Objective To investigate the role of bone morphogenetic protein 2 (BMP-2) combined with hypoxic microenvironment in chondrogenic phenotype differentiation of bone marrow mesenchymal stem cells (BMSCs) of rat in vitro. Methods BMSCs were harvested from 4-week-old female Sprague Dawley rats. BMSCs at passage 2 were divided into 4 groups according different culture conditions: normoxia control group (group A), normoxia and BMP-2 group (group B), hypoxia control group (3% oxygen, group C), and hypoxia and BMP-2 group (group D). Then the cellular morphology was observed under inverted phase contrast microscope. Alcian blue immunohistochemical staining was used to detect the glycosaminoglycans (GAG), Western blot to detect collagen type II and hypoxia-inducible factor 1α (HIF-1α), and RT-PCRto detect the expressions of chondrogenic related genes, osteogenic related genes, and hypoxia related genes. Results At 21 days after induction of BMP-2 and hypoxia (group D), BMSCs became round, cell density was significantly reduced, and lacuna-l ike cells were wrapped in cell matrix, while the changes were not observed in groups A, B, and C. Alcian blue staining in group D was significantly bluer than that in other groups, and staining became darker with induction time, and the cells were stained into pieces of deeply-stained blue at 21 days. Light staining was observed in the other groups at each time point. The expression level of collagen type II protein in group D was significantly higher than those in other groups (P lt; 0.05). HIF-1α protein expression levels of groups C and D were significantly higher than those of groups A and B (P lt; 0.05). The expressions of collagen II α1 (COL2 α1) and aggrecan mRNA (chondrogenic related genes) were highest in group D, while the expressions of COL1 α1, alkaline phosphatase, and runt-related transcri ption factor 2 mRNA (osteogenic related genes) were the highest in group B (P lt; 0.05). Compared with groups A and B, HIF-1α (hypoxic related genes) in groups C and D significantly increased (P lt; 0.05). Conclusion BMP-2 combined with hypoxia can induce differentiation of BMSCs into the chondrogenic phenotype, and inhibit osteoblast phenotype differentiation. HIF-1α is an important signaling molecule which is involved in the possible mechanism to promote chondrogenic differentiation process.
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 observe effects of the direct impaction onthe cell survival and the bone formation of the tissue engineered bone modified by the adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP2) gene and to verify the feasibility of the impacted grafting with it. Methods The marrow stromal cells (MSCs) were separated from the canine bone marrow and were cultured. MSCs were transfected with the Adv-hBMP2 gene and combined with the freeze-dried cancellous bone (FDB) to form the tissue engineered bone. Four days after the combination, the tissue engineered bone was impacted in a simulated impactor in vitro and implanted in the mouse. The cell survivals were evaluated with SEM 1 and 4 days after the combination, immediately after the impaction, and 1 and 4 days after the impaction, respectively. The bone formation and the allograft absorption were histologically evaluated respectively. Results There were multiple layers of the cells and much collagen on FDB before the impaction. Immediately after the impaction, most of the cells on the direct contact area disappearedand there was much debris on the section. Some of the cells died and separatedfrom the surface of FDB at 1 day, the number of the cells decreased but the collagen increased on the surface at 4 days. Histologically, only the fibrous tissue was found in FDB without the cells, the bone formation on FDB was even in distribution and mass in appearance before the impaction, but declined and was mainly on the periphery after the impaction in the AdvhBMP2 modified tissue-engineered bone. Conclusion The simulated impaction can decrease the cells survival and the bone formation of the AdvhBMP-2 modified tissue-engineered bone. The survival cells still function well.It is feasible to use the tissue engineered bone in the impaction graft.
Objective To review the research progress of bone morphogenetic protein (BMP) and the liability of ossification of the posterior longitudinal ligament (OPLL). Methods Recent literature concerning BMP and the liability of OPLL was reviewed, analysed, and summarized. Results The single nucleotide polymorphisms (SNPs) of BMP gene may produce a minor cumulative effect and increase individual susceptibility to OPLL. A variety of environmental factors can promote the occurrence and development of OPLL by increasing the expression of BMP gene. Conclusion The SNPs of BMP gene may increase individual susceptibility to OPLL. However, interaction of cumulative effect of the SNPs and environmental factors can promote the liability to OPLL.
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 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.
