## 3 Results and Discussion  
The random image was taken (Figure 1), hung on the wall, like in Antoni's Blowup, and gazed at for days. Then the little details began to pop up, inspiring thought-provoking observations. The AFM image shown in Figure 1 represents a handful of fixed hMSCs challenged with HAp nanoparticles. The cells adhering onto the bottom of the cell culture well display lower aspect ratios than those of spindly fibroblasts, including the control hMSCs (Figure 2), which may be indicative of their being impelled to enter the osteoblastic differentiation path after the exposure to bone mineral particles. Osteoblasts are derived from MSCs in a process paralleled by the expression of bone morphogenetic proteins, members of the Wingless pathways, and proteins including Runt-related transcription factors 2, Distal-less homeobox 5, and Osterix [38]. In the presence of HAp and other calcium phosphate nanoparticles, hMSCs have demonstrated the overwhelming tendency to transform to osteoblasts rather than to adipocytes or chondrocytes [39; 40; 41]. Accompanying this transition toward an osteoprogenitor lineage, the pronounced degree of elongation of hMSCs gets reduced, and cells begin to increasingly adopt a round appearance and the squared shapes characteristic for  
Figure 1: The randomly taken atomic force microscope (AFM) image of hMSCs challenged with HAp nanoparticles, used as the starting point for inquiry into the interaction between nanoparticles and cells. osteoblasts [42]. The differentiation process, however, usually requires timescales longer than 72 h [43], meaning that the majority of cells must have maintained their mesenchymal phenotype by the point of their fixation, and that no endogenous mineral nodules have yet begun to form. Imaged under the AFM, the cells also display typical features of fixed cells, primarily in terms of the lack of distinct cytoskeletal fibers, the reason being the crosslinking of the membrane proteins during the fixation procedure and the resulting stiffening of the cell membrane, which, as such, becomes more pronounced than the actomyosin microfibers [44]. The cell sizes vary and in this image decrease along the diagonal connecting the bottom left corner with the upper right one. HAp nanoparticle agglomerates visible in the image vary in size, too. Since the primary particles in this experiment were well under 100 nm in size (Figure 3e), such elementary particular entities would be invisible at the resolution of 185 nm employed to capture both the cells and the particles, with the entire image shown in Figure 1 having dimensions of 93.40 x 93.40 \(\upmu\)m. Therefore, all the distinguishable particulate entities in Figure 1 are agglomerates of HAp nanoparticles varying in size and shape. The most prominent and largest of them, visible near the upper central edge of the image frame, is magnified in Figure 3a. This nanoparticle cluster is -6.3 x 4.1 \(\upmu\)m in size and falls right around the 5 \(\upmu\)m upper limit for the nonspecific uptake pathway, also calledropinocytosis [45]. This size limit is significantly higher than the 50-500 nm upper limits established for the two specific, receptor-mediated uptake pathways, namely clathrin-mediated endocytosis (<200 nm) and caveolin-mediated endocytosis (<500 nm) [46], as well as for the clathrin- and caveolin-independent uptake routes, such as the lipid-raft mediated and ARF6-mediated GEEC pathways. Despite looking very bright, this specific nanoparticle aggregate has only a 1.7 \(\upmu\)m height, meaning that it is likely to be prone to macropinocytosis, at least by the sizable cells specializing in the uptake of particles and particle debris, such as macrophages or osteoclasts. Based solely on AFM imaging, however, it is impossible to precisely conclude whether this particle lies on the inside or the outside of the cell.