The association between iron overload and osteoporosis has been found in many diseases, such as hemochromatosis, β-thalassemia and sickle cell anemia with multiple blood transfusion. One of the contributing factors is iron toxicity to osteoblasts. Some studies showed the negative effects of iron on osteoblasts; however, the effects of two biological available iron species, i.e., ferric and ferrous, on osteoblasts are elusive. Since most intracellular ionized iron is ferric, osteoblasts was hypothesized to be more responsive to ferric iron. Herein, ferric ammonium citrate (FAC) and ferrous ammonium sulfate (FAS) were used as ferric and ferrous donors. Our results showed that both iron species suppressed cell survival and proliferation. Both also induced osteoblast cell death consistent with the higher levels of cleaved caspase 3 and caspase 7 in osteoblasts, indicating that iron induced osteoblast apoptosis. Iron treatments led to the elevated intracellular iron in osteoblasts as determined by atomic absorption spectrophotometry, thereby leading to a decreased expression of genes for cellular iron import and increased expression of genes for cellular iron export. Effects of FAC and FAS on osteoblast differentiation were determined by the activity of alkaline phosphatase (ALP). The lower ALP activity from osteoblast with iron exposure was found. In addition, ferric and ferrous differentially induced osteoblastic and osteoblast-derived osteoclastogenic gene expression alterations in osteoblast. Even though both iron species had similar effects on osteoblast cell survival and differentiation, the overall effects were markedly stronger in FAC-treated groups, suggesting that osteoblasts were more sensitive to ferric than ferrous.

One of the potential contributing factors for iron overload-induced osteoporosis is the iron toxicity on bone forming cells, osteoblasts. In this study, the comparative effects of Fe 3+ and Fe 2 + on osteoblast differentiation and mineralization were studied in UMR-106 osteoblast cells by using ferric ammonium citrate and ferrous ammonium sulfate as Fe 3+ and Fe 2+ donors, respectively. Effects of 1,25 dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] and iron chelator deferiprone on iron uptake ability of osteoblasts were examined, and the potential protective ability of 1,25(OH) 2 D 3 , deferiprone and extracellular calcium treatment in osteoblast cell survival under iron overload was also elucidated. The differential effects of Fe 3+ and Fe 2+ on reactive oxygen species (ROS) production in osteoblasts were also compared. Our results showed that both iron species suppressed alkaline phosphatase gene expression and mineralization with the stronger effects from Fe 3+ than Fe 2+ . 1,25(OH) 2 D 3 significantly increased the intracellular iron but minimally affected osteoblast cell survival under iron overload. Deferiprone markedly decreased intracellular iron in osteoblasts, but it could not recover iron-induced osteoblast cell death. Interestingly, extracellular calcium was able to rescue osteoblasts from iron-induced osteoblast cell death. Additionally, both iron species could induce ROS production and G0/G1 cell cycle arrest in osteoblasts with the stronger effects from Fe 3+ . In conclusions, Fe 3+ and Fe 2+ differentially compromised the osteoblast functions and viability, which can be alleviated by an increase in extracellular ionized calcium, but not 1,25(OH) 2 D 3 or iron chelator deferiprone. This study has provided the invaluable information for therapeutic design targeting specific iron specie(s) in iron overload-induced osteoporosis. Moreover, an increase in extracellular calcium could be beneficial for this group of patients.

Background Recently we have generated recombinant human osteopontin (rhOPN) using a plant platform (Nicotiana benthamiana) and demonstrated, when coated on culture plates, its osteogenic induction capacity of human periodontal ligament (PDL) cells. The aim of this study is to elucidate the molecular mechanism underlying the rhOPN‐induced osteogenic differentiation of human PDL cells. Methods Full length rhOPN (FL‐OPN) and three constructs of OPN containing integrin binding domain (N142), calcium binding domain (C122) and mutated calcium‐binding domain (C122δ) were generated from N. benthamiana. Human PDL cells were isolated from extracted third molars and cultured on FL‐OPN, N142, C122, or C122δ‐coated surfaces. Real‐time PCR and Western blot analyses were used to determine mRNA and protein expression. In vitro calcification was determined by Alizarin red staining. A chemical inhibitor and RNAi silencing were used to elucidate signaling pathways. In silico analyses were performed to predict the protein‐protein interaction. In vivo analysis was performed using a rat calvaria defect model. Results Human PDL cells seeded on FL‐OPN and C122‐coated surfaces significantly increased both mRNA and protein expression of osterix (OSX) and enhanced in vitro calcification. Soluble FL‐OPN as well as a surface coated with N142 did not affect OSX expression. Inhibition of activin receptor‐like kinase (ALK‐1) abolished the induction of osterix expression. In silico analysis suggested a possible interaction between the calcium binding domain (CaBD) of OPN and ALK‐1 receptor. C122, but not C122δ coated surfaces, induced the expression of p‐Smad‐1 and this induction was inhibited by an ALK‐1 inhibitor and RNAi against ALK‐1. In vivo data showed that 3D porous scaffold containing C‐122 enhanced new bone formation as compared to scaffold alone. Conclusion The results suggest that next to full length OPN, the CaBD of OPN, if coated to a surface, induces osteogenic differentiation via interaction with ALK‐1 receptor.