OSTEOBLASTIC PHENOTYPE OF RAT MARROW STROMAL CELLS CULTURED IN THE PRESENCE OF DEXAMETHASONE, BETA-GLYCEROLPHOSPHATE, AND L-ASCORBIC-ACID

We investigated the effects of the time course of addition of osteogen ic supplements dexamethasone, beta-glycerolphosphate, and L-ascorbic a cid to rat marrow stromal cells, and the exposure time on the prolifer ation and differentiation of the cells. It was the goal of these exper iments to determine the time point for supplement addition to optimize marrow stromal cell proliferation and osteoblastic differentiation. T o determine this, two studies were performed; one study was based on t he age of the cells from harvest, and the other study was based on the duration of exposure to supplemented medium. Cells were seen to proli ferate rapidly at early time points in the presence and absence of ost eogenic supplements as determined by H-3-thymidine incorporation into the DNA of replicating cells. These results were supported by cell cou nts ascertained through total DNA analysis. Alkaline phosphatase (ALP) activity and osteocalcin production at 21 days were highest for both experimental designs when the cells were exposed to supplemented mediu m immediately upon harvest. The ALP levels at 21 days were six times g reater for cells maintained in supplements throughout than for control cells cultured in the absence of supplements for both studies, reachi ng an absolute value of 75 x 10(-7) mu mole/min/cell. Osteocalcin prod uction reached 20 x 10(-6) ng/cell at 21 days in both studies for cell s maintained in supplemented medium throughout the study, whereas the control cells produced an insignificant amount of osteocalcin. These r esults suggest that the addition of osteogenic supplements to marrow-d erived cells early in the culture period did not inhibit proliferation and greatly enhanced the osteoblastic phenotype of cells in a rat mod el. (C) 1998 Wiley-Liss, Inc.