## 1 Introduction
Graphene-oxide (GO) enriched materials have raised a growing interest in many research areas, due to the capacity of GO to promote cell adhesion, proliferation, and differentiation [27; 28; 29]. The GO was demonstrated to be able to guarantee the viability and to enhance osteogenic differentiation of stem cells when compared with traditional substrates or scaffolds [27; 28; 29; 30]. miRNAs are regulators of bone development influencing the progression of differentiation through regulating Bone Morphogenetic Protein (BMP) signaling [31] targeting both SMAD5, a transcription factor that activates early osteoblast differentiation markers and RUNX2 which influences downstream genes for the osteoblast phenotype [32; 33] and affects osteoclastin expression [34]. BMP2 signaling appears to release osteo-specific mRNAs from miRNA inhibition, upregulating only a small number of miRNAs early in differentiation and downregulating the vast majority [35]. There is some overlap of miRNAs from the cardiogenesis, myogenesis, neurogenesis, and hematopoiesis pathways. miR-133 and miR-135 act in concert to regulate osteoblastogenesis in MSCs. MiR-133a and miR-135 inhibit differentiation [36]. The purpose of the present in vitro study was to investigate the osteoregenerative potential of ammonia-functionalized graphene-oxide (G-N)-coated equine bone substitute materials and melatonin on hDPSC. The innovative aspect of the present study was to assess the potential osteoinductive properties of these new hybrid materials and therefore their ability to improve bone regeneration. The idea to coat the equine bone block with G-N rather than with simple GO was born by the aim to enrich with amino groups the graphene-oxide coating in order to mimic proteins (due to the simultaneous presence of carboxylic and amine moieties on the graphitic surface), widely present in the extracellular matrix during bone formation. Furthermore, in a recent study, the use of amine-functionalized graphene filler for different biomedical applications evidenced the promotion of cell conductivity and consequently better cell growth and proliferation [37]. [PERSON] et al. reported that the amine-functionalized graphene used in combination with bone cement has been reported in vivo as an excellent healing agent for fractured bone, as evident from the calcination in just 20 days against no healing in the control [37]. Melatonin was associated with a G-N-enriched equine bone block in order to evaluate a possible synergic effect due to the capacity of melatonin to activate the differentiated cells, i.e., osteoclasts--to initiate bone resorption--and osteoblasts. The osteoregenerative potential of these association was evaluated following different measurements. The degree of proliferation and mostly of differentiation of hDPSC was determined by the isolation, quantification, and expression of microRNAs, particularly miR-133a/b, miR-135a, miR-206, miR-29b, and miR-let-7b, which are proper markers of bone growth promotion. Furthermore, real-time PCR analysis was performed to analyze the expression of RUNX2 and SMAD5 genes, bone formation-linked transcriptional factors. Finally, the levels of osteocalcin, released in the culture medium, were assessed to evaluate the bone turnover stage and histological analyses.