Calcium, lanthanum, pyrophosphate, and hydroxyapatite: a comparative study in fibroblast mitogenicity

Calcium-containing crystals and elevated levels of calcium chloride (CaCl2) and lanthanum chloride (LaCl3) have been previously reported to enhance the proliferative activity of cultured fibroblasts. We have investigated the relative mitogenicity of these agents, whether they function via precipitation on the cell surface and whether they interact with one another. Confluent cultures of newborn foreskin fibroblasts provided with fresh medium containing 10% fetal bovine serum (FBS) in the presence of hydroxyapatite (HA), pyrophosphate (PPi), LaCl3 (La), or additional CaCl2 (Ca) were all stimulated more than control cultures provided with fresh medium and 10% FBS alone as assessed by cell counts 5 days later. Increases in cell yield above the original confluent cell density were 316% for La, 271% for Ca, 189% for HA, 131% for PPi, and 45% for controls. Addition of fresh medium containing 10% FBS and epidermal growth factor or fresh medium containing 20% FBS as additional points of reference yielded increases of 204 and 107%, respectively, over original confluent density. Stimulation induced by La or Ca was significantly greater (P less than 0.001) than the stimulation induced by each of the other treatments. The same treatments added to confluent cultures without a change of medium also renewed mitotic activity, with La and Ca again the most mitogenic and approximately doubling the pretreatment cell yields. Cultures incubated in an inverted position to avoid cell contact with precipitates in the medium were also stimulated by La and Ca, but not by HA and PPi. When added to confluent cultures simultaneously supplemented with optimal additional Ca, La decreased Day 5 cell yields in a dose-dependent manner at low concentrations (0.03-0.2 mM) but increased cell yields over those obtained with 0.2 mM LaCl3 again in a dose-dependent manner at higher concentrations. Thus, while HA and PPi act via precipitation on the cell surface, the more mitogenic agents La and Ca function in solution and appear to stimulate cell division by different nonadditive mechanisms. These findings suggest multiple mechanisms of membrane participation in mitogen responsiveness and in density-dependent inhibition of growth.