Objective To study the effect of adenovirus bone morphogenetic protein 2 gene(Ad-BMP-2) transfer inducing mesenchymal stem cells (MSCs) compounded with fibrin gel on repair of rabbit cartilage defect. Methods ①BMP-2 and collagen type Ⅱ in MSCs transferred by Ad-BMP-2 were examined by RT-PCR, aniline dyeing and immunohistochemical analysis in vitro. ②MSCs were cultured in fibrin gel for 9 days, and were examined with electron microscope. ③Fortytwo rabbits suffering from cartilage defect were divided into 3 groups:the defects were treated with Ad-BMP-2 transfer inducing MSCs compounded with fibrin in group A, with MSCs compounded with fibringel in group B and with no implants in group C as control. HE and aniline dyeing, immunohistochemical analysis and biomechanics study were carried out in the 4th, 8thand 12th weeks. Results ①The positive results were observed for BMP-2 and collagen type Ⅱ with RT-PCR on the 3rd day and 5th day respectively, being statisticallysignificant difference when compared with control group(P＜0.05). ②Ad-BMP-2 transfer inducing MSCs cultured in fibrin gel were positively stained by aniline dyeing and immunohistochemstry. ③The therapy effect of group A was better than that of the other two groups in histology, biochemistry and biomechanics, and the biomechanic and histological features of repaired cartilage were similar to those of the natural cartilage. Conclusion Ad-BMP-2 can induce the expressionof collagen type Ⅱ and mucopolysaccharide in MSCs by secreting BMP-2, and can reconstruct articular cartilage defects better when compounded with fibrin gel.
ObjectiveTo investigate the effect of cyclic stretch stress on the osteogenic differentiation of human cartilage endplate-derived stem cells (CESCs). MethodsCESCs were isolated from the endplate cartilage tissues by the method of agarose suspension culture system. The endplate cartilage tissue was harvested for immunohistochemical staining. Flexercell-4000TM Tension Plus system was used to apply cyclic stretch on CESCs at a frequency of 1 Hz and at a stretch rate of 10% for 1, 6, 12, or 24 hours (experimental group). No stretch stress was performed on CESCs in the same culture condition (control group). After mechanical loading, the protein expression of bone morphogenetic protein 2 (BMP-2) was measured by Western blot, and gene expressions of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and SOX9 were detected by real-time fluorescent quantitative PCR. ResultsImmunohistochemical staining showed BMP-2 protein expression in chondrocytes. The continuous cyclic stretch stress of 10% can increase the expression of BMP-2 protein in CESCs. Significant differences were observed in the expressions of BMP-2 protein (P<0.05) between 2 groups at the other time points except at 1 hour (P>0.05), in a time-dependent manner. The real-time fluorescent quantitative PCR indicated that the gene expressions of Runx2 and ALP showed an increasing tendency with time in the experimental group when compared with the control group, but there was down-regulated expression of SOX9. Significant difference was found in mRNA expressions of Runx2 and ALP at 12 and 24 hours and in mRNA expressions of SOX9 at 6, 12, and 24 hours between 2 groups (P<0.05), in a time-dependent manner. ConclusionCyclic stretch stress may induce osteogenic differentiation of CESCs by regulating the expressions of some genes related osteogenesis in CESCs.
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.
