## 4 Discussion
At 14 days a statistically significant modulation was not found, except an upregulation for the G-N10 condition without melatonin in the GM condition. Instead at 14 days in DM, mir-29b was downregulated in G-N10 with and without melatonin. Finally, at 21 days of both GM and DM, it was possible to notice a statistically significant upregulation in the presence of G-N2 bone blocks compared to Ctrl. Instead, in DM in the presence of G-N10 plus melatonin, mir-29b was upregulated in G-N10 with melatoninin compared to Ctrl. These results suggest that the osteogenic stimulation action of miR-29b could begin later than other miRNAs, as it is a promoter of the later osteogenesis. This latter result could be explained considering that maybe the G-N2 is related to a differentiation caused by the presence of both blocks and melatonin. Conversely, the G-N10 plus melatonin, at 7 and 21 days in DM, drove the cells to a late or terminal differentiation stage. MiR-let-7b in vitro significantly promoted osteogenesis and counteracted adipogenesis of MSCs, by targeting high-mobility group AT-hook 2 (HMGA2) expression. [PERSON] and colleagues [49] detected the dynamic miRNA expression pattern in femurs of mice and found that miR-let-7 is a positive regulator of bone development. Gain and loss of-function analysis showed that miR-let-7 can promote osteogenic differentiation, suppress adipogenesis of human adipose-derived mesenchymal stem cells in vitro, and enhance bone formation in vivo as shown by the detection of expression of RUNX2, BSP, OC, and Col1a1 (late marker of osteogenesis), as well as ALP and OPN (early marker of osteogenesis). Results on miR-let-7b expression on hDPSCs cultured in GM and DM at 7 days demonstrate an increasing upregulation trend in the presence of both bone blocks and melatonin. MiR-let-7b expression at 7 days in GM was statistically significantly upregulated in the presence of both G-N2 and G-N10 conditions compared to Ctrl. At 7 days in DM, in the presence of G-N10 with and without melatonin, miR-let-7b was upregulated compared to Ctrl. At 21 days of hDPSCs culture in both GM and DM, miR-let-7b was statistically significantly upregulated in the presence of the G-N2 bone blocks alone, compared to Ctrl, even if in G-N10 GM condition it was upregulated with melatonin. However, overall, the data collected show an upregulation of miR-let-7b, confirming its role in enhancing the differentiation process of hDPSCs. The activation of the osteogenic signaling described above was also confirmed in our study by the increased upregulation of SMAD5 and RUNX2 in the presence of G-N-coated equine bone block and melatonin. Similarly, both RUNX2 and SMAD5 were upregulated in the differentiation condition at 14 days while in the growth condition at 21 days. This discrepancy could be due to the lack of the effect of differentiation factors present in differentiation medium. Moreover, it is feasible to suppose a temporal wave of gene expression as the signaling was activated at 14 days, while at 21 days it was extinguished. Noteworthy, the G-N-coated bone block and melatonin seemed to have a similar outcome without an additive effect. This outcome let us suppose that bone blocks and melatonin could lead to a push towards the osteogenic process through RUNX2. The gene expression behavior showed by SMAD5 strengthened the hypothesis of an osteogenic process mediated by RUNX2 which targeted SMAD5 downstream. Osteoclain, representing a late-stage marker of differentiation, was assessed. Our data showed that after 14 days of culture osteocllin levels were significantly increased both in the presence of G-N and G-N plus melatonin both in growth and differentiation conditions. This outcome clearly confirms that the presence of the bone block or melatonin are a key factor for pushing hDPSCs towards the differentiation process. The histological analysis was performed through a specific technique able to provide a section of the hard tissues, metals, or non-demineralized tissues, and polymerized with methacrylate resin [50].