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Effects of Culture Time, Fetal Bovine Serum, Progesterone, Testosterone and Estradiol on Alkaline Phosphatase Activity, Extracellular Matrix Mineralization and Osteoblast Viability
Author(s) -
Lemos José E.S.,
Brancaglião Lucas F.C.,
Bonfá Ana L.O.,
Rocha Nícolas F.,
Gonçalves Vítor M.,
Montrezor Luís H.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.723.6
Subject(s) - endocrinology , medicine , alkaline phosphatase , fetal bovine serum , polycystic ovary , chemistry , osteoblast , viability assay , testosterone (patch) , hormone , extracellular matrix , biology , andrology , in vitro , enzyme , biochemistry , insulin , insulin resistance
Understanding the interactions between ovarian and plasma changes induced by polycystic ovary syndrome (PCOS) and bone metabolism is essential for the comprehension of the pathophysiological mechanisms involving these organs. Ovarian steroids are involved in activities related to bone synthesis and reabsorption. Knowledge of the actions of the main ovarian steroids on the viability of osteoblasts in culture, which mimic the conditions of PCOS, is relevant to a better understanding of the functions of the cells in this endocrine disease that affects more than 10% of women of reproductive age. The goal of this study was to develop a cell culture model mimicking polycystic ovary syndrome that can be used for studies on culture conditions and the effects of steroids progesterone (P4), testosterone (T) and estradiol (E2) on cell viability, alkaline phosphatase activity and extracellular matrix mineralization of osteoblasts. We plated 5 × 10 3 cells in each well of 96‐well culture plates with 100 μL of modified Eagle's medium supplemented or not with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were maintained at 37° C under a humid atmosphere of 5% CO 2 and 95% air for 72 h. P4, T and E2 were used in different concentrations: control (without hormones), 10 −8 M and 10 −7 M mimicking the normal plasma concentrations in adult female rats and 10 −6 M and 10 −5 M, mimicking plasma concentrations in adult female rats with induced PCOS. To this end, each of the steroids were added to the wells of culture both separately and together (all in the same well). Our results show the cell viability of undifferentiated and differentiated osteoblasts in the osteogenic medium, the alkaline phosphatase activity and mineralization of the extracellular matrix in cells cultured for different periods of time and challenged with different concentrations of P4, T, E2, and the combination of all three added to the culture medium. Furthermore, FBS influences the culture conditions. From these results, we propose that the culture system described can be used to study the viability of osteoblasts, the activity of alkaline phosphatase and mineralization of the extracellular matrix in conditions similar to PCOS and, thus, it can be suggested that ovarian steroids, in isolation and in combination, modulate bone formation in vitro . Support or Funding Information CNPq and FUNADESP