• Basic Science

Erythropoietin (EPO) is a hematopoietic growth factor stimulating the formation of red blood cells. EPO is notoriously known as a doping substance in high-performance sports, and in cycling in particular. In the clinical setting, this erythropoiesis-stimulating agent is utilized to treat anemia, especially if caused by a lack of endogenous EPO production due to chronic renal failure. In recent years, the non-hematopoietic functions of EPO, also known as pleiotropic functions, have been intensively investigated. Of interest for orthopedics and musculoskeletal tissue engineering, the non-hematopoietic capabilities of EPO include osteogenic and angiogenic potencies. The objectives of the present thesis were to address and investigate the efficacy of EPO in regenerating bone and facilitating bone healing. The first paper investigated the effectiveness of continuous low-dose systemic EPO administration to enhance bone formation in an autograft posterolateral spinal fusion model in rabbits. We observed an increased bone volume and neo-vascularization compared with saline-treated controls after six weeks of observation. The second paper set out to investigate the cellular mechanisms of the osteogenic action of EPO and to describe the dose-response relationship in vitro. Human mesenchymal stromal cells (hMSCs) were exposed to a wide range of EPO-concentrations for up to three weeks. The lowest effective dose was 20 IU/ml EPO, and a proportional dose-response relationship was observed. Hence, the highest tested concentration of 100 IU/ml EPO yielded the most pronounced osteogenic effect. Regarding the cellular ways of action, two cell membrane receptors were observed, namely the EPO receptor (EPOR) and the cytokine receptor common beta subunit (CD131). Furthermore, the osteogenic effect was mediated via three intracellular signaling pathways: TOR serine-threonine kinase (mTOR), Janus kinase 2 (JAK2), and phosphatidylinositol 3-kinase (PI3K). The third paper was designed to accelerate clinical progress. Before the clinical implementation of EPO it was necessary to test EPO in a large-animal model. Systemic EPO administration can cause severe adverse events such as thromboembolisms. A single, locally administered, low-dose approach was therefore chosen. Bone formation was assessed in a porcine calvarial defect model. The defects were treated with EPO or placebo and in combination either with autologous bone graft, a commercially available collagen carrier, or a polycaprolactone scaffold. After five weeks of observation, an increased bone volume after EPO treatment was observed in the collagen carrier group. The excellent regenerative potential of the autograft was underlined by the fact that the bone volume did not significantly differ from that of the healthy reference bone. At the other end of the spectrum, bony ingrowth into the PCL scaffold was sparse both with and without EPO, which suggests the need to investigate other types of scaffold material or modified PCL constructs. In conclusion, bony ingrowth and vascularization of three-dimensional scaffolds for bone tissue regeneration remains a challenge. The described pleiotropic functions of EPO may overcome this limitation of skeletal tissue engineering in the future. EPO could potentially facilitate neovascularization, and the migration of cells that are directed into the core of the scaffold will facilitate bony ingrowth. Moreover, EPO promotes a direct and indirect osteogenic stimulation of hMSCs. A clinically safe dose enhanced bone healing in a large-animal model. This is encouraging news for the potential direct clinical utilization of EPO. EPO is therefore a promising growth factor in regenerative medicine.

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