Objective To study the effect of autogenous bone marrow on guided bone regeneration (GBR),and evaluate the repairing ability of GBR in bone defect with autogenous bone marrow. Methods Ten mm segmental defects were produced in both radii of 18 rabbits. The defect was bridged with a silicon tube. Autogenous bone marrow was injected into the tube on the experimental group at 0, 2,4 weeks after operation, and peripheralblood into the control group at thesame time. The X-ray, gross, histological and biochemical examinations were observed invarious times. Results The new bone formation of experimental group was prior to that of control group; calcium and alkaline phosphatase of experimental groupwere higher than those of control group. The experimental group had all been healed at the tenth week, but no one healed in control group. Conclusion It can be conclude that autogenous bone marrow can stimulate bone formation and facilitate GBR in bone defect.
Objective To explore the effective autologous bone marrow stem cell dosage for treatment of severe lower limb ischemia. Methods From December 2003 to December 2004, 22 cases of bilateral lower limb ischemia were treated with autologous bone morrow cell transplantation. All the patients were randomly divided into two groups according to ischemia degree. In group A(severe ischemia side), the amount of transplanted autologous bone marrow cells was more than 1×108, and ingroup B(mild ischemia side), the amount was less than 1×105. A series of subjective indexes, such as improvement of pain, cold sensation and numbness, and objective indexes, such as increase of ankle/brachial index (ABI) and transcutaneous oxygen pressure (TcPO2), angiography, amputation rate, and improvement of foot wound healing were used to evaluate the effect of autologous bone marrow stem cells implantation. Results The rates of pain relief were 90.0% in group A and 16.7% in group B (Plt;0.01); the rates of cold sensation relief were 90.5% in group A and 5.3% in group B（Plt;0.01）;the improvement of numbness was 62.5% in group A and 9.1% in group B（Plt;0.01）. Increase of ABI was 31.8% and 0 in groups A and B respectively（Plt;0.01） at 4 weeks after implantation. Increase of TcPO2was 94.4% and 11.1% in groups A and B respectively（Plt;0.01） at 4 weeks after implantation. Twelve cases of angiography showed rich new collateral vessels in 100% of the limbs in group A while no remarkable new collateral vessel in group B. The amputation rates were 4.5% in group A and 27.3% in group B（Plt;0.05） at 4 weeks after implantation. The rate of improvement of foot wound healing was 75% in group A and there was no changein wound healing in group B after 4 weeks of implantation. Conclusion The effectiveness of autologous bone marrow stem cell implantation depends on the number of implanted stem cells. Effectiveness is expected in most patients if the implanted stem cell is more than 1×108, whereas there would be little effect if the cell number is less than 1×105.
ObjectiveTo investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD+) levels.MethodsThe bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H2O2 to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H2O2 treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H2O2 simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD+ and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-β-galactosidase (SA-β-gal) staining and TUNEL staining] were detected after 24 hours of treatment.ResultsThe rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased (P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H2O2, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD+ and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-β-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant (P<0.05).ConclusionNMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD+ levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.
Objective To analyze the changes of gene expression profiles during the process that human bonemarrow mesenchymal stem cells (hBMSCs) are induced to differentiate into cardiomyogenic cells with 5-azacytidine (5-aza). Methods hBMSCs were isolated from marrow of obsolete ribs and induced with 5-aza. Then immunocytochemicalstaining was used to detect the expressions of α-actin, cardiac troponin T (cTnT), and connexin 43, and the percentage ofcTnT positive cells was tested with flow cytometry. In the process of differentiation, variation of gene expression was screenedwith Genechi ps Operating System of human gene expression profiles. And the differentially expressed genes were functionallyanalyzed and hierarchical clustered. Results When BMSCs were induced in vitro with 5-aza, part of the cells turnedinto myogenic cells morphologically. Before induction, immunocytochemical staining for α-actin and cTnT showed sl ightpositive and for connexin 43 showed negative. While after 3 weeks of induction, immunocytochemical staining for α-actin,cTnT, and connexin 43 showed all positive. With flow cytometry, the percentage of cTnT positive cells was 7.43% ± 0.02%before induction, but it was 49.64% ± 0.05% after induction. During differentiation, 1 814 differentially expressed geneswere reported by gene chi ps. Of them, 647 genes were divided into 5 groups with hierarchical clustering. They had variousbiological functions, involving signal transduction, cell metabol ism, prol iferation, differentiation, development, andtopogenesis. Conclusion hBMSCs can differentiate into cardiomyogenic cells with the induction of 5-aza in vitro. Multi plegenes related with signal transduction, transcri ption, and growth factors are involved during this process.
