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 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 construct a co-expressing vector of human bone morphogenetic protein 2 (BMP-2) and osteoprotegerin (OPG) and to determine the expression of BMP-2 and OPG in myoblast C2C12. METHODS: Using the isolated total RNA from osteosacoma cell line MG63 as a template, the cDNA encoding region of human OPG was amplified by reverse transcription-polymerase chain reaction (RT-PCT) method and cloned into sites EcoR 1 and BamH I of mammalian expressing vector pIRES2-EGFP, and the cDNA encoding region of human BMP-2 was cloned into endonucleases site BstX I. Then the recombinant plasmid pIRES2-BMP-2-OPG was transformed into C2C12 cell line, the expression of OPG and BMP-2 were determined by Western blot assay. RESULTS: The sequence of OPG cDNA obtained was the same as that reported, recombinant plasmid pIRES2-BMP-2-OPG was constructed successfully. Human OPG and BMP-2 co-expression cell line C2C12 was selected and confirmed by Western blot analysis. CONCLUSION: The co-expressing vector of OPG and BMP-2 is constructed and can expressed stably in myoblast C2C12. The co-expression of human OPG and BMP-2 may be logical approach for treatment of osteoporosis and bone metastasis.
Objective To investigate the effects of exogenous bone morphogenetic protein(BMP) and transforming growth factor-β(TGF-β) on biomechanical property for ulna of fracture healing.Methods Thirty-six adult rabbits were made the model of right ulnar fracture and treated locally with TGF-β/PLA, BMP/PLA，TGF-β+BMP/PLA or PLA(as control group). Fracture healing was evaluated by measurement of the mechanical parameters and geometric parameters.Results As compared with control group, the geometric parameters, the bending broken load, the ultimatebending strength, the bending elastic modulus, the ultimate flexural strength, the flexural elastic modulus, the ultimate compressing strength, the compressingelastic modulus, and the ultimate tensile strength for ulna of fracture healingincreased significantly in the treatment groups(P＜0.01). These parameters were higher in TGF-β+BMP/PLA group than in TGF-β/PLA group or in BMP/PLA group andin TGF-β/PLA group than in BMP/PLA group(P＜0.05). There was no significant difference in bone density between the treatment groups and control group. Conclusion Local application of exogenous TGF-β and BMP canincrease the callus formation and enhance biomechanical strength of bone after fracture healing. A combination of TGF-β and BMP has synergetic effect in enhancing fracture healing.
ObjectiveTo research the effect of recombinant adenovirus-bone morphogenetic protein 12 (Ad-BMP-12) transfection on the differentiation of peripheral blood mesenchymal stem cells (MSCs) into tendon/ligament cells. MethodsPeripheral blood MSCs were isolated from New Zealand rabbits (3-4 months old) and cultured in vitro until passage 3. The recombinant adenoviral vector system was prepared using AdEasy system, then transfected into MSCs at passage 3 (transfected group); untransfected MSCs served as control (untransfected group). The morphological characteristics and growth of transfected cells were observed under inverted phase contrast microscope. The transfection efficiency and green fluorescent protein (GFP) expression were detected by flow cytometry (FCM) and fluorescence microscopy. After cultured for 14 days in vitro, the expressions of tendon/ligament-specific markers were determined by immunohistochemistry and real-time fluorescent quantitative PCR. ResultsGFP expression could be observed in peripheral blood MSCs at 8 hours after transfection. At 24 hours after transfection, the cells had clear morphology and grew slowly under inverted phase contrast microscope and almost all expressed GFP at the same field under fluorescence microscopy. FCM analysis showed that the transfection efficiency of the transfected group was 99.57%, while it was 2.46% in the untransfected group. The immunohistochemistry showed that the expression of collagen type Ι gradually increased with culture time in vitro. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of the tendon/ligament-specific genes (Tenomodulin, Tenascin-C, and Decorin) in the transfected group were significantly higher than those in untransfected group (0.061±0.013 vs. 0.004±0.002, t=-7.700, P=0.031; 0.029±0.008 vs. 0.003±0.001, t=-5.741, P=0.020; 0.679±0.067 vs. 0.142±0.024, t=-12.998, P=0.000). ConclusionAd-BMP-12 can significantly promote differentiation of peripheral blood MSCs into tendon/ligament fibroblasts and enhance the expressions of tendon/ligament-specific phenotypic differentiation, which would provide the evidence for peripheral blood MSCs applied for tendon/ligament regeneration.
