## 5 Cells and Polymer-Nanocomposite Interaction
### Cell Morphology  
Cell morphology studies allow for the identification of the shape, structure, and size of cells. Therefore, the regular examination of the cell morphology in culture is mandatory for a successful cell culture on biomaterials within a bio-hybrid system. The cell shape might be evaluated by using several commercial reagents that label cell membranes without affecting cell viability, such as the Lipophilic Tracers or Carbocyanine dyes [147]. The cytoskeleton architecture is directly correlated with the cell morphology. Therefore, the visualization of the F-actin filaments or the microtubules organization gives good indication of the form of the cells seeded on biomaterials. Thus, Phalloidin is a toxin belonging to a class of phallotoxins, found in the mushroom Amanita phalloides, that attack the host cells by a tight and selective binding with the F-actin filaments. On this basis, commercially available phalloidin containing fluorescent tags might be used to visualize F-actin widely with fluorescent microscopy [148]. Cytoplasmic microtubules can be visualized by indirect immunofluorescence staining of chemically fixed cells with tubulin antibody-conjugated with a fluorescent dye by using a fluorescence microscopy [137; 149]. The staining of F-actin and microtubules of cells seeded on biomaterials might be used to define the cell shape index [150]. This parameter is useful to establish if the biomaterial affects or influences the morphology of the cells. In order to investigate the cells morphology, scanning electron microscopy (SEM) is also used. SEM produces images by probing the sample with a focused electron beam that is scanned across the area of the specimen. During the interaction, the electron beam loses energy by a variety of mechanisms, converting it into alternative forms, which provide signals carrying information about the properties of the specimen surface, such as its topography and composition. SEM is able to image bulk samples up to many centimeters in size and has a great depth of field. Therefore, it can produce images that are good representations of the three-dimensional shape of the sample. SEM has improved resolution capacities with respect to the optical microscope, high depth of focus, simplified interpretation of the images due to the 3D impression, and use of different contrast mechanisms for the creation of images. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Stem Cell Types** & **Biomaterial** & **Differentiation** & **Ref.** \\ \hline \begin{tabular}{c} hBMSCs \\ iPSCs \\ ESCs \\ \end{tabular} & \begin{tabular}{c} Electrospun PLLA \\ Electrospun PLLA + HAP \\ \end{tabular} & No differentiation (stemess maintenance) & [72] \\ \begin{tabular}{c} iPSCs \\ ESCs \\ \end{tabular} & \begin{tabular}{c} Electrospun PLLA + HAP \\ \end{tabular} & Osteogenic & [65] \\ \hline \begin{tabular}{c} hUCMSCs \\ \end{tabular} & Film PLLA & Epiblast-like & [10] \\ \begin{tabular}{c} Film PLLA + MWCNTs \\ Film PLLA + O\({}_{2}\) \\ \end{tabular} & \begin{tabular}{c} Primitive endoderm-like \\ Acquisition of spheroids conformation \\ \end{tabular} & [10; 147] \\ \hline \begin{tabular}{c} hASCs \\ \end{tabular} & \
\begin{tabular}{c} Film PLLA \\ Film PLLA + O\({}_{2}\) \\ \end{tabular} &
\begin{tabular}{c} Morphology maintenance \\ Acquisition of spheroids conformation \\ \end{tabular} & [147] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Biological effects of poly-l-lactide (PLLA) polymer in different form (electrospun fibers and film), in combination with different nanofillers [hydroxyapatite (HAP) or multiwalled carbon nanotubes (MWCNTs)] or after oxygen (O\({}_{2}\)) modification in different stem cell types. One of the major problems with the use of SEM on biological materials is the sample preparation. Since the SEM operates in high vacuum levels, biological samples must be dehydrated before the analysis.