Effect of Dexamethasone and 1,25(OH)2D3 on Proliferation and Osteogenic Differentiation of Cultured Human Bone Marrow Stromal Cells / 대한내분비학회지

BACKGROUND: It is crucial, in the case of regenerating bone by tissue-engineering technique, that osteoblast progenitors are proliferated and induced to differentiate to osteoblasts sequentially at the proper time. Osteoblasts can be obtained from bone itself or from osteoblast progenitors in bone marrow, even though the amount of human marrow stromal cells in marrow aspirate is usually scanty. These cells, however, have been known demonstrate the potential to easily proliferate and differentiate in osteoblasts, chondroblasts or adipocytes according to different microenvironmental factors. We evaluated the effect of dexamethasone and 1,25(OH)2D3 on the proliferation, differentiation, and mineralization of human marrow stromal cells in vitro. METHODS: We used twelve bone marrow aspirates obtained from different healthy bone marrow donors. Culture plates were randomly divided into the following four experimental groups; group 1 was cultured with control medium only, group 2 with control medium containing 1,25(OH)2D3, group 3 with control medium containing dexamethasone, and group 4 with control medium containing both 1,25(OH)2D3 and dexamethasone. 3H-thymidine uptake, protein content of cell lysates, alkaline phosphatase activities and alkaline phosphatase histochemistries were measured. Alizarin Red-S staining and quantification of dissolved dye were also performed. RESULTS: Combined stimulation of marrow stromal cells with both 1,25(OH)2D3 and dexamethasone was found to be effective to maintain stable long-term culture of the cells and to increased differentiation and mineralization of the cells. Synthesis and mineralization of matrix were highest when the cells were stimulated with 1,25(OH)2D3 alone during the early culture phase. However, 1,25(OH)2D3 shortened the lifespan of the cells. Interestingly, mineralization was higher in female donor cells than in male donor cells when stimulated with dexamethasone alone or with both dexamethasone and 1,25(OH)2D3. Neither 1,25(OH)2D3 nor dexamethasone affected cell proliferation. CONCLUSION: Our results suggest that the synergistic effect of dexamethasone and 1,25(OH)2D3 is important in maintaining long-term culture and differentiation of human marrow stromal cells. It is preferable to administer 1,25(OH)2D3 after the attachment of cultured osteoblasts to biomaterials has been established, since it could shorten cell survival despite the great increase of mineralization at the early culture phase.

Interaction of TGF-beta1 and rhBMP-2 on Human Bone Marrow Stromal Cells Cultured in Collagen Gel Matrix

Transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) are abundant proteins in the bone matrix. However, their interaction in controlling osteoblast differentiation is not clearly understood. In this study, HBMSCs were cultured in collagen gel matrix with different condition of exogenous rhBMP-2 and TGF-beta1 in order to determine the interaction of BMP-2 and TGF-beta1 on human bone marrow stromal cells (HBMSCs) differentiation. The cultured cells were analyzed for cell proliferation, alkaline phophatase (ALP) activity and mineralization stainning with Von-Kossa. The cells treated with TGF-beta1 exhibited a higher rate of cell growth than those without. However, the cells cultured in collagen gel matrix showed a lower rate of cell growth than the cells cultured in a monolayer. To investigate the effects of both cytokines on osteoblast differentiation, the cells were treated with 0, 1, 5, 10 ng/ml of TGF-beta1 for 2 days. This was followed by culturing with 0, 1, 5, and 10 ng/ml of TGF-beta1 and 100 ng/ml of rhBMP-2 together for 3 days with the alkaline phosphatase (ALP) activity measured. The cells treated with 1 ng/ml of TGF-beta1 responded efficiently to rhBMP-2 and expressed ALP activity with a level equivalent to that exhibited by cells that were not treated with TGF-beta1. The cells treated with 5 and 10 ng/ml of TGF-beta1 showed a dramatic decrease in ALP activity. The cells treated with 10ng/ml of TGF-beta1 followed by rhBMP-2 alone exhibited an intermediate ALP activity. The cells treated with 100 ng/ml of rhBMP-2 demonstrated Von-Kossa positive solid deposits after 3 weeks, while there were few Von-Kossa positive solid deposits when the cells treated with 10 ng/ml of TGF-beta1. These results show that TGF-beta1 inhibits the effects of rhBMP-2 on the osteoblast differentiation of HBMSCs in a dose dependant manner. Furthermore, the effects of TGF-beta1 on HBMSCs are reversible. This suggest that TGF-beta1 and rhBMP-2 are coordinately controlled during the osteoblast differentiation of HMBSCs.