Objective To construct the recombinant adenovirus bearing human transforming growth factor β1(TGF-β1) and bone morphogenetic protein 7 (BMP-7) genes, and investigate its co-expression in the marrow stromalstemcells (MSCs) and bioactivity effect. Methods Using the replication defective adenovirus AdEasy as a carrier, MSCs were infected by the high-titer-level recombinant adenovirus taking TGF-β1 and BMP-7 genes. Immunocytochemistry, in situ hybridization，reverse transcription-polymerase chain reaction (RT-PCR), and hexuronic acid level test were used to detect the coexpression of the exogenous genes and to analyze their effect transfection on directive differentiation of MSCs. Results The immunocytochemistry staining showed that the brown coarse grains were situated in the cytoplasm of the most MSCs 72 h after infection. Procollagen ⅡmRNA in the cells was detected by the in situ hybridization, and the content of hexuronic acid in the culture mediumwas significantly increased 10 days after infection compared with the level before infecton (Plt;0.01). Conclusion The recombinant adenovirus bearing human TGF-β1 and BMP-7 genes can be constructed, and the exogenous gene can be coexpressed in MSCs, which may offer a novel approach to thelocal combination gene therapy for repairing joint cartilage defects.
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 effect of dexamethasone, recombinant human fibroblast growth factor (rhFGF) and recombinant human bone morphogenetic protein 2 (rhBMP-2) on the proliferation and differentiation of marrow stromal stem cells (MSCs) for their further application in tissue engineering. Methods MSCs were isolated and cultured in vitro, and then exposed to different dose of dexamethasone (10-8 mol/L,10-7 mol/L,10 -6 mol/L), rhFGF (50 ng/ml,200 ng/ml,500 ng/ml) and rhBMP-2 (50 ng/ml,500 ng/ml,1 000 ng/ml) respectively. The total protein and alkaline phosphatase (ALP) activity of each group was measured on 4th and 7th day. Results Exposure of MSCs with 10-6mol/L dexamethasone inhibited protein synthesis without obvious effects on ALP expression. The application of rhFGF significantly promoted cell proliferation but inhibited ALP activity. In comparison, ALP expression was significantly enhanced by treatment of rhBMP-2 at concentration of 500 ng/ml，1 000 ng/ml. Conclusion The exposure of dexamethasone as well as rhBMP-2 to MSCs with an appropriate concentration promotes osteogenic expression without reverse effects on cell proliferation, which indicates the great potential value in cell-based strategy of bone tissue engineering.
Objective To explore the significance and the relationshi p between osteoporosis and the mRNA expressions of vascular endothel ial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2) in nontraumatic avascular necrosis of the femoral head (NONFH), so as to provide a theoretical basis for the pathogenesis and the cl inical treatment of NONFH. Methods Sixty-nine specimens of femoral head were collected from voluntary donators undergoing total hi p arthroplasty, including 37 cases of NONFH (NONFH group) and 32 cases of fresh femoral neck fracture (control group). In NONFH group, there were 26 males and 11 females with an average age of 57.3 years (range, 43-75 years), including 19 cases of steroid-induced avascular necrosis of the femoral head (ANFH), 16 cases of alcohol ic ANFH, and 2 cases of idiopathicANFH; according to Ficat staging system, there were 23 cases at stage III and 14 cases at stage IV. In control group, there were 23 males and 9 females with an average age of 58.6 years (range, 46-79 years). The NO level of serum, the Q value of femur, and the bone mineral density (BMD) of weight-bearing area were measured firstly. The bone tissues were harvested from weightbearing necrosis area and healthy area. The pathological change was observed by HE staining, the percentage of empty bone lacuna and the percentage of trabecular bone area were calculated. The mRNA expressions of VEGF and BMP-2 in femoral head were detected through in situ hybridization technique. Results There were significant differences (P lt; 0.05) in the NO level of serum, the Q value of femur, and the BMD between NONFH group and control group. In NONFH group, the femoral head showed irregular shape, the articular cartilage exfol iated and collapsed. In weight-bearing necrosis area, the bone trabeculae were sparse and non-intact with a great number of empty lacuna; necrotic bone trabeculae were decomposed and absorbed; no obvious bone regeneration and repair were observed. In weight-bearing healthy area, the fat cells in bone marrow showed prol iferation and hypertrophy. In control group, the femoral head had normal appearance, intact articular cartilage, and intact bone trabeculae with a regular arrange, and osteocytes were clearly seen. There were significant differences in the percentage of empty bone lacuna and the percentage of trabecular bone area between NONFH group and control group (P lt; 0.05). The mRNA expressions of VEGF and BMP-2 were positive in 2 groups. The positive area ratio, the absorbance value, and integral absorbancevalue of VEGF mRNA and BMP-2 mRNA in NONFH group were significantly lower than those in control group (P lt; 0.05);the grey scales of VEGF mRNA and BMP-2 mRNA in NONFH group were significantly higher than that in control group (P lt;0.05). Conclusion The pathological stage of osteoporosis may play an important role in the mechanism of the NONFH. The decrease of mRNA expressions of VEGF and BMP-2 in femoral head of NONFH is important reason that affect its bone mass, osteoporosis, rehabil itation, and reconstruction. It may be benefit to the reparative process of the necrosis femoral head to increase the mRNA expressions of VEGF and BMP-2 in the femoral head.
