## 4 Discussion  
Bone regeneration is still a challenge, particularly in maxillofacial sites. In this context, the use of biomaterials combined with stem cell cultures is giving promising results. Some of the authors of the present study have recently shown that scaffolds cultured with mesenchymal stem cells (MSC) and/or their derivatives, as conditioned medium (CM), extracellular vesicles (EV) or EV engineered with polyethylenimine (PEI-EV) improve bone regeneration. It is worth noting that EVs have been isolated, in the present as well as in the previous referred studies, by using ExoQuick TC commercial \begin{table}
\begin{tabular}{|c|c|c|c|c|c|} \hline  & \(\Delta\)MDD\({}^{\prime}\)\({}_{\rm peak}\) & \(\Delta\)MDD\({}^{\prime}\)\({}_{\rm mean}\) & \(\Delta\)MDD\({}^{\prime}\)\({}_{\rm fwhm}\) & \(\Delta\)MDD\({}^{\prime}\)\({}_{\rm low}\) & \(\Delta\)MDD\({}^{\prime}\)\({}_{\rm high}\) \\ \hline COL & 5.0 (4.3) & -9.8 (5.6) & 4.8 (3.4) & 5.0 (2.8) & 14.5 (8.8) \\ \hline COL/hPDLSCs & -1.8 (1.1) & -1.3 (2.6) & 4.8 (2.5) & -6.0 (0.3) & 3.5 (5.1) \\ \hline COL/hPDLSCs/CM & 3.3 (3.9) & 2.8 (3.8) & 5.3 (1.4) & -2.5 (2.7) & 8.0 (5.0) \\ \hline COL/hPDLSCs/EVs & 2.0 (9.7) & 7.5 (8.6) & 2.3 (6.6) & 5.3 (2.9) & 9.8 (5.1) \\ \hline COL/hPDLSCs/PEI-EVs & -4.0 (6.0) & -1.4 (0.5) & 0.4 (1.4) & -1.8 (1.1) & -1.0 (1.7) \\ \hline PLA & 4.8 (1.0) & 6.5 (1.2) & 0.5 (2.5) & 6.0 (1.3) & 7.0 (6.6) \\ \hline PLA/hGMSCs & 2.5 (1.9) & 2.0 (2.9) & 3.0 (3.3) & -1.0 (0.8) & 5.0 (6.1) \\ \hline PLA/hGMSCs/CM & -9.3 (3.9) & -8.5 (3.4) & -6.5 (2.8) & -2.0 (0.6) & \(-15.0\) (6.3) \\ \hline PLA/hGMSCs/EVs & 5.3 (7.0) & 10.3 (5.9) & 0.5 (2.3) & 9.8 (4.1) & 10.8 (8.0) \\ \hline PLA/hGMSCs/FEI-EVs & 16.8 (6.8) & 15.8 (6.8) & -11.0 (7.4) & 26.8 (1.6) & 4.8 (14.1) \\ \hline \end{tabular}
\end{table} 
Table 1: Bone relative mass density distribution (MDD\({}^{\prime}\))—results. Instead of the absolute indices, the following relative parameters were studied in each rat included in the groups of study: \(\Delta\)_MDD\({}^{\prime}\)\({}_{i}\)=MDD\({}^{\prime}\)\({}_{i,ctr}-\)MDD\({}^{\prime}\)\({}_{i,test}\)_, where i defines each index between peak, mean, fwhm, low and high. Data are shown as mean (standard deviation) for each group of study. agglutinant polymer. This means that the presence of residual agglutinating polymer cannot be excluded although, as reported in the Materials and Methods section, the engineering step could contribute to its removal and microscopic data [16; 18] did not highlight the presence of polymer residue. Nevertheless, no evidence of cytotoxicity related to these residues has been evidenced [16; 18] and [PERSON] et al. [53] demonstrated that ExoQuick TC isolation preserves biological activities of EV. In particular, both in vitro and in vivo results on rat calvarial bone defects showed greater expression of osteogenic markers in scaffolds grown with human-derived SC and with PEI-EV, also associated with increased protein levels of vascular endothelial growth factor (VEGF) and VEGF 2 receptor (VEGF2). Histological examination confirmed these deductions. Furthermore, the morphometric analysis deriving from microCT investigation confirmed on rat calvarial defects that scaffolds cultured with hSCs and PEI-EV exhibited increased bone regeneration and greater integration [20]. In this context, synchrotron radiation-based X-ray microtomography (microCT) has shown to be a powerful technique, able to achieve the analysis not only of bone microarchitecture but also of local mineralization at different hierarchical length scales, including the imaging of the lacuno-canalicular network [21; 22; 50; 54].