THE EFFECT OF METHOTREXATE ON MOUSE BONE-CELLS IN CULTURE

Objective. We have recently shown that administration of long-term, lo w-dose methotrexate (MTX) causes severe osteopenia in female rats. Thi s osteopenia is characterized both by decreased osteoblast function wi thout a decrease in osteoblast numbers, and by increased bone resorpti on that is believed to represent a physiologic remodeling response by osteoclasts. The present study investigates the effects of varying dos es of MTX on mouse bone cells in culture. Methods. Cells were obtained by sequential digestion of neonatal mouse calvariae, and cultured wit h fetal calf serum (10% for osteoblast-like cells and 2% for osteoclas t-like cells). After 1 week, MTX was added to each culture in concentr ations of 0.6 mu M, 0.4 mu M, 0.2 mu M, 0.1 mu M, 1 nM, and 0.5 nM. Al l experiments were done on 24 wells for each MTX concentration and for the controls. The effect on osteoblastic cells was assessed, at 7 day s, by cell counts and by measurement of lysate alkaline phosphatase an d supernatant osteocalcin levels, and, at 21 days, by analysis of the calcified matrix production, which was cultured with ascorbic acid and beta-glycerophosphate. For osteoclastic cells, cell count and lysate acid phosphatase levels were determined. Results. Levels of osteoblast ic cells and lysate alkaline phosphatase were not changed by any of th e concentrations of MTX. Matrix calcification and supernatant osteocal cin levels were diminished by MTX in a dose-responsive manner. Osteocl ast-like cell numbers and acid phosphatase levels were not significant ly affected by MTX. Conclusion. These results suggest that diminished mouse osteoblastic cell function occurs with very low mean concentrati ons of MTX, in a dose-responsive manner. The mechanism seems to be ina bility of the cell to synthesize and calcify matrix, possibly through defective osteocalcin production. Thus, low-dose MTX may have an impor tant impact on bone density by slowing osteoblastic matrix production.