Mechanism of action of ?-glycerophosphate on bone cell mineralization

Abstract

Experiments were performed to determine whether beta-glycerophosphate (beta-GP) promoted mineralization in vitro by modulating bone cell metabolic activity and/or serving as a local source of inorganic phosphate ions (Pi). Using MC3T3-E1, ROS 17/2.8, and chick osteoblast-like cells in the presence of beta-GP or Pi, we examined mineral formation, lactate generation, alkaline phosphatase (AP) activity, and protein and phospholipid synthesis. Neither beta-GP nor Pi modulated any of the major biosynthetic activities of the bone cells. Thus, we found no change in the levels of phospholipids, and the total protein concentration remained constant. Measurement of lactate synthesis showed that beta-GP did not effect the rate of anaerobic glycolysis. Evaluation of medium Pi levels clearly indicated that beta-GP was hydrolyzed by bone cells; within 24 hours, almost 80% of 10 mM beta-GP was hydrolyzed. It is likely that this local increase in medium Pi concentration promoted rapid mineral deposition. Chemical, energy dispersive X-ray, and Fourier transform infrared analysis of the mineral formed in the presence of beta-GP showed that it was nonapatitic; moreover, mineral particles were also seen in the culture medium itself. Experiments performed with a cell-free system indicated that mineral particles formed spontaneously in the presence of AP and beta-GP and were deposited into a collagen matrix. We conclude that medium supplementation with beta-GP or Pi should not exceed 2 mM. If this value is exceeded, then there will be nonphysiological mineral deposition in the bone cell culture.

Mineralization of Decalcified Bone Occurs Under Cell Culture Conditions and Requires Bovine Serum But Not Cells

“…3), and this supraphysiological level of ionic phosphate caused the nonspecific precipitation of mineral within both the cartilage and collagen matrices of the tibia. A similar nonspecific precipitation of mineral has been seen by other investigators using medium containing 10 mM b-glycerophosphate, and in each instance the mineral precipitation has been associated with the hydrolysis of the b-glycerophosphate by phosphatases and the associated massive increase in medium levels of phosphate [14][15][16][17].…”

“…In an earlier study, Chung et al [17] had proposed not exceeding 2 mM Pi in the culture medium in order to prevent nonphysiological mineral formation. When DMEM supplemented with 15% FBS and containing 2 mM phosphate was tested, we succeeded in remineralizing a demineralized tibia in all sites where mineral is normally found in a nondemineralized tibia (Fig.…”

“…Previous studies have shown that over 70% of the bglycerophosphate added to osteoblastic cell cultures is hydrolyzed to phosphate within 24 hours by cell-associated alkaline phosphatase [17], and that the accompanying massive increase in medium phosphate results in the nonspecific deposition of calcium phosphate mineral in the matrix [14][15][16][17]. Since there was clearly nonspecific deposition of mineral in tibias incubated in medium containing 10 mM b-glycerophosphate (particularly in the cartilaginous ends), it seemed likely that the hydrolysis of b-glycerophosphate to phosphate also occurred in the present studies as a result of endogenous alkaline phosphatase activity in the FBS.…”

“…Using the approach of Chung et al [17], we decided to boost medium phosphate to 2 mM so that we could more directly control medium phosphate lev- Fig. 4.…”

“…The role of the bglycerophosphate is generally accepted to be as a source of phosphate for mineral growth, but it may affect mineral deposition in other ways as well [15]. Alkaline phosphatase secreted by bone cells efficiently hydrolyzes the added 10 mM b-glycerophosphate [16,17] and increases medium phosphate in osteoblast cultures to 7-9 mM within 24 hours [17]. Khouja et al [14] have shown that serum, in the absence of cells, is also able to hydrolyze b-glycerophosphate, presumably because of endogenous action of serum alkaline phosphatase.…”