ObjectiveTo investigate whether desferrioxamine (DFO) can enhance the homing of bone marrow mesenchymal stem cells (BMSCs) and improve neovascularization in random flaps of rats.MethodsBMSCs and fibroblasts (FB) of luciferase transgenic Lewis rats were isolated and cultured. Forty 4-week-old Lewis male rats were used to form a 10 cm×3 cm rectangular flap on their back. The experimental animals were randomly divided into 4 groups with 10 rats in each group: in group A, 200 μL PBS were injected through retrobulbar venous plexus; in group B, 200 μL FB with a concentration of 1×106 cells/mL were injected; in group C, 200 μL BMSCs with a concentration of 1×106 cells/mL were injected; in group D, cells transplantation was the same as that in group C, after cells transplantation, DFO [100 mg/(kg·d)] were injected intraperitoneally for 7 days. On the 7th day after operation, the survival rate of flaps in each group was observed and calculated; the blood perfusion was observed by laser speckle imaging. Bioluminescence imaging was used to detect the distribution of transplanted cells in rats at 30 minutes and 1, 4, 7, and 14 days after operation. Immunofluorescence staining was performed at 7 days after operation to observe CD31 staining and count capillary density under 200-fold visual field and to detect the expressions of stromal cell derived factor 1 (SDF-1), epidermal growth factor (EGF), fibroblast growth factor (FGF), and Ki67. Transplanted BMSCs were labeled with luciferase antibody and observed by immunofluorescence staining whether they participated in the repair of injured tissues.ResultsThe necrosis boundary of ischemic flaps in each group was clear at 7 days after operation. The survival rate of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Laser speckle imaging showed that the blood perfusion units of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Bioluminescence imaging showed that BMSCs gradually migrated to the ischemia and hypoxia area and eventually distributed to the ischemic tissues. The photon signal of group D was significantly stronger than that of other groups at 14 days after operation (P<0.05). CD31 immunofluorescence staining showed that capillary density in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). The expressions of SDF-1, EGF, FGF, and Ki67 in groups C and D were significantly stronger than those in groups A and B, and in group D than in group C. Luciferase-labeled BMSCs were expressed in the elastic layer of arteries, capillaries, and hair follicles at 7 days after transplantation.ConclusionDFO can enhance the migration and homing of BMSCs to the hypoxic area of random flap, accelerate the differentiation of BMSCs in ischemic tissue, and improve the neovascularization of ischemic tissue.
ObjectiveTo study the effect and mechanism of lipopolysaccharide (LPS) on osteoclasts formation and its bone resorption function.MethodsBone marrow-derived macrophages (BMMs) were extracted from the marrow of femur and tibia of 4-week-old male C57BL/6 mice. Flow cytometry was used to detect BMMs. The effect of different concentrations of LPS (0, 100, 200, 500, 1 000, 2 000 ng/mL) on BMMs activity was examined by cell counting kit 8 (CCK-8) activity test. In order to investigate the effect of LPS on osteoclastogenesis, BMMs were divided into macrophage colony-stimulating factor (M-CSF) group, M-CSF+receptor activator of nuclear factor κB ligand (RANKL) group, M-CSF+RANKL+50 ng/mL LPS group, M-CSF+RANKL+100 ng/mL LPS group. After the completion of culture, tartrate resistant acid phosphatase (TRAP) staining was used to observe the formation of osteoclasts. In order to investigate the effect of LPS on the expression of Connexin43, BMMs were divided into the control group (M-CSF+RANKL) and the LPS group (M-CSF+RANKL+100 ng/mL LPS); and the control group (M-CSF+RANKL), 50 ng/mL LPS group (M-CSF+RANKL+50 ng/mL LPS), and 100 ng/mL LPS group (M-CSF+RANKL+100 ng/mL LPS). The expressions of Connexin43 mRNA and protein were detected by Western blot and real-time fluorescent quantitative PCR, respectively. In order to investigate the effect of LPS on osteoclast bone resorption, BMMs were divided into M-CSF group, M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group. Bone absorption test was used to detect the ratio of bone resorption area.ResultsThe flow cytometry test confirmed that the cultured cells were BMMs, and CCK-8 