Objective To investigate the effect of growth differentiation factor 7 (GDF-7) on the tenogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro, to provide evidence for improving the efficacy of BMSCs on tendon repair. Methods BMSCs were isolated from bone marrow tissue of green fluorescent protein rats by density gradient centrifugation method. Chondrogenic, osteogenic, and adipogenic differentiation assays were used to demonstrate the multi-differentiation potential of the BMSCs. BMSCs at passage 3 were cultured and divided into 4 groups according to different concentrations of GDF-7 (0, 12.5, 25.0, and 50.0 ng/mL): group A, B, C, and D, respectively. After cultured for 2 weeks in vitro, the mRNA expressions of scleraxis, tenomodulin, tenascin C, and collagen type I were detected by real-time fluorescent quantitative PCR method, the protein expressions of tenomodulin, tenascin C, and collagen type I by immunocytochemistry staining in 4 groups, and the protein expressions of tenomodulin by Western blot in groups A and C. Results BMSCs had osteogenic, chondrogenic, and adipogenic differentiation potentials. The mRNA expressions of tenomodulin in groups B, C, and D were 2.85, 3.41, and 3.07 times higher than that in group A, respectively; the mRNA expressions of scleraxis in groups B, C, and D were 2.13, 1.50, and 2.56 times higher than that in group A, respectively; and the mRNA expressions of tenascin C in groups B, C, and D were 2.45, 2.86, and 1.88 times higher than that in group A, respectively. There were significant differences between groups B, C, D and group A (P lt; 0.05), while there was no significant difference among groups B, C, and D (P gt; 0.05). The mRNA expressions of collagen type I in groups B and C were 1.92 and 2.45 times higher than that in group A, showing significant differences between groups B, C and group A (P lt; 0.05), but no significant difference between groups A and D (P gt; 0.05). Immunocytochemistry staining showed that the protein expressions of tenomodulin, tenascin C, and collagen type I were detected in groups B, C, and D but not in group A. The results were further confirmed by Western blot results which showed higher protein expression of tenomodulin in group C than in group A. Conclusion GDF-7 can be used to promote tenogenic differentiation of rat BMSCs in vitro.
Objective To study the influence of autologous bone mesenchymal stem cells (BMSCs) on myocardial structure and cardiac function after being implantated into acute infarcted myocardial site. Methods Bone marrow was aspirated from the posterosuperior iliac spine of Guizhou Xiang swine. After being isolated, cultured and co cultured with 5 azacytidine, either autologous BMSCs (total cells 2×10 6, experimental group, n =12), or a comparable volume of culture medium (control group, n =12), was injected into the left anterior descending(LAD) branch of coronary artery just distal to the ligation site of the LAD. The same volume of BMSCs or culture medium was injected into several spots in the infarcted myocardium. Echocardiographic measurements were performed three or six weeks after implantation to assess the myocardial structure and cardiac function. Results Left ventricular function, including eject fraction(EF), fractional shortening and wall thickening, were higher in experimental group when compared with control group. The thickness of the ventricular wall and septum was also found increased while the left ventricular chamber size was smaller in experimental group. Conclusion Implantation of BMSCs into the infarcted myocardium is believed to attenuate the remodeling process, inhibit the extent of wall thinning and dilatation of the ventricular chamber. BMSCs implantation may also improve the contractile ability of the myocardium and cardiac function.
Objective To study the vascularization of the compositeof bone morphogenetic protein 2 (BMP-2) gene transfected marrow mesenchymal stem cells (MSCs) and biodegradable scaffolds in repairing bone defect. Methods Adenovirus vector carrying BMP-2 (Ad-BMP-2) gene transfected MSCs and gene modified tissue engineered bone was constructed. The 1.5 cm radial defect models were made on 60 rabbits, which were evenly divided into 4 groups randomly(n=15, 30 sides). Different materials were used in 4 groups: Ad-BMP-2 transfected MSCs plus PLA/PCL (group A), AdLacz transfected MSCs plus PLA/PCL (group B), MSCs plus PLA/PCL (group C) and only PLA/PCL scaffolds (group D). The X-ray, capillary vessel ink infusion, histology, TEM, VEGF expression and microvacular density counting(MVD) were made 4, 8, and 12 weeks after operation. Results In group A after 4 weeks, foliated formed bones image was observed in the transplanted bones, new vessels grew into the bones, the pores of scaffolds were filled with cartilage callus, osteoblasts with active function grew around the microvessels, and VEGF expression and the number of microvessels were significantly superior to those of other groups, showing statistically significant difference (Plt;0.01); after 8 weeks, increasingly more new bones grew in the transplanted bones, microvessels distended and connected with each other, cartilage callus changed into trabecular bones; after 12 weeks, lamellar bone became successive, marrow cavity recanalized, microvessels showed orderly longitudinal arrangement. In groups B and C, the capability of bone formation was weak, the regeneration of blood vessels was slow, after 12 weeks, defects were mostly repaired, microvessels grew among the new trabecular bones. In group D, few new vessels were observed at each time, after 12 weeks, broken ends became hardened, the defectedarea was filled with fibrous tissue. Conclusion BMP-2 gene therapy, by -upregulating VEGF expression, indirectly induces vascularization ofgrafts,promotes the living of seed cells, and thus accelerates new bone formation.
