Effects Of Exogenous Metal Ions On Proliferation, Differentiation And Mineralized Function Of Primary Osteoblasts In Vitro

By using methods of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), alkaline phosphatase (ALP) activity assay, oil red O stain and alizarin red-S stain, we studied the effects of metal ions (Fe³⁺, Fe²⁺; Cu²⁺, Cu⁺; Pr³⁺, Gd³⁺) on the proliferation, differentiation and calcification of primary osteoblasts (OBs), and the effects of mixed metal elements (MME) during total flavonoids or icariin from Epimedium koreanum preventing osteoporosis. The results showed that:1. Both Fe³⁺ and Fe²⁺ (1×10^-91×10^-4 mol/L) promoted the proliferation of OBs, but turned to inhibit at a concentration of 1×10-3 mol/L. Most concentrations of Fe³⁺ and Fe²⁺ had inhibitory effects on the differentiation of OBs. Fe³⁺ inhibited adipocytic trans-differentiation of OBs. At low concentrations, Fe²⁺ showed promotive effects on adipocytic trans-differentiation of OBs, which turned to weaken with increasing concentration. At concentrations of 1×10^-6 and 1×10^-5 mol/L, Fe²⁺ showed inhibitory effects. And then it showed promotive effect at a concentration of 1×10^-4 mol/L. For the formation of mineralized matrix nodules, a converse trend had been detacted between Fe³⁺ and Fe²⁺.2. The effects of Cu²⁺ and Cu⁺ on the proliferation and differentiation were complicated, depending on concentration and culture time. Both Cu²⁺ and Cu⁺ promoted the adipocytic trans-differentiation of OBs at all tested concentrations. Both Cu²⁺ and Cu⁺ show inhibitory or no effects on the formation of mineralized matrix nodules of OBs, except that Cu²⁺ promoted the formation of mineralized matrix nodules of OBs at a concentration of 1×10-7 mol/L.3. Pr³⁺ and Gd³⁺ decreased the vability of OBs, which was related to incubation time, and both of them increased the vability at a concentration of 1×10^-4 mol/L. The effects on ALP activity depended on concentration and culture time. Pr³⁺ promoted the mineralization at day 17 and has no effect at day 21. Gd³⁺ inhibited the mineralization except 1×10^-4 mol/L, and the effects were attenuate by prolonging the incubation time. The promotive effects of Pr³⁺ and Gd³⁺ on the adipocytic transdifferentiation were dose-dependent. 4. The efficiency of TF or icariin from Epimedium koreanum could be disturbed by administration of MME (Zn²⁺/Ca²⁺/Mn²⁺), and both synergistic and antagonistic effects weregenerated. There may be a potential cooperative action between flavonoids and trace metal elements on the proliferation and differentiation of primary OBs by forming metal complexes.The results suggested that concentration, culture time, species and valence state of ions were key factors for switching the biological effects of different metal ions (from toxicity to activity, from damage to protection, or from down-regulation to up-regulation). We deduced that the varying arrangements of the electrons influenced the coordination action with related proteins and factors, and subsequently disturbed bone metabolism in various ways.