Objective To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1). Methods The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture. Results The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased (P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day (P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points (P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity (P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased (P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups (P>0.05). Conclusion Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.
Bone tissue engineering is considered as one of the most promising way to treat large segmental bone defect. When constructing bone tissue engineering graft in vitro, suitable bioreactor is usually used to incubate cell-scaffold complex under perfusion to obtain bone tissue engineering graft with good repair efficiency. However, the theoretical model for growth rate of single cell (especially for stem cell) during this process still has many defects. The difference between stem cells and terminally differentiated cells is always ignored. Based on our previous studies, this study used self-made perfusion apparatus to apply different modes and strengths of fluid shear stress (FSS) to the cells seeded on scaffolds. The effects of FSS on the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) were investigated. The regression analysis model of the effect of FSS on the single-cell growth rate of MSCs was further established. The results showed that 0.022 5 Pa oscillatory shear stress had stronger ability to promote proliferation and osteogenic differentiation of MSCs, and the growth rate of a single MSC cell under FSS was modified. This study is expected to provide theoretical guidance for optimizing the perfusion culture condition of bone tissue engineering grafts in vitro.
ObjectiveTo investigate the effect of micro RNA (miR)-335-5p regulating bone morphogenetic protein 2 (BMP-2) on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodshBMSCs were cultured in vitro and randomly divided into control group (group A), miR-335-5p mimics group (group B), miR-335-5p mimics negative control group (group C), miR-335-5p inhibitor group (group D), and miR-335-5p inhibitor negative control group (group E). After grouping treatment and induction of osteogenic differentiation, the osteogenic differentiation of cells in each group was detected by alkaline phosphatase (ALP) and alizarin red staining; the expressions of miR-335-5p and BMP-2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) mRNAs were detected by real-time fluorescence quantitative PCR analysis; the expressions of Runx2, OPN, OCN, and BMP-2 proteins were detected by Western blot.ResultsCompared with group A, the relative proportion of ALP positive cells and the relative content of mineralized nodules, the relative expressions of BMP-2, miR-335-5p, OPN, OCN, Runx2 mRNAs, the relative expressions of Runx2, OPN, OCN, and BMP-2 proteins in group B were significantly increased (P<0.05); the above indexes in group D were significantly decreased (P<0.05); the above indexes between groups C, E and group A were not significantly different (P>0.05).ConclusionmiR-335-5p can up-regulate BMP-2 expression and promote osteogenic differentiation of hBMSCs.
ObjectiveTo investigate the specific microRNA (miRNA) in osteogenic and chondrogenic differentiations of C3H10T1/2 cells. MethodsC3H10T1/2 cells were induced to differentiate into osteoblasts and chondrocytes.Specific miRNA more than 2 fold change and 2 average normalized probe signal between C3H10T1/2 and C3H10T1/2-derived osteoblast,and between C3H10T1/2 and C3H10T1/2-derived chondrocytes were screened out by miRNA microarray,and verified by real-time fluorescence quantitative PCR (RT-qPCR). ResultsAlkaline phosphatase expression of osteogenic induced group was significantly higher than that of control group at 7 days after induced (P<0.05).RT-qPCR results showed the expressions of Runx2,serine protease (Sp7),collagen type I,and osteopontin (OPN) genes were significantly increased at 7,14,and 21 days after induced when compared with before induced (P<0.05).Western blot results showed the expressions of Runx2,Sp7,collagen type I,and OPN proteins of osteogenic induced group were significantly higher than those of control group at 21 days after induced (P<0.05).The expressions of SOX9,collagen type Ⅱ,Aggrecan,and Has2 were significantly increased at 5,10,and 15 days after induced when compared with before induced (P<0.05).The expressions of SOX9,collagen type 2,Aggrecan,and Has2 proteins of chondrogenic induced group were significantly higher than those of control group at 15 days after induced (P<0.05).Totally,10 osteogenic and 3 chondrogenic miRNA more than 2 fold change and 2 average normalized probe signal were screened out by miRNA microarray.RT-qPCR results of these specific miRNAs were similar to microarray results except miR-455-3p. ConclusionSpecific miRNAs are screened out by microarray and it is a good foundation for the future study on miRNA functional verification and target gene prediction.
