Preliminary study on the mechanism by which exosomes derived from human exfoliated deciduous teeth improve the proliferation and osteogenic inhibitory effect of glucocorticoid-induced BMSCs.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a disease characterized by a collapsed femoral head caused by the overuse of glucocorticoids. Dysfunction of bone marrow mesenchymal stem cells (BMSCs) is an important pathological feature of SONFH. In this study, we investigated whether exosomes from SHEDs (stem cells from human exfoliated deciduous teeth) have a therapeutic effect on glucocorticoid-induced inhibition of proliferation and osteogenesis in BMSCs, and elucidated the underlying mechanisms involved. Primary dental pulp cells were isolated and cultured from human deciduous tooth pulp, SHEDs were isolated and purified by the limiting dilution method and exosomes were isolated from the supernatants of SHEDs by ultracentrifugation. The cell surface markers CD31, CD34, CD45, CD73, CD90 and CD105 were detected by flow cytometry. A Cell-Counting-Kit-8 assay was used to detect cell activity. ALP and Alizarin Red staining were used to identify osteogenic differentiation ability, and exosomes were identified using transmission electron microscopy, NanoFCM and Western blotting. PKH67 fluorescence was used to track the uptake of exosomes by BMSCs. Transcriptome analysis combined with quantitative real-time PCR was used to explore the underlying mechanism involved. Exosomes secreted by SHEDs can be endocytosed by BMSCs, and can partially reverse the inhibitory effects of glucocorticoids on the viability and osteogenic differentiation of BMSCs. Transcriptome sequencing analysis revealed that the differentially expressed mRNAs regulated by SHED-derived exosomes were enriched mainly in signaling pathways such as the apoptosis pathway, the PI3K-Akt signaling pathway, the Hippo signaling pathway and the p53 signaling pathway. qPCR showed that SHED-derived exosomes reversed the dexamethasone-induced upregulation of HGF and ITGB8 expression and the inhibition of EFNA1 expression, but further increased the dexamethasone-induced downregulation of IL7 expression. In conclusion, SHED-derived exosomes partially reversed the inhibitory effects of glucocorticoids on BMSC proliferation and osteogenesis by inhibiting the expression of HGF, ITGB8 and IL7, and upregulating the expression of EFNA1.

Liu F ，Wang X ，Xu J ，Lu Y ，Bai Y ，Lv J ... -  《-》

Osteogenic Effect of tsRNA-10277-Loaded Exosome Derived from Bone Mesenchymal Stem Cells on Steroid-Induced Osteonecrosis of the Femoral Head.

Steroids are known to inhibit osteogenic differentiation and subsequent bone formation in bone mesenchymal stem cells (BMSCs). However, little is known about the role of BMSC exosomes (Exos) and tRNA-derived small RNAs (tsRNAs) in steroid-induced osteonecrosis of the femoral head (SONFH). The objective of this study was to characterize the tsRNA expression profiles of plasma Exos collected from SONFH patients and healthy individuals using small RNA sequencing and further explore the effect of BMSC Exos carrying specific tsRNAs on osteogenic differentiation. Based on insights from small RNA sequencing, five differentially expressed (DE) tsRNAs were selected for quantitative real-time polymerase chain reaction (qRT-PCR). The regulatory networks associated with interactions of the tsRNAs-mRNA-pathways were reconstructed. The osteogenesis and adipogenesis in BMSCs were detected via ALP and oil red O staining methods, respectively. A total of 345 DE small RNAs were screened, including 223 DE tsRNAs. The DE tsRNAs were enriched in Wnt signaling pathway and osteogenic differentiation. We identified five DE tsRNAs, among which tsRNA-10277 was significantly downregulated in plasma Exos of SONFH patients compared to that in healthy individuals. Dexamethasone-induced BMSCs were associated with an increased fraction of lipid droplets and decreased osteogenic differentiation, whereas BMSC Exos restored the osteogenic differentiation of that. After treatment of tsRNA-10277-loaded BMSC Exos, the lipid droplets and osteogenic differentiation ability were found to be decreased and enhanced in dexamethasone-induced BMSCs, respectively. An altered tsRNA profile might be involved in the pathophysiology of SONFH. tsRNA-10277-loaded BMSC Exos enhanced osteogenic differentiation ability of dexamethasone-induced BMSCs. Our results provide novel insights into the osteogenic effect of BMSC Exos carrying specific tsRNAs on SONFH.