activity test proved that the 100 ng/mL LPS could promote the proliferation of BMMs, showing significant differences when compared with the 0, 200, 500, 1 000, and 2 000 ng/mL LPS (P<0.05). TRAP staining showed no osteoclast formation in M-CSF group. Compared with M-CSF+RANKL group, the osteoclasts in M-CSF+RANKL+50 ng/mL LPS group and M-CSF+RANKL+100 ng/mL LPS group were larger with more nuclei, while the osteoclasts in M-CSF+RANKL+100 ng/mL LPS group were more obvious, and the differences in the ratio of osteoclast area between groups were statistically significant (P<0.05). Western blot result showed that the relative expression of Connexin43 protein in LPS group was significantly higher than that in control group (P<0.05). Real-time fluorescent quantitative PCR showed that the relative expression of Connexin43 mRNA in control group, 50 ng/mL LPS group, and 100 ng/mL LPS group increased gradually, and the differences between groups were statistically significant (P<0.05). Bone resorption test showed that osteoclast bone resorption did not form in M-CSF group, but the ratio of bone resorption area increased gradually in M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group, and the differences between groups were statistically significant (P<0.05).ConclusionLPS at concentration of 100 ng/mL can promote the expression of Connexin43, resulting in increased osteoclastogenesis and enhanced osteoclastic bone resorption.
OBJECTIVE To investigate the effect of percutaneous bone marrow graft for the management of nonunion of tibia. METHODS From March 1996 to June 2000, 56 cases with nonunion of tibia were treated by autogenous bone marrow graft. Among them, there were 38 males and 18 females, aged from 19 to 72 years. A marrow needle was inserted into the site of the nonunion under the X-ray, the autogenous bone marrow was injected into the site of nonunion. Compression bandage and appropriate immobilization material were applied after operation. This procedure was repeated every month, 2 or 3 times was needed. RESULTS 56 patients were followed-up for 4 months to 4 years and 2 months, averaged 2.8 years. Fracture healed in 53 cases and X-ray displayed fracture line disappeared and a great deal of continuous callus formed, and nonunion in 3 cases. CONCLUSION Percutaneous autogenous bone marrow graft can play a role in osteogenesis at the site of nonunion. It is easy to aspirate bone marrow and the operation is simple. It has clinical application value for the satisfactory effect.
Objective To evaluate the therapeutic results of percutaneous injection of autogeous bone marrow for simple bone cyst and to analyze the prognostic factors of the treatment. Methods From March 2000 to June 2005, 31 patients with simple bone cysts were treated by percutaneous injection of autogeous bone marrow. Of 31 patients, there were 18 males and 13 females, aged 5 years and 7 months to 15 years. The locations were proximal humerus in 18 cases ,proximal femur in 7 cases and other sites in 6 cases. Two cases were treated with repeated injections. The operative process included percutaneous aspiration of fluid in the bone cysts and injection of autogenous bone marrow aspirated fromposterior superior iliac spine. The mean volume of marrow injected was 40 ml(30-70 ml).Results No complications were noted during treatment. Thirty patients were followed for an average of 2.2 years(1.5 years) with 2 cases out of follow-up. After one injection of bone marrow, 9 cysts(29.0%) were healed up completely, 7 cysts(226%)basically healed up,13 cysts (41.9%)healed up partially and 2 (6.5%) had no response.The satisfactory and effective rates were 67.7% and 93.5% respectively. There was significant difference between active stagegroup and resting stage group(P＜0.05). There were no statistically significant difference in therapeutic results between groups of different ages, lesion sites or bone marrow hyperplasia(Pgt;0.05). Conclusion Percutaneous injection of autogeous bone marrow is a safe and effective method to treat simple bone cyst, but repeated injections is necessary for some patients. The therapeutic results are better in cysts at resting stage than those at active stage.
Objective
To investigate the protective effect of the antioxidant glutathione (GSH) on the steroid-induced imbalance between osteogenesis and adipogenesis in human bone marrow mesenchymal stem cells (BMSCs).
