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Effects of a Hydroxyapatite-based Biomaterial on Gene Expression in Osteoblast-like Cells

¹ Department of Biochemistry and Molecular Biology and
² Research Center for the Study of Periodontal Diseases, University of Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy

View larger version (26K): [in a new window]   Figure 1. Time- and dose-dependent reduction in proliferation of Biostite®-treated cells. (A) SaOS-2 (open bars) and MG-63 (patterned bars) cell lines were plated at 2 x 106 and 3 x 104/mL, respectively, and, after 24 hrs, exposed to increasing Biostite® concentrations, as described in the METHODS section. After 48 and 72 hrs of treatment, cells were counted. Data represent the mean ± SEM obtained in a triplicate experiment run in duplicate (N = 6). Results indicate a significant dose-dependent decrease in cell counts at 48 hrs (F = 9.5, P > 0.01 for SaOS-2; F = 48.6, P > 0.001 for MG-63) and 72 hrs (F = 27.4, P > 0.001 for SaOS-2; F = 42.7, P > 0.001 for MG-63) for both cell lines. Asterisks over the bars indicate significant differences with respect to baseline (0) values (*P < 0.05; **P < 0.01; ***P < 0.001). Symbols () between bars indicate significant variations between concentrations (P < 0.05; P < 0.01; P < 0.001). (B) MG-63 cells were cultured for 48 hrs in the presence of Biostite® concentrations in powder form (diamonds) and its conditioned medium (triangles). Data represent the mean ± SEM (N = 6). A dose-dependent decrease in cell count is evident for both Biostite® forms. Asterisks indicate significant differences with respect to baseline (0) values (*P < 0.05; **P < 0.01). (C) MG-63 cells (plated at 1 x 104/mL and grown as in A) were cultured in the presence of 500 µg/mL of Biostite® and counted at baseline (0), and after 1, 2, 3, and 6 days of treatment. Solid symbols/bars represent Biostite®-treated cells, open symbols/bars represent untreated control cells. Data represent the mean ± SEM (N = 6). Differences in cell counts were statistically significant between groups at days 1, 2, and 3 of treatment, but not at day 6. Asterisks indicate significant differences between groups at each observation interval (*P < 0.05; **P < 0.01; ***P < 0.001, ANOVA).

View larger version (46K): [in a new window]   Figure 2. Genes differentially expressed in response to Biostite®. MG-63 cells (3 x 104/mL) were seeded in T175 flasks and cultured for 48 hrs with and without Biostite® (500 µg/mL) (n = 3 for treated and control cells). RNA extraction, purification, 32P-labeled cDNA synthesis and hybridization conditions were detailed in the METHODS section and in the APPENDIX. Expressed genes in Cancer (black bars) and Cell interaction (patterned bars) arrays are defined as up-regulated and down-regulated when expression levels showed more than a two-fold increase and reduction, respectively, after treatment, compared with untreated controls. Relative values were averaged for the results obtained in two hybridization experiments (see METHODS/APPENDIX for technical details). The number of genes affected by Biostite® is shown with respect to the expressed genes which were detected in each array (inset).

View larger version (20K): [in a new window]   Figure 3. RT-PCR analysis of Biostite®-modulated genes. Plasminogen activator inhibitor-1 (PAI1), tenascin, and osteonectin RNA levels in MG-63 cells exposed for 48 hrs to 500 µg/mL of Biostite® (+). RT-PCR products were subjected to electrophoresis on 2% agarose gel and visualized by UV exposure. A representative analysis is shown on the left. The level of specific bands was normalized for the ß-actin cDNA content, and the results from three experiments are shown on the right as mean values ± SEM of controls (open bars) and treated cells (patterned bars) (*P < 0.05; **P < 0.01; ***P < 0.001, ANOVA). Similar results have been obtained by normalization with respect to GAPDH cDNA (data not shown).

View larger version (19K): [in a new window]   Figure 4. Biostite® modulates protein levels and differentiation. (A) Western blot analysis of MG-63 cells treated with (+) and without (-) Biostite® (500 µg/mL) for 72 hrs. Total cell lysates (50 µg) were subjected to electrophoresis on 4-12% gradient PAGE and transferred to filters probed with antibodies against polycystin-2, NF-B, BMP-8, and ß-actin. Polycystin-2 expression levels were normalized for the ß-actin levels and expressed as ratios of O.D. arbitrary units. Bars express mean values ± SEM (n = 4). A significant increase in polycystin-2 levels was observed in Biostite®-treated cells (patterned bars), as compared with controls (open bars) (*p < 0.05, ANOVA). (B) ALP activity in MG-63 cells cultured with and without Biostite® (500 µg/mL) for 24 and 72 hrs. ALP activity was determined colorimetrically, corrected for the lysate protein content measured according to Bradford, and expressed as Units/mg protein. Bars express mean values ± SEM of results from three experiments. A significant increase in ALP was observed in Biostite®-treated cells (patterned bars) compared with controls (open bars) (**p < 0.01, ANOVA).