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The successful vascularisation of complex tissue engineered constructs for bone regeneration is still a major challenge in the field of tissue engineering. In this context, co-culture systems of endothelial cells and osteoblasts represent a promising approach to advance the formation of a stable vasculature as well as an excellent in vitro model to identify factors that positively influence bone healing processes, including angiogenesis. Under physiological conditions, the activation phase of angiogenesis is mainly induced by hypoxia or inflammation. Inflammatory cells such as macrophages secrete proinflammatory cytokines and proangiogenic growth factors, finally leading to the formation of new blood vessels. The aim of this study was to investigate if macrophages might positively influence the formation of microvessel-like structures via inflammatory mechanisms in a co-culture system consisting of human outgrowth endothelial cells (OECs) and primary osteoblasts. Treatment of co-cultures with macrophages (induced from THP-1) resulted in a higher number of microvessel-like structures formed by OECs compared to the co-culture. This change correlated with a significantly higher concentration of the proangiogenic VEGF in cell culture supernatants of triple-cultures and was accompanied by an increase in the expression of different proinflammatory cytokines, such as IL-6, IL-8 and TNFα. In addition, the expression of E-selectin and ICAM-1, adhesion molecules which are strongly involved in the interaction between leukocytes and endothelial cells during the process of inflammation was also found to be higher in triple-cultures compared to the double co-cultures, documenting an ongoing proinflammatory stimulus. These results raise the possibility of actively using pro-inflammatory stimuli in a tissue engineering context to accelerate healing mechanisms.

Macrophage-mediated angiogenic activation of outgrowth endothelial cells in co-culture with primary osteoblasts