Sox9-Increased miR-322-5p Facilitates BMP2-Induced Chondrogenic Differentiation by Targeting Smad7 in Mesenchymal Stem Cells.

Bone morphogenetic protein 2 (BMP2) induces effective chondrogenesis of mesenchymal stem cells (MSCs) by promoting Sox9 expression. However, BMP2 also induces chondrocyte hypertrophy and endochondral ossification by upregulating Smad7 expression, which leads to the disruption of chondrogenesis. In addition, Smad7 can be inhibited by Sox9. Therefore, the underlying mechanism is not clear. Currently, an increasing number of studies have shown that microRNAs play a pivotal role in chondrogenic and pathophysiological processes of cartilage. The purpose of this study was to determine which microRNA is increased by Sox9 and targets Smad7, thus assisting BMP2 in maintaining stable chondrogenesis. We found that miR-322-5p meets the requirement through next-generation sequencing (NGS) and bioinformatic analysis. The targeting relationship between miR-322-5p and Smad7 was confirmed by dual-luciferase reporter assays, qPCR, and western blotting (WB). The in vitro study indicated that overexpression of miR-322-5p significantly inhibited Smad7 expression, thus causing increased chondrogenic differentiation and decreased hypertrophic differentiation, while silencing of miR-322-5p led to the opposite results. Flow cytometry (FCM) analysis indicated that overexpression of miR-322-5p significantly decreased the rate of early apoptosis in BMP2-stimulated MSCs, while silencing of miR-322-5p increased the rate. A mouse limb explant assay revealed that the expression of miR-322-5p was negatively correlated with the length of the BMP2-stimulated hypertrophic zone of the growth plate. An in vivo study also confirmed that miR-322-5p assisted BMP2 in chondrogenic differentiation. Taken together, our results suggested that Sox9-increased miR-322-5p expression can promote BMP2-induced chondrogenesis by targeting Smad7, which can be exploited for effective tissue engineering of cartilage.

Silencing Smad7 potentiates BMP2-induced chondrogenic differentiation and inhibits endochondral ossification in human synovial-derived mesenchymal stromal cells.

BACKGROUND: Bone morphogenetic protein 2 (BMP2) is a promising chondrogenic growth factor for cartilage tissue-engineering, but it also induces robust endochondral ossification. Human synovial-derived mesenchymal stromal cells (hSMSCs) have attracted great interest due to their poor potential for differentiation into osteogenic lineages. Smad7 plays a significant in the endochondral ossification. In this study, we explored a new method to amplify the BMP2-induced chondrogenic differentiation of hSMSCs by downregulating Smad7 and applying a cellular scaffold. METHODS: hSMSCs were isolated from human knee joint synovium from 3 donors through adhesion growth. In vitro and in vivo models of the chondrogenic differentiation of hSMSCs were established. Transgenic expression of BMP2 and silencing of Smad7 and Smad7 was achieved by adenoviral vectors. The osteogenic differentiation was detected by alkaline phosphatase staining, alizarin red staining, and RT-PCR analysis of the osteogenic genes RUNX2, Osterix, and Osteocalcin. The chondrogenic differentiation was detected by Alcian blue staining and RT-PCR analysis of the chondrogenic genes SOX9, COL2, and aggrecan. Hypertrophic differentiation was detected by the markers COL10 and MMP13. A subcutaneous stem cell implantation model was established with polyethylene glycol citrate-co-N-isopropylacrylamide (PPCN) scaffolds and athymic nude mice (3/group, 4-6 week-old female) and evaluated by micro-CT, H&E staining, and Alcian blue staining. An immunohistochemistry assay was used to detected COL1 and COL2, and an immunofluorescence assay was used to detect COL10 and MMP13. RESULTS: These hSMSCs identified by flow cytometry. These hSMSCs exhibited lower osteo-differentiation potential than iMads and C3H10T1/2-cells. When Smad7 was silenced in BMP2-induced hSMSCs, the chondrogenic differentiation genes SOX9, COL2, and aggrecan were enhanced in vitro. Additionally, it silencing Smad7 led to a decrease in the hypertrophic differentiation genes COL10 and MMP13. In subcutaneous stem cell implantation assays, immunofluorescence and immunohistochemical staining demonstrated that silencing Smad7 increased the number of COL2-positive cells and decreased the expression of COL1, COL10, and MMP13. CONCLUSION: This study suggests that the application of hSMSCs, cell scaffolds, and silencing Smad7 can potentiate BMP2-induced chondrogenic differentiation and inhibit endochondral ossification. Thus, inhibiting the expression of Smad7 in BMP2-induced hSMSC differentiation may be a new strategy for cartilage tissue-engineering.

