Effect of Thymosins on Human Osteoblast Osteocalcin Secretion in vitro

Objectives: To determine if thymosin β4 and thymosin β10 can stimulate the secretion of osteocalcin, the major non-collagenous protein of the bone matrix, by human osteoblasts in culture. Methods: Human adipose-derived adult stem cells (Zen-Bio, Research Triangle Park, NC) were cultured in pre-adipocyte medium for one week to approximately 60% confluence. In one experiment, cells were cultured for an additional 24 or 48 hours in serum-free pre-adipocyte medium containing 0, 0.01, 0.1, 1.0, or 10 μg/ml thymosin β4 or thymosin β10 (Phoenix Pharmaceuticals, Burlingame, CA). In addition cells were cultured for the same time in osteoblast differentiation medium. In a second study, stem cells were cultured for one week in osteoblast differentiation medium to promote osteoblast characteristics. These cells were then treated for an additional 24 or 48 hours with thymosin β4 or thymosin β10 as described. Osteocalcin concentrations in the culture medium samples were determined by ELISA (Human osteocalcin instant ELISA, eBioscience, San Diego, CA). Results: Control concentrations of osteocalcin averaged 1.0 ng/ml, compared to the average ostecalcin concentration of 3.0 ng/ml in normal human sera. Thymosin β4 and thymosin β10 had the greatest effect on the secretion of osteocalcin after cells had been in the osteoblast differentiation medium for one week, and then treated with thymosins for 48 hours. In cells treated with 0.1 μg/ml thymosin β10, osteocalcin levels were 1.4 ng/ml compared to 1.1 ng/ml in cells cultured in osteoblast differentiation medium. Cells treated with 1.0 μg/ml thymosin β4 showed an osteocalcin secretion level of 1.2 ng/ml. Conclusions: In this pilot study using adipose-derived human adult stem cells, thymosin β4 and thymosin β10 may have an effect on stimulating the secretion of osteocalcin from osteoblasts. This result has implications for the use of thymosins in stimulating the activity of osteoblasts.