ObjectiveTo study the effect of transforming growth factor β3 (TGF-β3), bone morphogenetic protein 2 (BMP-2), and dexamethasone (DEX) on the chondrogenic differentiation of rabbit synovial mesenchymal stem cells (SMSCs). MethodsSMSCs were isolated from the knee joints of 5 rabbits (weighing, 1.8-2.5 kg), and were identified by morphogenetic observation, flow cytometry detection for cell surface antigen, and adipogenic and osteogenic differentiations. The SMSCs were cultured in the PELLET system for chondrogenic differentiation. The cell pellets were divided into 8 groups: TGF-β3 was added in group A, BMP-2 in group B, DEX in group C, TGF-β3+BMP-2 in group C, TGF-β3+DEX in group E, BMP-2+DEX in group F, and TGF-β3+BMP-2+DEX in group G; group H served as control group. The diameter, weight, collagen type II (immuohistochemistry staining), proteoglycan (toluidine blue staining), and expression of cartilage related genes [real time quantitative PCR (RT-qPCR) technique] were compared to evaluate the effect of cytokines on the chondrogenic differentiation of SMSCs. Meanwhile, the DNA content of cell pellets was tested to assess the relationship between the increase weight of cell pellets and the cell proliferation. ResultsSMSCs were isolated from the knee joints of rabbits successfully and the findings indicated that the rabbit synovium-derived cells had characteristics of mesenchymal stem cells. The diameter, weight, collagen type II, proteoglycan, and expression of cartilage related genes of pellets in groups A-F were significantly lower than those of group G (P<0.05). RT-qPCR detection results showed that the relative expressions of cartilage related genes (SOX-9, Aggrecan, collagen type II, collagen type X, and BMP receptor II) in group G were significantly higher than those in the other groups (P<0.01). Meanwhile, with the increase of the volume of pellet, the DNA content reduced about 70% at 7 days, about 80% at 14 days, and about 88% at 21 days. ConclusionThe combination of TGF-β3, BMP-2, and DEX can make the capacity of chondrogenesis of SMSCs maximized. The increase of the pellet volume is caused by the extracellular matrix rather than by cell proliferation.
OBJECTIVE: To explore the distribution and effect of endogenic bone morphogenetic protein (BMP) in repairing rabbit skull with tissue engineered bone. METHODS: The autologous osteoblast-like cells were instantly implanted onto polyglycolic acid (PGA) matrix coated with collagen. The rabbit skull defect models were established by resection of bilateral 1.5 cm x 1.0 cm full-thickness parietal bone in 18 New Zealand rabbits, which were randomly divided into two groups. In one group, the composite of osteoblast- like cells and PGA matrix were grafted into the defect on one side of the skull as experimental group I, leaving the same defect area on the other side as control group without any graft implanted. In the other group, simply PGA was done in the same way as experimental group II. The tissue samples were harvested at 3, 8 and 14 days postoperatively and examined by histological and immunohistochemistry methods. The concentrations of BMP in different regions of the samples were measured using computer image analysis system. RESULTS: After 3 days of operation, the BMP positive cells were found in the matrix of experimental group I. At 8 days postoperatively, the formation of new bone on experimental group I was prior to that of experimental group II and control group. On the 14th day, bone trabecula was formed on the experimental group I, but there was only fibrous tissue on control group. The concentration of BMP on the experimental group I and II were higher than that of corresponding region on control side. CONCLUSION: The osteoblast-like cells instantly implanted onto PGA matrix can synthesize and secrete BMP. It may be one of the reasons of tissue engineered bone inducing new bone regeneration that localizing endogenic BMP in bone defect area, increasing the concentration of endogenic BMP and improving its distribution by tissue engineering technique.
