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BACKGROUND: Cornelia de Lange syndrome is a genetic disease with multiple developmental defects, of which NIPBL is the main pathogenic gene. OBJECTIVE: To investigate the effect of NIPBL gene on the proliferation and osteogenic differentiation of bene marrow mesenchymal stem cells. METHODS: The NIPBL+/- mice were constructed by NIPBL-Loxp and Cre mice and used as experimental group, and the wild-type NIPBL+/+ mice served as control group. Mouse bone marrow mesenchymal stem cells were isolated and cultured in the two groups. Cell proliferation was detected using cell counting kit-8 assay when thecells were passed to the third generation. Osteoblastic differentiation was then compared between two groups after osteogenesis induction. RESULTS AND CONCLUSION: The proliferation capacity of bone marrow mesenchymal stem cells in the experimental group was lower than that in the control group (P < 0.05). The activity of alkaline phosphatase in the experimental group was significantly lower than that in the control group on the 7th day of osteogenic induction (P < 0.05). The expression levels of osteogenic genes and proteins (Runx2 and OCN) in the experimental group were lower than those in the control group after osteogenic induction (P < 0.05). On the 21st day of osteogenic induction, results from alizarin red staining indicated there were more red calcium nodules in the control group than the experimental group under inverted microscope. These findings suggest that NIPBL gene knockout can reduce the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.
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BACKGROUND: Bone tissue engineering has provided a novel ideal for treating bone defects in clinic. This study is the first to combine traditional Chinese medicine with the nanostructures of tissue-engineered scaffolds in order to explore and construct a new bone tissue substitute material for the treatment of bone defects. OBJECTIVE: To investigate the osteogenic activity of icariin (ICA)/hydroxyapatite (HA)/poly(lactic-co-glycolic acid) (PLGA) composite scaffolds. METHODS: A HA/PLGA composite scaffold was prepared by physical blending of HA and PLGA, and was then soaked in ICA solution of different concentrations to obtain the HA/ICA/PLGA scaffold. Rabbit bone marrow mesenchymal stem cells were used to evaluate the cell adhesion, proliferation, osteogenesis and cytotoxicity of the composite scaffold. The cell adhesion, proliferation and cytotoxicity were detected by MTT method. The activities of alkaline phosphatase and osteocalcin were detected by ELISA. The expression levels of osteogenic genes and proteins were detected by fluorescence quantitative PCR and western blot assay, respectively. RESULTS AND CONCLUSION: Adding appropriate amount of HA into PLGA could improve the mechanical strength of the scaffold, and 10% HA had the best effect with tensile strength of (1.67±0.37) MPa, and compression modulus of (4.17±1.62) MPa, and nanostructure would be formed on the surface of the scaffold. The nanostructure could promote the adhesion of bone marrow mesenchymal stem cells on the surface of the scaffold. ICA did not affect the proliferation of bone marrow mesenchymal stem cells on the composite scaffold. However, the HA/PLGA composite scaffold soaked in 1.00 µmol/L ICA aqueous solution had the optimal osteogenic differentiation function, and the expression levels of alkaline phosphatase, osteocalcin, osteogenic related genes and proteins (Runx-2 and COL I) were increased. The ICA/HA/PLGA scaffold had no cytotoxicity. These results suggest that HA (10%)/ICA (1.00 µmol/L)/PLGA scaffold has good mechanical properties, osteogenesis and biocompatibility, which has the potential to be a favorable scaffold for bone tissue engineering.
