A Comparison of the Biological Activities of Human Osteoblast hFOB1.19 Between Iron Excess and Iron Deficiency

Abstract

Bone metabolism has a close relationship with iron homeostasis. To examine the effects of iron excess and iron deficiency on the biological activities of osteoblast in vitro, human osteoblast cells (hFOB1.19) were incubated in a medium supplemented with 0-200 μmol/L ferric ammonium citrate and 0-20 μmol/L deferoxamine. The intracellular iron was measured by a confocal laser scanning microscope. Proliferation of osteoblasts was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptotic cells were detected using annexin intervention V/PI staining with a flow cytometry. Alkaline phosphatase (ALP) activity was measured using an ALP assay kit. The number of calcified nodules and mineral area was evaluated by von Kossa staining assay. The expressions of type I collagen and osteocalcin of cultured osteoblasts were detected by reverse transcriptase polymerase chain reaction and Western blot. Intracellular reactive oxygen species (ROS) was measured using the oxidation-sensitive dye 2,7-dichlorofluorescin diacetate by flow cytometry. The results indicated that excessive iron inhibited osteoblast activity in a concentration-dependent manner. Low iron concentrations, in contrast, produced a biphasic manner on osteoblasts: mild low iron promoted osteoblast activity, but serious low iron inhibited osteoblast activity. Osteogenesis was optimal in certain iron concentrations. The mechanism underlying biological activity invoked by excessive iron may be attributed to increased intracellular ROS levels.

Wnt5a is a key target for the pro-osteogenic effects of iron chelation on osteoblast progenitors

“…Our study is in line with a number of other studies that have shown an osteoblast-supporting effect of DFO. [26][27][28]44,45 Interestingly, osteoblasts treated with 50 mM DFO showed a somewhat decreased mineralization potential as compared to 25 mM DFO, indicating that this concentration might already exert toxic effects. In fact, we found a reduced cell viability in murine cells treated with 50 mM DFO, while cell vitality was not affected at 25 mM.…”

“…Depending on the cell type, other studies have also shown toxic effects of DFO in concentrations above 30 mM. 27,28 In contrast, human periodontal ligament cells do not seem to be particularly sensitive to DFO, as even concentrations of up to 200 mM still exerted pro-osteogenic effects. 26 In our hands, DFO reduced human BMSC viability at similar concentrations as murine BMSC (i.e., 50 mM, data not shown).…”

“…In other studies, different sources of iron are found, for example ferric ammonium citrate. 28 However, all studies have shown suppressive effects of iron on osteoblast differentiation, suggesting that the exact chemical composition may not be of relevance for in vitro studies. This is further supported by our study showing that ferric ammonium citrate and holo-transferrin also suppressed osteoblast differentiation.…”

“…[21][22] In contrast, osteoblast function is suppressed by iron excess in osteoblast cell lines, [23][24][25] while iron chelation using deferoxamine (DFO) seems to exert positive effects on osteoblast maturation. [26][27][28] In addition, bone regeneration during distraction osteogenesis and after radiation therapy has been shown to be promoted in the presence of DFO. [29][30] However, some studies have reported an inhibitory effect of DFO on osteoblasts and a suppression of alkaline phosphatase (ALP).…”

Astragalus Polysaccharide Attenuated Iron Overload-Induced Dysfunction of Mesenchymal Stem Cells via Suppressing Mitochondrial ROS

“…More and more evidence shows that iron overload is correlated with bone mass loss and osteoporosis. Accumulation of iron has been shown to induce the apoptosis of osteoblasts [29,30]. Excess iron also can lead to toxic effects on the proliferation and differentiation of mesenchymal stem cells.…”