Calcium and phosphate supplementation promotes bone cell mineralization: Implications for hydroxyapatite (HA)-enhanced bone formation

Abstract

Organic phosphate, in particular beta-glycerophosphate (beta-GP), has been used to induce mineralization in cell culture systems. It serves as a source of inorganic phosphate when hydrolyzed by alkaline phosphatase. This study examined the effect of supplemental calcium and phosphate as well as the influence of various metabolic inhibitors on mineralization in a rat osteoblast-like cell-culture system. Mineralization was induced by supplementation of 1.8 mM of Ca(+2) and 5 mM of beta-GP or Pi. Mineral deposits associated with in vitro mineralization were revealed under SEM and TEM. Levamisole (10-100 microM) inhibited alkaline phosphatase activity and effectively reduced mineral formation. Actinomycin (500 ng/mL) and cycloheximide (50 microg/mL) also reduced mineral depositions by blocking RNA synthesis and protein synthesis, respectively. Levamisole and beta-GP did not appear to influence DNA synthesis. Spontaneous precipitation of calcium phosphate mineral was not detected in the culture medium with calcium and phosphate supplements in the absence of cell culture. The findings suggest that an elevated concentration of calcium and phosphate is crucial for in vitro mineralization. Furthermore, the mineralization process is associated with biologic events rather than with a spontaneous precipitation of calcium phosphate mineral. In view of the degradation potential of hydroxyapatite (HA)-coated implants, these results may be a viable indication that HA enhances bone formation through a similar mechanism.

Osteoblast response to zirconia‐hybridized pyrophosphate‐stabilized amorphous calcium phosphate

“…Recently, amorphous calcium phosphate has been stabilized by pyrophosphate ions (P 2 0 7 -4 ) and hybridized with zirconium (Zr-ACP) to retard its dissolution and subsequent conversion to HAP in aqueous environments [50,51]. In addition, Zr-ACP allows for controlled release of calcium and phosphate ions; factors that have been postulated to increase osteoblast differentiation and mineralization in vitro [52][53][54][55].…”

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

“…The majority of these studies have utilized an osteoblastic cell line in culture media that typically contain ionic calcium levels between 1.8 mM [Minimum Essential Medium (MEM), aMEM, and Dulbecco's Modified Eagle Medium (DMEM)] and 2.6 mM [Biggers, Gwatkin and Judah b (BGJ/b) Medium, Fitton Jackson Modification], ionic phosphate levels of 0.9 mM (DMEM) to 1.6 mM (BGJ/b), and 10 mM b-glycerophosphate [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. 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].…”

Effect on growth and osteoblast mineralization of hydroxyapatite-zirconia (HA-ZrO 2 ) obtained by a new low temperature system

“…Since calcium and phosphate ions are regulators of in vitro osteoblastic differentiation and mineralization [50][51][52], ceramic scaffold material capable of releasing these ions may also provide suitable osteoinductive properties for accelerating bone healing. According to the EDAX analysis, HZ650 showed high levels of calcium and phosphate.…”