Objective To construct recombinant lentiviral vectors of porcine bone morphogenetic protein 2 (BMP-2) gene and to detect BMP-2 gene activity and bone marrow mesenchymal stem cells (BMSCs) osteogenetic differentiation so as to lay a foundation of the further study of osteochondral tissue engineering. Methods BMSCs were isolated from bone marrow of 2-month-old Bama miniature porcines (weighing, 15 kg), and the 2nd generation of BMSCs were harvested for experiments. The porcine BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and was used to transfect BMSCs at multiplicity of infection (MOI) of 10, 25, 50, 100, and 200, then the optimal value of MOI was determined by fluorescent microscope and inverted phase contrast microscope. BMSCs transfected by BMP-2 recombinant lentiviral vectors served as experimental group (BMP-2 vector group); BMSCs transfected by empty vector (empty vector group), and non-transfected BMSCs (non-transfection group) were used as control groups. RT-PCR, immunohistochemistry staining, and Western blot were performed to detect the expressions of BMP-2 mRNA and protein. Then the BMSCs osteogenesis was detected by alkaline phosphatase (ALP) staining, ALP activities, and Alizarin red staining. Results The recombinant lentiviral vectors of porcine BMP-2 gene was successfully constructed and identified by RT-PCR and gene sequencing, and BMSCs were successfully transfected by BMP-2 recombinant lentiviral vectors. Green fluorescent protein could be seen in the transfected BMSCs, especially at MOI of 100 with best expression. The immunohistochemistry staining and Western blot showed that BMSCs transfected by BMP-2 recombinant lentiviral vectors could express BMP-2 protein continuously and stably at a high level. After cultivation of 2 weeks, the expression of ALP and the form of calcium nodules were observed. Conclusion The porcine BMP- 2 gene lentiviral vector is successfully constructed and transfected into the BMSCs, which can express BMP-2 gene and protein continuously and stably at a high level and induce BMSCs differentiation into osteoblasts.
ObjectiveTo explore the potential role of WNT6 in the proliferation, differentiation, and migration of bone marrow mesenchymal stem cells (BMSCs). MethodsMouse BMSCs were cultured to the cell fusion of 30%-50%, and divided into different groups. WNT6 knockdown included 3 experiment groups:cells transfected with WNT6 specific short hairpin RNA (shRNA) (group A1), cells transfected with control shRNA group (group B1), and nontransfected cells (group C1). WNT6 over-expression included 3 groups:cells transfected with WNT6 recombinant plasmid (group A2), cells transfected with blank vector (group B2), and non-transfected cells (group C2). After transfection, the stably transfected cells were cultured for 48 hours. Cell morphology was observed under inverted microscope; real-time fluorescent quantitative PCR was used to analyze WNT6 mRNA levels; Western blot was used to detect WNT6 and Ki67 protein expressions; cell proliferation was assayed by MTT method, and cell migration was detected by Transwell assay. After cells were cultured in osteogenic differentiation medium for 12 days, the alkaline phosphatase (ALP) activity and calcium deposits were detected by biochemical determination. ResultsThe inverted microscope observation showed that the cell morphology were similar among groups A1, B1, C1, and A2, B2, C2. The WNT6 mRNA and protein levels, Ki67 protein level, cell proliferation, cell migration, ALP activity, and calcium deposition in group A1 were all significantly lower than those in groups B1 and C1 (P<0.05), but there was no significant difference between groups B1 and C1 (P>0.05). On the contrary, the above indexes in group A2 were all significantly higher than those in groups B2 and C2 (P<0.05), but no significant difference was shown between groups B2 and C2 (P>0.05). ConclusionWNT6 can promote the proliferation and migration, as well as can enhance osteogenic differentiation ability in mouse BMSCs.