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Objective::To investigate the role and mechanism of Testudinis Carapax et Plastrum aqueous extract in promoting osteogenic differentiation of mouse preosteoblast cell line(MC3T3-E1) by regulating nuclear transcription factor-κB(NF-κB) inflammation microenvironment. Method::MC3T3-E1 cells were cultured in vitro, and osteogenic induction (OI) was performed. Testudinis Carapax et Plastrum was prepared and treated the cells. Cells were devided into control group, osteogenic induction group and Testudinis Carapax et Plastrum (20 mg·L-1)with osteogenic induction group. The proliferation of MC3T3-E1 was detected by cell counting kit-8 (CCK-8), and the optimum concentration of intervention was determined. MC3T3-E1 differentiation and osteogenic mineralization were assayed using alkaline phosphatase (ALP) and Alizarin red staining (ARS), respectively. The expressions of NF-κB p65, NF-κB p105, interleukin-6(IL-6), ALP and Collagen-Ⅰ(COL-Ⅰ) mRNA were detected by Real-time PCR. Result::The results of CCK-8 showed that the proliferation of MC3T3-E1 did not change statistically with time, but it showed an upward trend, while the proliferation at 20 mg·L-1 was more obvious than other groups. The ALP and ARS showed that the positive staining rate of osteogenic induction group and Testudinis Carapax et Plastrum with osteogenic induction group were higher than control group.Real-time PCR results showed that on the 7th day in culture, the expression of NF-κB p105 and IL-6 mRNA in Testudinis Carapax et Plastrum with osteogenic induction group was significantly lower than that in control group (P<0.01), and the expression of ALP and COL-Ⅰ mRNA was significantly upregulated(P<0.05), on the 14th day, the expression of NF-κB p65, NF-κB p105 and IL-6 mRNA in Testudinis Carapax et Plastrum with osteogenic induction group was significantly lower than that in control group (P<0.01). The expression of ALP and COL-Ⅰ mRNA was significantly increased (P<0.05, P<0.01). Conclusion::Testudinis Carapax et Plastrum aqueous extract can promote osteogenic differentiation of MC3T3-E1 via a mechanism associated with the regulation of inhibition of NF-κB inflammatory microenvironment.
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Objective@#This study investigated the effects of interleukin-1β (IL-1β) on human synovial fluid-derived mesenchymal stem cells (hSFMSCs) in the temporomandibular joint.@*Methods @#hSFMSCs from synovial fluid samples of temporomandibular disorder (TMD) patients were cultured in vitro. hSFMSCs were divided into three groups with different concentrations of rhIL-1β in complete medium (α-MEM cell culture medium + 10% FBS + 1× GlutaMAX): 0 ng/mL IL-1β group, 1 ng/mL IL-1β group and 10 ng/mL IL-1β group. Changes in the rate of colony formation, growth curve, cell cycle and apoptosis of hSFMSCs under IL-1β stimulation were evaluated. The osteogenic, adipogenic and chondrogenic potential of the cells were also determined using histochemical and real-time fluorescence quantitative PCR methods. @*Results @#No significant differences in growth or proliferation capacity were observed in any IL-1β-stimulated group in comparisons of the colony-formation rate (F = 0.665, P=0.548), growth curve (F=0.001, P=0.999), cell cycle (FG1=0.773, PG1=0.503; FS =0.636, PS =0.562) or apoptosis (F=0.196, P=0.827) of the cells. However, the multidifferentiation capacity of hSFMSCs was affected in the inflammatory environment. Mineralized nodule and lipid clusters decreased significantly, and the gene expression levels of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), peroxisomal proliferative receptor G2 (PPARG2) and lipoprotein lipase (LPL) were suppressed significantly in IL-1β-mediated induction medium (P < 0.05). In general, cartilage pellets formed in all the IL-1β-mediated chondrogenic differentiation groups. The gene levels of sex-determining region Y-related high-mobility group box-9 (SOX9) and collagen II were decreased (P < 0.05), while that of matrix metalloproteinase 13 (MMP13) was increased significantly in the presence of IL-1β (P < 0.05). @*Conclusion@# IL-1β directly affects the multidifferentiation potential of hSFMSCs but not their cell growth or proliferation ability.
