Influence of manganese ions on cellular behavior of human osteoblasts in vitro

Abstract

Divalent cations like Mn 2+ are known to strongly influence the integrin affinity to ligands and – in consequence – cell adhesion to extracellular matrix proteins. Therefore, divalent cation supplementation of biomaterials could be a promising approach to improve the ingrowth and the integration of implants. We were interested, whether manganese ions affect cellular functions like spreading, proliferation as well as gene expression in human osteoblasts. MG-63 osteoblastic cells were cultured in DMEM with 10% FCS. MnCl 2 was added at a concentration range of 0.01–0.5 mM for 24 h and 48 h. Spreading (cell area in μm 2) of PKH26-stained cells (cell membrane dye) was analyzed using confocal microscopy. Cell proliferation was measured by flow cytometry. Quantification of the phosphorylation status of signaling proteins was estimated using the Bio-Plex 200 system. Gene expression of osteogenic markers at the mRNA and protein level was analyzed by quantitative real time RT-PCR and Western blot, respectively. The results demonstrated that at higher concentrations of Mn 2+ cells revealed a spindle shaped morphology. Further analyses indicated a reduced spreading, proliferation as well as phosphorylation of signaling proteins due to the influence of Mn 2+ in a concentration-dependent manner. Although expression of bone sialo protein (BSP) at the mRNA level increased both after 24 h and 48 h in the presence of manganese, no increased expression of BSP was detected at the protein level. The expression of alkaline phosphatase (ALP) and collagen 1 (Col 1) mRNA decreased at >0.1 mM MnCl 2. We speculate that the effect of manganese cations on cell functions is strongly concentration-dependent and the release of manganese when incorporated in a biomaterial surface has to be thoroughly adjusted.