Effects of ipriflavone-loaded mesoporous nanospheres on the differentiation of endothelial cells and their modulation by macrophages.

RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez) ; Angiogenic biomaterials for bone repair are being designed to promote vascularization and optimize tissue regeneration. The use of nanoparticles of bioactive materials loaded with different drugs represents an interesting strategy to stimulate osteogenesis and angiogenesis and to inhibit bone resorption. Ipriflavone (IP) prevents osteoporosis by inhibiting osteoclast activity and promoting preosteoblast differentiation into mature osteoblasts. Since endothelial progenitor cells (EPCs) are involved in the formation of blood vessels which are necessary for tissue regeneration, the isolation and characterization of porcine EPCs have been carried out in this work to evaluate the in vitro effects of unloaded (NanoMBGs) and IP-loaded nanospheres (NanoMBG-IPs) designed to stimulate osteogenesis. Because different signals between vascular and nonvascular cells are also essential to initiate angiogenic events, the potential modulating role of macrophages has been also evaluated by studying the expression of vascular endothelial growth factor receptor 2 (VEFGR2) as a specific marker for EPC differentiation under different culture conditions: a) EPCs in monoculture treated with NanoMBGs or NanoMBG-IPs, b) EPCs treated with conditioned media from basal, proinflammatory M1 and reparative M2 macrophages previously treated with NanoMBGs or NanoMBG-IPs, c) EPCs cocultured with macrophages in the presence of NanoMBGs or NanoMBG-IPs, and d) EPCs cocultured with M2d angiogenic macrophages. Moreover, the endocytic mechanisms by which these nanospheres are incorporated by EPCs have been identified by using six endocytosis inhibitors (i.e. wortmannin, genistein, cytochalasin B, cytochalasin D, phenylarsine oxide and chlorpromazine) ...