[Role of M2 Macrophage Exosomes in Osteogenic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells under High-Glucose and High-Insulin].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

[Human umbilical cord mesenchymal stem cells co-cultured with dental pulp cells in vitro and its effect on cell biological behaviors].

PURPOSE: To investigated the effect of human umbilical cord mesenchymal stem cells (hUCMSCs) and human dental pulp cells (hDPCs) on cell biological behaviors by co-culture system in vitro. METHODS: hUCMSCs and hDPCs were obtained by primary culture. A culture system of hUCMSCs and hDPCs induced by BMP2 was established in vitro. hUCMSCs and hDPCs were co-cultured at the ratio of 1:1, 1:5 and 5:1. The optimum ratio of each group was selected to further experiment. The formation of calcium nodule was stained by alizarin red staining at 21 day. The expression of DSPP，ALP，DMP1，OCN，VEGF，HGF and Nanog gene was detected by real-time quantitative PCR at 7 day and 14 day. 1:1 group and hUCMSCs, hDPCs group were selected for alizarin red staining at 21 day according to PCR results. Statistical analysis was performed using SPSS 21.0 software package. RESULTS: Calcified nodules formation in 1:1 group was significantly higher than in hUCMSCs group (P<0.05), close to that in hDPCs. qPCR showed that the mRNA expression of DSPP, ALP, DMP1, OCN, VEGF and HGF in 1:1 group was significantly higher than that in hUCMSCs (P<0.05); mRNA expression of Nanog in 1:1 group was significantly lower than in hUCMSCs group (P<0.05). The results of alizarin red staining showed that the OD value of 1:1 group was significantly higher than that of hUCMSCs group (P<0.05). CONCLUSIONS: The cells can be induced to differentiate into odontoblastoid-like cells and the mRNA expression of angiogenic factors was stimulated by hUCMSCs co-culure wih hDPCs.

Glycan Stimulation Enables Purification of Prostate Cancer Circulating Tumor Cells on PEDOT NanoVelcro Chips for RNA Biomarker Detection.

A glycan-stimulated and poly(3,4-ethylene-dioxythiophene)s (PEDOT)-based nanomaterial platform is fabricated to purify circulating tumor cells (CTCs) from blood samples of prostate cancer (PCa) patients. This new platform, phenylboronic acid (PBA)-grafted PEDOT NanoVelcro, combines the 3D PEDOT nanosubstrate, which greatly enhances CTC capturing efficiency, with a poly(EDOT-PBA-co-EDOT-EG3) interfacial layer, which not only provides high specificity for CTC capture upon antibody conjugation but also enables competitive binding of sorbitol to gently release the captured cells. CTCs purified by this PEDOT NanoVelcro chip provide well-preserved RNA transcripts for the analysis of the expression level of several PCa-specific RNA biomarkers, which may provide clinical insights into the disease.

[miRNA profile of the human dental pulp cells during odontoblast differentiation induced by BMP-2].

PURPOSE: To screen and verify the differentially expressed microRNAs (miRNAs) during the differentiation of human dental pulp cells (hDPCs) to odontoblasts induced by BMP-2. METHODS: The isolated hDPCs were cultured in vitro and induced by BMP-2. The levels of ALP, DMP-1 and DSPP were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). The potential characteristics of hDPCs were investigated by miRNA microarray and highly expressed miRNAs were selected with bio-information software for predicting target genes and their biological functions. Then the results were validated using qRT-PCR analysis for the selected miRNAs. Statistical analysis was performed using SPSS 18.0 software package. RESULTS: The expression of ALP, DSPP, and DMP-1 showed significantly higher levels in BMP-2 induced groups compared to the control group(P<0.05). A total of 36 miRNAs were changed (i.e. 20 miRNAs were up-regulated and 16 were down-regulated). The results of qRT-PCR were consistent with the microarray results. GO categories revealed that they were mainly associated with biological process(37.8%), cellular component (29%), molecular function(33%), while the function of other 0.2% genes remained unknown. CONCLUSIONS: This study identified differential expression of miRNAs in BMP-2-induced odontoblastic differentiation of hDPCs, thus contributing to further investigations of regulatory mechanisms and biological effect of target genes in BMP-2-induced odontoblastic differentiation of hDPCs.