Objective To construct the recombined DNA pcDNA3.1-hBMP-2 and transfect into human marrow stromal stem cells (MSCs) in vitro, and to explore theeffects of transfection on cellular proliferation and expression of vascular endothelial growth factor (VEGF). Methods The expression of human bone morphogenetic protein 2(hBMP-2) in these cells after transfection was determined by in situ hybridization and immunohistochemical analysis and Western blot analysis. The changes of cell proliferation were observed by flow cytometry. The effects of BMP-2 gene transfection on expression of VEGF in the cells were analyzed by in situ hybridization of VEGF cDNA probe. Results Stable expressionof hBMP-2 in pcDNA3.1-hBMP-2 transfected MSCs was confirmed in the levels of mRNA and protein.Cellular proportion in S period increased, which indicated that the synthesis of cell DNA increased. The expression of VEGF in the cells increased obviously. Conclusion With the help of lipofectamine, the pcDNA3.1-hBMP-2 were transfected into human MSCs successfully. hBMP-2 plays an important role in promoting cellular proliferation and vascular generation during bone repair.
ObjectiveTo investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein 9 (BMP-9) and erythropoietin (EPO) genes co-transfection on osteogenic differentiation of adipose-derived stem cells (ADSCs) in vitro. MethodsThe inguinal adipose tissue was harvested from 4-month-old New Zealand rabbits, ADSCs were isolated with enzyme digestion and adherence method, and multipotent differentiation capacity was identified. The 3rd generation ADSCs were divided into 5 groups: normal cells (group A), empty plasmid control group (group B), BMP-9 or EPO recombinant adenovirus transfected cells (groups C and D), BMP-9 and EPO recombinant adenovirus co-transfected cells (group E). The inverted phase contrast microscope was used to observe the cell growth at 7 days; the expression of cell fluorescence was observed under a fluorescence microscope at 14 days, and viral transfection efficiency was calculated at 48 hours; Western blot was used to detect the expressions of BMP-9 and EPO proteins at 14 days. The expression of alkaline phosphatase (ALP) activity was detected at 3, 7, and 14 days after osteogenic induction, and alizarin red staining was used to detect calcium nodules formation and real-time fluorescence quantitative PCR to detect the expressions of osteopontin (OPN) and osteocalcin (OCN) at 3 weeks. ResultsAt 7 days after transfected, some cells showed oval, round, and irregular shape under the inverted phase contrast microscope in groups A and B; a few fusiform cells were observed in groups C and D; oval cells increased obviously, and there were only few round cells in group E. The fluorescence microscope observation showed that BMP-9 and EPO, BMP-9/EPO recombinant adenovirus could stably transfected ADSCs, with transfection efficiency of 80%-93%. The expressions of BMP-9 and EPO proteins significantly higher in group E than the other groups by Western blot (P < 0.05). The ALP activity significantly increased in group E when compared with that in the other groups at 3, 7, and 14 days after osteogenic induction (P < 0.05); the number of calcium nodules in group E was significantly more than that in the other groups (P < 0.05). Real-time fluorescence quantitative PCR showed that OPN and OCN genes expressions were significantly higher in group E than other groups (P < 0.05), and in groups C and D than groups A and B (P < 0.05). ConclusionRecombinant adenovirus-mediated BMP-9 and EPO genes can transfect ADSCs, which can stably express in ADSCs, BMP-9/EPO genes co-transfection can more promote the expressions of osteoblast-related genes and protein than non-transfected and single gene transfection.
Objective To study the effect of direct bone morphogenetic protein 2 (BMP-2) gene therapy mediated by adenovirus on repairing bone defect. Methods The radial defect models were made on 60 rabbits, which were evenly divided into 4 groups randomly. The 4 groups were treated with different materials: group A, adenovirus carrying BMP-2 gene (AdBMP-2) plus bovine cancellous bone (BCB); group B, reconstructed BMP-2 plus BCB; group C, AdLacz plus BCB; and group D, only BCB scaffolds. The X-ray, histological examination, biomechanics analysis, and immunohistochemical staining were made 4, 8, and 12 weeks after the operation. Results Group A gained better effect in the volume of new bones, the anti-bending intensity of the healing bone, and the expression of BMP-2 than those of group B. The defect in group A was healed. No new bones were observed in group C and group D. Conclusion Direct BMP-2 gene therapy is easy to perform and has veryb osteoinduction ability. It is a good method to repair segmental bone defects.