Iron Overload Induced TNF-α Bearing Monocyte Microparticles Enhance Osteogenesis of MSC

Abstract

Introduction: Heterotopic ossification (HO) occurs at a high frequency in severe trauma induced injuries. Whether haemorrhage induced local iron overload after trauma related to enhanced osteogenesis is virtually unclear. In this study, our first objective was to determine the in vitro effect of iron on microparticles (MPs) generation by monocytes cell line. And to find out what possible mechanisms underlying iron-induced monocyte MP generation, this including iron overload induced apoptosis and oxidative stress in relation with MPs generation. Furthermore, to examine effect of excessive iron induced monocyte MPS on mesenchymal stromal cell osteogenesis.

Methods and results: By using monocyte cell line, we showed that excessive iron induced monocyte MPs generation in dose dependent manner, iron increased reactive oxygen species (ROS) production (37.5-300 μM). Apoptotic cells were found to be significantly increased under iron treatment (75 μM-300 μM, 48 hours) as showed by AnnexinV/PI assay. Secondly, we found that MSC engulfed iron induced monocyte MPs and enhance the osteogenesis as showed by mineralisation assay and alkaline phosphatase assay. By using western blotting method, we found excessive iron upregulated TNF-α gene and protein expression of monocyte. In order to confirm it is TNF-α caused the enhanced osteogenesis effect, we using monocyte TNF-α knock down model. Our data showed that iron induced monocyte MPs from TNF-α knock down cells had a lower stimulation ability on osteogenic differentiation of MSCs. To further confirm this data, we using antibody neutralisation assay, anti-TNF-α had anti osteogenic effect on TNF-α induced osteogenic differentiation of MSCs.

Conclusions: Our findings suggest that iron-overload induces monocyte MPs which may caused by ROS induced cells apoptosis. Furthermore iron induced monocyte MPs which contain TNF-α may contribute to etiotopic bone formation in HO.