Estrogen and phenol red free medium for osteoblast culture: study of the mineralization ability

Abstract

To design an estrogen and phenol red free medium for cell culture and check its effectiveness and safety on osteoblast growth it is necessary to maintain the estrogen receptors free for tests. For this purpose, we tested some modifications of the traditional culture media: estrogen depleted fetal bovine serum; estrogen charcoal stripped fetal bovine serum and phenol red free α-MEM. The aim of this work is to examine the effects of its depletion in the proliferation, differentiation, and toxicity of mesenchymal stromal cells differentiated into osteoblasts to obtain an effective interference free culture medium for in vitro studies, focused on non-previously studied estrogen receptors. We performed viability tests using the following techniques: MTT, alkaline phosphatase specific activity, formation of mineralized matrix by Alizarin technique and analysis of SEM/EDX of mineralized nodules. The results showed that the culture media with estrogen free α-MEM + phenol red free α-MEM did not impact viability, alkaline phosphatase activity and mineralization of the osteoblasts culture compared to control. In addition, its nodules possess Ca/P ratio similar to hydroxyapatite nodules on the 14th and 21st day. In conclusion, the modified culture medium with phenol red free α-MEM with estrogen depleted fetal bovine serum can be safely used in experiments where the estrogen receptors need to be free.

Keywords: Estrogen depleted; Fetal bovine serum; Osteoblasts; Phenol red free; α-MEM.

Fig. 1

Cell viability of osteoblasts cultures incubated during 1, 7 and 14 days with Ct (α-MEM + FBS); α-MEM + ES FBS; α-MEM + CS FBS; PRF α-MEM + FBS; PRF α-MEM + ES FBS and PRF α-MEM + CS FBS. Osteoblasts (2 × 10⁴ cells/well) were cultured in 24-well plates, and the results were expressed as percentage of the average of three experiments performed in triplicate compared to control. *p value < 0.05 versus control. The error bars represent the standard deviation of the data set

Fig. 2

Levels of alkaline phosphatase activity in osteoblast cultures after 7, 14 and 21 days of incubation with Ct (α-MEM + FBS); α-MEM + ES FBS; α-MEM + CS FBS; PRF α-MEM + FBS; PRF α-MEM + ES FBS and PRF α-MEM + CS FBS. Osteoblasts (2 × 10⁴ cells/well) were cultured in 24-well plates, and the results were expressed as percentage of the average of three experiments performed in triplicate compared to control. *p value < 0.05 versus control. The error bars represent the standard deviation of the data set

Fig. 3

Formation of mineralized matrix in osteoblast cultures after 14 and 21 days of incubation with Ct (α-MEM + FBS); α-MEM + ES FBS; α-MEM + CS FBS; PRF α-MEM + FBS; PRF α-MEM + ES FBS and PRF α-MEM + CS FBS. Osteoblasts (2 × 10⁴ cells/well) were cultured in 24-well plates, and the results were expressed as Alizarin Red absorbances’ average at 405 nm (a.u.) of three experiments performed in triplicate compared to control. *p value < 0.05 versus control. The error bars represent the standard deviation of the data set

Fig. 4

SEM images at the magnification of 1000× of mineralized surfaces after 14 (a–f) and 21 (g–I) days of incubation with Ct (α-MEM + FBS) (Fig. 4a, g, respectively); α-MEM + ES FBS (Fig. 4b, h, respectively); α-MEM + CS FBS (Fig. 4c, i, respectively); PRF α-MEM + FBS (Fig. 4d, j, respectively); PRF α-MEM + ES FBS (Fig. 4e, k, respectively) and PRF α-MEM + CS FBS (Fig. 4f, l, respectively). Osteoblasts (2 × 10⁴ cells/well) were cultured in 24-well plates

Fig. 5

SEM images at the magnification of 3000× of mineralized surfaces after 14 (a–f) and 21 (g–I) days of incubation with Ct (α-MEM + FBS) (Fig. 5a, g, respectively); α-MEM + ES FBS (Fig. 5b, h, respectively); α-MEM + CS FBS (Fig. 5c, i, respectively); PRF α-MEM + FBS (Fig. 5d, j, respectively); PRF α-MEM + ES FBS (Fig. 5e, k, respectively) and PRF α-MEM + CS FBS (Fig. 5f, l, respectively). Osteoblasts (2 × 10⁴ cells/well) were cultured in 24-well plates