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 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.
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.
ObjectiveTo investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein 9 (BMP-9) and erythropoietin (EPO) genes co-transfection on osteogenic differentiation of adipose-derived stem cells (ADSCs) in vitro. MethodsThe inguinal adipose tissue was harvested from 4-month-old New Zealand rabbits, ADSCs were isolated with enzyme digestion and adherence method, and multipotent differentiation capacity was identified. The 3rd generation ADSCs were divided into 5 groups: normal cells (group A), empty plasmid control group (group B), BMP-9 or EPO recombinant adenovirus transfected cells (groups C and D), BMP-9 and EPO recombinant adenovirus co-transfected cells (group E). The inverted phase contrast microscope was used to observe the cell growth at 7 days; the expression of cell fluorescence was observed under a fluorescence microscope at 14 days, and viral transfection efficiency was calculated at 48 hours; Western blot was used to detect the expressions of BMP-9 and EPO proteins at 14 days. The expression of alkaline phosphatase (ALP) activity was detected at 3, 7, and 14 days after osteogenic induction, and alizarin red staining was used to detect calcium nodules formation and real-time fluorescence quantitative PCR to detect the expressions of osteopontin (OPN) and osteocalcin (OCN) at 3 weeks. ResultsAt 7 days after transfected, some cells showed oval, round, and irregular shape under the inverted phase contrast microscope in groups A and B; a few fusiform cells were observed in groups C and D; oval cells increased obviously, and there were only few round cells in group E. The fluorescence microscope observation showed that BMP-9 and EPO, BMP-9/EPO recombinant adenovirus could stably transfected ADSCs, with transfection efficiency of 80%-93%. The expressions of BMP-9 and EPO proteins significantly higher in group E than the other groups by Western blot (P < 0.05). The ALP activity significantly increased in group E when compared with that in the other groups at 3, 7, and 14 days after osteogenic induction (P < 0.05); the number of calcium nodules in group E was significantly more than that in the other groups (P < 0.05). Real-time fluorescence quantitative PCR showed that OPN and OCN genes expressions were significantly higher in group E than other groups (P < 0.05), and in groups C and D than groups A and B (P < 0.05). ConclusionRecombinant adenovirus-mediated BMP-9 and EPO genes can transfect ADSCs, which can stably express in ADSCs, BMP-9/EPO genes co-transfection can more promote the expressions of osteoblast-related genes and protein than non-transfected and single gene transfection.
ObjectiveTo investigate the effect of cyclic stretch stress on the osteogenic differentiation of human cartilage endplate-derived stem cells (CESCs). MethodsCESCs were isolated from the endplate cartilage tissues by the method of agarose suspension culture system. The endplate cartilage tissue was harvested for immunohistochemical staining. Flexercell-4000TM Tension Plus system was used to apply cyclic stretch on CESCs at a frequency of 1 Hz and at a stretch rate of 10% for 1, 6, 12, or 24 hours (experimental group). No stretch stress was performed on CESCs in the same culture condition (control group). After mechanical loading, the protein expression of bone morphogenetic protein 2 (BMP-2) was measured by Western blot, and gene expressions of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and SOX9 were detected by real-time fluorescent quantitative PCR. ResultsImmunohistochemical staining showed BMP-2 protein expression in chondrocytes. The continuous cyclic stretch stress of 10% can increase the expression of BMP-2 protein in CESCs. Significant differences were observed in the expressions of BMP-2 protein (P<0.05) between 2 groups at the other time points except at 1 hour (P>0.05), in a time-dependent manner. The real-time fluorescent quantitative PCR indicated that the gene expressions of Runx2 and ALP showed an increasing tendency with time in the experimental group when compared with the control group, but there was down-regulated expression of SOX9. Significant difference was found in mRNA expressions of Runx2 and ALP at 12 and 24 hours and in mRNA expressions of SOX9 at 6, 12, and 24 hours between 2 groups (P<0.05), in a time-dependent manner. ConclusionCyclic stretch stress may induce osteogenic differentiation of CESCs by regulating the expressions of some genes related osteogenesis in CESCs.
Objective To summarize the regulations of Hedgehog signal ing pathway on the prol iferation and multidifferentiation of mesenchymal stem cells (MSCs). Methods The related l iterature in recent years concerning the regulations of Hedgehog signal ing pathway on the biological characteristics of MSCs was reviewed and analyzed. Results Hedgehog signal ing pathway promoted the prol iferation of MSCs, and played a major role in the induction of osteogenic and chondrogenic differentiations, but it inhibited the adi pocytic differentiation. Conclusion The regulations of Hedgehog signal ing pathway in MSCs multidifferentiation and prol iferation could be used as the new therapeutic targets of tissue ischemia, osteoporosis, achondroplasia, obesity, and so on.
