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Objective:To investigate the combined effect of fluoride exposure and low nutrition on osteogenesis and osteoclastic differentiation in rats.Methods:SD rats were divided into four groups by the method of random number table, namely normal nutrition group, low nutrition treatment group, fluoride exposure group and co-treatment of fluoride and low nutrition group according to 2 × 2 factorial experimental design, 8 rats in each group, half male and half female. Five months after the experiment, immunohistochemistry was used to test the expression levels of femoral alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL). Analysis of variance of factorial design was used to determine the interaction between fluoride exposure and low nutrition on osteogenesis and osteoclastic differentiation.Results:The immunohistochemical results of bone tissue showed that there were significant differences in the expression levels of osteogenesis differentiation markers ALP and Runx2 between different groups ( F = 25.98, 17.77, P < 0.001). Compared with normal nutrition group (0.005 2 ± 0.002 7, 0.003 1 ± 0.001 4), the expression levels of ALP and Runx2 in fluoride exposure group were higher (0.019 5 ± 0.005 0, 0.014 4 ± 0.004 4, P < 0.05). There was no significant difference between low nutrition treatment group (0.002 6 ± 0.001 8, 0.004 4 ± 0.003 2) and co-treatment of fluoride and low nutrition group (0.003 6 ± 0.000 7, 0.002 9 ± 0.000 8, P > 0.05). The expression levels of ALP and Runx2 in co-treatment of fluoride and low nutrition group were lower than those of fluoride exposure group ( P < 0.05). There were significant differences in the expression level osteoclastic differentiation marker of RANKL and the ratio of RANKL/OPG ( F = 10.50, 31.05, P < 0.001). Among them, the RANKL/OPG ratio (0.115 3 ± 0.039 5) in fluoride exposure group was lower than that in normal nutrition group (1.426 3 ± 0.777 2), and the RANKL expression level and RANKL/OPG ratio (0.019 5 ± 0.007 7, 7.258 7 ± 3.674 3) in co-treatment of fluoride and low nutrition group were higher than those in normal nutrition group (0.004 4 ± 0.002 5, 1.426 3 ± 0.777 2, P < 0.05). However, there was no significant difference in the RANKL expression level and RANKL/OPG ratio (0.004 0 ± 0.001 9, 2.022 3 ± 0.753 7) in low nutrition treatment group ( P > 0.05). The expression level of RANKL and the ratio of RANKL/OPG in the co-treatment of fluoride and low nutrition group were higher than those in low nutrition treatment group and fluoride exposure group ( P < 0.05). The 2 × 2 analysis of variance of factorial design showed that fluoride exposure and low nutrition had interaction on ALP, Runx2, RANKL expression levels and RANKL/OPG ratio ( F = 4.38, 19.39, 22.12, 108.00, P < 0.05), antagonistic effect on ALP and Runx2 expression, synergistic effect on RANKL expression and RANKL/OPG ratio. Conclusions:In rat bone tissue, fluoride exposure promotes osteogenesis differentiation, inhibits osteoclastic differentiation dominated by active osteogenic function. The interaction between fluoride and low nutrition on osteogenesis and osteoclastic differentiation is antagonistic osteogenesis differentiation and synergistic promotion of osteoclastic differentiation. Normal nutrition conditions are material basis of osteogenesis differentiation, and low nutrition is the inducement of enhanced osteoclastic differentiation.
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Objective:To investigate the effect and potential mechanism of serum exosome-derived cirC_0009362 on the osteogenic differentiation of human bone marrow mesenchymalstem cells (hBMSCs) .Methods:Serum samples from patients with osteoporosis (OP) were collected and exosomes were isolated. The expression level of circ_0009362 in exosomes was detected by qRT-PCR. hBMSCs osteogenesis was induced and the expression of circ_0009362 and miR-29b-3p was detected. The interaction between circ_0009362 and miR-29b-3p was detected by dual luciferase reporter assay. Alkaline phosphatase (ALP) kit was used to detect ALP activity and alizarin red (ARS) staining was used to detect calcium deposition.Results:Compared with healthy control group, the expression of circ_0009362 in serum exosomes of OP patients was increased, and the expression of circ_0009362 was decreased after inducing hBMSCs osteogenesis (all P<0.05) . The ALP activity and the percentage of calcium deposition in hBMSCs were decreased by exosomes, and this effect was achieved by secreting circ_0009362. The effect of exosomes was partially offset by circ_0009362 expression in knockdown exosomes (all P<0.05) . The expression of miR-29b-3p was increased after inducing hBMSCs osteogenesis ( P<0.05) . Circ_0009362 had a targeting relationship with miR-29b-3p, and exosomes inhibited the expression of miR-29b-3p by secreting circ_0009362. The ALP activity and the percentage of calcium deposition in hBMSCs were promoted by overexpression of miR-29b-3p, which was partially offset by exosomes (all P<0.05) . Conclusion:Serum exosomes of OP patients inhibit the osteogenic differentiation of hBMSCs by secreting circ_0009362 to down-regulate the expression of miR-29b-3p.
