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.
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.
To study the method of isolating and culturing synovium-derived MSCs (SMSCs), and to investigate its multiple differentiation potential in vitro. Methods Three 2-month-old Changfeng hybrid swines weighing 8-10 kg (male and female) were used. SMSCs were harvested from the synovium of swine knee joints and cultured in vitro. When the SMSCs at passage 3 reached confluence, basic culture medium was removed, and the multi ple differentiationpotential of SMSCs was demonstrated in specific induction media (experimental group). The cells at passage 3 cultured with basic culture medium served as control group. After 21 days of chondrogenic differentiation, the cells underwent toluidine blue staining, immunohistochemistry staining and real-time fluorescence quantitative PCR detection. After 10 and 21 days of osteogenic differentiation, the cells underwent ALP staining and Al izarin red staining, respectively. After 21 days of adipogenic differentiation, the cells underwent Oil red O staining. Results SMSCs displayed long and thin or polygonal morphology 24 hours after culture. They prol iferated fast 48 hours after culture and presented large number of spindle-shaped cells with few globular cells 72 hours after culture. For the experimental group 21 days after chondrogenic induction, the cells were positive for toluidine blue staining with the formation of Aggrecan outside the cells; the immunohistochemistry staining revealed the expression of Col II; the real-time fluorescence quantitative PCR detection showed that the expressions of Col II A1, Aggrecan and SOX9 mRNA of the experimental group were greater than that of control group (P lt; 0.05). The cells were positive for ALP staining 10 days after osteogenic induction, and positive for Al izarin red staining 21 days after osteogenic induction, with the formation of calcium nodules. Oil red O staining displayed the formation of l i pid droplets inside the cells 21 days after adi pogenic induction. For the control group, the results of all the staining assays were negative except the ALP staining presenting with sl ight positive result. Conclusion SMSCs can be isolated from knee joint of swine and proliferate and differentiate into osteogenic, adi pogenic and chondrogenic cells in vitro. SMSCs may be a promising source of seed cells for tissue engineering.
Objective Dexamethasone is one of the basic agents which could induce osteogenic differentiation of mesenchymal stem cells. To investigate the optimal concentration of dexamethasone in osteogenic differentiation of adiposederivedstem cells (ADSCs) so as to provide the theoretical basis for further bone tissue engineering researches. Methods FiveNew Zealand rabbits (2-3 kg) of clean grade, aged 3 months and male or female, were obtained. ADSCs were isolated from the subcutaneous adipose tissue of inguinal region, and cultured with collagenase digestion, then were detected and identified by CD44, CD106 immunofluorescence staining and adi pogenic differentiation. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 105 cells/mL, then the cells were treated with common cultural medium (group A) and osteogenic induced medium containing 0 (group B), 1 × 10-9 (group C), 1 × 10-8 (group D), 1 × 10-7 (group E), 1 × 10-6 (group F), and 1 × 10-5 mol/ L (group G) dexamethasone, respectively. The cell prol iferation and the mRNA expressions of osteocalcin (OC) and core binding factor α1 (Cbfα1) were detected by MTT and RT-PCR, respectively. The activity of alkal ine phosphatase (ALP) was measured, and the percentage of mineral area was calculated. The mineral nodules were also detected by al izarin red staining. Results ADSCs mostly presented fusiform and polygon shape with positive expression of CD44 and negative expression of CD106. The result of oil red O staining was positive after ADSCs treated with adipogenic induced medium. The result of MTT revealed that the absorbance (A) value decl ined with the ascending of the concentration of dexamethasone, and there was significant difference in A value between groups D and E at 5 and 7 days after osteogenic induction (P lt; 0.05). The mRNA expressions of OC and Cbfα1 reached the peak in groups E and D at 7 days after osteogenic induction, respectively. The activity of ALP and the percentage of mineral area had the maximum value in group D at 14 days, then decl ined gradually. There was no significant difference in the mRNA expressions of OC and Cbfα1, the activity of ALP, and the percentage ofmineral area between groups D and E (P gt; 0.05), but significant differences were found between groups D and E and other groups (P lt; 0.05). After 14 days, the cells of group G died, and the result of al izarin red staining was positive in groups B, C, D, E, and F. Conclusion When the concentration of dexamethasone in osteogenic medium is 1 × 10-8 mol/L, it could not only reduce the inhibitive effect on cells prol iferation, but also induce osteogenic differentiation of ADSCs more efficiently.
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.
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.
Objective Calcium phosphate bioceramics has a broad appl ication prospect because of good biocompatibil ity, but porous scaffolds with complex shape can not be prepared by the traditional methods. To fabricate porous calcium phosphate ceramics by rapid prototyping and to investigate the in vitro osteogenic activities. Methods The porous calcium phosphate ceramics was fabricated by rapid prototyping. The bone marrow mesenchymal stem cells (BMSCs)were isolated from bone marrow of Beagle canine, and the 3rd passage BMSCs were seeded onto the porous ceramics. The cell/ceramics composite cultured in osteogenic medium were taken as the experimental group (group A) and the cell/ceramics composite cultured in growth medium were taken as the control group (group B). Meanwhile, the cells seeded on the culture plate were cultured in osteogenic medium or growth medium respectively as positive control (group C) or negative control (group D). After 1, 3, and 7 days of culture, the cell prol iferation and osteogenic differentiation on the porous ceramics were evaluated by DNA quantitative analysis, histochemical staining and alkal ine phosphatase (ALP) activity. After DiO fluorescent dye, the cell adhesion, growth, and prol iferation on the porous ceramics were also observed by confocal laser scanning microscope (CLSM). Results DNA quantitative analysis results showed that the number of BMSCs in all groups increased continuously with time. Plateau phase was not obvious in groups A and B, but it was clearly observed in groups C and D. The CLSM observation indicated that the activity of BMSCs was good and the cells spread extensively, showing good adhesion and prol iferation on the porous calcium phosphate ceramics prepared by rapid prototyping. ALP quantitative analysis results showed that the stain of cells on the ceramics became deeper and deeper with time in groups A and B, the staining degree in group A were ber than that in group B. There was no significant difference in the change of the ALP activity among 4 groups at the first 3 days (P gt; 0.05); the ALP activity increased obviously in 4 groups at 7 days, group A was significantly higher than other groups (P lt; 0.05) and groups C, D were significantly higher than group D (P lt; 0.05). Conclusion The porous calcium phosphate ceramics has good cytocompatibil ity and the designed pores are favorable for cell ingrowth. The porous ceramicsfabricated by rapid prototyping has prominent osteogenic differentiation activity and can be used as a choice of scaffolds for bone tissue engineering.
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.
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 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.