The aim of this study was to investigate if chemically produced nanotopography on titanium (Ti) surface induces osteoblast differentiation of cultured human bone marrow mesenchymal stem cells (hMSCs) by regulating the expression of microRNAs (miRs). It was demonstrated that Ti with nanotopography induces osteoblast differentiation of hMSCs as evidenced by upregulation of osteoblast specific markers compared with untreated (control) Ti at day 4. At this time-point, miR-sequencing analysis revealed that 20 miRs were upregulated (>2 fold) while 20 miRs were downregulated (>3 fold) in hMSCs grown on Ti with nanotopography compared with control Ti. Three miRs, namely miR-4448, -4708 and -4773, which were significantly downregulated (>5 fold) by Ti with nanotopography affect osteoblast differentiation of hMSCs. These miRs that directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (BMP-2) osteogenic signal, were upregulated by Ti with nanotopography. Overexpression of miR-4448, -4708 and 4773 in MC3T3-E1 pre-osteoblasts noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and therefore repressed the gene expression of key bone markers. Additionally, it was observed that the treatment with BMP-2 displayed a higher osteogenic effect on MC3T3-E1 cells grown on Ti with nanotopography compared with control Ti, suggesting that the BMP-2 signaling pathway was more effective on this surface. Taken together, these results indicate that a complex regulatory network involving a miR-SMAD-BMP-2 circuit governs the osteoblast differentiation induced by Ti with nanotopography.

Edited by Joel GottesfeldMicroRNAs (miRs) and Hox transcription factors have decisive roles in postnatal bone formation and homeostasis. In silico analysis identified extensive interaction between HOXA cluster mRNA and microRNAs from the miR-23a cluster. However, Hox regulation by the miR-23a cluster during osteoblast differentiation remains undefined. We examined this regulation in preosteoblasts and in a novel miR-23a cluster knockdown mouse model. Overexpression and knockdown of the miR-23a cluster in preosteoblasts decreased and increased, respectively, the expression of the proteins HOXA5, HOXA10, and HOXA11; these proteins' mRNAs exhibited significant binding with the miR-23a cluster miRNAs, and miRNA 3-UTR reporter assays confirmed repression. Importantly, during periods correlating with development and differentiation of bone cells, we found an inverse pattern of expression between HoxA factors and members of the miR-23a cluster. HOXA5 and HOXA11 bound to bone-specific promoters, physically interacted with transcription factor RUNX2, and regulated bone-specific genes. Depletion of HOXA5 or HOXA11 in preosteoblasts also decreased cellular differentiation. Additionally, stable overexpression of the miR-23a cluster in osteoblasts decreased the recruitment of HOXA5 and HOXA11 to osteoblast gene promoters, significantly inhibiting histone H3 acetylation. Heterozygous miR-23a cluster knockdown female mice (miR-23a Cl WT/ZIP ) had significantly increased trabecular bone mass when compared with WT mice. Furthermore, miR-23a cluster knockdown in calvarial osteoblasts of these mice increased the recruitment of HOXA5 and HOXA11, with a substantial enrichment of promoter histone H3 acetylation. Taken together, these findings demonstrate that the miR-23a cluster is required for maintaining stagespecific HoxA factor expression during osteogenesis.Mammalian homeobox (Hox) 3 developmental transcription factors have diverse roles in bone development and postnatal bone formation (1-3). These roles include skeletal element formation (4), anterior-posterior homeotic development (4 -10), and limb and axial skeleton formation (11)(12)(13)(14)(15). In fact, cooperation among Hox genes is critical in skeletal development (2, 13, 16 -18). Moreover, Leucht et al. (19) reported that the Hox gene expression status influences the process of adult bone regeneration. A high-throughput ChIP-sequencing study revealed that HOXD13 binds numerous genes that act in key pathways required for early limb and skeletal patterning (20). Furthermore, Wan and Cao (21) reported that a SMAD-HOX association is required to decipher the mechanism of bone morphogenetic protein signaling in osteoblast growth and differentiation. In previous studies, we demonstrated that selective recruitment of HOX transcription factors to bone-specific chromatin at specific stages of osteoblast maturation mediates gene activation (1,(22)(23)(24). Hence, multiple levels of transcriptional and epigenetic regulation by HOX proteins must be examined to define the complete m...

bStudies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis. D entinogenesis is the process by which dentin, the major mineralized tissue of teeth, is formed through progressive cytodifferentiation of progenitor cells to mature odontoblasts (1). Multiple layers of gene regulation, including those by microRNA (miRNA), orchestrate the physiologic process of dentinogenesis in a stage-specific manner (2). Progenitor cells, including dental papilla cells or dental follicle cells, derived from the ectomesenchyme of the cranial neural crest, differentiate into preodontoblasts and produce predentin. Predentin stimulates further differentiation of the cells it surrounds, giving rise to mature odontoblasts that produce dentin. Odontoblast secretion of dentin extracellular matrix proteins, including dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1), aids in the process of mineralization that forms primary dentin. However, the mechanisms of odontoblast-specific gene regulation by miRNA during dentinogenesis are not clearly understood.miRNAs are endogenous, noncoding RNAs implicated in posttranscriptional RNA silencing (3-9). The importance of miRNAs in skeletogenesis has been shown in mice by loss-offunction analysis of proteins involved in miRNA processing (Drosha and DGCR8), maturation (Dicer), and silencing (argonaute 2; AGO2), which revealed embryonic lethality and severe developmental defects upon loss of these proteins (10-15). Furthermore, cartilage-specific deletion of Dicer led to accelerated differentiation and subsequent cell death (11), whereas osteoblast-and osteoclast-specific deletion increased bone mass (13,16). Current studies on miRNA regulation of gene expression indicate a key role for this process in tooth development (17)(18)(19)(20) and in controlling cellular signaling (18,(21)(22)(23)(24)(25) and differentiation (2,26). However, ...