![]() ![]() Nanoparticle cell uptake studies in vitro or in vivo require nanoparticles to be dispersed in biological media. The workflow presented enables accurate nanoparticle dispersion analysis of particles suspended in more complex biological media. Automated analysis of these images was achieved through machine learning using two readily accessible freeware CellProfiler and ilastik. In order to use this approach statistically relevant large image datasets were collected from appropriately prepared samples of nanoparticle suspensions by implementing an automated imaging protocol. Here we present an alternative analysis which utilises electron microscopy. These biological environments often contain a number of components such as salts, vitamins and proteins which can lead to difficulties when using traditional techniques to monitor dispersion. In order to further advance studies in both nanomedicine and nanotoxicology, we need to continue to understand the dispersion of nanoparticles in biological fluids. The specific results and overall workflow described enable accurate nanoparticle agglomerate analysis of particles suspended in aqueous media containing other potential confounding components such as salts, vitamins and proteins. Automated analysis of images of agglomerates was achieved by machine learning using two free open-source software tools: CellProfiler and ilastik. The need to collect large, statistically relevant datasets by imaging vacuum dried, plunge frozen aliquots of suspension was accomplished by developing an automated STEM imaging protocol implemented in an SEM fitted with a transmission detector. Here we present an alternative analysis and associated workflow which utilises electron microscopy. For nanomedicinal and nanotoxicological research this is complicated by the often complex nature of the biological dispersant and ultimately this leads to severe limitations in the analysis of the nanoparticle dispersion by light scattering techniques. This study shows the ability to grow oriented tissue-engineered cocultures with significant increases in osteogenesis over monoculture conditions.For many nanoparticle applications it is important to understand dispersion in liquids. Taken together, we conclude that coculture of hMSCs with ECs is an effective strategy to promote osteogenesis on electrospun scaffolds, and aligned fibers could be introduced to regenerate bone tissues with oriented topography without significant deleterious effects on hMSCs differentiation. More importantly, cocultured cells on both random and aligned scaffolds had significantly higher ALP activities than monoculture groups, which indicated that coculture with HUVECs provided a larger relative contribution to the osteogenesis of hMSCs compared with fiber alignment. The aligned structure of PCL fibers strongly influenced the morphology and orientation of hMSCs and HUVECs however, fiber alignment was observed to not affect alkaline phosphate (ALP) activity or mineralization of hMSCs compared with random scaffolds. ![]() Coculture results showed that hMSCs and HUVECs adhered to and proliferated well on both scaffolds. After optimizing cell ratio, a hMSCs/HUVECs ratio (90:10) was considered to be the best combination for osteogenic differentiation. Inspired by the aligned fibrillar nanostructures in human cancellous bone, we fabricated polycaprolactone (PCL) electrospun fibers with aligned and random morphology, cocultured human MSCs with human umbilical vein ECs (HUVECs), and finally investigated how these two factors modulate osteogenic differentiation of human MSCs (hMSCs). ![]() Moreover, bone regeneration is especially challenging due to its unique structural properties with aligned topographical cues, with which stem cells can interact. One strategy for improving the survival and osteogenesis of tissue-engineered bone grafts involves the coculture of endothelial cells (ECs) with mesenchymal stromal cells (MSCs). The lack of vascular networks leads to insufficient oxygen and nutrients supply, which compromises the survival of regenerated bone. Vascularization is a critical process during bone regeneration. ![]()
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