Uptake, translocation, and risk assessment of PAHs in contaminated soil-air-vegetable systems based on a field simulation experiment.

Vegetable consumption is a potential toxin exposure pathway for humans. Studies have recognized that vegetables can uptake organic contaminants via roots and translocate pollutants to their aerial parts. However, the aerial parts might also directly uptake polycyclic aromatic hydrocarbons (PAHs) from contaminated soils. This has not been extensively studied. The aim of this study was to explore the uptake and translocation of PAHs in contaminated soil-air-vegetable systems. Sixteen individual PAHs in contaminated soils, vegetable roots, and leaves were identified using GC-MS. The results showed that the average PAH concentrations both in roots and leaves from the reference soil, the moderately contaminated soil, and the heavily polluted soil increased as expected. PAHs with log KOW < 5 accumulated more easily in roots and leaves. Using a Pearson correlation analysis, isomer ratios, and a principal component analysis (PCA), it was found that the contaminated soil not only caused PAH accumulation in roots, but also increased the PAH concentration in leaves. Quantitatively, the absorption of PAHs in roots in the moderately contaminated soil (70.3 ng m-3) was approximately twice that of the reference soil (40.8 ng m-3). The PAHs absorbed by vegetable roots in the heavily polluted soil (74.7 ng m-3) was only slightly higher than that of the moderately polluted soil. In addition, the PAH dose volatilized into the air from the reference soil, the moderately contaminated soil, and the heavily polluted soil also showed an increasing trend. The incremental lifetime cancer risk (ILCR) indicated that adult females had a higher cancer risk via vegetable consumption than other groups. Although vegetable consumption had a slight effect on cancer risk for some groups in the present study, the cancer risk of PAHs caused by eating vegetables grown in heavily contaminated soil still requires attention.

Risk of childhood overweight or obesity associated with excessive weight gain during pregnancy: a meta-analysis.

PURPOSE: Several investigations have shown that the risk of childhood overweight and obesity may be associated with excessive gestational weight gain (GWG), but the study samples were limited and results were mixed; hence we conducted this meta-analysis to assess the explicit association between them and present evidence. METHODS: We searched PubMed, EMbase, Ovid, Web of knowledge and Cochrane library for relevant studies. Bibliographies from retrieved articles were also manually searched. Cohort studies that presented results by OR or RR with 95 % CI for the association of maternal excessive GWG and child overweight/obesity were included. The pooled estimates were calculated by fixed-effect model or random-effect model according to the heterogeneity. RESULTS: Twelve cohort studies were identified, and the combined OR of excessive GWG and childhood overweight/obesity was 1.33 (95 % CI 1.18-1.50) with evidence of heterogeneity (P = 0.000, I (2) = 71.1 %). Adjustment of maternal BMI, investigation area, age of children, research type, and omission of any single study had little effect on the pooled estimate. After eliminating the effect of publication bias by trim and fill analyses, the result still remained statistically significant (OR 1.21, 95 % CI 1.05-1.40). CONCLUSIONS: This meta-analysis suggests that the risk of childhood overweight/obesity is significantly associated with excessive gestational weight gain.