Objective To investigate the effects of the recombinanthuman bone morphogenetic protein 2 (rhBMP-2) and/or the osteogenic agents on proliferation and expression of the osteoblast phenotype differentiation of the SD rat mesenchymal stem cells（MSCs）. Methods The rat MSCs were cultured in vitro and were randomly divided into the experimental groups(Groups A-I) and the control group. In the experimental group, MSCs were induced by rhBMP2 in different doses (10, 50, 100 and 200 μg/L) in Groups BE, the osteogenic agent alone (Group A) and by the combined use of rhBMP-2 ［in different doses (10,50, 100 and 200 μg/L)］ and the osteogenic agent in Groups F-I. The MTT colorimetric assay was used to evaluate the proliferation, and the activities of alkaline phosphatase (ALP) and osteocalcin (OC) were observed at 3, 6, 9, 12 days, respectively. Results The inverted phase contrast microscopy showed that MSCs by primary culture for 12 hours were adhibited, with a fusiform shape at 48 hours. At 4 days they were polygonal or atractoid, and were spread gyrately or radiately at 6 days. At 10 days, they were spread at the bottom of the bottle.The statistical analysis showed that the expression of the osteoblast phenotype differentiation of MSCs could be induced in the experimental groups. The proliferation of MSCs could be enhanced in a dosedependent manner in GroupsB-E. The expression of the osteoblast phenotype differentiation, which was tested by the activities of ALP and OC, was significantly higher in Groups F-I than in Groups A-E. Conclusion The combined use of rhBMP-2 and the osteogenic agents can enhance the MSC proliferation and induce an expressionand maintenance of the osteoblast phenotype differentiation of the rat MSCs.
Objective To study the method of inducing human marrow mesenchymal stem cells (MSCs) into osteoblasts directionally and to identify osteogenesis characteristics. Methods MSCs were isolated from adult marrow using density gradient separation method and were cultured in conditioned medium containing Dex 10 -8 mol/L，β-GP 10 mmol/L，and AA 50 μg/ml. The MSCs attachment formed soon and passage 3 cells were chosen to check osteogenesis characteristics, including alkaline phosphatase assay with modified calcium-cobalt staining method, type Ⅰ collagen assay with immunohistochemistry, osteopontin and osteonectin assay with in situ hybridization and calcium nodes assay with Von Kossa staining. Results Passage 3 MSCs had typical appearance of osteoblasts and could be passaged continuously till passage 10. The rate of ALP expression was 85%. The expressions of collagen type Ⅰ, osteopontin and osteonectin were positive and calcium nodeswere seen by Von Kossa staining. Conclusion We have successfully induced human MSCs into osteoblasts; the induced cells have typical osteogenesis characteristics.
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.
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 study the immunogenicity of human bone marrow mesenchymal stem cells (BMSCs) and the suppression ability to the proliferation of peripheral blood mononuclear cell (PBMC) during osteogenic, chondrogenic, and adipogenic differentiations. MethodsBMSCs were isolated from bone marrow of healthy donors and were induced to osteogenic, chondrogenic, and adipogenic differentiations for 7, 14, and 21 days. The expressions of human leukocyte antigen (HLA) class I and class II were detected by flow cytometry. PBMC were isolated from peripheral blood of healthy donors and were co-cultured with BMSCs at a ratio of 10∶1 for 5 days. The suppression ability of undifferentiated and differentiated BMSCs to proliferation of PBMC were detected by flow cytometry. ResultsThe HLA class I expression was observed but almost no expression of HLA class II was seen in undifferentiated BMSCs. There was no obviously change of the HLA class I and class II expressions during osteogenic and chondrogenic differentiations (P>0.05), and a low expression of HLA class II was kept. The HLA class I expression gradually increased at 14 and 21 days after adipogenic differentiation, showing significant differences when compared with the value at 0 and 7 days (P<0.05);the HLA class II expression also gradually increased at 7, 14, and 21 days after adipogenic differentiation, showing significant differences when compared with the value at 0 day (P<0.05). There was no proliferation of PBMC without the stimulation of CD3 and CD28 microspheres and significant proliferation was observed when CD3 and CD28 microspheres were added, and undifferentiated BMSCs could significantly inhibit the proliferation of PBMC. There was no obvious change of the ability of BMSCs to inhibit the proliferation of PBMC during osteogenic and chondrogenic differentiations (P>0.05);and the ability of BMSCs to inhibit the proliferation of PBMC was gradually weakened at 7, 14, and 21 days after adipogenic differentiation, showing significant differences among different time points (P<0.05). ConclusionBMSCs maintain low immunogenicity and strong immune suppression ability during osteogenic and chondrogenic differentiations, which are suitable for allogenic tissue engineering repair and cell transplantation. However, increased immunogenicity and decreased immune suppression ability after adipogenic differentiation may not be suitable for allogenic tissue engineering repair and cell transplantation.
