AbstractAngiogenically stimulated alternative monocytes (aMO2) could be established as cellular release system accelerating the endothelialization of polymers rendering their surfaces hemocompatible in a short-term study. However, for their clinical application it is essential that aMO2 do not switch back to the MO1 state sustaining their capability as cellular release system over an extended period of time. We explored whether aMO2 can maintain their differentiation state over 21 days in a mono- and in a co-culture with HUVEC. In comparison, the influence of recombinant VEGF-A165 on the endothelialization of biomaterials was assessed including endothelial cell (HUVEC) density, organisation of the endothelial cytoskeleton, cytokine secretion profile and release of prostacyclin, thromboxaneA2 and matrix metalloproteinases. In mono-culture aMO2 secreted high amounts of VEGF and other growth factors/cytokines. Co-cultured with HUVEC, aMO2 accelerated the formation of a confluent HUVEC monolayer. Furthermore, no pro-inflammatory cytokines were found, neither in aMO2-mono, nor in co-cultures with HUVEC indicating that the majority of the aMO2 remained stable in their aMO2 state during the 21 days of cultivation. In contrast, the addition of recombinant VEGF-A165 instead of the co-culture with aMO2 resulted in the formation of stress fibres, dissociated marginal filament bands, and a detachment of HUVEC. In addition, the profile of bioactive agents of HUVEC (e.g. prostacyclin, thromboxaneA2, matrix metalloproteinases, IFN-γ and TNF-α) was influenced by the VEGF-A165 treatment inducing the detachment of HUVEC. In conclusion, in co-culture with HUVEC aMO2 remained stable in their type 2 state over 21 days confirming the suitability of aMO2 as biological release system for the endothelialization of biomaterial surfaces with constant release of angiogenic factors but without secretion of pro-inflammatory cytokines over three weeks. Therefore, this endothelialization approach seems to be appropriate to improve the hemocompatibility of cardiovascular implant materials in vitro, and proved to be superior to the use of recombinant VEGF-A165.