## 3 Results  
Synchrotron-based high resolution tomographic (microCT) analyses were performed on rat calvaria of the following groups of study:  
1. COL (N = 4): rats grafted with COL;
2. COL/hPDLSCs (N = 4): rats grafted with COL enriched with hPDLSCs;
3. COL/hPDLSCs/CM (N = 4): rats grafted with COL enriched with hPDLSCs and CM;
4. COL/hPDLSCs/EVs (N = 4): rats grafted with COL enriched with hPDLSCs and EVs;
5. COL/hPDLSCs/PEI-EVs (N = 4): rats grafted with COL enriched with hPDLSCs and PEI-EVs;* PLA (N = 4): rats grafted with PLA;
* PLA/hGMSCs (N = 4): rats grafted with PLA enriched with hGMSCs;
* PLA/hGMSCs/CM (N = 4): rats grafted with PLA enriched with hGMSCs and CM;
* PLA/hGMSCs/EVs (N = 4): rats grafted with PLA enriched with hGMSCs and EVs;
* PLA/hGMSCs/PEI-EVs (N = 4): rats grafted with PLA enriched with hGMSCs and PEI-EVs. MicroCT images of chosen scaffolds and some representative repaired sites in retrieved samples are shown in Figure 1. The 3D reconstructions of the COL and the PLA scaffolds before grafting were shown in Figure 1a,d, respectively. Moreover, representative 3D reconstructions of defects grafted with the sole biomaterial (Figure 1b for the COL and Figure 1e for the PLA) and with the biomaterial seeded with stem cells and engineered extracellular vesicles were also shown (Figure 1c for the COL/hPDLSCs/PEI-EVs and Figure 1f for the PLA/hGMSCs/PEI-EVs). All tissues, except for mineralized bone and residual scaffolds, were made virtually transparent. From the simple 3D reconstructions of the repaired defects, volume mismatches in terms of the amount of newly formed bone were evident between defects grafted with the sole biomaterials and those also treated with stem cells and engineered extracellular vesicles. In particular, the latter showed greater volumes and connectivity of newly formed bone than the former group, in agreement with previous morphometric studies on the same samples [20]. However, the present study was focused on the study of the relative bone mineral density distribution (MDD\({}^{r}\)), describing and quantifying the calcium concentration and distribution (weight %) in the different groups of study. Indeed, independently from the volumetric growth of the newly formed bone in the different conditions, it is fundamental to evaluate the mineral density of the regenerated bone, strictly related to the kinetics of bone remodeling, as very recently shown by some of the authors [50]. In particular, this study was performed with an approach proposed by [PERSON] [52] and was focused on the mineralized bone portion. The method was supported by the parameters indicated in Figure 2a. In order to verify the sensitivity of the method, we first studied the distribution of both the mean relative mass density (MDD\({}^{r}\)mean) and the full width at half maxima of the distribution (MDD\({}^{r}\)fwhm) in the whole set of \(\mathit{ctr}\) sites (outside the defect), as shown with the support of box plots in Figure 2b-c. The high variability of the mean and of the fwhm values is motivated by the extremely high sensitivity of the synchrotron-based phase-contrast microCT combined with the use of different rats having different MDD in the studied calvarial site. Moreover, for the complete \(\mathrm{ctr}\) and test sets of data, we plotted the dispersion graphs describing mean vs. fwhm dependency, the two regression equations and their coefficient of determination \(R^{2}\) (Figure 2d). \(R^{2}\) values were found to be extremely low (\(\mathrm{R^{2}_{\mathrm{ctr}}}\) = 0.07; \(\mathrm{R^{2}_{\mathrm{test}}}\) = 0.20); thus, the high variability of the mean and of the fwhm values combined to their low correlation motivated us to investigate, instead of the absolute indices, the following relative parameters in each rat included in the groups of study:  
\[\Delta\mathit{MDD^{r}}_{i}=\mathit{MDD^{r}}_{i,\mathit{ctr}}-\mathit{MDD^{r}}_ {i,\mathit{test}},\]  
where \(i\) defines each parameter between peak, mean, fwhm, low and high.