ObjectiveTo summarize the research progress of the effects and mechanisms of Hedgehog signaling pathway in regulating bone formation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). MethodsThe related literature concerning the regulations and mechanism of Hedgehog signaling pathway in osteogenic differentiation of BMSCs and bone formation in vivo, in vitro, and ex vivo studies in recent years was analyzed and summarized. ResultsThe in vitro studies indicate that Hedgehog signaling pathway can promote osteogenic differentiation of BMSCs via activation of key molecules Smoothened (Smo) and Gli1 which are downstream of Hedgehog signaling, and Hedgehog signaling can activate mTORC2-Akt signaling by upregulation of insulin-like growth factor which has similar effects. Hedgehog signaling regulates osteoblast differentiation via activation of Hh-Smo-Ptch1-Gli signaling pathway and inhibition of Hh-Gαi-RhoA stress fibre signaling. Hedgehog signaling can regulate key molecules of osteogenesis Runx2 for promoting osteogenic differentiation and matrix mineralization by synergism of bone morphogenetic protein and Wnt signaling, and promotes bone formation and repair and healing for bone defect and bone graft model in vivo. ConclusionHedgehog signaling can regulate bone formation and osteogenic differentiation of BMSCs via activation of Hedgehog signaling and other signaling pathways. Hedgehog signaling pathway may be a potential target for developing treatment for bone related diseases of osteoporosis and fracture healing disorders.
ObjectiveTo study the immunological properties of osteogenically differentiated umbilical cord blood derived mesenchymal stem cells (UCB-MSCs). MethodsUCB-MSCs were isolated from the umbilical cord vein, and were expanded; the cells at passage 3 were osteogenically induced for 2 weeks in vitro. The expressions of human leukocyte antigen I (HLA-I) and HLA-Ⅱ molecules were observed by flow cytometry analysis before and after osteogenic induction. Peripheral blood T lymphocytes were isolated and cultured with osteoblastic induced or non-osteoblastic induced UCB-MSCs in different cell concentrations of 1×102, 1×103, 1×104, and 1×105 cells/well. The intake value of 3H-thymidine was calculated with luminescence counter. Then T lymphocytes were pretreated with PHA, and co-cultured with osteoblastic induced and non-osteoblastic induced UCB-MSCs as described above. IL-2 was further added to test the reversed effect of T lymphocytes proliferation stimulated by UCB-MSCs. Finally, to investigate whether the immunomodulatory effects on T lymphocytes proliferation depend on direct or indirect cell contact, the Transwell chamber culture system of UCB-MSCs and T lymphocytes was established. ResultsFlow cytometry analysis showed that non-osteoblastic induced UCB-MSCs expressed HLA-I but did not express HLA-Ⅱ; the expression of HLA-Ⅱ increased in osteoblastic induced UCB-MSCs. No T lymphocyte response was stimulated by non-osteoblastic induced UCB-MSCs, but osteoblastic induced UCB-MSCs could stimulate the proliferation of allogeneic T lymphocytes, especially after IFN-γ treatment. Non-osteoblastic induced UCB-MSCs of 1×104 and 1×105 cells/well could suppress the proliferation of T lymphocytes evoked by PHA, and this suppression could be reversed by the addition of IL-2. While osteoblastic induced UCB-MSCs did not have such suppressive effect. The results of the Transwell culture system also showed that non-osteoblastic induced UCB-MSCs could obviously inhibit the proliferation of T lymphocytes, but the osteoblastic induced UCB-MSCs could not. ConclusionThe immunological properties of UCB-MSCs will change accordingly after osteogenic induction, so UCB-MSCs might not be suitable for the seed cells of bone tissue engineering.
Objective To clarify the trends of expression levels of several up-regulated micro RNA (miRNA) in tissues of atrophic bone nonunion and mRNAs and proteins of their related target genes in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and to explore their biological functions. Methods The hBMSCs were isolated from bone marrow of il iac bone by gradient centrifugation, and cultured. Osteogenic culture medium was used for osteogenic differentiation of the 4th generation of hBMSCs. The changes of corresponding miRNAs, mRNA and protein expression levels of related target genes were observed at 0 hour, 12 hours, 1 day, 2 days, 4 days, 7 days, and 14 days, by quantitative real-time PCR and Western blot. Results In the process of hBMSCs osteogenic differentiation, the mRNA and protein expression levels of osteoblastic target genes [alkal ine phosphatase l iver/bone/kidney (ALPL), bone morphogeneticprotein 2 (BMP-2), and platelet-derived factor alpha polypeptide (PDGF-A)] at most time points increased significantly whencompared with the values at 0 hour except that of BMP-2 decreased at 12 hours and 1 day, with maximum changes at 1 to 7 days. The miRNA expression levels, mRNA and protein expression levels changed significantly at different time points, while the trends of hsa-miRNA-149 and hsa-miRNA-654-5p changes were negatively correlated with the trends of ALPL and BMP-2 mRNA and protein expression changes respectively (P lt; 0.05). There was no obviously negative correlation between the trends of hsa-miRNA-221 change and PDGF-A change (P gt; 0.05). Conclusion In the osteogenic differentiation process of hBMSCs, hsa-miRNA-149 and hsa-miRNA-654-5p are closely related with the mRNA and protein regulation of ALPL and BMP-2, respectively.