To study the effect of autogeneic PRP on prol iferation and osteogenetic differentiation of human adipose-derived stem cells (ADSCs) in vitro. Methods ADSCs were isolated from adipose tissue obtained from donor undergoing l iposuction and were cultured, and growth condition of the cells was observed by inverted microscope. ADSCs at passage 3 were cultured in adipogenic or chondrogenic medium and underwent identification, immunofluorescence staining observations for CD29 and CD44 were performed. ADSCs at passage 3 were divided into 2 groups: PRP group cultured by osteogenic induction culture medium containing 10 mL/L PRP, and control group cultured by osteogenic induction culture medium without PRP. Then growth condition of the cells was observed by inverted microscope. MTT method was used to observe cell prol iferation activity 1, 2, 3, 4 and 5 days after culture. ALP activity detection was conducted 7, 14, 21 and 28 days after culture. ALP staining was performed on PRP group 7 and 14 days after culture. Al izarin red staining was performed on PRP group 14 days after culture to detect the formation of calcium nodule. Results Under the inverted microscope, most ADSCs at passage 3 were spindle-shaped and the doubl ing time was about 35 hours. Adipogenic and chondrogenic differentiation were confirmed, and the cells were positive for CD29 and CD44 immunofluorescence staining. MTT method revealed the absorbance value of PRP group at 1, 2, 3, 4 and 5 days was 0.137 ± 0.015, 0.219 ± 0.023, 0.367 ± 0.031, 0.586 ± 0.039 and 0.948 ± 0.046, respectively, and in the control group, it was 0.081 ± 0.009, 0.115 ± 0.012, 0.162 ± 0.017, 0.242 ± 0.025 and 0.356 ± 0.032, respectively, suggesting there were significant differences between two groups (P lt; 0.01). At 7 days after osteogenic induction, PRP group was positive for ALP staining, grey-black cell plasm and black precipitate were evident; the positive cells increased
ObjectiveTo investigate the effect of Notch signaling pathway important target Hey1 expression on the differentiation and proliferation of C3H10T1/2 cells induced by bone morphogenetic protein 9 (BMP-9). MethodsHey1 lentivirus and Hey1 short hairpin RNA lentivirus were constructed and used to infect C3H10T1/2 cells to change the expression level of Hey1 in C3H10T1/2 cells. C3H10T1/2 cells infected with LV-Blank (empty plasmid) as control. The Hey1 expression levels of different groups were detected by fluorescence microscope, real-time fluorescence quantitative PCR, and Western blot. The C3H10T1/2 cells with different Hey1 expression level were induced by BMP-9 conditioned medium (BMP-9+C3H10T1/2 group, BMP-9+C3H10T1/2-Hey1 group, and BMP-9+C3H10T1/2-shHey1 group); the cells of control groups (C3H10T1/2 group and C3H10T1/2-Blank group) were cultured with normal medium. The mRNA and protein expression levels of osteogenesis related transcription factors (Runx2, osteopontin, and osteocalcin) were detected at 48 hours by real-time fluorescence quantitative PCR and Western blot assay. The cells proliferation and cycles were detected by MTT assay at 4, 5, 6, and 7 days and flow cytometry at 4, 5, and 10 days. The alkaline phosphatase (ALP) activity was analyzed by ELISA and observed by ALP staining at 4 and 7 days. ResultsC3H10T1/2 cell lines with different Hey1 expression levels were successfully established. In osteogenesis compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 enhanced the mRNA and protein expressions of transcription factors (Runx2, osteopontin, and osteocalcin), and the expression of osteogenic differentiation marker (ALP) (P < 0.05); however, inhibition of Hey1 expression significantly decreased the above indexes (P < 0.05). In cell proliferation activity compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 increased absorbance (A) value in MTT assay and pecentage of G2+S cells in cytometry assay, but inhibition of Hey1 expression significantly decreased the indexes (P < 0.05). ConclusionExpression of Hey1 is the important link in the osteogenic differentiation process of C3H10T1/2 cells induced by BMP-9, and plays an important role in the regulation of early cell proliferation.