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Steroid-induced osteonecrosis of the femoral head (SONFH) is a kind of disease caused by hip joint dysfunction. Long-term or high-dose application of hormone drugs leads to impaired blood supply to the femoral head and the death of a variety of bone stromal cells, resulting in the structural change of trabecular bone in the load-bearing area of the femoral head and irreversible collapse of the femoral head. Early manifestations were hip pain and discomfort, and tenderness in the inguinal region, in the middle stage, pain affects activity; in the later stage, the hip joint space become narrowed, the motion of the hip joint is reduced, and the motion is limited. SONFH has greatly affected the quality of life of patients, so modern medicine is dedicated to improve the symptoms of patients through early intervention, enabling SONFH to get effective treatment and reducing the pressure of life and economic burden of patients. Bone marrow mesenchymal stem cells (BMSCs) have become a focus in the early treatment of SONFH due to its osteogenic differentiation. BMSCs are stem cells with multi-directional differentiation potential, with self-proliferation and differentiation characteristics, which can differentiate into bone, provide mechanical support for the necrotic area and secrete a variety of growth factors to support hematopoiesis, slow down the progress of disease, and extend the service life of their own joints. The unreasonable use of hormones mainly inhibits the osteogenic activity of BMSCs, resulting in the occurrence of SONFH. This paper reviews the research on BMSC osteogenic differentiation in recent years, hoping to improve the therapeutic effect of SONFH.
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Objective @# To explore the effect of miR-21 on human periodontal ligament stem cells (PDLSCs) proliferation and osteogenesis and to provide a theoretical basis for the stem cell treatment of periodontitis.@*Methods@#hPDLSCs were isolated and cultured with the enzymatic tissue block method, and surface molecules (CD34, CD45, CD90 and CD105) were detected by flow cytometry. An miR-21 mimics (pre-miR-21) and inhibitor (anti-miR-21) were transfected into hPDLSCs by Lipofectamine 2000. The experiment groups: mimics-NC group, mimics group, inhibitor group, and inhibitor-NC group. The transfection efficiency of miR-21 was determined by qRT-PCR. Proliferation was detected by CCK-8 and flow cytometry. The osteogenic differentiation ability of hPDLSCs was determined by alizarin red staining. Western blot was used to detect the protein expression of osteogenic related genes: Runx2.@*Results@#The mRNA expression of miR-21in the mimics group was significantly higher than that in the mimics-NC group; additionally, the expression in the inhibitor group was significantly weaker than that in the inhibitor-NC group (P < 0.05). hPDLSCs proliferation and the S phase cell ratio in the mimics group were stronger than those in the mimics-NC group(P < 0.05); those in the inhibitor group were weaker than those in the inhibitor-NC group (P < 0.05). After alizarin red staining, the mimics group was found to have more mineralized modules than mimics-NC group, and the inhibitor group had fewer than that in the inhibitor-NC group. Runx2 protein expression in the mimics group was higher than that in the mimics-NC group (P <0.05), and expression was lower in the inhibitor group than in the inhibitor-NC group (P < 0.05).@*Conclusion@#miR-21can promote the proliferation and osteogenesic differentiation of hPDLSCs.
