OBJECTIVE: To study the effect of simvastatin on the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphates (ALP) activity in the primary cultured bone marrow stromal cells, and to elucidate the mechanism of the anabolic osteogenetic effect of simvastatin. METHODS: Bone marrow stromal cells in femur and tibia of adult mouse were cultured in vitro. after treated with different concentrations of simvastatin (0, 0.1, 0.2, 0.5 and 1.0 mumol/L) or recombinant human BMP-2 for 72 hours, ALP activity of bone marrow stromal cells was determined. BMP-2 expression of bone marrow stromal cells was analyzed by using immunocytochemistry and Western blotting. RESULTS: After treated with simvastatin for 72 hours, BMP-2 expression increased, while little BMP-2 expression could be observed in the control group. ALP activity also increased in a dose-dependent manner; t-test showed that ALP activity in the group which concentrations of simvastatin were 0.5 mumol/L (t = 2.35, P = 0.041), 1.0 mumol/L (t = 2.348, P = 0.041) had significant difference when compared with control group. CONCLUSION: Simvastatin lead to high expression of BMP-2 in bone marrow stromal cells, via the increased auto- or para-crine of BMP-2, and ALP activity increased. These may be parts of the mechanism on the anabolic osteogenetic effect of simvastatin.
OBJECTIVE To investigate the ectopic osteogenesis of bone marrow stromal cells (MSC) induced by bone morphogenetic protein(BMP) in vitro and in vivo, providing the experimental evidence for making an artificial bone with its own capacity of bone formation. METHODS MSC were separated and cultured from bone marrow of Wistar rats, MSC were co-cultured with BMP in vitro (cultured in plate and diffuse chamber). Artificial coral hydroxyapatites (CHA) with MSC and BMP were implanted into dorsal muscles of Wistar rats, their bone formation were observed by morphological examination, histochemistry and immunohistochemistry. RESULTS Only cartilaginous matrix were produced by MSC in vitro (cultured in plate and diffuse chamber), and both cartilaginous and bone matrix production within the combined grafts were seen. The bone formation of experimental groups (CHA + BMP + MSC) was ber than that of control A(CHA + MSC) and control B(CHA). CONCLUSION It may be possible to produce an artificial bone with its own capacity of bone formation by combined graft (CHA + BMP + MSC). There may be multiple factors as well as BMP inducing bone formation both in the whole body and the location of the implantation. Further research on these factors will have the significance for making the ideal artificial bone.
Objective To observe the main biological characteristics and chondrogenesis potency of bone marrow -derived stromal cells(MSCs) after cytokinesinduction or gene modification in vitro. Methods MSCs from an adult New Zealand white rabbit were isolated and cultivated, and then MSCs were divided into the common medium group(Group A, 15%FBS in DMEM), the induced group by cytokines (Group B), the transfected group(Group C)with adenovirus-hepatocyte growth factor transgene (adHGF). The medium of group B consisted of transforming growth factor-β1(TGF-β1,10 ng/ml), basic fibroblast growth factor(bFGF,25 ng/ml) addexamethasone (DEX,10-7mol/L) with 15%FBS in DMEM. Cartilage slices wereobtained from femoral condyles and patellar grove in the same rabbit. The minced cartilage was digested in Ⅱ collagenase (3 mg/ml) to obtain chondrocytes(Group D). The change of cell appearance, proliferation capacity, glycosaminoglycans(GAG), immunohistochemical staining for type Ⅰ, Ⅱ collagen were observed during the 5th passage MSCs and MSCs after induction or gene modification. Expression of mRNA for type Ⅰ and Ⅱ collagen was detected by RT-PCR. Results Primary MSCs proliferated as shortspindle shape, while the 5th MSCs showed longspindle shape. Positive stain of type Ⅰ collagen could be found in groups A, B and C, while positivestain of type Ⅱ collagen was shown in groups B and D. The content of GAG in group B was higher than that in group A, but there was no significant difference between them（Pgt;0.05）, and there was significant difference between groups A and D（Plt;0.05）. No significant difference was noted in groups A,B and C on proliferation by MTT（Pgt;0.05）,except that of at the fourth day after transfection between groups A and C（Plt;0.05）. RT-PCR demonstrated that MSCs always had higher levelsof mRNA type Ⅰ collagen in groups A, B and C. The expression of mRNA type Ⅱ collagen was identified in groups B and D, and only low levels of mRNA type Ⅱ collagen in group C. Conclusion The above results indicate MSCs have a natural tendency of osteogenic differentiation in vitro culture, and also demonstrate the chondrogenic potency with the technique of cytokines induction or gene modification after passage. MSCs can be transfected efficiently being seed cells in tissue engineered bone or cartilage to accept target genes such as adHGF, and have a higher levels of expression in vitro, which lasted 4 weeks at least.