ObjectiveTo systematically review the correlation between the T538C polymorphism in bone morphogenetic protein 4 (BMP-4) and the risk of non-syndromic cleft lip with or without cleft palate (NSCL/P). MethodsWe electronically searched databases including PubMed, The Cochrane Library, EMbase, CBM, CNKI, VIP, and WanFang Data from inception to November 2014, to collect case-control studies of the correlation between the T538C polymorphism in BMP-4 and the risk of NSCL/P. Two reviewers independently screened literature according to the inclusion and exclusion criteria, extracted data and assessed the risk of bias of included studies. Then, meta-analysis was performed using RevMan 5.2 software. ResultsA total of 6 case-control studies involving 926 cases and 1 110 controls were included. The results of meta-analysis showed that:there was no significant association between the T538C polymorphism in BMP-4 gene and the risk of NSCL/P (C vs. T:OR=1.14, 95% CI 0.78 to 1.66; CC vs. TT:OR=0.75, 95% CI 0.50 to 1.11; CC vs. TT:OR=1.53, 95% CI 0.69 to 3.37; CC vs. CT+TT:OR=1.80, 95% CI 0.96 to 3.38; CC+CT vs. TT:OR=0.90, 95% CI 0.57 to 1.43). Subgroup analysis based on ethnicity showed that, the T538C polymorphism in BMP-4 gene was associated with increased risk of NSCL/P in Asian population (C vs. T:OR=1.54, 95% CI 1.26 to 1.87; CC vs. TT:OR=2.91, 95% CI 1.88 to 4.52; CC vs. CT+TT:OR=2.99, 95% CI 1.99 to 4.49), but decreased risk of NSCL/P in Latin populations (C vs. T:OR=0.69, 95% CI 0.50 to 0.96; CT vs. TT:OR=0.52, 95% CI 0.40 to 0.68; CC+CT vs. TT:OR=0.52, 95% CI 0.35 to 0.78). ConclusionAvailable evidence suggests that the T538C polymorphism in BMP-4 gene may be associated with increased risk of NSCL/P in Asians and decreased risk of NSCL/P in Latinas. Due to limited quality and quantity of the included studies, more high quality studies are needed to verify the above conclusion.
Objective To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro. Methods SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 ± 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining. Results SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells. Conclusion It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.
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.
ObjectiveTo explore the effects on osteogenic differentiation of adipose derived stem cells (ADSCs) by simultaneously down-regulating Noggin combined with up-regulating bone morphogenetic protein 14 (BMP-14) in vitro. MethodsPrimary ADSCs were isolated and expanded in vitro from 5 Sprague Dawley rats (weighing, 250-300 g). ADSCs were transfected with lentiviral (Lv)-enhanced green fluorescent protein in group A (control group), with Lv-BMP-14 in group B, and with Lv-BMP-14 and Lv-Noggin shRNA in group C. BMP-14 and osteogenesis-related genes[collagen type I, alkaline phosphatase (ALP), and osteocalcin (OCN)] mRNA expression levels were detected by real time fluorescence quantitative PCR at 3, 7, and 14 days after transfection. Alizarin red staining for calcium nodules was also employed to assess the osteogenic ability of co-transfected ADSCs. ResultsAt 3 days after transfection, no significant difference was found in BMP-14 mRNA expression among groups P>0.05). At 7 and 14 days after transfection, BMP-14 mRNA expression was significantly higher in group C than groups A and B, and in group B than group A (P<0.05). At 3 days after transfection, collagen type I, ALP, and OCN mRNA expressions of group C were significantly higher than those of groups A and B (P<0.05), but no significant difference was shown between groups A and B P>0.05). At 7 and 14 days, collagen type I, ALP, and OCN mRNA expressions were higher in group C than groups A and B, and in group B than group A, showing significant difference (P<0.05) except collagen type I mRNA expression at 7 days between groups A and B P>0.05). The results of alizarin red staining showed that the amount of calcium nodules presented an increased tendency in the order of group A, group B, and group C. ConclusionBMP-14 is capable of enhancing osteogenic differentiation of ADSCs. A combination of inhibiting Noggin gene expression and enhancing BMP-14 gene expression in ADSCs can significantly strengthen osteogenic differentiation capability, showing significant synergistic effect.
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 identify the expression of nerve growth factor (NGF) and its high affinity receptor (tyrosine kinase receptor A, TrkA) during bone induction by recombined human bone morphogenetic protein 2 (rhBMP-2) by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) and to discuss the role of NGF on the bone induction of the BMP. Methods Thirty-six ICR mice were divided into the experimental groupand the control group at random. rhBMP-2 /collagen sponge and collagen sponge were implanted into the right thigh muscle pouches of the mice in the experimental group and the control group, respectively. The tissues in the implanted site of the two groups were removed on the 7th, 14th and 21st day after the implantation. Histological, immunohistochemical and RT-PCR analyses were performed to detect osteoinductive effects of rhBMP-2 and the expression of NGF and TrkA. Results Gross observation showed that a solid lump was found in theright thigh in the experimental group on the 7th day and became harder on the 14th and 21st day, which was not found in the control group. rhBMP-2/collagen sponge displayed a potent ability to induce bone formation, while immunostaining for NGF and TrkA was observed in the course of osteoinduction by rhBMP-2. On the 7th day in the experimental group, NGF positive immunostaining reached the peak in thestage of chondrogenesis and there were a large number of cells expressing NGF, including fibroblasts, chondroblasts, chondrocytes, and osteoblasts; then, therewas a decrease in the number of the positivecells and in the intensity of immunostaining on the 14th and 21st day. Staining of TrkA wassimilar to that of NGF. The expression level of the mRNA of NGF during the course of bone induction peaked 7 days after the implantation and then decreased. Conclusion rhBMP-2/collagen is a kind of satisfactory osteoinductive material, and many different kinds of cells induced by rhBMP-2 can express NGF and TrkA, which suggests that NGF may play an important role in the osteogenesis initiated by exogenous BMP through direct and indirect pathways.