ObjectiveTo investigate the bone regeneration potential of cell-tissue engineered bone constructed by human bone marrow mesenchymal stem cells (hBMSCs) expressing the transduced human bone morphogenetic protein 2 (hBMP-2) gene stably. MethodsThe full-length hBMP-2 gene was cloned from human muscle tissues by RT-PCR and connected into a vector to consturct a eukaryotic expression system. And then the gene expression system was transduced to hBMSCs with lipidosome. hBMSCs were transfected by hBMP-2 gene (experimental group) and by empty plasmid (negative control group), untransfected hBMP-2 served as blank control group. RT-PCR, dot-ELISA, immunohistochemical analysis and ALP activity were performed to compare and evaluate the situation of hBMP-2 expression and secretion after transfection. hBMSCs transfected by hBMP-2 gene were seeded on hydroxyapatite (HA) and incubated for 4 days to construct the hBMP-2 gene modified tissue engineered bone, and then the tissue engineered bone was observed by the inverted phase contrast microscope and scanning electron microscope. Then the hBMP-2 gene modified tissue engineered bone (group A, n=3), empty plasmid transfected hBMSCs seeded on HA (group B, n=3), hBMSCs suspension transfected by hBMP-2 gene (group C, n=3), and hBMP-2 plasmids and lipidosome (group D, n=3) were implanted into bilateral back muscles of nude mice. The osteogenic activity was detected by HE staining and alcian blue staining after 4 weeks. ResultsAt 48 hours and 3 weeks after transfection, RT-PCR and dot-ELISA results indicated that the transfected hBMSCs could express and secrete active and exogenous hBMP-2 stably. The immunohistochemical staining was positive, and the ALP activity in the transfected hBMSCs was significantly higher than that in two control groups (P < 0.05). The transfected hBMSCs had a good attaching and growing on the three-demension suface of HA under inverted phase contrast microscope and scanning electron microscope. In vivo study indicated that a lot of new bone formation was obviously found at 4 out of 6 sides of back muscles in group A. Some new bone formation at both sides of back muscles was observed in 1 of 3 mice in group B. No new bone formation was found in group C. A few new bone formation was observed at one side of back muscles in group D. ConclusionThe tissue engineered bone constructed by hBMP-2 gene modified hBMSCs and HA is able to express and secrete active hBMP2 stably and can promote new bone formation effectively in muscles of nude mice.
Objective To review the recent advances in transforming growth factor-β(TGF-β) super family study and its role in new bone formation. Methods The latest original articles related to this subject were retrieved extensively,especially the effect of TGF-β, bone morphogenetic proteins(BMPs) and activin(ACT) on distractionosteogenesis. Results TGF-β, BMPs and ACT play important roles in prompting new bone formation and each of them has different effects. Among them, TGF-β can stimulate the proliferation of osteoblast and synthesis ofextra cellular medium; BMPs can initiate the differentiation of interstitial cell toosteocyte; then ACT displays the combine effect of above two factors. Conclusion TGF-β superfamily can regulate new bone formation and thus shorten the course of mandibular distraction osteogenesis.
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.
Objective Melatonin (MLT) can increase the expression of cartilage-derived growth factor and stimulate the synthesis of cartilage matrix. To investigate the prevention and treatment effects of MLT on damaged cartilage through observing the expressions of bone morphogenetic protein 2 (BMP-2) and interleukin 1β (IL-1β) in articular cartilage of the rats with osteoarthritis (OA). Methods Forty SPF 4-week-old male SD rats (weighing 120-150 g) were randomly divided into 4 groups (n=10): normal control group (group A), OA group (group B), OA/pinealectomy group (group C), and OA/ pinealectomy/MLT group (group D). The rats of group A served as a control without treatment. The rats of groups B, C, andD underwent left knee joint injection of 0.2 mL 4% papain solution 1 time every other day for 2 weeks for establ ishing OAmodel. Two weeks after papain injection, the rats of groups C and D were exposed to continuous l ight for 24 hours (intensity of illumination: 500 lx) for creating pinealectomy models. And at the next day after pinealectomy model establ ishing, the rats of group D were treated with intra-articular injections of 0.2 mL 20 mg/mL MLT solution 4 times a week for 4 weeks. At 1 week after last MLT injection, the venous blood samples were taken in groups A, B, and C to test the level of serum MLT by ELISA, respectively, and then the specimens of left cartilage of femoral condyle were harvested for macroscopic, histological, and immunohistochemical examinations in 4 groups. Results The OA and pinealectomy models of rats were successfully establ ished, and all rats survived. There were significant differences in the serum MLT level among groups A, B, and C, and among different time points at the same group (P lt; 0.05). In group A, articular cartilage surface was smooth and elastic, and chondrocytes arranged regularly. In groups B and C, articular cartilage surface was rough, cartilage defects and subchondral bone exposure were observed in some areas, and chondrocytes arranged irregularly. In group D, cartilage surface was more smooth than that in groups B and C, and the degrees of cartilage defect and subchondral bone exposure decreased with regular arrangment of chondrocytes. There were significant differences in Mankin scores and integral absorbance values among 4 groups (P lt; 0.05). Conclusion Exposure to continuous l ight can accelerate degeneration process of articular cartilage of OA rats. Injections of 0.2 mL MLT solution (20 mg/mL) by intra-articular for 4 weeks can inhibit the progress of cartilage defects. Upregulationof anabol ic factor of BMP-2 as well as down-regulation of catabol ic factors of IL-1β is associated with cartilage repairin the pathological features of OA.