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Objective@#To compare the in vitro biocompatibility of bone marrow mesenchymal cells on polyetheretherketone (PEEK) and titanium (Ti) surfaces.@*Methods @#PEEK and Ti foils with thicknesses of 1 mm and diameters of 10 mm were prepared. First, bone marrow mesenchymal cells were separated and purified by the whole bone marrow adherent culture method in vitro. Then, osteogenesis-induced bone marrow mesenchymal cells were cultivated on the surfaces of the PEEK and Ti foils. Scanning electron microscopy (SEM), the Alamar Blue test, an alkaline phosphatase (ALP) kit and Alizarin Red staining were used to analyze calcium nodules and compare the adhesion, proliferation and osteogenic differentiation ability of bone marrow mesenchymal cells on the surfaces of the PEEK and Ti foils.@*Results @# ① The morphology of the bone marrow mesenchymal cells cultured on the PEEK and Ti foils at 1 h, 4 h and 24 h showed no significant differences. ② In the 1 h, 3 h, 1 d and 3 d cultures of the bone marrow mesenchymal cells inoculated on the surfaces of the foils, the number of living cells in the PEEK group was greater than that in the Ti group (P < 0.05). ③ In the 7 d and 14 d osteogenesis-induced cultures of the inoculated bone marrow mesenchymal cells, the ALP activity of the PEEK group cells was significantly greater than that of the Ti group cells (P < 0.05). ④ Semiquantitative analysis after Alizarin Red staining showed that the mineralization degree of the bone marrow mesenchymal cells induced by osteoblasts was greater in the PEEK group than in the Ti group (P < 0.05). @*Conclusion@#PEEK has better in vitro biocompatibility than Ti and can better promote cell adhesion, proliferation and osteogenic differentiation compared with Ti, and so it is expected to become a new dental implant material.
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Objective@#To investigate the effect of PR domain zinc finger protein 9 (PRDM9), one of the histone methylated transferases, on osteogenic differentiation ability of periodontal ligament mesenchymal stem cells (PDLSC).@*Methods@#PDLSC with PRDM9 gene knocked down by PRDM9 shRNA using recombinant lentiviral vector were allocated into the PRDM9sh group, and the transfected shRNA was as the control group. The gene expression efficiency was evaluated by reverse transcription polymerase chain reaction (RT-PCR). Alkaline phosphatase activity (ALP), alizarin red staining, mineralization and osteocalcin, which belongs to osteogenic differentiation markers detected by RT-PCR and Western blotting to detect the osteogenic differentiation ability of stem cells from periodontal ligaments in vitro. In vivo, PRDM9sh and control group cells was transplanted into the dorsal dermal to explore the osteogenesis. The area percentage of new osteogenic tissue was calculated by image pro software and statistically analyzed.@*Results@#RT-PCR results showed that the relative expression of PRDM9 gene in PRDM9sh (0.460±0.017) was significantly lower than that in control group (1.000±0.107) (P<0.05). The results of ALP activity determined at 5 days postinduction in a significant decrease in PRDM9sh cells (0.762±0.063) compared with control group (1.225±0.058) (P<0.01). Alizarin red staining induced by osteogenesis at 2 weeks and 3 weeks showed that the staining of PRDM9sh was significantly lighter than that in control group. Quantitative calcium analysis results showed that the calcium ion concentration induced by osteogenesis at 2 weeks and 3 weeks [(0.071±0.004), (0.075±0.001)] in PRDM9sh was significantly lower than that in control group at 2 weeks and 3 weeks [(0.282±0.006), (0.485+0.004)] (P<0.01). RT-PCR results showed that the relative expression of osteocalcin mRNA in PRDM9sh (1.059±0.148) was significantly lower than that in control group at 2 weeks (2.542±0.190) (P<0.01). Western blotting results showed that osteocalcin expression in PRDM9sh was significantly lower than that in control group at 1 and 2 weeks after osteogenesis induction. Animal transplantation experiments results indicated that PRDM9 significantly inhibited the osteogenesis of PDLSC in vivo, and the proportion of osteogenic area calculated showed that the osteogenic capacity of PRDM9sh [(3.8±2.41)%] was significantly lower than that in control group [(24.54±7.06)%](P<0.05).@*Conclusions@#Depletion of PRDM9 repressed the osteogenic differentiation of stem cells from periodontal ligament in vitro and in vivo.
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Objective:To investigate the induction and differentiation potential of ADSCs by tissue culture method,and to preliminary study on the origin of ADSCs.Methods:Using adipose tissue culture method to culture human ADSCs.The third generation of ADSCs for the adipogenic and osteogenesis differentiation,and staining by oil red O and alizarin red S.HE staining was performed after the seventh day culture of adipose tissue.Results:The primary human ADSCs were successfully cultured with adipose tissue culture method.ADSCs cultured to the eighth generation,still maintained a good proliferation ability and cell morphology.ADSCs can be successfully induced into adipose cells and bone cells.ADSCs were mainly distributed around the mesenchymal vascular and connective tissue,by HE staining of adipose tissue after seven days of culture.Conclusion:The cells that were cultured with adipose tissue have the potential to adipogenic and osteogenesis differentiation.The ADSCs were mainly distributed around the mesenchymal vascular and connective tissue.
