## 5 Cells and Polymer-Nanocomposite Interaction
### Cell Differentiation  
To be successful, tissue engineering must guarantee the correct function of the cells. This includes the stem cell properties, the stem cell specification process, and their differentiation. The different cell status is strictly correlated with the final application of the tissue engineering biohybrid system [10; 151; 4]. In particular, cell differentiation, as the process through committed progenitor cells evolves toward a specialized cell reaching its mature form and function, represents a goal standard of the tissue engineering. (Table 2)  
Currently, the biological status of a cell might be evaluated with many innovative strategies that give a landscape information about the genes and proteins expressed in a given cell type, such as the genome-wide gene expression profiling or the proteome-wide expression profiling [152; 153; 154]. Alternatively, as stem, committed and differentiated cells are specifically correlated to a biological function, the monitoring of the correct status might be studied by evaluating a more restricted number of genes and proteins. In fact, as these molecules are specifically expressed in a type of cell with a specific phenotype (e.g., stem cells, committed, and differentiated), they are considered as the identification markers to be monitored. In this case, techniques as real-time polymerase chain reaction (Real-Time PCR or qPCR) (to measure the gene expression), Western blotting (to measure the expression of proteins), and the immunofluorescence (to visualize the proteins into the cells) might be helpful to evaluate the expression of such markers. Western blotting is a broadly analytical technique used to identify specific proteins in a biological sample, based on the capability of antibodies to react with a specific target protein. Then, the complex target protein-antibody is detected by a secondary antibody conjugated with a dye or an enzyme that may be easily measured. Real-Time PCR measures the amplification of a DNA target during the procedure. Generally, the template is the cDNA obtained by the purified and retro-transcribed mRNAs of given cells (e.g., cells cultured on a biomaterial). Immunofluorescence and colorimetric staining for monitoring specific differentiation markers are also helpful for addressing the changes of phenotype of cells cultured on biomaterials. For instance, Alizarin Red S and Von Kossa staining are classical assays for evaluating the osteogenic differentiation [65], whereas the Oil Red staining is the most useful assay for evaluating the audiogenic differentiation process [65]. The above-mentioned techniques were, for example, used to investigate the effect of nanocomposite fibrous materials of PLLA loaded with 1 or 8 wt % of calcium-deficient nanohydroxyapatite (d-HAp) on human-bone-marrow-derived mesenchymal stem cells, murine-induced pluripotent stem cells, and murine embryonic stem cells. The d-Hap incorporated in the PLLA polymer matrix by electrospinning consisted of needle-like particles, with width of 10-30 nm and length of 50-100 nm. We have demonstrated that the culture of the above-mentioned stem cell types on PLLA/d-HAp nanocomposites in the absence of soluble osteogenic factors induced the expression of the osteogenic markers. This differentiation process was absent on all stem cell types grown on neat PLLA cultures [71]. The techniques described in this review allowed us to demonstrate that the active interaction between the human umbilical cord matrix stem cells and PLLA and PLLA/Multi Walled Carbon Nanotubes (MWCNTs) nanocomposite films, resulted in the stem cell acquisition of a spheroid conformation and in the transition of the stem cell fate. For instance, the F-actin staining allowed to visualize the spheroids conformation of the cells and how they directly respond to the surface and the bulk properties (e.g., electric, dielectric, and thermal) of neat and nanocomposite PLLA films (mechanotransduction axis).