Objective To investigate the effect of various concentration of platelet-rich plasma (PRP) on osteogenic differentiation of rabbit skeletal muscle-derived stem cells (SMSCs) cultured in vitro. Methods Blood drawn from the central ear arteries of 9 one-year-old New Zealand white rabbits weighing 2.5-3.0 kg (male and female) was used to prepare PRP (Landesberg method). Full blood count and platelet count in PRP were tested. Soleus muscle of right hindl imb in rabbit was obtained and used to culture SMSCs in vitro. The cells at passage 3 were randomly divided into different groups: the experimental groups in which the cells were treated by conditioned culture media with various concentrations of autologousPRP (6.25%, 12.50%, 25.00%, 50.00%), and the control group in which the cells were treated with the media without PRP. At different time points after intervention, osteogenetic activity of the cells was detected by ALP staining observation, ALP activity detection was conducted, al izarin red staining for calcium nodules and immunofluorescence staining for osteocalcin were performed, and core binding factor α1 (Cbfα1) of osteogenic gene expression was tested by RT-PCR. Results The full blood PRP count and the platelet count in PRP was (3.06 ± 0.46) × 105/μL and (18.08 ± 2.10) × 105/μL, respectively. ALP staining: the cells in all the experimental groups were positive for the staining with many black sediment particles in cytoplasm; the cells in the control group were negative staining. ALP activity: all the experimental groups were higher than the control group (P lt; 0.05), the experimental group at 12.50% was superior to other experimental groups at each time point (P lt; 0.05). Al izarin red staining: at 14 days after culture, orange-red calcium nodules were evident in all the experimental groups; no orange-red calcium nodules were observed in the control group with a mineral ization rate of zero; there were significant difference between the experimental groups and the control group in terms of mineral ization rate (P lt; 0.05), the experimental group at 12.50% had a higher mineral ization rate than other experimental groups (P lt; 0.05). Immunofluorescence staining for osteocalcin: at 7 days after culture, the experimental groups were positive for the staining with yellow fluorescence in cytoplasm, and the result of the control group was negative. RT-PCR detection: no obvious changes of the gene expression were noted at 4, 12, and 24 hoursafter culture in the control group; the gene expression in all the experimental groups was significant superior to that of control group, especially at 12 hours, and the expression in the experimental group at 12.50% was the highest. Conclusion PRP can obviously promote the osteogenic differentiation of SMSCs cultured in vitro in a concentration-dependent manner, and the 12.50% is proved to be the ideal concentration.
ObjectiveTo investigate whether miR-93-5p suppresses osteogenic differentiation of mouse mesenchymal stem cells (C3H10T1/2) by targeting Smad5, a predicted target in silicon. MethodsSmad5 3'-UTRluciferase vector (pmiR-RB-REPORTTM) was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-93-5p on Smad5 3'-UTR-luciferase activity to identify whether Smad5 was the target gene of miR-93-5p. miR-93-5p mimics (group M), miR-93-5p inhibitor (group In), miR-93-5p mimics negative control (group MC), and miR-93-5p inhibitor negative control (group InC) were transfected into the C3H10T1/2 cells, respectively, and followed by induction of osteogenic differentiation. After 48 hours, the real-time fluorescent quantitative PCR (qRTPCR) and Western blot assays were performed to detect the relative expressions of Smad5 mRNA and protein. At 14 days, to realize the regulation role of miR-93-5p in osteogenic differentiation, the extracellular calcium deposition during the osteogenesis of C3H10T1/2 cells was tested by Alizarin red staining. ResultsDual-luciferase reporter gene assay showed that miR-93-5p could combine with Smad5 mRNA 3'-UTR specificity, and inhibited its luciferase activity (P<0.05). After 48 hours, no significant difference was shown in the relative expression of Smad5 mRNA between group M and group MC as well as between group In and group InC by qRT-PCR assay (P>0.05); however, the results of Western blot assay showed that the relative expression of Smad5 protein was significantly decreased in group M and increased in group In when compared with groups MC and InC (P<0.05). At 14 days after osteogenic induction, Alizarin red staining showed that the extracellular calcium deposition of group M was obviously less than that of group MC, and it was obviously more in group In than in group InC. ConclusionSmad5 may be the target gene of miR-93-5p. And miR-93-5p can suppress osteogenic differentiation of C3H10T1/2 cells by directly targeting Smad5.