Matrix vesicles (MtVs), a type of extracellular vesicles (EVs), are secreted by osteoblasts and initiate bone mineralization. Recent evidence suggests that MtVs are also involved in intercellular communication, inhibiting osteoclast formation, and promoting bone repair. These findings suggest that MtVs might hold therapeutic potential for osteoporosis; however, their effects on osteoporosis have incompletely remained understood. In this study, we investigated the effects of mineralized mouse osteoblastic MC3T3-E1 cell-derived collagenase-released EVs (MC-CREVs) on bone loss in ovariectomized mice, using C2C12 myotube-derived EVs (MT-EVs) as a control. MC-CREVs approximately 100 nm in size were isolated from collagenase-digested mineralized MC3T3-E1 cultures by density gradient ultracentrifugation. Systemic administration of MC-CREVs restored trabecular bone mineral density, bone volume, and trabecular thickness in ovariectomized mice, while the number of osteoclasts on the trabecular bone surface was decreased. In vitro experiments, MC-CREVs suppressed osteoclast formation and osteoclast marker gene expression induced by RANKL more effectively than MT-EVs in RAW264.7 cells. The miRNA array analyses showed that miR-1224-5p was abundantly expressed in MC-CREVs. In conclusion, we demonstrated that systemic administration of MC-CREVs is effective for trabecular bone loss presumably through suppression of bone resorption in ovariectomized mice.

Similar to embryonic stem cells, induced pluripotent stem (iPS) cells can differentiate into various cell types upon appropriate induction, and thus, may be valuable cell sources for regenerative medicine. However, iPS cells have not been reported to differentiate into odontogenic cells for tooth regeneration. Here we demonstrated that neural crest-like cells (NCLC) derived from mouse iPS cells have the potential to differentiate into odontogenic mesenchymal cells. We developed an efficient culture protocol to induce the differentiation of mouse iPS cells into NCLC. We confirmed that the cells exhibited neural crest (NC) cell markers as evidenced by immunocytochemistry, flow cytometry, and real-time reverse transcription-polymerase chain reaction. Further, in recombination cultures of NCLC and mouse dental epithelium, NCLC exhibited a gene expression pattern involving dental mesenchymal cells. Some NCLC also expressed dentin sialoprotein. Conditioned medium of mouse dental epithelium cultures further enhanced the differentiation of NCLC into odontoblasts. These results suggest that iPS cells are useful cell sources for tooth regeneration and tooth development studies.

During tooth development, inner enamel epithelial (IEE) cells differentiate into enamel-secreting ameloblasts, a polarized and elongated cellular population. The molecular underpinnings of this morphogenesis and cytodifferentiation, however, are not well understood. Here, we show that Rho-associated coiled-coil-containing protein kinase (ROCK) regulates ameloblast differentiation and enamel formation. In mouse incisor organ cultures, inhibition of ROCK, hindered IEE cell elongation and disrupted polarization of differentiated ameloblasts. Expression of enamel matrix proteins, such as amelogenin and ameloblastin, and formation of the terminal band structure of actin and E-cadherin were also perturbed. Cultures of dental epithelial cells revealed that ROCK regulates cell morphology and cell adhesion through localization of actin bundles, E-cadherin, and β-catenin to cell membranes. Moreover, inhibition of ROCK promoted cell proliferation. Small interfering RNA specific for ROCK1 and ROCK2 demonstrated that the ROCK isoforms performed complementary functions in the regulation of actin organization and E-cadherin-mediated cell-cell adhesion. Thus, our results have uncovered a novel role for ROCK in amelogenesis.