Objective As one of the adult stem cells, adi pose-derived stem cells (ADSCs) have become an important seed cell source for tissue engineering recently. But whether the thawed cryopreserved ADSCs could be used to tissue engineered bone remains unknown. To investigate the effect of cryopreservation on the growth and osteogenesis of ADSCs invitro. Methods The ADSCs were isolated from the adipose aspirates by collagenase digestion method. For the experimental group, the 2nd generation cells were stored with a simple method of cryopreservation by slow cool ing with dimethyl sulphoxide as a cryoprotectant and rapid thawing. After cryopreserved in l iquid nitrogen for 4 weeks, ADSCs were recovered and cultured in osteogenic media, with non-cryopreserved ADSCs as the control group. The osteogenic differentiation was evaluated by alkal ine phosphatase (ALP) staining and Al izarin red O staining at 2 and 3 weeks respectively. The cell growth and osteogenesis of ADSCs were further determined using DNA assay and the ALP activity and calcium content were measured. Results The survival percentage of the cryopreserved cells was 90.44% ± 2.62%. The cell numbers and ALP activity increased with osteogenic induction time, and reach plateaus at 7 days and 11 days, respectively. The ALP staining and Al izarin red O staining results were both positive at 2 weeks and 3 weeks after osteogenic induction, respectively. And no significant difference in the cells number, ALP activity, and calcium content were found between experimental group and control group (P gt; 0.05). Conclusion Cryopreservation does not affect the growth and osteogenesis of ADSCs, and the cryopreserved ADSCs can be used as cell source for tissue engineered bone.
ObjectiveTo investigate the role of the forkhead/Fox transcription factor 2 (Foxc2) over-expression in regulating osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by Wnt-β-catenin signaling pathways in vitro so as to provide the experimental basis for repairing osteonecrosis of the femoral head. MethodsThe recombinant lentivirus carrying green fluorescent protein (group A) or Foxc2 (group B) were used to transfect the fifth generation rabbit BMSCs, and untransfected BMSCs served as a control (group C). The cell viability was measured with water soluble tetrazolium-1 (WST-1) regent at 72 hours after transfection. After 2 weeks of transfection, the expression of β-catenin in BMSCs was detected by real time fluorescence quantitative PCR, Western blot, and immunofluorescence staining. Meanwhile, the β-catenin inhibitors XAV-939 (0, 0.1, and 1.0 μmol/L) was added in group B; at 2 weeks after osteogenic and adipogenic induction, the gene and protein expressions of collagen type I (COL I), osteocalcin (OCN), and peroxisome proliferator activated receptor gamma 2 (PPARγ-2) were detected by real time PCR and Western blot. ResultsWST-1 results showed that the cell viability of group B (130.85%±0.15%) was significantly higher than that of group A (100.45%±0.35%) (t=7.500, P=0.004) at 72 hours after transfection. At 2 weeks after transfection, the gene and protein expressions of β-catenin in group B were significantly higher than those in group A (P<0.01). After XAV-939 was added in group B, the mRNA and protein expressions of OCN and COL I gradually decreased; the mRNA and protein expressions of PPARγ-2 significantly increased (P<0.05), showing a dose-dependent manner. ConclusionThe over-expression of Foxc2 gene in BMSCs may promote osteogenic differentiation by Wnt-β-catenin signaling pathway.
ObjectiveTo investigate the regulatory effect of simvastatin on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at middle/late stages by p38MAPK pathway under condition of osteoinductive environment. MethodsThe bone marrow of bilateral femur and tibia were harvested from 20 4-week-old female Sprague Dawley rats. BMSCs were isolated and cultured with whole bone marrow culture method; the second generation of cells were randomly divided into 5 groups: control group (complete medium, CM), simvastatin group (simvastatin medium, SIM), osteogenic induction group (osteogenic induction medium, OM), simvastatin and osteogenic induction group (simvastatin+osteogenic induction medium, OM+SIM), and blocker group (SB203580+simvastatin+osteogenic induction medium, OM+SIM+SB). MTT assay was used to detect the cell activity in CM group and SIM group at 2, 3, 4, 5, and 6 days, ELISA method to measure the content of alkaline phosphatase (ALP) in OM group and OM+SIM group at 7 and 14 days. The mRNA and protein expressions of osteocalcin (OCN) were detected by real-time quatitative PCR and Western blot after 1, 12, and 24 hours of osteogenic induction at 21 and 28 days. The protein expressions of phospho-p38 (p-p38) and p38 in OM group, OM+SIM group, and OM+SIM+SB group were detected by Western blot at the best induction time of simvastatin. ResultsMTT assay showed that no significant difference was found in absorbance (A) value between CM group and SIM group at each time point (P > 0.05), indicating no effect of 1×10-7 mol/L simvastatin on cell viability. ELISA results showed that ALP content significantly increased in OM+SIM group when compared with OM group at 7 and 14 days; the ALP content was significantly higher at 7 days than 14 days in OM group and OM+SIM group (P < 0.05). OCN mRNA and protein expressions at 12 hours were significantly higher than those at other time points in each group (P < 0.05), and the expressions of OM+SIM group was significantly higher than those of OM group (P < 0.05). The best induction time of simvastatin was 12 hours. At 12 hours after blocking intervention, the p-p38/p38 in OM+SIM+SB group was significantly lower than that in OM group and OM+SIM group (P < 0.05), and the p-p38/p38 in OM+SIM group was significantly higher than that in OM group (P < 0.05). ConclusionSimvastatin can increase the mRNA and protein expression levels of OCN and the protein of p-p38 in osteogenic differentiation of BMSCs at middle/ late stages, and its best induction time is 12 hours.