Objective To study the differenation of adult marrow mesenchymal stem cells(MSCs) into vascular endothelial cells in vitro and to explore inducing conditions. Methods MSCs were isolated from adult marrow mononuclear cells by attaching growth. MSCs were divided into 4 groups to induce: the cells seeded at a density of 5×103/cm2 in 2% and 15% FCS LDMEM respectively (group1 and group 2)， at a density of 5×104/cm2 in 2% and 15% FCS LDMEM respectively (group 3 and group 4); vascular endothelial growth factor(VEGF) supplemented with Bovine pituitary extract was used to induce the cell differentiation. The differentiated cells were identified by measuring surfacemarks (CD34, VEGFR2, CD31 and vWF ) on the 14th day and 21st day and performed angiogenesis in vitroon the 21st day.The cell proliferation index（PI）of different inducing conditions were measured. Results After induced in VEGF supplemented with Bovine pituitary extract, the cells of group 3 expressed the surface marks CD34, VEGFR-2, CD31 and vWF on the 14th day, the positive rates were 8.5%, 12.0%, 40.0% and 30.0% respectively, and on the 21st day the positive ratesof CD34 and VEGFR2 increased to 15.5％ and 20.0％, while the other groups did not express these marks; the induced cells of group 3 showed low proliferating state(PI was 10.4%) and formed capillary-like structure in semisolid medium. Conclusion Adult MSCs can differentiate into vascular endothelial cellsafter induced by VEGF and Bovine pituitary extract at high cell densities and low proliferatingconditions，suggesting that adult MSCs will be ideal seed cells forthe therapeutic neovascularization and tissue engineering.
Objective To investigate the effect of combined therapy of granulocyte colony stimulating factor (G-CSF) and bone marrow mesenchymal stem cells (BMSCs) carrying hepatocyte growth factor (HGF) gene on the angiogenesis of myocardial infarction (MI) in rats and the mechanisms of the synergistic effect. Methods BMSCs were aspirated from the femur and tibia of 3-week-old Sprague Dawley (SD) male rats. The third generation of BMSCs were harvested and transfectedwith Ad-HGF. The MI models were establ ished in 44 SD male rats (weighing 200-250 g) by l igating the left coronary artery. At 4 weeks after l igation, the shorting fraction (FS) of the left ventricle being below 30% was used as a criteria of model success. The BMSCs (5 × 107/ mL) transfected with Ad-HGF were transplanted into the infarct zone of 12 SD rats, and the expression of HGF protein was detected by Western blot method at 2, 7, and 14 days after transplantation. At 4 weeks, the other 32 SD rats were randomly divided into 4 groups (n=8). The 0.1 mL normal sal ine was injected into the infarct zone in control group; 0.1 mL normal sal ine was injected combined with intraperitoneal injection G-CSF [100 μg/ (kg•d)] for 5 days in G-CSF group; 0.1 mL BMSCs (5 × 107/ mL) transfected with Ad-HGF was injected into the infarct zone in HGF group; 0.1 mL BMSCs (5 × 107/ mL) transfected with Ad-HGF was injected combined with intraperitoneal injection G-CSF [100 μg/ (kg•d)] for 5 days in combined therapy group. At 2 weeks after transplantation, heart function was detected by cardiac ultrasound and hemodynamic analysis, and then myocardial tissue was harvested to analyse the angiogenesis of the infarct zone, and the expression of VEGF protein by immunofluorescence staining. Results The expression of HGF protein in vivo was detected at 2 days and 7 days of BMSCs transfected with Ad-HGF transplantation. There was no significant difference in left ventricular systol ic pressure (LVSP), left ventricular end-diastol ic pressure (LVEDP), dP/dtmax, and FS between G-CSF group and control group (P gt; 0.05). When compared with the control group, LVEDP decreased significantly; LVSP, FS, and dP/dtmax increased significantly (P lt; 0.05) in HGF group and combined therapy group. When compared with HGF group, FS and dP/dtmax increased significantly in combined therapy group (P lt; 0.05). Immunofluorescence staining showed that the vascular endothel ial cells were observed in myocardial infarction border zone. The vascular density and the expression of VEGF protein were significantly higher in combined therapygroup than in other 3 groups (P lt; 0.05). Conclusion The combined therapy of G-CSF and BMSCs carrying HGF gene has a synergistic effect and can enhance infarct zone angiogenesis through inducing the expression of VEGF protein.
