Concentrated growth factor, a novel autologous plasma extract, contained various growth factors which promoted tissue regeneration. In this study, we aimed to investigate the biological effects of concentrated growth factor on human dental pulp stem cells. The microstructure and biocompatibility of concentrated growth factor scaffolds were evaluated by scanning electron microscopy. Cell proliferation and migration, odontoblastic and endothelial cell differentiation potential were assessed after exposing dental pulp stem cells to different concentrations (5%, 10%, 20%, 50%, or 80%) of concentrated growth factor extracts. The results revealed that concentrated growth factor scaffolds possessed porous fibrin network with platelets and leukocytes, and showed great biocompatibility with dental pulp stem cells. Higher cell proliferation rates were detected in the concentrated growth factor-treated groups in a dose-dependent manner. Interestingly, in comparison to the controls, the low doses (<50%) of concentrated growth factor increased cell migration, alkaline phosphatase activity, and mineralized tissue deposition, while the cells treated in high doses (50% or 80%) showed no significant difference. After stimulating cell differentiation, the expression levels of dentin matrix protein-1, dentin sialophosphoprotein, vascular endothelial growth factor receptor-2 and cluster of differentiation 31 were significantly upregulated in concentrated growth factor-supplemented groups than those of the controls. Furthermore, the dental pulp stem cell-derived endothelial cells co-induced by 5% concentrated growth factor and vascular endothelial growth factor formed the most amount of mature tube-like structures on Matrigel among all groups, but the high-dosage concentrated growth factor exhibited no or inhibitory effect on cell differentiation. In general, our findings confirmed that concentrated growth factor promoted cell proliferation, migration, and the dental pulp stem cell-mediated dentinogenesis and angiogenesis process, by which it might act as a growth factor-loaded scaffold to facilitate dentin-pulp complex healing.

MicroRNAs (miRNAs) are the most abundant RNA species found in serum, and recently, several miRNAs have been found to be associated with osteoporosis. However, the development of such associated miRNAs into diagnostic and therapeutic targets remains unaddressed, mostly because of a lack of functional validation. Here, we identified circulating miR-338 associated with postmenopausal osteoporosis, and performed functional validation in vivo and in vitro . Methods : We collected the serum from postmenopausal osteoporosis patients (N=15) and female volunteers of the same age but with normal bone density (N=15) and examined the enrichment of miR-338 cluster. We also confirmed such enrichment using mice subjected to ovariectomy at different stages. We employed primary bone marrow stromal cells from mice and the MC-3T3 cell line along with CRISPR, RNA-seq and ChIP-qPCR to validate the biological function of secreted miR-338 cluster on osteoblastic differentiation and their upstream regulators. Moreover, we generated miR-338 knockout mice and OVX mice injected with an inhibitor against miR-338 cluster to confirm its biological function in vivo . Results : We observed a significant enrichment of miR-338 cluster in postmenopausal osteoporosis patients. Such enrichment was also prominent in serum from mice subjected to ovariectomy and was detected much earlier than bone density decreases revealed by micro-CT. We also confirmed the presence of an estrogen-dependent Runx2 / Sox4 /miR-338 positive feedback loop that modulated osteoblast differentiation, providing a possible explanation for our clinical findings. Moreover, deletion of the miR-338 cluster or direct intravenous injection of an miR-338 cluster inhibitor significantly prevented osteoporosis after ovariectomy. Conclusion : Circulating miR-338 cluster in the serum could serve as a promising diagnostic and therapeutic target for postmenopausal osteoporosis patients.

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WW domain-containing E3 Ub-protein ligase 2 (WWP2) belongs to the homologous to E6AP C-terminus (HECT) E3 ligase family. It has been explored to regulate osteogenic differentiation, chondrogenesis, and palatogenesis. Odontoblasts are terminally differentiated mesenchymal cells, which contribute to dentin formation in tooth development. However, it remained unknown whether WWP2 participated in odontoblast differentiation. In this study, WWP2 was found to be expressed in mouse dental papilla cells (mDPCs), odontoblasts, and odontoblastic-induced mDPCs by immunohistochemistry and Western blotting. Besides, WWP2 expression was decreased in the cytoplasm but increased in the nuclei of differentiation-induced mDPCs. When Wwp2 was knocked down, the elevated expression of odontoblast marker genes ( Dmp1 and Dspp) in mDPCs induced by differentiation medium was suppressed. Meanwhile, a decrease of alkaline phosphatase (ALP) activity was observed by ALP staining, and reduced formation of mineralized matrix nodules was demonstrated by Alizarin Red S staining. Overexpression of WWP2 presented opposite results to knockdown experiments, suggesting that WWP2 promoted odontoblastic differentiation of mDPCs. Further investigation found that WWP2 was coexpressed and interacted with KLF5 in the nuclei, leading to ubiquitination of KLF5. The PPPSY (PY2) motif of KLF5 was essential for its physical binding with WWP2. Also, cysteine 838 (Cys838) of WWP2 was the active site for ubiquitination of KLF5, which did not lead to proteolysis of KLF5. Then, KLF5 was confirmed to be monoubiquitinated and transactivated by WWP2, which promoted the expression of KLF5 downstream genes Dmp1 and Dspp. Deletion of the PY2 motif of KLF5 or mutation of Cys838 of WWP2 reduced the upregulation of Dmp1 and Dspp. Besides, lysine (K) residues K31, K52, K83, and K265 of KLF5 were verified to be crucial to WWP2-mediated KLF5 transactivation. Taken together, WWP2 promoted odontoblastic differentiation by monoubiquitinating KLF5.