Methods
The BMSCs were isolated from the proximal femur bone marrow from 3 patients of femoral neck fracture and were separated, cultured, and purificated by density gradient centrifugation and adherent wall methodin vitro. The third generation BMSCs were divided into 5 groups: group A, BMSCs (1×105 cells/mL); group B, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone; group C, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+5 μmol/L GSH; group D, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+10 μmol/L GSH; group E, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+50 μmol/L GSH. After cultured for 7 days, the reactive oxygen species expression was detected by flow cytometry; the superoxide dismutase (SOD) and Catalase mRNA expressions were determined by RT-PCR; the peroxisome proliferator-activated receptors γ (PPAR-γ), CCAAT/enhancer-binding family of proteins (C/EBP), Runx2, and alkaline phosphatase (ALP) mRNA expressions were evaluated by real-time fluorescence quantitative PCR. After cultured for 21 days, Oil red O staining was used to observe the adipogenesis differentiation of cells, and the expressions of related proteins were detected by Western blot.
Results
The reactive oxygen species expression in group B was obviously higher than in the other groups, in group C than in groups A, D, and E, and in groups D, E than in group A, all showing significant differences between groups (P<0.05); but there was no significant difference between groups D and E (P>0.05). The oil red O staining positive cells in group B were obviously more than the other groups, and groups C, D, E, and A decreased sequentially, the absorbance (A) values had significant differences between groups (P<0.05). RT-PCR detection showed that the relative expressions of SOD and Catalase mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E (P<0.05), but there was no significant difference among groups A, D, and E (P>0.05). Real-time fluorescence quantitative PCR detection showed that the relative expressions of PPAR-γ and C/EBP mRNA in group B were significantly higher than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A (P<0.05); but there was no significant difference between groups D and E (P>0.05). The relative expressions of Runx2 and ALP mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A (P<0.05); but there was no significant difference between groups D and E (P>0.05). Western blot detection showed that the relative expression of PPAR-γ and C/EBP protein in group B was significantly higher than those in the other groups, and groups C, D, E, and A decreased sequentially, all showing significant differences between groups (P<0.05). The relative expression of Runx2 and ALP protein in group B was significantly lower than those in the other groups, and groups C, D, E, and A increased sequentially, all showing significant differences between groups (P<0.05).
Conclusions
GSH can inhibit the adipogenesis differentiation and enhance the osteogenic differentiation of human BMSCs by reducing the intracellular reactive oxygen species level; and in a certain range, the higher the concentration of GSH, the more obvious the effect is.
The purpose of this study was to investigate the effect of biaxial tensile strain on the osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. The rBMSCs were isolated from tibia and femur of 4 weeks-old Sprague-Dawley (SD) rats. The rBMSCs were cultured in DMEM-LG complete culture medium and grew to subconfluence in the cell culture device for loading tensile strain. The biaxial tensile strain was applied to the rBMSCs for periods of 2, 4 and 6 hours every day, respectively, lasting 3 days. The amplitude of biaxial tensile strain applied to the rBMSCs were 1%, 2% and 5% respectively, at a frequency of 1 Hz. Unstrained rBMSCs were used as blank control (control group). The rBMSCs cultured with DMEM-LG complete culture medium containing 100 nmol/L β-Estradiol (E2) were used as positive control. The mRNA expression of alkaline phosphatase (ALP), collagen typeⅠ (ColⅠ), Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) was examined with real-time quantitative PCR and the protein expression of ALP, ColⅠ, Runx2 and OCN was detected with Western blot method. The results showed as follws: (1) The mRNA and protein expression of the ALP, ColⅠ, Runx2, OCN were significantly higher in rBMSCs of the E2 group than those in the control group (P＜0.05). (2) The mRNA and protein expression level of the ALP, Runx2 were higher markedly in the 1% tensile strain groups than those in the control group (P＜0.05), but lower than those in the E2 group (P＜0.05). (3) The mRNA and protein expression level of the ALP, ColⅠ, Runx2, OCN were significantly higher in the 2% tensile strain groups than those in the control group (P＜0.05), and the mRNA and protein expression level of ColⅠ and Runx2 in the group applied with 2% amplitude of tensile strain for 4 h/d was significantly higher than those in E2 group (P＜0.05). (4) The mRNA and protein expression level of the ALP, ColⅠ, Runx2 were significantly higher in the groups applied with 5% amplitude of tensile strain for 2 h/d or for 4 h/d than those in the control group (P＜0.05). In our study, E2 and mechanical stimulation played an important role in the regulation of differentiation of rBMSCs into osteoblasts, and the manner applied with the 2% amplitude of tensile strain for 4 h/d, lasting 3 days was an optimal stimulus for up-regulating the mRNA and protein expression of ALP, ColⅠ, Runx2, OCN of rBMSCs.