Abstract To examine the effects of porous tricalcium phosphate (TCP) combined with autogenous red bone marrow (BM) in therepar of bone defects, 21 cases of bone defects were implanted with the above prepared composite material, 17 cases had benign or low-grade malignant tumors and 4cases had old fractures. Serial X-ray films were taken after surgery. The results showed that new bone formation was seen between the interface of the implantand surrounding host bone after 6 weeks, and osseous union developed after 12 weeks. Evident osteogenesis in all patients was observed after a follow-up for 1～3 years. TCP-BM is both osteoconductive and osteoinductive. Its function was similar to the fresh autogenous cancellous bone graft and TCP-BM had the potencyof promoting the repair of osseous defect. It was suggested that TCP-BM might be an ideal material for treating bone defects.
ObjectiveTo explore the effect and mechanisms of bone marrow mesenchymal stem cells (BMSCs) on healing quality of acetic acid-induced gastric ulcer. MethodsForty-eight clean grade male Wistar rats were used to establish the model of gastric ulcer with acetic acid and were randomly divided into 3 groups after 3 days of modeling, 16 rats each group. After the abdominal cavity was open and stomach was pulled out, no treatment was given in group A, 150 μL phosphate buffered saline (PBS) and 150 μL BMSCs at passage 4+PBS (1×108 cells/100 μL) were injected into the gastric wall surrounding the ulcer at 5 different points in groups B and C respectively. After 10 days, the ulcer area was measured, the mucosal thickness and the number of dilated glands were tested in the regenerative mucosa by histological method. And the expression of vascular endothelial growth factor (VEGF) was detected at ulcerative margin by immunohistochemical method. ResultsThe ulcer area in group C was significantly smaller than that of groups A and B (P<0.01), but no significant difference was found between groups A and B (P>0.05). HE staining showed that group C had thicker regenerative gastric mucosa, less dilated glands, and more regular mucosal structure than groups A and B, showing significant differences in regenerative gastric mucosa thickness and dilated glands number (P<0.01), but no significant difference between groups A and B (P>0.05). Immunohistochemical staining showed that the positive expression of VEGF in the ulcer margin mucosa of group C was significantly higher than that of groups A and B. The integral absorbance (IA) value of VEGF expression in group C was significantly higher than that in groups A and B (P<0.01), but no significant difference between groups A and B (P>0.05). ConclusionBMSCs can accelerate ulcer healing by the secretion of VEGF, and improve the quality of ulcer healing.
