The effect of methotrexate on mouse bone cells in culture

Abstract

Objective. We have recently shown that administration of long-term, lowdose methotrexate (MTX) causes severe osteopenia in female rats. This osteopenia is characterized both by decreased osteoblast function without a decrease in osteoblast numbers, and by increased bone resorption that is believed to represent a physiologic remodeling response by osteoclasts. The present study investigates the effects of varying doses of MTX on mouse bone cells in culture.Methods. Cells were obtained by sequential digestion of neonatal mouse calvariae, and cultured with fetal calf serum (10% for osteoblast-like cells and 2% for osteoclast-like cells). After 1 week, MTX was added to each culture in concentrations of 0.6 pM, 0.4 pM, 0.2 jdf, 0.1 jdf, 1 IN, and 0.5 nM. All experiments were done on 24 wells for each MTX concentration and for the controls. The effect on osteoblastic cells was assessed, at 7 days, by cell counts and by measurement of lysate alkaline phosphatase and supernatant osteocalcin levels, and, at 21 days, by analysis of the calcified matrix production, which was cultured with ascorbic acid and Fglycerophosphate. For osteoclastic cells, cell count and lysate acid phosphatase levels were determined.Resuks. Levels of osteoblastic cells and lysate alkaline phosphatase were not changed by any of the concentrations of MTX. Matrix calcification and supernatant osteocalcin levels were diminished by MTX in a dose-responsive manner. Osteoclast-like cell numbers and acid phosphatase levels were not significantly affected by MTX. Conclusion.These results suggest that diminished mouse osteoblastic cell function Occurs with very low mean concentrations of MTX, in a dose-responsive manner. The mechanism seems to be inability of the cell to synthesize and calcify matrix, possibly through defective osteocalcin production. Thus, low-dose MTX may have an important impact on bone density by slowing osteoblastic matrix production.Methotrexate (MTX) is a folate antagonist that is commonly used in the treatment of rheumatoid arthritis (RA) and other inflammatory diseases. It is known that administration of MTX in high doses to treat childhood leukemias and breast cancer has been associated with the development of bone pain, severe osteopenia, and fractures (14). The inhibition of conversion of folic acid to tetrahydrofolic acid by competitive inhibition of the enzyme, dihydrofolate reductase, and the subsequent inhibition of DNA synthesis, accounts for the antiproliferative effects of MTX on cancer cells, and may account for the osteopenia observed in these patients (1). Limited human data on high-dose regimens ( 2 4 , however, point to stimulation of bone resorption, rather than decrease in bone formation, as the etiologic mechanism of osteopenia in these patients. Our previous studies investigated the skeletal effects of long-term, low-dose MTX in rats, using a regimen similar to that used for inflammatory disease states. In those studies, we demonstrated significant osteopenia, which was characterized by diminished osteoblast fu...

Skeletal morbidity in childhood acute lymphoblastic leukaemia

“…Osteoclasts, however, were less affected, as the numbers of osteoclasts and the extent of their activity were no different from that observed in untreated rats. 41,42 In vivo data support this latter observation, as markers of bone resorption were suppressed to a much lesser extent than markers of bone formation during treatment of childhood ALL with chemotherapy. Age-related bone loss may be due to an inadequate supply of osteoblast precursor cells and a resultant decline in osteoblast numbers.…”

Bone Mass Change During Methotrexate Treatment in Patients with Juvenile Rheumatoid Arthritis

“…A study reported localized osteopenia and bone pain in two elderly patients affected by RA and treated with MTX [13]: however, given the patients' age, we cannot exclude other pathogenetic mechanisms underlying skeletal alterations. A few studies on small animals, and one study on synovial and bone tissues of patients submitted to surgery for rheumatoid articular lesions, demonstrated the presence of MTX in both trabecular and cortical bone [12,[23][24][25][26][27]. This was linked to the possibility of bone damage, on the basis of inhibition of osteoblastic activity and stimulation of osteoclastic recruitment, resulting in a net increase in bone resorption.…”

Density and structural changes in the bone of growing rats after weekly alendronate administration with and without a methotrexate challenge

“…Therefore, the small growth loss was more likely due to direct effects of MTX on bone remodeling, matrix synthesis, and calcification. 35 In the central diaphysis of the femur, MTX led to an increase in BMD, which also may be due to failure of normal cortical remodeling and reshaping induced by the treatment. These effects did not, under our conditions, lead to a significant change in femoral bone strength.…”