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Aim To investigate the drug-containing serum of Rhizoma drynariae on osteogenesis differentiation of bone marrow mesenchymal stem, and discuss the possible mechanism.Methods BMSCs were cultured in media with different concentrations of medicine containing serum.BMSCs proliferation ability was detected in 3,5,7,9 days by CCK-8.ALP activity was detected after 7,10,14 days′ induction.After 3 weeks culturing, alizarin red staining was performed to observe the formation of calcium nodules.The expression of β-catenin,LRP5,RUNX-2 and Osteriex mRNA were detected using RT-PCR.The protein expression of β-catenin,LRP5 was detected using Elisa method.Results Rhizoma drynariae drug-containing serum could obviously promote the proliferation of BMSCs and calci-fied nodule formation.Besides, the ALP activity was improved in a certain period of time.The expression of β-catenin,LRP5,GSK-3β,RUNX-2 and Osteriex mRNA were significantly up-regulated,and the protein expression of β-catenin,LRP5 was up-regulated too.The expression of GSK-3β was down-trgulated.Conclusions Rhizoma drynariae drug-containing serum promotes mineralization and osteogenic differentiationof BMSCs, and the mechanism is closely related with activating WNT/beta-catenin signaling pathway, raising the beta-catenin, LRP5, RUNX-2, and Osteriex mRNA expression, beta-catenin, LRP5 protein expression,and down-regulation of GSK-3β mRNA expression.
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Objective:To transfect the non-viral vector polyethylenimine (PEI)mediated miR-2861 mimic into the MC3T3-E1 cell line,and to explore the transfection efficiency of PEI/miR-2861 complex and its effects on the proliferation and osteogenesis differentiation in pre-osteoblasts. Methods:The proper amount of PEI was blended with miR-2861 mimic and negative control (NC)separately in a ratio of N∶P=10∶1,and they were divided into experiment group and NC group. The NC/PEI complex acted as the NC group was used to eliminate the interference of osteogenesis from the addition of double-stranded RNA mimic.MTT assay was used to determine the optimal concentration of PEI/miR-2861 mimic complex.The fluorescence imaging technique and bulge-loop RT-PCR were used to detect the transfection efficiency and mRNA expression of miRNA-2861 in the cells with different concentrations (10,30, 50,and 100 nmol · L-1 ), separately.The osteogenesis ability of MC3T3 cells was identified with RT-PCR and Alizarin red staining with the selected concentration of PEI/miR-2861 by transient transfection.Results:Compared with blank control group,the proliferation rates of MC3T3 cells in 100 nmol·L-1 PEI/miR-2861 group was decreased significantly at 72 h (P < 0. 05 ). With the increasing of transfected concentration the transfection efficiency of miRNA/PEI complex was increased gradually.The results of Alizarin red staining and quantitative analysis showed that calcium deposits were more and bigger in experiment group after induced for 21 d,while both in blank control group and NC group they were less.Conclusion:The miRNA-2861 mimic can be effectively transfected into the MC3T3-E1 cell line and expresses with a high level,which is mediated by PEI as the gene vector.miR-2861 mimic has a certain ability of promoting osteogenesis differentiation of MC3T3-E1 cells.
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Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β(TGF-β) superfamily and play critical roles in skeletal development, bone formation and stem cell differentiation.BMP9 is one of the most osteogenic BMPs, promoting osteogenesis differentiation of periodontal ligament stem cells ( PDLSCs) both in vitro and in vivo.Recently, signal transduc-tion studies have revealed that BMP-Receptor-Smads and BMP-Receptor-Mitogen activated protein kinase ( MAPK) play vital roles in BMP9 which promote PDLSCs osteogenesis differentiation.Several studies revealed that transcription factors closely associated with os-teogenesis differentiation are found in the downstream of the Smads and MAPK pathways.This review aims to summarize our current knowledge of BMP9-mediated osteogenesis by presenting recently completed works which may help us to further elucidate these path-ways.