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: A rare huge desmoplastic fibroma on thoracic wall in 1 female case of 25 years old was resected, and the accompanying huge thoracic wall defect, ribs and soft tissues were repaired by tissue engineered bone and pedicled flap. The paper aims to explore the clinical results of early stage after operation. METHODS: Autogeneic bone marrow stromal cells (MSC) were obtained from bone marrow puncture of iliac bone and isolated and cultured according to the Houghton’s methods, MSC were directively induced and differentiated to osteoblasts. Allogeneic ribs were made to the bio-derived bone scaffold materials after treatment of decell, deantigen, decalcification and dry freezing. 5 x 10(6)/ml MSC were cocultured with the bio-derived bone for 6 days in vitro. After intact resection of tumor, the diaphragm flap was applied to repair pleural cavity, the three defect ribs were repaired by tissue engineered bone and the soft tissue defect was repaired by transfer of pedicled ipsilateral abdominal flaps. RESULTS: The patient recovered well with first intention. Followed up for 3 months, tissue engineered ribs were matured in vitro and the heart and pulmonary functions were improved markedly. CONCLUSION: The tissue engineered bone constructed by autogeneic MSC is advantageous in individual treatment.
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 study the biocompatibility on bioactive glass ceramics (BGC) and polylactic acid (PLA) combined with cultured bone marrow stromal cells (BMSCs) in bone tissue engineering. METHODS BMSCs were cultured combined with BGC and PLA in vitro, and the morphological characters, cell proliferation, protein content, and alkaline phosphatase activity were detected. RESULTS: BMSCs could be attached to and extended on both BGC and PLA, and normally grown, proliferated, had active function. BGC could promote cell proliferation. CONCLUSION The results show that both BGC and PLA have good biocompatibility with BMSCs, they can be used as biomaterials for cell transplantation in tissue engineering.
Objective To explore the differences between transient osteoporosis of the hi p (TOH) and bone marrow edema (BME) associated with osteonecrosis of the femoral head (ONFH) in terms of cl inical practice and imaging. Methods From January 2006 to February 2008, 5 hips of TOH in 5 cases (1 male and 4 females, aged 29-42 years) and 67 hips of BME associated with ONFH in 63 cases (53 males and 10 females, aged 18-70 years) were analyzed. According to ARCO classification, there were 23 hi ps of stage II, 43 hi ps of stage III, and 1 hi p of stage IV. The induced factors, the degree of pain, the duration of pain and commemorative symptoms were compared. The X-ray, MRI and ECT were also compared. Results There were no differences in induced factors, the degree of pain, joint effusion and ECT between TOH and BME associated with ONFH. TOH had no risk factors, antecedent symptoms and commemorative pain. There were 2 hips with TOH which showed osteopenia on X-ray films. The location of edema was in the superior part or the whole femoral head.A total of 65 hips with BME associated with ONFH had related causes of disease, 10 had antecedent pain and 59 had change of hardening on X-ray films. The X-ray films showed sclerotic area and the edema changes on MRI surrounded the necrotic lesion. The pain just amel iorated when BME disappeared. Conclusion There are differences between TOH and BME associated with ONFH on symptoms, X-ray films and MRI.
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.
Objective The biological treatment of intervertebral disc degeneration becomes a research hotspot in recentyears. It is necessary to find an effective approach to induce bone marrow mesenchymal stem cells (BMSCs) differentiate to disc cells which could make appl ication of cell transplantation as a treatment of intervertebral disc degeneration. To investigate the effects of the recombinant plasmid pcDNA3.1IE-SOX9Flag on differentiation of rabbit BMSCs into nucleus pulposus-l ike cells. Methods The eukaryotic expression vector of pcDNA3.1IE-SOX9Flag was constructed. Rabbit BMSCs were isolated and cultured from one-month-old New Zealand white rabbits and were induced into osteogenetic cells in the osteogenesis supplement medium; and the cell surface markers were detected by flow cytometry. The cells at the 3rd passage were randomly divided into 3 groups: in transfected group, the cells were transfected with recombinant plasmid pcDNA3.1IE-SOX9Flag; in negative control group, the cells were transfected with plasmid pcDNA3.1; and in blank control group, the cells were treated with the media without recombinant plasmid. After selected by G418 for 7 days, the cells were harvested and RT-PCR was employed to assay SOX9 mRNA and collagen type II gene (Col2al) mRNA expressions in BMSCs. The expression of SOX9 protein was assayed by Western blot and collagen type II expression was also observed by immunohistochemical staining. Results The SOX9 eukaryotic expression vector was constructed successfully. The BMSCs after 5 days of osteogenetic induction were positive for the alkal ine phosphatase staining. What was more, CD44 expression was positive but CD34 and CD45 expressions were negative. The transfection efficiency was 34.32% ± 1.75% at 72 hours after transfection. After 2 weeks of transfection, BMSCs turned to polygonal and ell iptical. And the cell prol iferation was gradually slow which was similar to the growth characteristic of nucleus pulposus cells. RT-PCR identification showed that SOX9 mRNA and Col2al mRNA expressions were positive in transfected group, and were negative in 2 control groups. Western blot detection showed that SOX9 protein expressed in transfected group but did not express in the control groups. At 2 weeks after transfection, the result of the immunohistochemicalstaining for collagen type II was positive in transfected group. Conclusion The recombinant plasmid pcDNA3.1IE-SOX9Flag can be successfully transfected into rabbit BMSCs, the transfected BMSCs can differentiate into nucleus pulposus-l ike cells, which lays a theoretical foundation for treatment of intervertebral disc degeneration with BMSCs transplantation.