O-4: The Interaction of Bioactive Glass Nanoparticles with Mesenchymal Stem Cells In Vitro

Background: Bioactive glass (BG) nanoparticles are amongst the most promising class of biomaterials for hard tissue regeneration because of their distinctive properties of rapid bone bonding, controlled biodegradability and their ability to stimulate new bone growth. Despite the vast interest in BG scaffolds in medical applications, the synthesis of bioactive glass nanoparticles is still facing numerous challenges. In addition, despite the great potential of BG’s as porous scaffolds for bone regeneration, concerns have arisen on their longterm fate in the body as small particles may be released after implantation. This could lead to undesirable reactions with the surrounding cells, hence investigations on such nanoparticles is crucial. BG nanoparticles, also have the potential to be injected directly into the defect site to allow healing and regeneration of bone tissue. As mesenchymal stem cells (MSCs) are precursors to osteoblasts, the effect of nanoparticles on their behaviour is critical. In this study 80S20C (80 mol% SiO2 and 20 mol% CaO) mono dispersed BG nanoparticles have been synthesised and characterised for the first time. The combined utilization of nanoparticles and human MSCs in regenerative medicine requires particle uptake into the cells. Hence, the BG nanoparticle’s uptake and distribution inside MSCs was assessed using confocal microscopy and transmission electron microscopy (TEM). The effect of the BG nanoparticles on cell viability, metabolic activity and proliferation as a result of particle uptake was also determined. Materials and Methods: The Stöber process was applied to produce sol-gel derived BG nanoparticles. To follow the internalisation and intracellular distribution of the BG nanoparticles inside MSCs (Lonza, UK) in 3D, cells were exposed to BG nanoparticles at a concentration of 100μg/ml in cell medium (Dublecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin (all from Invitrogen, UK) for 24 hours. For confocal microscopy the actin cytoskeleton was stained with Alexa Fluor conjugated phaloidin (molecular probes, UK) and particles were labeled with FITC. TEM was also used to monitor the uptake and distribution of the nanoparticles (100μg/ml) inside MSCs after 24 hours exposure. Cells were fixed, osmicated and the samples were embedded in resin and sectioned. The effect of the nanoparticles on cell viability and proliferation was determined by exposing MSCs to three different BG nanoparticle concentrations: 100, 150 and 200 μg/ ml in cell medium (DMEM) for 24 hours and their response monitored over of the period of 1, 4 and 7 days using LIVE/DEAD (Molecular Probes, UK), MTT (Sigma, UK), total DNA using Hoechst (Sigma, UK) and Lactate dehydrogenase Cytotox-oneTM (Promega, UK). Results: The processing route for the synthesis of the BG nanoparticles was successful as it resulted in spherical and dense particles with a composition of SiO2 and CaO. The uptake and intracellular localization of the BG nanoparticles inside MSCs was confirmed by confocal microscopy and TEM. The uptake of the 100μg/ml of BG nanoparticles by MSCs was captured, for the first time, using confocal. The nanoparticles were then seen in the cell cytoplasm. Also, TEM demonstrated nanoparticles entrapped in endosomes after 24 hours exposure. Furthermore, the dissolution behaviour and breakup of the nanoparticles inside cells were also observed using TEM. No significant levels of cytotoxicity were observed for the nanoparticles at all concentrations following a range of cytotoxicity assays. At the concentrations of 100 and 150 μg/ml, the particles were seen to increase metabolic activity of human MSCs. The effect of the BG nanoparticles on MSC differentiation will also be presented. Conclusion: Spherical bioactive glass nanoparticles of 80S20C composition with controlled size were produced. In this study we successfully showed that BG nanoparticles were able to internalize inside MSCs. The viability and proliferation assays confirmed that none of the BG nanoparticles concentrations tested induced any major toxicity to MSCs.