Objective To investigate tissue engineered spinal cord which was constructed of bone marrow mesenchymal stem cells (BMSCs) seeded on the chitosan-alginate scaffolds bridging the both stumps of hemi-transection spinal cord injury (SCI) in rats to repair the acute SCI. Methods BMSCs were separated and cultured from adult male SD rat. Chitosan-alginate scaffold was produced via freeze drying, of which the structure was observed by scanning electron microscope (SEM) and the toxicity was determined through leaching l iquor test. Tissue engineered spinal cord was constructed by seeding second passage BMSCs on the chitosan-alginate scaffolds (1 × 106/mL) in vitro and its biocompatibil ity was observed under SEM at 1, 3, and 5 days. Moreover, 40 adult female SD rats were made SCI models by hemi-transecting at T9 level, and were randomly divided into 4 groups (each group, n=10). Tissue engineered spinal cord or chitosan-alginate scaffolds or BMSCs were implanted in groups A, B, and C, respectively. Group D was blank control whose spinal dura mater was sutured directly. After 1, 2, 4, and 6 weeks of surgery, the functional recovery of the hindl imbs was evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating score. Other indexes were tested by wheat germ agglutinin-horseradish peroxidase (WGA-HRP) retrograde tracing, HE staining and immunofluorescence staining after 6 weeks of surgery. Results Chitosan-alginate scaffold showed three-dimensional porous sponge structure under SEM. The cells adhered to and grew on the surface of scaffold, arranging in a directional manner after 3 days of co-culture. The cytotoxicity of chitosan-alginate scaffold was in grade 0-1. At 2, 4, and 6 weeks after operation, the BBB score was higher in group A than in other groups and was lower in group D than in other groups; showing significant differences (P lt; 0.05). At 4 and 6 weeks, the BBB score was higher in group B than in group C (P lt; 0.05). After 6 weeks of operation, WGA-HRP retrograde tracing indicated that there was no regenerated nerve fiber through the both stumps of SCI in each group. HE and immunofluorescence staining revealed that host spinal cord and tissue engineering spinal cord l inked much compactly, no scar tissue grew, and a large number of neurofilament 200 (NF-200) positive fibers and neuron specitic enolase (NSE) positive cells were detected in the lesioned area in group A. In group B, a small quantity of scar tissue intruded into non-degradative chitosan-alginate scaffold at the lesion area edge, and a few of NSE flourescence or NF-200 flourescence was observed at the junctional zone. The both stumps of SCI in group C or group D were filled with a large number of scar tissue, and NSE positive cells or NF-200 positive cells were not detected. Otherwise, there were obviously porosis at the SCI of group D. Conclusion The tissue engineered spinal cord constructed by multi-channel chitosan-alginate bioscaffolds and BMSCs would repair the acute SCI of rat. It would be widely appl ied as the matrix material in the future.
Objective To construct the lentiviral vector containing homo sapiens forkhead box C2 (Foxc2) gene and to detect its expression in bone marrow mesenchymal stem cells (BMSCs) of rabbits. Methods Human Foxc2 gene coding region fragment was obtained by RT-PCR and then cloned into the plasmid of LV-green fluorescent protein (GFP) to prepare Foxc2 lentiviral plasmid. Foxc2 lentiviral plasmid, pGC-LV, pHelper1.0, and pHelper2.0 were co-transfected into 293T cells to obtain recombinant virus containing Foxc2 gene. The lentiviral titer was detected. BMSCs were isolated from bone marrow of rabbit and infected with Foxc2 recombined lentiviral, then the optimum multiplicity of infection (MOI) was determined by detecting the intensity of fluorescence expression. The expression of Foxc2 in the infected BMSCs was determined at 1, 3, and 7 days after transfection by inverted fluorescence microscope and Western blot. After osteogenic induction, Alizarin red staining was done to observe the formation of mineralized nodule. Results The Foxc2 recombinant lentiviral vector was constructed and was confirmed by restriction enzyme digestion and sequencing analysis. It could efficiently transfect 293T cells and express in 293T cells. The lentiviral titer was 2 × 108 TU/mL. The optimum MOI was 200. The inverted fluorescence microscope observation showed that the Foxc2 gene expressed in 84.5% ± 4.8% of infected BMSCs at 3 days after transfection. The expression of Foxc2 in infected BMSCs was stable and high, and increased gradually within 7 days after transfection by Western blot. At 2 weeks after osteogenic induction, Alizarin red staining showed that there were a large number of red calcified matrix deposition in the cytoplasm. Conclusion Foxc2 recombined lentivirus with high viral titer is successfully constructed and packaged, and the Foxc2 gene can be transfected into BMSCs with stable and high expression of Foxc2 in infected cells, and these cells may be applied for gene therapy of avascular necrosis of the femoral head.
