## 4 Discussion  
Raman and TGA measurements proved the presence of ammonia-functionalized graphene-oxide, G-N, on the equine bone block. In particular, the amount of G-N was directly dependent on the concentration of G-N in the dispersion used for the coating protocol. The coating was demonstrated to be relatively homogenous, especially for the more concentrated sample. Mesenchymal stem cells (MSCs) are multipotent osteogenic stem cells that are induced to differentiate into osteoblasts via BMP signaling [46]. So far, most of the miRNAs characterized in osteogenesis appear to maintain the MSC-state [46], which is lost upon BMP2 signaling [35]. MiR-133 functions by targeting p27 (a cell cycle regulator), Hox10a, and RUNX2, which induces expression of alkaline phosphatase and osteoclast, and miR-135 targets SMAD5, which activates expression of early osteoblast differentiation markers [35]. p27 is upregulated in response to BMP2, which downregulates miR-133 and miR-135, and SMAD5 together with C/EBP can bind to RUNX2 and increase its transcriptional activity [47]. RUNX2, along with osteoclast and b-catenin, is essential for osteoblast formation [46]. It was found out that miR-133a at 7 days in GM is so downregulated that it is not measurable in some experimental points. Indeed, we found that in GM in the presence of G-N10 plus melatonin miR-133a was not expressed, while in G-N2 and G-N10 it was downregulated in these conditions compared to Ctrl. A similar outcome was shown by the miR-133b expression in GM condition, at 7 days, which resulted so strongly downregulated for G-N2 and G-N2 + Mel that it was not detectable in some experimental points, indeed for G-N10 and G-N10 + Mel which is statistically significantly downregulated compared to Ctrl. MiR-135 was highly downregulated in all time point experimental GM/DM conditions in the presence of G-N2 and G-N10. Our results achieved on hDPSCs cultured in DM show the downregulation of miR-133a after 7, 14, and 21 days of hDPSCs. MiR-133b was strongly downregulated after 7 days of culture in GM in the presence of G-N10 plus melatonin. Furthermore, the downregulation seemed to rely on the type of functionalized block with a decreasing expression from equine bone blocks to G-N10 bone blocks. The expression of miR-135a was undetermined at 7 and 14 days of culture in DM in the presence of G-N10 + Mel, but at 21 days we observed a downregulation trend dependent on the presence of both G-N and melatonin. Taken together, these data suggest that the downregulation observed for miR-133a, miR-133b, and miR-135, which normally inhibits osteoblast differentiation, strongly supports the differentiation process through the activation of RUNX2 and SMAD5. Another inhibitor of osteogenesis is miR-206 (also involved in myogenesis), which was highly expressed in the osteoprogenitor cells of the periodondrium and was significantly downregulated on initiation of osteogenesis. [PERSON] and collaborators [47] showed that knockdown of miR-206 in primary mouse osteoblasts was sufficient to induce differentiation, while over-expression of miR-206 inhibited differentiation and affected bone formation rate, leading to decreased bone mass independent of a shift from osteogenesis to myogenesis. As osteogenesis proceeds, more miRNAs are upregulated, with maximal expression peaking during the mineralization stage when non-collagen proteins that facilitate the last step in terminal differentiation are secreted [48]. After 14 days of hDPSCs culture in GM, miR-206 was downregulated in the G-N2- and G-N10-coated bone blocks with and without melatonin compared to Ctrl. At 7 and 21 days, no statistically significant differences in the distinct experimental conditions were underlined. miR-29b also targets HDAC4, which decreases RUNX2 expression via deacetylation [48]. MiR-29b expression was upregulated at 7 and 14 days of hDPSCs GM culture in the presence of G-N10 with and without melatonin compared to Ctrl; while in DM we found an upregulation only for G-N10 plus melatonin compared to Ctrl, at 7 days.