**5. Conclusions**

**4.3. Atomic force microscopy (AFM)**

cell spread is collected.

198 Cytotoxicity

Atomic force microscopy (AFM) is based on a laser reflected off a cantilever onto a scanning surface of the examined object and quantitative information about surface morphology and

AFM is a crucial technique for determining cell interactions on the surface of the tested material. If material exhibits high biocompatibility, the surface of the material will allow cells to attach (interaction between cell-surface integrin receptors) and adsorb extracellular matrix (ECM) proteins. Surface properties, such as wettability, roughness or surface charge, are important for cellular attachment and lamellipodium/filopodium formation. The AFM measurement provides information on cellular morphology changes and lamellipodium/filopodium permissiveness. The measurement of atomic force microscopy of living cells can be performed in PBS and provides information on cell height, total cell surface area, attachment angle and extension of lamellipodia/filopodia. It is also possible to measure fixed cell (in 4% paraformaldehyde) topography and examine filopodia and lamellipodia. An interesting example is the analysis of H4 and PC12 cell lines plated on different materials—glass, polystyrene (PSt), silicon (Si), nanocrystalline diamond (NCD) and cubic silicon carbide (3C-SiC). In the latter study, AFM analysis demonstrated that the type of the surface determined cell height/area, attachment angle and the reduction of the lamellipodium/filopodium area. Cell-substrate interaction was different for H4 and PC12 cell lines, e.g., for H4 cells; the most negative interaction was recorded for glass, the most positive for 3C-SiC, while PC12 cells had the most negative interaction with glass, but the best with 3C-SiC and PSt. The authors concluded that AFM analysis indicated that neural cell interactions with 3C-SiC resulted in the optimal cell viability, morphology and interaction of cells with 3C-SiC surface [1]. Frewin et al. [1] published the results of AFM analysis concerning cellular interaction on graphene. The experiments focused on cytoskeleton organisation and the determination of the number of contact sites, and AFM technology can provide valuable information on the mechanism of cellular adhesion and proliferation on graphene surface. Different methods of graphene preparation, for example, mechanical cleaving, chemical synthesis and chemical vapour deposition (CVD) on metals or epitaxial growth on SiC, not only give graphene different electrical, optical or morphological properties, but also different biocompatibility. For example, the biocompatibility of a single

graphene layer produced by CVD on Cu was higher in comparison with SiO<sup>2</sup>

analysis clearly exhibited differences in cell growth on the two surface variants [85].

ied on human osteoblasts and mesenchymal stem cells [1, 84]. In another study, epitaxially grown graphene films on (0001) 6H-SiC substrates were evaluated in cellular response experiments using AMF analysis. It was found that HaCaT (human keratinocytes) after 72-h culture on graphene and 6H-SiC surfaces exhibited similar morphology to cells cultured on the PSt control. On the other hand, the MTT assay suggested better biocompatibility for 6H-SiC than for the graphene surface. Moreover, different preparation of graphene surfaces (first one without any further surface treatment, and the second one additionally disinfected by immersion in ethanol) resulted in more homogeneous and increased cell adhesion on ethanol-sterilised graphene surface [1]. Our study also confirmed the undeniable value of AMF analysis in the experiment involving the MAC-T cell line seeded on different surfaces (glass, glass coated with poly-D-lysine) (**Figure 7**). In the aforementioned study, surface analysis and cell height

/Si surfaces stud-

The present overview describes and compares widely used biocompatibility/cytotoxicity assays in nanomaterial studies. Due to the type of nanoparticles and their properties, applicability of popular assays used for engineered nanomaterial screening might be limited. The significant numbers of false-positive or false-negative signals are generated [16]. The tendency of nanoparticles to:


affect the results obtained in popular assays, thus classic cytotoxicity assays alone are not sufficient to evaluate nanomaterial biocompatibility.

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General Cytotoxicity and Its Application in Nanomaterial Analysis

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