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Objective To explore the effect of dexamethasone(Dex)treatment on the expression of interleukin(IL)?6 and IL?8 in human peri?odontal ligament cells(hPDLCs). Methods hPDLCs were subjected to one of the following treatments for 24 h or 48 h:10-9 mol/L Dex;10-6 mol/L Dex;10μg/mL Porphyromonas gingivalis(P. g)?lipopolysaccharide(LPS);10-9 mol/L Dex+10μg/mL P. g?LPS;10-6 mol/L Dex+10μg/mL P. g?LPS;or 0.1%absolute ethyl alcohol(control). Protein and mRNA expression was detected using ELISA and real?time PCR ,respectively. Results At 24 h and at 48 h,IL?6 and IL?8 protein expression in the 10-9 mol/L Dex?and 10-6 mol/L Dex?treated groups was significantly lower than that in the control group(P<0.05). At 48 h,IL?6 mRNA expression in the 10-9 mol/L Dex?and 10-6 mol/L Dex?treated groups was signifi?cantly lower than that in the control group(P<0.05),while IL?8 mRNA expression in the 10-6 mol/L Dex?treated group was significantly higher than that in the control group(P<0.05). At 24 h and at 48 h,IL?6 protein and mRNA expression in the 10-9 mol/L Dex+10μg/mL P. g?LPS?treated group and the 10-6 mol/L Dex+10μg/mL P. g?LPS?treated group was significantly lower than that in the 10μg/mL P. g?LPS?treated group (P<0.05). At 24 h,IL?8 protein expression in the 10-9 mol/L Dex+10μg/mL P. g?LPS?treated group and the 10-6 mol/L Dex+10μg/mL P. g?LPS?treated group was significantly lower than that in the 10μg/mL P. g?LPS?treated group(P<0.05),while no such significant difference exist?ed at 48 h. At 48 h,IL?8 mRNA expression in the 10-6 mol/L Dex+10μg/mL P. g?LPS?treated group was significantly higher than that in the 10μg/mL P. g?LPS?treated group(P<0.05). Conclusion Dex inhibits the innate and P. g?LPS?induced expression of IL?6 in hPDLCs. However, Dex exerts profound effects on IL?8 expression,and treatment with high doses of Dex may promote IL?8 expression over an extended period.
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BMP-2 is a well-known TGF-beta related growth factor, having a significant role in bone and cartilage formation. It has been employed to promote bone formation in some clinical trials, and to differentiate mesenchymal stem cells into osteoblasts. However, it is difficult to obtain this protein in its soluble and active form. hBMP-2 is expressed as an inclusion body in the bacterial system. To continuously supply hBMP-2 for research, we optimized the refolding of recombinant hBMP-2 expressed in E. coli, and established an efficient method by using detergent and alkali. Using a heparin column, the recombinant hBMP-2 was purified with the correct refolding. Although combinatorial refolding remarkably enhanced the solubility of the inclusion body, a higher yield of active dimer form of hBMP-2 was obtained from one-step refolding with detergent. The refolded recombinant hBMP-2 induced alkaline phosphatase activity in mouse myoblasts, at ED₅₀ of 300-480ng/ml. Furthermore, the expressions of osteogenic markers were upregulated in hPDLSCs and hDPSCs. Therefore, using the process described in this study, the refolded hBMP-2 might be cost-effectively useful for various differentiation experiments in a laboratory.
Subject(s)
Animals , Humans , Mice , Alkalies , Alkaline Phosphatase , Cartilage , Detergents , Heparin , Inclusion Bodies , Mesenchymal Stem Cells , Methods , Myoblasts , Osteoblasts , Osteogenesis , Solubility , Stem Cells , Transforming Growth Factor betaABSTRACT
Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily, acting as important regulators in embryogenesis and bone and cartilage formation and repair.The complexity of BMPs action has early been unveiled by generous cell and molecular biology approaches and transgenic animal studies.Genetic engineering allows for the mass production of BMPs for clinical use of bone repair, but the desirable scaffold material is also needed to ensure controlled release and maximum biological activity of BMPs at the surgical site.Currently, BMP-2 and BMP-7 have been approved by United States Food and Drug Administration for clinical therapy in long bone fracture, spinal fusion and oral and maxillofacial surgery.The results are satisfactory, but the side effects have been also widely concerned.The functions and clinical applications of BMPs in bon repair are reviewed.
