Matrix mineralization in MC3T3-E1 cell cultures initiated by beta-glycerophosphate pulse

MC3T3-E1 cells, grown in the presence of serum and ascorbate, express alkal ine phosphatase and produce an extensive collagenous extracellular matrix t hat can be mineralized by the addition of beta-glycerophosphate (beta-GP), In the present work, we study the influence of concentration and duration o f beta-GP treatment on the mineralization pattern in 4-week-old cell cultur es. Amount and structure of mineral deposition were monitored by von Kossa staining, light, and electron microscopy, as well as small-angle X-ray scat tering (SAXS) of unstained specimens, SAXS measures the total surface of th e mineral phase and is therefore preferentially sensitive to very small cry stals (typically <50 nm), It was used to determine the ratio (M) of small c rystals to collagen matrix. A variety of mineralization patterns was observ ed to occur simultaneously, some associated with collagen within nodules or in deeper layers of the cultures and some independent of it. At a beta-GP concentration of 10 mmol, mineralization was initiated after about 24 h and continued to increase, irrespective of whether the high level of beta-GP w as maintained or reduced to 2 mmol, With shorter pulses (<24 h), no signifi cant mineralization was observed in the week following beta-GP pulse. With continuous treatment at 5 mmol beta-GP, the first signs of mineralization w ere detected 14 days after the beginning of treatment in the 4-week-old cul tures, but no mineralization at all occurred at lower beta-GP concentration s. When cells were grown without ascorbic acid for 4 weeks, only two cell l ayers without collagen matrix were found, In these cultures, no mineralizat ion detectable by SAXS could be induced with beta-GP, These data indicate t hat, in viable cells, high doses of beta-GP are essential for the nucleatio n of mineral crystals, but not for the progression of mineralization once c rystals had been nucleated. In contrast, when 4-week-old cell cultures were devitalized, M was found to increase immediately, even at 2 mmol beta-GP, These results suggest that, in MC3T3-E1 cell cultures, cell viability is es sential for prevention of spontaneous mineralization of the extracellular m atrix. (Bone 23:511-520; 1998) (C) 1998 by Elsevier Science Inc. All rights reserved.