## 3 Results and Discussion
### Uptake of HAp Nanoparticles Can Facilitate the Cell Adhesion and Proliferation on the Surface
The results of an analysis carried out on a sample of 16 cells for each sample group demonstrated a definitely larger adhesion force of hMSCs subjected to the uptake of HAp, compared to the control cellular population, as indicated by the almost three-times higher ratio of the average side length of the cell contours to the average opening angle (Figure 9c), which is directly proportional to the adhesion force (Figure 9a). Figure 8: Control cells (**a**) and cells treated with the nanoparticles (**b**) stained for their nuclei (blue) and collagen production (red). Proliferation of control cells reseeded in cell culture dishes and cells that underwent the uptake of HAp nanoparticles prior to reseeding (**c**). hMSC viability of population subjected to HAp nanoparticle uptake relative to the viability of the control population (**d**). Data points in (**c**,**d**) represent averages (n = \(2-4\)), error bars represent standard deviation, and \(p\)-values represent the confidence intervals for the statistical significance of the difference of the value of the sample group compared to the control. These findings suggest that infusing the cells with the mineral nodules can be expected to commit the cell to a particular location, at least until these intracellular nodules do not become secreted and fused with the extracellular mineral phase being built. Since HAp nanoparticles, upon internalization, alter the actin cytoskeleton architecture in the direction of increased cell adhesion favoring osteogenic differentiation, and through increased Ca\({}^{2+}\) levels activate Ca\({}^{2+}\)-mediated signaling cascades, e.g., Ras/cAMP/Rap1/MAPK, which also set the stem cells on the osteogenic differentiation pathway [89], this scenario is quite realistic and is supported by the observed morphological semblance of HAp-exposed hMSCs and round osteoblastic cells evidenced in Figure 1. This effect, however, is expected to be greatly dependent and perhaps even antagonistic, depending on the cell type, the nanoparticle dose, and the cell seeding density. Moreover, the normally inverse relationship between the metabolic activity and the proliferation rate often predisposes the treated cells to display opposite effects relative to the control at different incubation time points. For example, the proliferation of cells seeded on HAp-containing polymeric surfaces with distinct topographic features was initially slowed because of their enhanced differentiation and augmented metabolic activity, but it later caught up with the control, and even surpassed it [90]. Osteoblasts originating from MSCs, however, may be immune to this antagonism; an earlier study, for example, demonstrated the faster proliferation of such cells than that of their undifferentiated MSC precursors [91]. HAp owes its excellent cellular uptake propensities to multiple effects, including the bioactivity of its elemental composition [92], the low net surface charge [93], the superb adsorption capacity [94], and the fine release of calcium ions [95]; the exocytotic activators, fostering the opening of the complementary endocytic pathways [96]. HAp does not have the mechanical rigidity of metallic nanoparticles or oxides such as silica, which is known to be a physical factor favoring the uptake [97], but it has a diffusive surface character thanks to its hydroxylated, clay-like interface [98; 99; 100],  
Figure 9: Schematization of the discrete adhesion model proposed by [PERSON] et al., and applied to estimate the force of adhesion of the cells to the underlying surface (**a**). The model predicts the direct dependence of the adhesion force on the cell dimensions defined by the cell side length, \(d\), and on the surface tension, \(\sigma\). The surface tension can be further expressed as inversely proportional to the opening angle at the cell corners, \(\phi\), as direct adhesion sites. A representative hMSC stained for f-actin in red and the nucleus in blue, along with the derived parameters \(d\) and \(\phi\) (**b**).