalcium scaffold granules seeded with cGMP-hBM-MSC derived from (A) donors #1 to donor #6 respectively. Regions adjacent to the scaffold (s) contained newly formed osteoid bone (b) stained more homogeneously pink relative to the surrounding fibrous tissue (ft) and contained numerous osteocytes within lacunae (arrows). A representative section of the control implant of hydroxyapatite ß-tricalcium scaffold granules without cells revealed scaffold (s) and fibrous tissue (ft) only. (B) Histogram of the histological section area governed by scaffold (grey column), stromal fibrous tissue (purple column) or bone osteoid matrix (pink column) showing significant bone formation (§p<0.05). Donor heterogeneity with regard to the relative amount of bone formed showed statistically significant differences (*p < 0.05). Scale bar = 100 μm.</p

    Downregulation of type I collagen restored ex-vivo matrix mineralization.

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    <p>(A) Histogram of RT-PCR determined <i>COL1A2</i> mRNA downregulation following treatment of donor #6 cells with Interferon gamma (IFN-Îł) or TGF-Ăź1 signaling inhibitor SB431542 for three days before treatment with osteogenic medium for one or two weeks. *p <0.05, **p <0.005. (B) Representative photomicrographs of Alizarin Red S staining after two-week treatment of cells with maintenance medium (MM) or osteogenic medium (OM) using donor #1 cells as a positive control compared to donor #6 cells and donor #6 cells pre-treated with MM supplemented with 40U/mL INF-Îł or 2 ÎĽM SB431542 for three days. Bar = 100 ÎĽM.</p

    Inter-donor heterogeneity for OM-PL-induced ALZ<sup>+</sup>, VK<sup>+</sup>, <i>MKI67</i><sup>+</sup> <i>ex-vivo</i> phenotypes.

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    <p>After two weeks of osteogenic induction in OM-PL, the hBM-MSCs were stained for matrix mineralization. (A) Representative photomicrographs (10X) of donor-specific cGMP-hBM-MSC populations positive for Alizarin red S stain (ALZ+) and histogram of eluted dye staining intensity measurement at 562 nm (*p < 0.05). (B) Representative photomicrographs (10X) of donor-specific cGMP-hBM-MSC populations positive for Von Kossa stain (VK+) and histogram of positively stained area quantified using Image J software (*p < 0.05). (C) Histogram showing the extent of gene upregulation of <i>MKI67</i> determined after induction with OM-PL for two weeks (dark columns) or one week (light columns). Measurements from triplicate determinations that were statistically significant are indicated (*p < 0.05).</p

    <i>Ex-vivo</i> osteogenic signature gene expression could cluster donor-specific cGMP-hBM-MSC populations according to bone forming potential.

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    <p>The gene upregulation profiles of the six donor-specific populations induced for osteogenic differentiation with 1W-OM-PL were subjected to cluster analysis: (A) The dendrogram derived using all twelve osteogenic biomarker genes without prior selection for significance in osteogenic function led to (B) a plot of correlation coefficients between donors and their bone forming potential which revealed no significant relationship (r<sup>2</sup> = 0.104, p = 0.596). (C) Dendrogram from cluster analysis restricted to the five osteogenic signature genes. Using a Euclidian distance, single linkage algorithm the dendogram indicated closest similarity between bone-forming donors #1 and #2. (D) The plot of correlation coefficients versus bone-forming potential suggested that most associations appeared to constitute a regression slope (closed circles), but there was an outlier association between donors #4 and #6 (open circle) preventing overall correlation (r<sup>2</sup> = 0.225, p = 0.420). The above cluster analysis was repeated but with modification to accommodate outlying observations. (E) Dendrogram from cluster analysis excluding data from outlying donor #6, restricted to the five osteogenic signature genes, using a Pearson centroid linkage and uncentered correlation distance. (F) The resulting plot of donor correlation coefficients versus bone forming potential confirmed a presumed regression line relationship between bone-forming potential and gene expression for cells from the five congruent donors (r<sup>2</sup> = 0.996, p = 0.0169). (G) The dendrogram resulting from a Euclidian distance, single linkage algorithm with donor#6 <i>COL1A2</i> induction adjusted to 6.63-fold enhanced similarity between bone-forming donors. (H) The corresponding plot of donor correlation coefficients versus bone-forming potential showed a strong linear relationship between <i>ex vivo</i> gene expression and <i>in vivo</i> bone-forming potential in cells from all donors (r<sup>2</sup> = 0.948, p = 0.0051) with closely clustered bone-forming donors #1, #2, #3, #6 (red box).</p
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