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Objective To study the isolation,culture, adipogenic and osteogenic induction Tupaia bone marrow mesenchymal stem cells(BM-MSCs).Method The BM-MSCs from tupaia were isolated and expended by combination of gradient centrifugation and adherence culture , then subcultured and observed for morphology under inverted phase contrast microscope.BM-MSCs were induced to adipocytes .and osteoblasts in vitro Result Cells were spindle or triangle-shaped, and clone proliferation .Cells were successfully induced into adipocytes .and osteoblasts Conclusions The method of isolation BM-MSCs from tupaia by combination of gradient centrifugation and adherence culture is simple and feasible , BM-MSCs have differentiation potential into adipocytes and osteoblasts .
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PURPOSE: To determine if stem cells transplanted directly into a bone defect of rabbit tibias have osteogenic induction potential. MATERIALS AND METHODS: Immature white New Zealand rabbits underwent tibial osteotomies, and were divided into three groups according to the implant material used: stem cells embedded in agar (group 1); agar alone (group 2); nothing (group 3). For all rabbits, radiographs were taken weekly for 8 weeks, and histological studies of the newly formed-bone were performed. CM-Dil was used to label the stem cells prior to transplantation to ascertain whether or not the newly formed bone was derived from the transplanted stem cells. RESULTS: Fibroblasts and osteoblasts (osteoid matrix-forming cells) derived from the stem cells were identified by electron microscopy. Interspersed enchondral ossification (probably induced by osteogenic cells from the remaining periosteum and marrow) and pure osteoids (created directly from the osteoblasts originating from the transplanted stem cells) were identified. Fluorescent-labeled cells were conspicuous in the new bones until 6 weeks after surgery, which indicates that the new bones were induced by the stem cells. CONCLUSION: The osteogenic induction potential of the undifferentiated stem cell has promise for therapeutic application, which may be used for the treatment of bone defects in the future.
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Rabbits , Agar , Fibroblasts , Microscopy, Electron , Osteoblasts , Osteotomy , Periosteum , Stem Cells , TibiaABSTRACT
Objective To evaluate the effects of adeno-associated virus-enhanced green fluorescent protein (AAV-EGFP)on the biologic behavior of rabbit's bone marrow stromal cells(BMSCs)by means of a simple method of culturing and osteogenic induction in vitro so as to find an ideal viral vector and cell tracing mark for tissue en- gineering.Methods Total bone marrow culture was conducted to obtain rabbit BMSCs which were then induced in the osteogenic direction.The morphology of the cells was observed continuously,and their surface antigen and ossification were detected by alkali phosphatase stain and Von Kossa stain.On the basis of the above results, AAV-EGFP was transfected into the induced cells.The morphologic changes of the cells,the expression time and intensity of fluorescent light were observed.The transfection efficiency was detected to find the best multiplicity of infection(MOI)value.The cell growth curves were drawn to evaluate the biologic effects of AAV-EGFP on the cyto-activity.Results The morphology and purity of the rabbit BMSCs obtained were good.The ossification of the cells was significant after osteogenic induction.The best MOI value was found to be 1?10~5.The expression intensity of fluorescent light was strong with the expression time more than eight weeks so that the fluorescent light could be observed after cell generations.The transfection efficiency of AAV was high without significant biologic effects on the cyto-activity.Conclusions The total bone marrow culture and in vitro cell induction can satisfy the requirements for seeding cells in tissue engineering.AAV is an ideal viral vector for tissue engineering.Transfection of AAV-EGFP to cells could be an ideal method for cell tracing mark.
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0.05)),forming interconnected nodular aggregates on the surface of the surface of the dish,and increasing ALP activity.Conclusion The model for inducing MSCs from young adult SD rat bone marrow to become osteoblasts in vitro has been established.The proliferation competence of cell was not affected by the osteogenic supplements used in our experiment and massive seed cells could be obtained quickly.