STUDIES OF THE LEVAMISOLE INHIBITORY EFFECT ON RAT STROMAL-CELL COMMITMENT TO MINERALIZATION

The ability of Levamisole to decrease mineralization in skeletal tissu e is usually related to its effect on alkaline phosphatase (ALP). Howe ver, Levamisole is also suspected to diminish mineralization by an add itional mechanism which is unrelated to the ALP control of apatite cry stal growth. To delineate the time in differentiation during which Lev amisole inhibits mineralization, a tissue culture model system of bone marrow stromal cells was used. Secondary cultures of stromal cells we re propagated in osteoprogenitor cell (OPC) induction medium for three weeks, followed by measurement of calcium precipitation. In situ ALP assays at pH 7.6 were also performed. When cells were cultured with 0. 2 mM Levamisole for three weeks, Day 20 values of calcium precipitates were lower than in controls, but Day 20 ALP values were paradoxically higher. The correlation between calcium and ALP within each group was low. The correlation slightly improved, in uninhibited cultures, when Day 21 calcium values were matched with earlier Day 12 ALP values. Th is suggested the existence of a Levamisole-sensitive mechanism for min eralization inhibition effective prior to the culture's mineralization stage. To focus on this early effect on mineralization Levamisole was added to stromal cultures on different days and removed on Day 12. Le vamisole decreased Day 21 mineralization when added on Days 0, 3, 5, a nd 7, but not when added on Day 9. The Levamisole-induced inhibition o f mineralization was accompanied by an increase in Day 12 ALP specific activity, compared to controls, when added from Day 5 and thereafter. The results indicate that part of the ability of stromal cells to min eralize is determined during the first week of culture. The early inhi bitory effect of Levamisole on mineralization was associated with incr eased Day 12 ALP activity. (C) 1993 Wiley-Liss, Inc.