Objective To study the possibil ity of bone marrow mesenchymal stem cells (BMSCs) differentiation into tenocytes (TCs) under strain stimulation by co-culture of BMSCs-small intestinal submucosa (SIS) composites in vitro. Methods BMSCs were isolated by adherent culture from the bone marrow of 1-week-old SD rats. Inducing method of multiple differentiation and flow cytometry were appl ied to identify the cells. The stress-strain curve of SIS was measured with Instron machine. Purified BMSCs (2nd passage, 2.5 × 105 cells/cm2) were seeded on SIS (3 cm × 1 cm at size) and cultured for 2 daysand then continued for another 5 days under strain stimulation (stretching frequency was 0.02 Hz, action time was 15 minutes/ hour and 12 hours/day, strain ampl itude was 5%) as experimental group, while the BMSCs-SIS composites were sustained static culture as control group. TCs were isolated from tail of 1-week-old SD rats. TCs-SIS composites were cultured under non-strained as positive control group. Scanning electron microscope (SEM) was used to examine the morphological changes of BMSCs after strain stimulation. The contents of Scleraxis and Tenomodulin in supernatant were tested by ELISA kit. Results The BMSCs could be induced to differentiate into osteoblasts and l ipocytes, and showed the results of CD34-, CD45-, and CD90+, which were accorded with the biological characteristics of BMSCs. The failure test of SIS showed that the average elastic strain was 39.5%. SEM observation showed that the strain-stimulated BMSCs had the TCs-l ike morphological characteristics. The contents of Scleraxis and Tenomodul in in supernatant of experimental group, control group, and positive control group were (3.56 ± 0.91) μmol/L and (4.27 ± 1.10) μmol/L, (0.23 ± 0.14) μmol/L and (0.16 ± 0.10) μmol/L, and (14.73 ± 2.30) μmol/L and (10.65 ± 1.51) μmol/L, respectively. There were significant differences among 3 groups (P lt; 0.05). Conclusion Appropriate strain stimulation could induce BMSCsdifferentiate into TCs, and the best conditions of strain stimulation need more experiments.
Objective To investigate the possibility of establishing the human bone marrow mesenchymal stem cells (hMSCs) bank as to provide an alternative source for the seed cells of tissue engineering. Methods The cell surface antigensof the purified, expanded hMSCs and the ones following cryopreservation were detected by flow cytometry, cultured in a special medium to induce the ostoegenic and chondrocytic- differentiation. Morphology was studied by light and electronic microscopes. The detection of alkaline phosphatase, collagen type Ⅰ, osteocalcin, and collagen type Ⅱ were also performed by immunochemistry and molecular biology.Results The phenotype and expansion possibility of hMSCs after cryopreservation were remained. It could expand for 10 generations. The doubling time was 40 h.Conclusion The bank of hMSCs is inipiently established and can provide eligible seed cells for tissue engineering.