ObjectiveTo investigate the effect of tissue interface stiffness change on the spreading, proliferation, and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs), and to find the suitable stiffness range for stem cell differentiation. MethodsBone marrow of male Sprague Dawley rats (4 weeks old) were selected to isolate and culture BMSCs by whole bone marrow cell adherent method. The third generation BMSCs (1×105 cells/mL) were inoculated into the ordinary culture dishes covered with polyacrylamide hydrophilic gel (PA) which elastic modulus was 1, 4, 10, 40, and 80 kPa (cells seeded on PA), and ordinary culture dish (75 MPa extreme high elastic modulus) as control. Spreading of cells in different stiffness of PA was observed under light microscope. The elastic modulus values of 4, 10, and 40 kPa PA were selected as groups A, B, and C respectively; the ordinary culture dish (75 MPa extreme high elastic modulus) was used as control group (group D). Cell counts was used to detect the growth conditions of BMSCs, alkaline phosphatase (ALP) kit to detect the concentration of ALP, alizarin red staining technique to detect calcium deposition status, and real-time quatitative PCR technique to detect the expressions of bone gla protein (BGP), Runx2, and collagen type I mRNA. ResultsWith increased PA stiffness, BMSCs spreading area gradually increased, especially in 10 kPa and 40 kPa. At 1 and 2 days after culture, the growth rate showed no significant difference between groups (P > 0.05); at 3-5 days, the growth rate of groups B and C was significantly faster than that of groups A and D (P < 0.05), but difference was not statistically significant between groups A and D (P < 0.05); at 5 days, the proliferation of group C was significantly higher than that of group B (P < 0.05). ALP concentrations were (53.69±0.89), (97.30±1.57), (126.60±14.54), and (12.93±0.58) U/gprot in groups A, B, C, and D respectively; groups A, B, and C were significantly higher than group D, and group C was significantly higher than groups A and B (P < 0.05). Alizarin red staining showed that the percentages of calcium nodules was 20.07%±4.24% in group C; group C was significantly higher than groups A, B, and D (P < 0.05). The expression levels of BGP and collagen type I mRNA were significantly higher in groups A, B, and C than group D, and in group C than groups A and B (P < 0.05). The expression level of Runx2 mRNA was significantly higher in groups B and C than group D, and in group C than group B (P < 0.05), but no significant difference was found between groups A and D (P > 0.05). ConclusionPA elastic modulus of 10-40 kPa can promote the proliferation and osteogenic differentiation of BMSCs, and the higher the stiffness, the stronger the promoting effect.
Objective To investigate the protective effect of the antioxidant glutathione (GSH) on the steroid-induced imbalance between osteogenesis and adipogenesis in human bone marrow mesenchymal stem cells (BMSCs). Methods The BMSCs were isolated from the proximal femur bone marrow from 3 patients of femoral neck fracture and were separated, cultured, and purificated by density gradient centrifugation and adherent wall methodin vitro. The third generation BMSCs were divided into 5 groups: group A, BMSCs (1×105 cells/mL); group B, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone; group C, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+5 μmol/L GSH; group D, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+10 μmol/L GSH; group E, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+50 μmol/L GSH. After cultured for 7 days, the reactive oxygen species expression was detected by flow cytometry; the superoxide dismutase (SOD) and Catalase mRNA expressions were determined by RT-PCR; the peroxisome proliferator-activated receptors γ (PPAR-γ), CCAAT/enhancer-binding family of proteins (C/EBP), Runx2, and alkaline phosphatase (ALP) mRNA expressions were evaluated by real-time fluorescence quantitative PCR. After cultured for 21 days, Oil red O staining was used to observe the adipogenesis differentiation of cells, and the expressions of related proteins were detected by Western blot. Results The reactive oxygen species expression in group B was obviously higher than in the other groups, in group C than in groups A, D, and E, and in groups D, E than in group A, all showing significant differences between groups (P<0.05); but there was no significant difference between groups D and E (P>0.05). The oil red O staining positive cells in group B were obviously more than the other groups, and groups C, D, E, and A decreased sequentially, the absorbance (A) values had significant differences between groups (P<0.05). RT-PCR detection showed that the relative expressions of SOD and Catalase mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E (P<0.05), but there was no significant difference among groups A, D, and E (P>0.05). Real-time fluorescence quantitative PCR detection showed that the relative expressions of PPAR-γ and C/EBP mRNA in group B were significantly higher than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A (P<0.05); but there was no significant difference between groups D and E (P>0.05). The relative expressions of Runx2 and ALP mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A (P<0.05); but there was no significant difference between groups D and E (P>0.05). Western blot detection showed that the relative expression of PPAR-γ and C/EBP protein in group B was significantly higher than those in the other groups, and groups C, D, E, and A decreased sequentially, all showing significant differences between groups (P<0.05). The relative expression of Runx2 and ALP protein in group B was significantly lower than those in the other groups, and groups C, D, E, and A increased sequentially, all showing significant differences between groups (P<0.05). Conclusions GSH can inhibit the adipogenesis differentiation and enhance the osteogenic differentiation of human BMSCs by reducing the intracellular reactive oxygen species level; and in a certain range, the higher the concentration of GSH, the more obvious the effect is.