Figure 1.
Investigating the effect of the individual components of the osteogenic cocktail on 4T1 cell mineralization.
Alizarin red S and von Kossa staining are positive for calcium (red) and calcium phosphate (black/brown) respectively in the OC, OC&dex and βG groups on day 14. No positive staining was observed in the control, AA or dex groups. Representative images were taken at 100× magnification (n = 3) and the scale bar represents 500 µm. Control = regular growth media. AA = 50 µg/ml ascorbic acid. βG = 10 mM β-glycerophosphate. OC (osteogenic cocktail) = 50 µg/ml AA and 10 mM βG. Dex = 100 nM dexamethasone.
Figure 2.
Investigating the effect of increasing concentrations of β-glycerophosphate on 4T1 cell mineralization.
Representative images were captured at 100× magnification and the scale bars represent 500 µm. (A) Positive alizarin red S staining for calcium (red) was observed in the OC, 5 mM βG and 10 mM βG treated groups, beginning on days 14, 28 and 14 respectively. (B) Positive von Kossa staining for calcium phosphate (black/brown) was observed in the OC, 5 mM βG and 10 mM βG treated groups on day 28. (C) The calcium content of 4T1 cells as determined by the o-cresolphthalein calcium assay and normalized to protein. Increases in calcium were observed in the OC and 10 mM βG treated groups over time. Each point represents the mean amount of calcium measured in ppm normalized to protein measured in mg, +/− SEM, n = 3, two-way ANOVA. *P<0.05, ***P<0.001 vs. control at each time point. OC (osteogenic cocktail) = 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate. βG = β-glycerophosphate.
Figure 3.
Investigating the effect of increasing concentrations of inorganic phosphate on 4T1 cell mineralization.
Representative images were captured at 100× magnification and the scale bars represent 500 µm. (A) Positive alizarin red S staining for calcium (red) was observed in the OC treated group by day 14 and on day 7 for the AA&10 mM Pi, 5 mM Pi and 10 mM Pi treated groups. (B) Positive von Kossa staining for calcium phosphate (black/brown) was observed in the OC, AA&10 mM Pi, 5 mM Pi and 10 mM Pi on day 28. (C) The calcium content of 4T1 cells as determined by the o-cresolphthalein calcium assay normalized to protein. Increases in calcium were observed in the OC, AA&10 mM Pi, 5 mM Pi and 10 mM Pi groups over time, with the greatest increase detected in the 10 mM Pi group. Each point represents the mean amount of calcium measured in ppm normalized to protein measured in mg, +/− SEM, n = 3, two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001 vs. control at each time point. OC (osteogenic cocktail) = 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate. AA = 50 µg/ml ascorbic acid. Pi = inorganic phosphate.
Figure 4.
The effect of exogenous BMP2 on 4T1 cell mineralization.
Representative images were captured at 100× magnification and the scale bars represent 500 µm. Positive staining for calcium (red) and calcium phosphate (black/brown) were observed in the OC&BMP2 group by day 7 and increased in intensity by day 14, as shown by alizarin red S (A) and von Kossa (B) staining. Positive staining was detected in the OC group by day 14 using both alizarin red S and von Kossa stains. (C) The calcium content of 4T1 cells as determined by the o-cresolphthalein calcium assay normalized to protein. By day 14 the calcium levels of the OC&BMP2 group are 30-fold greater than the OC group. Each point represents the mean amount of calcium measured in ppm normalized to protein measured in mg, +/− SEM, n = 3, two-way ANOVA. **P<0.01 OC vs. control on day 14. ***P<0.001 OC&BMP2 vs. all other groups on days 7 and 14. OC (osteogenic cocktail) = 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate. BMP2 = 100 ng/ml human recombinant bone morphogenetic protein 2.
Figure 5.
Investigating the expression of several bone markers in mineralizing 4T1 cells using real-time RT-PCR.
(A) Alizarin red S and von Kossa staining of 4T1 cells after 28 days as viewed under a light microscope at 100× magnification (n = 3). The scale bar represents 500 µm. Positive staining was observed for calcium (red) and calcium phosphate (black/brown) using alizarin red S and von Kossa staining respectively, in the OC group and to a lesser extent in the OC&dex group. (B) There is a decrease in col1a1 (collagen type 1, alpha 1) mRNA expression in the OC&dex group compared to the control group from days 4–28. In contrast, there is increased expression of col1a1 in the OC group on days 11, 21 and 28. *P<0.05 OC vs. control on day 11. **P<0.01 OC&dex vs. control on days 11, 21 and 28. ***P<0.001 OC vs. control on days 21 and 28, also OC&dex vs. control on days 4, 7 and 14. (C) On day 21 there is an increase in the expression of bone sialoprotein (BSP) mRNA in the OC group compared to the control and OC&dex groups. ***P<0.001 OC vs. control and OC&dex groups on day 21. (D) No changes in the expression of Runx2 mRNA were detected between the different treatment groups over time. All real-time RT-PCR results are expressed in arbitrary units and normalized to the control samples at each time point. Each point represents the mean +/− SEM, n = 3, two-way ANOVA. OC (osteogenic cocktail) = 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate. Dex = 100 nM dexamethasone.
Figure 6.
Assessing mineralization of 4T1 cells grown in an osteogenic cocktail within 3D collagen scaffolds.
Representative images are shown at 400× and 40× magnifications and the scale bars represent 200 µm and 2000 µm respectively. (A) Positive hematoxylin and eosin (H&E) staining was observed at 400× magnification by day 14 and the intensity of the hematoxylin stain increased by day 28. Some positive alizarin red S staining for calcium (red) was detected by day 14. Complete positive staining with alizarin red S and von Kossa (including toluidine blue counterstain) was observed by day 28. (B) At 40× magnification the previously described patterns of staining are confirmed and minor contraction and disintegration of the scaffolds are observed over time. (C) Assessing cell viability of 4T1 cells grown within the 3D scaffolds using an alamar blue metabolic assay. An increase in cell viability was detected between day 14 and day 28. Each time point represent the mean % reduction in alamar blue +/− SEM, n = 3, students t-test. *P = 0.0284 day 28 vs. day 14. OC (osteogenic cocktail) = with 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate.