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
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[Role of M2 Macrophage Exosomes in Osteogenic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells under High-Glucose and High-Insulin].
To study the role of M2 macrophage-derived exosomes (M2-exo) in osteogenic differentiation and Hedgehog signaling pathway of mouse bone marrow mesenchymal stem cells (BMSCs) under in vitro high-glucose and high-insulin conditions.
RAW 264.7 cells were induced toward M2 macrophage polarization and then M2-exo were extracted and identified. Immunofluorescence assay was performed to detect the internalization of M2-exo by BMSCs. BMSCs were divided into the normal control group (Control group), the high-glucose and high-insulin group (HGI group), and the HGI with M2-exo intervention group (HGI+M2e group). BMSCs in the Control group were cultured in osteogenic inductive medium with 5.5 mmol/L glucose, but no insulin or M2-exo. BMSCs in the HGI group were cultured in osteogenic inductive medium with 25 mmol/L glucose and 174 nmol/L insulin. BMSCs in the HGI+M2e group were cultured in the same medium as that of the HGI group, with the additional treatment of 6, 30, 60 μg/mL M2-exo, respectively. After osteogenic induction for 7 days and 14 days, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively to assess the osteogenic differentiation potential of BMSCs from different groups. In addition, BMSCs in the Control group, HGI group, and HGI+M2e group treated with 30 μg/mL M2-exo were examined with qPCR after osteogenic induction for 14 days and Western blot after osteogenic induction for 21 days to assess the osteogenesis and the expression of Hedgehog pathway-related genes and proteins.
M2 macrophage polarization was induced successfully, with highly positive expression of CD206, the M2 polarization surface marker. The M2-exo had the typical structure of round or oval-shaped bilayered-membrane vesicles. The diameter distribution of M2-exo ranged from 50 to 125 nm (accounting for 99.14% of all M2-exo). M2-exo samples showed positive expression of exosomal markers CD9, CD63 and CD81 proteins. Immunofluorescence staining showed that M2-exo were taken up and internalized by BMSCs. After osteogenic induction for 7 days, the ALP activity of BMSCs in the HGI group was lower than that of the Control group. After interventions of 6 μg/mL, 30 μg/mL, and 60 μg/mL M2-exo, the ALP activity of the HGI+M2-exo group was significantly increased compared with that of the HGI group ( P<0.05). After osteogenic induction for 14 days, the number of mineralized nodules in the HGI group was lower than that in the Control group, and after intervention, only the HGI+M2e group treated with 30 μg/mL M2-exo showed higher level of mineralization than that in the HGI group ( P<0.05). qPCR analysis revealed that the expression levels of the osteogenesis-related genes, including Runx2, Alp and Ocn, and Hedgehog pathway-related genes, including Gli1, Smo and Ptch1, were downregulated in the HGI group, all being lower than those of the Control group to varying degrees, while 30 μg/mL M2-exo treatment could promote the up-regulation of these genes, showing significant difference in comparison with their expression levels in the HGI group ( P<0.05). In addition, Western blot analysis showed that the expression of the osteogenesis-related proteins, including RUNX2 and COL1A1, and GLI1, the Hedgehog signaling pathway protein, was down-regulated in the HGI group, while the expression of COL1A1 and GLI1 was up-regulated after 30 μg/mL M2-exo treatment, showing significant difference when compared with that of the HGI group ( P<0.05).
High glucose and high insulin had inhibitory effect on the osteogenic differentiation potential of BMSCs. After intervention with M2-exo, the Hedgehog signaling pathway in BMSCs was activated and the osteogenic differentiation potential was enhanced, suggesting that M2-exo might have therapeutic potentials for the treatment of diabetic bone disease.
Zhang C
，Bao LR
，Yang YT
，Wang Z
，Li Y
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SHED-derived conditioned exosomes enhance the osteogenic differentiation of PDLSCs via Wnt and BMP signaling in vitro.
The exosomes from human exfoliated deciduous teeth (SHED-Exos) have exhibited potential therapeutic role in dental and oral disorders. The biological effects of exosomes largely depend on cellular origin and physiological status of donor cell. In the present study, we explored the influence of conditioned exosomes from SHED with osteogenic induction on periodontal ligament stem cells (PDLSCs) in vitro. Conditioned SHED-Exos from a 3-day osteogenic supernatant were applied during PDLSCs osteogenic differentiation. We found that conditioned SHED-Exos had no cytotoxicity on PDLSCs viability assessed by CCK-8 assay. These SHED-Exos promoted PDLSCs osteogenic differentiation with deep Alizarin red staining, high alkaline phosphatase (ALP) activity and upregulated osteogenic gene expression (RUNX2, OPN and OCN). We further found BMP/Smad signaling and Wnt/β-catenin were activated by enhanced Smad1/5/8 phosphorylation and increased nuclear β-catenin protein expression. Inhibiting these two signaling pathways with specific inhibitors (cardamonin and LDN193189) remarkably weakened the enhanced osteogenic differentiation. Furthermore, Wnt3a and BMP2 were upregulated in SHED and SHED-Exos. Silencing Wnt3a and BMP2 in SHED-Exos partially counteracts the enhanced osteogenic differentiation. Our findings indicate that conditioned SHED-Exos-enhanced PDLSCs osteogenic differentiation was partly due to its carrying Wnt3a and BMP2. These data provide new insights into the use of SHED-Exos in periodontitis-induced bone defects therapy.
Wang M
，Li J
，Ye Y
，He S
，Song J
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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
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