OBJECTIVE To review the recent research progress of bone-marrow stromal stem cells (BMSCs) in the conditions of culture in vitro, chondrogenic differentiation, and the application in cartilage tissue engineering. METHODS: Recent original articles related to such aspects of BMSCs were reviewed extensively. RESULTS: BMSCs are easy to be isolated and cultivated. In the process of chondrogenesis of BMSCs, the special factors and interaction between cells are investigated extensively. BMSCs have been identified to form cartilage in vivo. One theory is the committed chondrocyte from BMSCs is only a transient stage. CONCLUSION: BMSCs are the alternative seeding cells for cartilage tissue engineering. The conditions promoting mature chondrocyte should be further investigated.
ObjectiveTo investigate the effect of transforming growth factorβ1 (TGF-β1) and basic fibroblast growth factor 1 (bFGF-1) on the cellular activities, prol iferation, and expressions of ligament-specific mRNA and proteins in bone marrow mesenchymal stem cells (BMSCs) and ligament fibroblasts (LFs) after directly co-cultured. MethodsBMSCs from 3-month-old Sprague Dawley rats were isolated and cultured using intensity gradient centrifugation. LFs were isolated using collagenase. The cells at passage 3 were divided into 6 groups: non-induced BMSCs group (group A), non-induced LFs group (group B), non-induced co-cultured BMSCs and LFs group (group C), induced BMSCs group (group D), induced LFs group (group E), and induced co-cultured BMSCs and LFs group (group F). The cellular activities and prol iferation were examined by inverted contrast microscope and MTT; the concentrations of collagen type Ⅰ and type Ⅲ were determined by ELISA; and mRNA expressions of collagen types I andⅢ, fibronectin, tenascin C, and matrix metalloproteinase 2 (MMP-2) were measured by real-time fluorescent quantitative PCR. ResultsA single cell layer formed in the co-cultured cells under inverted contrast microscope. Group F had fastest cell fusion ( > 90%). The MTT result indicated that group F showed the highest absorbance (A) value, followed by group D, and group B showed the lowest A value at 9 days after culture, showing significant difference (P < 0.05). Moreover, the result of ELISA showed that group F had the highest concentration of collagen type Ⅰ and type Ⅲ (P < 0.05); the concentration of collagen type Ⅲ in group E was significantly higher than that in group D (P < 0.05), but no significant difference was found in the concentration of collagen type Ⅰ between 2 groups (P > 0.05). The ratios of collagen type Ⅰ to type Ⅲ were 1.17, 1.19, 1.10, 1.25, 1.17, and 1.18 in groups A-F; group D was higher than the other groups. The real-time fluorescent quantitative PCR results revealed that the mRNA expressions of collagen type Ⅰ and type Ⅲ and fibronectin were highest in group F; the expression of tenascin C was highest in group D; the expression of MMP-2 was highest in group E; and all differencs were significant (P < 0.05). ConclusionDirectly co-cultured BMSCs and LFs induced by TGF-β1 and bFGF-1 have higher cellular activities, proliferation, and expressions of ligament-specific mRNA and protein, which can be used as a potential source for ligament tissue engineering.
Objective To summarize and review the heterogeneity of bone marrow derived stem cells (BMDSCs) and its formation mechanism and significance, and to analyze the possible roles and mechanisms in intestinal epithel ial reconstruction. Methods The related l iterature about BMDSCs heterogeneity and its role in intestinal epithel ial repair was reviewed and analyzed. Results The heterogeneity of BMDSCs provided better explanations for its multi-potency. The probable mechanisms of BMDSCs to repair intestinal epithel ium included direct implantation into intestinal epithel ium, fusion between BMDSCs and intestinal stem cells, and promotion of injury microcirculation reconstruction. Conclusion BMDSCs have a bright future in gastrointestinal injury caused by inflammatory bowl disease and regeneration.