Effects of graphene oxide and reduced graphene oxide nanomaterials on porcine endothelial progenitor cells.

Graphene oxide (GO) and reduced graphene oxide (rGO) have been widely used in the field of tissue regeneration and various biomedical applications. In order to use these nanomaterials in organisms, it is imperative to possess an understanding of their impact on different cell types. Due to the potential of these nanomaterials to enter the bloodstream, interact with the endothelium and accumulate within diverse tissues, it is highly relevant to probe them when in contact with the cellular components of the vascular system. Endothelial progenitor cells (EPCs), involved in blood vessel formation, have great potential for tissue engineering and offer great advantages to study the possible angiogenic effects of biomaterials. Vascular endothelial growth factor (VEGF) induces angiogenesis and regulates vascular permeability, mainly activating VEGFR2 on endothelial cells. The effects of GO and two types of reduced GO, obtained after vacuum-assisted thermal treatment for 15 min (rGO15) and 30 min (rGO30), on porcine endothelial progenitor cells (EPCs) functionality were assessed by analyzing the nanomaterial intracellular uptake, reactive oxygen species (ROS) production and VEGFR2 expression by EPCs. The results evidence that short annealing (15 and 30 minutes) at 200 °C of GO resulted in the mitigation of both the increased ROS production and decline in VEGFR2 expression of EPCs upon GO exposure. Interestingly, after 72 hours of exposure to rGO30, VEGFR2 was higher than in the control culture, suggesting an early angiogenic potential of rGO30. The present work reveals that discrete variations in the reduction of GO may significantly affect the response of porcine endothelial progenitor cells.

Effective Actions of Ion Release from Mesoporous Bioactive Glass and Macrophage Mediators on the Differentiation of Osteoprogenitor and Endothelial Progenitor Cells.

Due to their specific mesoporous structure and large surface area, mesoporous bioactive glasses (MBGs) possess both drug-delivery ability and effective ionic release to promote bone regeneration by stimulating osteogenesis and angiogenesis. Macrophages secrete mediators that can affect both processes, depending on their phenotype. In this work, the action of ion release from MBG-75S, with a molar composition of 75SiO2-20CaO-5P2O5, on osteogenesis and angiogenesis and the modulatory role of macrophages have been assessed in vitro with MC3T3-E1 pre-osteoblasts and endothelial progenitor cells (EPCs) in monoculture and in coculture with RAW 264.7 macrophages. Ca2+, phosphorous, and silicon ions released from MBG-75S were measured in the culture medium during both differentiation processes. Alkaline phosphatase activity and matrix mineralization were quantified as the key markers of osteogenic differentiation in MC3T3-E1 cells. The expression of CD31, CD34, VEGFR2, eNOS, and vWF was evaluated to characterize the EPC differentiation into mature endothelial cells. Other cellular parameters analyzed included the cell size and complexity, intracellular calcium, and intracellular content of the reactive oxygen species. The results obtained indicate that the ions released by MBG-75S promote osteogenesis and angiogenesis in vitro, evidencing a macrophage inhibitory role in these processes and demonstrating the high potential of MBG-75S for the preparation of implants for bone regeneration.

Effects of Ipriflavone-Loaded Mesoporous Nanospheres on the Differentiation of Endothelial Progenitor Cells and Their Modulation by Macrophages.

Angiogenic biomaterials are designed to promote vascularization and tissue regeneration. Nanoparticles of bioactive materials loaded with drugs represent an interesting strategy to stimulate osteogenesis and angiogenesis and to inhibit bone resorption. In this work, porcine endothelial progenitor cells (EPCs), essential for blood vessel formation, were isolated and characterized to evaluate the in vitro effects of unloaded (NanoMBGs) and ipriflavone-loaded nanospheres (NanoMBG-IPs), which were designed to prevent osteoporosis. The expression of vascular endothelial growth factor receptor 2 (VEGFR2) was studied in EPCs under different culture conditions: (a) treatment with NanoMBGs or NanoMBG-IPs, (b) culture with media from basal, M1, and M2 macrophages previously treated with NanoMBGs or NanoMBG-IPs, (c) coculture with macrophages in the presence of NanoMBGs or NanoMBG-IPs, and (d) coculture with M2d angiogenic macrophages. The endocytic mechanisms for nanosphere incorporation by EPCs were identified using six different endocytosis inhibitors. The results evidence the great potential of these nanomaterials to enhance VEGFR2 expression and angiogenesis, after intracellular incorporation by EPCs through clathrin-dependent endocytosis, phagocytosis, and caveolae-mediated uptake. The treatment of EPCs with basal, M1, and M2 macrophage culture media and EPC/macrophage coculture studies also confirmed the angiogenic effect of these nanospheres on EPCs, even in the presence of phagocytic cells.