Fang S ，He T ，Jiang J ，Li Y ，Chen P ... -  《Drug Design Development and Therapy》

Exosomes derived from human exfoliated deciduous teeth ameliorate adult bone loss in mice through promoting osteogenesis.

Wei J ，Song Y ，Du Z ，Yu F ，Zhang Y ，Jiang N ，Ge X ... -  《-》

Effect of Jagged-1 and Dll-1 on osteogenic differentiation by stem cells from human exfoliated deciduous teeth.

The aim of the present study was to determine the influence of Notch ligands, Jagged-1 and Dll-1, on osteogenic differentiation by stem cells from human exfoliated deciduous teeth. Notch ligands were immobilized on tissue culture surface using an indirect affinity immobilization technique. Cells from the remaining of dental pulp tissues from human deciduous teeth were isolated and characterized using flow cytometry and differentiation assay. Alkaline phosphatase (ALP) enzymatic activity, osteogenic marker gene expression, and mineralization were determined using ALP assay, real-time polymerase chain reaction, and alizarin red staining, respectively. The isolated cells exhibited CD44, CD90, and CD105 expression but lack of CD45 expression. Further, these cells were able to differentiate toward osteogenic lineage. The upregulation of HES-1 and HEY-1 was observed in those cells on Dll-1 and Jagged-1 coated surface. The significant increase of ALP activity and mineralization was noted in those cells seeded on Jagged-1 surface and these results were attenuated when cells were pretreated with gamma secretase inhibitor. The significant upregulation of ALP and collagen type I gene expression was also observed in those cells seeded on Jagged-1 surface. The inconsistent Dll-1 induced osteogenic differentiation was found and high Dll-1 immobilized dose (50 nM) slightly enhanced alkaline phosphatase enzymatic activity. However, the statistical significant difference was not obtained as compared to the hFc control. The surface immobilization of Notch ligands, Jagged-1 and Dll-1, likely to enhance osteogenic differentiation of SHEDs. However, Jagged-1 had more ability in enhancing osteogenic differentiation than Dll-1 in our model.

Sukarawan W ，Peetiakarawach K ，Pavasant P ，Osathanon T ... -  《-》

Comparative characterization of stem cells from human exfoliated deciduous teeth, dental pulp, and bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are used clinically in tissue engineering and regenerative medicine. The proliferation and osteogenic differentiation potential of MSCs vary according to factors such as tissue source and cell population heterogeneity. Dental tissue has received attention as an easily accessible source of high-quality stem cells. In this study, we compared the in vitro characteristics of dental pulp stem cells from deciduous teeth (SHED), human dental pulp stem cells (hDPSCs), and human bone marrow mesenchymal stem cells (hBMSCs). SEHD and hDPSCs were isolated from dental pulp and analyzed in comparison with human bone marrow (hBM)MSCs. Proliferative capacity of cultured cells was analyzed using a bromodeoxyuridine immunoassay and cell counting. Alkaline phosphatase (ALP) levels were monitored to assess osteogenic differentiation. Mineralization was evaluated by alizarin red staining. Levels of bone marker mRNA were examined by real-time PCR analysis. SHED were highly proliferative compared with hDPSCs and hBMSCs. SHED, hDPSCs, and hBMSCs exhibited dark alizarin red staining on day 21 after induction of osteogenic differentiation, and staining of hBMSCs was significantly higher than that of SHED and hDPSCs by spectrophotometry. ALP staining was stronger in hBMSCs compared with SHED and hDPSCs, and ALP activity was significantly higher in hBMSCs compared with SHED or hDPSCs. SHED showed significantly higher expression of the Runx2 and ALP genes compared with hBMSCs, based on real-time PCR analysis. In bFGF, SHED showed significantly higher expression of the basic fibroblast growth factor (bFGF) gene compared with hDPSCs and hBMSCs. SHED exhibited higher proliferative activity and levels of bFGF and BMP-2 gene expression compared with BMMSCs and DPSCs. The ease of harvesting cells and ability to avoid invasive surgical procedures suggest that SHED may be a useful cell source for application in bone regeneration treatments.

Kunimatsu R ，Nakajima K ，Awada T ，Tsuka Y ，Abe T ，Ando K ，Hiraki T ，Kimura A ，Tanimoto K ... -  《-》