Objective To elucidate whether glucose transporters-4 （GLUT-4） takes part in glucose uptake of mesenchymal stem cells （MSCs） and whether Akt gene improves translocation and expression of GLUT-4 in MSCs under hypoxic environment ex vivo. Methods MSCs, transfected by Akt gene and no, were cultured with normoxia (5% CO2) or hypoxia (94%N2, 1%O2 and 5% CO2) at 37 ℃ for 8 h. Glucose uptake was assayed by using radiation isotope 2-［3H］-deoxy-Dglucose (3H-G) and the expression of GLUT-4 protein and mRNA was assayed by immunocytochemistry, Western blot and RT-PCR, respectively. Results ①3 H-G intake of MSCs was significantly increased in hypoxiatransfection group than that in hypoxia-non-transfection 〔（1.39±0.13） fold, P＜0.05〕, but which was lower than that in normoxia-non-transfection group, P＜0.05. ②GLUT-4 was expressed by MSCs under any conditions. Compared with normoxia-non-transfection group, hypoxia decreased the expressions of GLUT-4 mRNA and protein significantly (P＜0.05). ③Compared with hypoxianontransfection group, the expression of GLUT-4 〔mRNA（1.756±0.152） fold, total protein in cell （1.653±0.312） fold, protein in plasma membrane （2.041±0.258） fold〕 was increased in hypoxia-transfection group significantly (P＜0.05), but which was lower than that in normoxianontransfection group (P＜0.05). ④There was significantly positive relation between 3H-G intake and GLUT-4 protein expression in plasma membrane (r=0.415, P=0.001).Conclusion GLUT-4 may take part in glucose uptake of MSCs, and the capability of Akt gene to improve MSCs anti-hypoxia may be finished by its role in increasing the expression and translocation of GLUT-4.
Objective Bone marrow mesenchymal stem cells (BMSCs), as replacement cells of Schwann cells, can increase the effect of peripheral nerve repair. However, it has not yet reached any agreement to add the appropriate number of seeded cells in nerve scaffold. To investigate the effect of different number of BMSCs on the growth of rat dorsal root gangl ia(DRG). Methods Three 4-week-old Sprague Dawley (SD) rats (weighing 80-100 g) were selected to isolate BMSCs, whichwere cultured in vitro. Three 1- to 2-day-old SD rats (weighing 4-6 g) were selected to prepare DRG. BMSCs at passage 3 were used to prepare BMSCs-fibrin glue complex. According to different number of BMSCs at passage 3 in fibrin glue, experiment was divided into group A (1 × 103), group B (1 × 104), group C (1 × 105), and group D (0, blank control), and BMSCs were cocultured with rat DRG. The axon length of DRG, Schwann cell migration distance, and axon area index were quantitatively evaluated by morphology, neurofilament 200, and Schwann cells S-100 immunofluorescence staining after cultured for 48 hours. Results Some long cell processes formed in BMSCs at 48 hours; migration of Schwann cells and axons growth from the DRG were observed, growing in every direction. BMSCs in fibrin glue had the biological activity and could effect DRG growth. The axon length of DRG and Schwann cell migration distance in groups A, B, and C were significantly greater than those in group D (P lt; 0.05). The axon length of DRG and Schwann cell migration distance in group C were significantly less than those in group B (P lt; 0.05), but there was no significant difference between group A and group C, and between group A and group B (P gt; 0.05). The axon area index in groups A and B was significantly greater than that in group D (P lt; 0.05), but there was no significant difference between group C and group D (P gt; 0.05); there was no significant difference in groups A, B, and C (P gt; 0.05). Conclusion In vitro study on DRG culture experiments is an ideal objective neural model of nerve regeneration. The effect of different number of BMSCs in fibrin glue on the growth of DRG has dose-effect relationship. It can provide a theoretical basis for the appropriate choice of the BMSCs number for tissue engineered nerve.
Objective To summarize the research progress of biological characteristics and advantages of Wharton’s jelly-mesenchymal stem cells (WJ-MSCs). Methods The related l iterature on the biological characteristics of WJ-MSCs,umbil ical cord blood MSCs (UBMSCs) and bone marrow MSCs (BMSCs) was extensively reviewed and analyzed. Results A large number of MSCs which are able to self-repl icate, self-renew and have high prol iferation and multipotent differentiation can be isolated from the Wharton’s jelly of umbil ical cord. WJ-MSCs have many advantages in isolation time, isolation efficience, expansion time, passage capacity, expansion capacity when compared with UBMSCs and BMSCs. Conclusion WJ- MSCs have numerous advantages of convenient and abundant sources, relatively pure, non-ethical issues, and so on, which can be used for cell transplant therapy, gene therapy, and the ideal seed cells of building tissue engineered organ, so they provide new ideas for tissue regeneration repair and reconstruction.