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The initial phase of fracture healing decides on success of bone regeneration and is characterized by an inflammatory milieu and low oxygen tension (hypoxia). Negative interference with or prolongation of this fine-tuned initiation phase will ultimately lead to a delayed or incomplete healing such as non-unions which then requires an effective and gentle therapeutic intervention. Common reasons include a dysregulated immune response, immunosuppression or a failure in cellular adaptation to the inflammatory hypoxic milieu of the fracture gap and a reduction in vascularizing capacity by environmental noxious agents (e.g. rheumatoid arthritis, smoking). The hypoxia-inducible factor (HIF)-1α is responsible for the cellular adaptation to hypoxia, activating angiogenesis and supporting cell attraction and migration to the fracture gap. Here, we hypothesized that stabilizing HIF-1α could be a cost-effective and low-risk prevention strategy of fracture healing disorders. Therefore, we combined a well-known HIF-stabilizer – deferoxamine (DFO) – and a less known HIF-enhancer – macrophage migration inhibitory factor (MIF) – to synergistically induce improved fracture healing. Stabilization of HIF-1α enhanced calcification and osteogenic differentiation of MSCs in vitro. In vivo, the application of DFO with or without MIF during the initial healing phase accelerated callus mineralization and vessel formation in a clinically relevant mouse-osteotomy-model in a compromised healing setting. Our findings provide support for a promising preventive strategy towards bone healing disorders in patients with a higher risk due to e.g. delayed neovascularization by accelerating fracture healing using DFO and MIF to stabilize HIF-1α.

Background The cytokines IL-4 and IL-10 have been implicated in the regulation of T cell proliferation and differentiation. IL-4 drives the development of Th2 cells from naive precursors and it is supposed to enhance T cell proliferation, whereas IL-10 has been suggested to support the differentiation of regulatory Th cells and to negatively affect T cell proliferation. Objectives To analyse the proliferation and stability of cytokine expression of individual cytokine-producing T cells. Methods We established the cellular affinity matrix technology for the immunofluorescent identification and isolation of IL-4- or IL-10-secreting cells. With this technology we purified IL-4- or IL-10-secreting and non-secreting Th cells, labelled them with the fluorescent dye CFSE to track their proliferation after antigen-specific or polyclonal restimulation, and analysed their cytokine expression upon later restimulation. Results Previously, we have shown that naive Th cells have to enter the initial S phase of the first cell cycle to respond to an IL-4 signal by production of IL-4 or IL-10 upon restimulation. By direct counterstaining of cytokines and DNA content we now tested whether in general cytokine production is restricted to certain cell cycle phases, which we found not to be the case. The isolated IL-4+ and IL-4- T cells showed the same proliferation behaviour over 6 days, and the sorted IL-10+ cells were not inhibited in their proliferation compared with the IL-10- T cells. Thus, neither IL-4 nor IL-10 production by an individual T cell directly influences the proliferation of the same cell. When we analysed the stability of cytokine memory of the sorted T cells in later restimulations, a substantial fraction of the purified cytokine-producing cells did not reexpress the cytokine that it had expressed in an earlier stimulation. Conclusion Thus, there is a certain degree of instability in the cytokine memory of Th cells, the molecular basis of which we are currently analysing.