Objective To study the expression changes of vascular endothel ial growth factor (VEGF), basic fibroblast growth factor (bFGF), and bone morphogenetic protein 2 (BMP-2) in femoral neck fracture, traumatic, and non-traumatic avascular necrosis of femoral head (ANFH), and to study the relationshi p between the expressions of VEGF, bFGF, BMP-2mRNA and bone mass so as to explore the pathogenesis of ANFH and provide the exprimental basis for individual treatment of ANFH. Methods Femoral head specimens were obtained from 59 donors undergoing total hip replacement, including 22 cases of traumatic ANFH (group A, 13 cases of Ficat stage III and 9 cases of Ficat stage IV), 19 cases of non-traumatic ANFH (group B, 11 cases of Ficat stage III and 8 cases of Ficat stage IV; 10 cases of steroid-induced ANFH, 7 cases of alcohol ic ANFH, and 2 cases of unexplained ANFH), and 18 cases of fresh femoral neck fracture (group C). There was no significant difference in the general data among 3 groups (P gt; 0.05). The bone mineral density (BMD) at weight-bearing area of the femoral head was measured with dual energy X-ray absorptiometry. The pathological changes were observed by using optical microscope and scanning electron microscope. The percentage of empty bone lacuna and the percentage of trabecular bone area were calculated. The expressions of VEGF, bFGF, and BMP-2 mRNA in femoral head were detected by use of in-situ hybridization technique. Results The BMD in groups A and B were significantly lower than that in group C (P lt; 0.05), and there was significant difference between group A and group B (P lt; 0.05). In the necrosis area of groups A and B, the bone trabecula was rarefactive and not of integrity, with a great number of empty bone lacuna. In healthy area, more fiber hyperplasia was observed in group A, the prol iferated and hypertrophic fat cells in the medullary cavity in group B. Scanning electron microscope showed that many osteocytes underwent fatty degeneration and necrosis, and that the prol iferation of fat cells in bone matrix was observed in groups A and B. While in group C, the femoral head had intact articular cartilage and intact bone trabeculae, and osteocytes were clearly seen. The percentage of empty bone lacuna was significantly higher (P lt; 0.05) and the percentage of trabecular bone area was significantly lower (P lt; 0.05) in groups A and B than group C; and there was significant difference in the percentage of empty bone lacuna between groups A and B (P lt; 0.05). The expressions of VEGF, bFGF, and BMP-2 mRNAwere significantly lower in groups A and B than group C (P lt; 0.05), and the expressions of BMP-2 and bFGF mRNA in group A were significantly higher than those in group B (P lt; 0.05). There were positive l inear correlation between the expressions of VEGF mRNA, bFGF mRNA, BMP-2 mRNA and the BMD and percentage of trabecular bone area, respectively. While there were significantly negative correlation between the expressions of VEGF mRNA, bFGF mRNA, BMP-2 mRNA and percentage of empty bone lacuna. Conclusion The repair capacity of local femoral head in traumatic ANFH is ber than that in non-traumatic ANFH. The expressions of VEGF mRNA, bFGF mRNA, and BMP-2 mRNA decl ine in traumatic and nontraumatic ANFH.
ObjectiveTo compare the osteogenic effect of bone marrow mesenchymal stem cells (BMSCs) transfected by adenovirus-bone morphogenetic protein 2-internal ribosome entry site-hypoxia inducible factor 1αmu (Ad-BMP-2-IRES-HIF-1αmu) and by Ad-cytomegalovirus (CMV)-BMP-2-IRES-human renilla reniformis green fluorescent protein 1 (hrGFP-1) single gene so as to optimize the source of osteoblasts. MethodsBMSCs were separated and cultured from 1-month-old New Zealand white rabbit. The BMSCs at passage 3 were transfected by virus. The experiment was divided into 4 groups (groups A, B, C, and D) according to different virus: BMSCs were transfected by Ad-BMP-2-IRES-HIF-1αmu in group A, by Ad-CMV-BMP-2-IRES-hrGFP-1 in group B, by Ad-CMV-IRES-hrGFP-1 in group C, and BMSCs were not transfected in group D. The optimum multiplicity of infection (MOI) (50, 100, 150, and 200) was calculated and then the cells were transfected by the optimum MOI, respectively. The expression of BMP-2 gene was detected by immunohistochemistry staining after transfected, the expressions of BMP-2 protein and HIF-1α protein were detected by Western blot method. The osteogenic differentiation potential was detected by alkaline phosphatase (ALP) activity and Alizarin red staining. ResultsThe optimum MOI of groups A, B, and C was 200, 150, and 100, respectively. The expression of BMP-2 was positive in groups A and B, and was negative in groups C and D by immunohistochemistry staining; the number of positive cells in group A was more than that in group B (P ﹤ 0.05). The expression of BMP-2 protein in groups A and B was significantly higher than that in groups C and D (P ﹤ 0.05), group A was higher than group B (P ﹤ 0.05). The expression of HIF-1α protein in group A was significantly higher than those in the other 3 groups (P ﹤ 0.05), no significant difference was found among the other 3 groups (P ﹥ 0.05). ALP activity in groups A and B was significantly higher than that in groups C and D (P ﹤ 0.05), group A was higher than group B (P ﹤ 0.05). Calcium nodules could be seen in groups A and B, but not in groups C and D; the number of calcium nodules in group A was higher than that in group B (P ﹤ 0.05). ConclusionThe expression of BMP-2 and osteogenic effect of BMSCs transfected by Ad-BMP-2-IRES-HIF-1αmu (double genes in single carrier) are higher than those of BMSCs transfected by Ad-CMV-BMP-2-IRES-hrGFP-1 (one gene in single carrier).
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 investigate the effect of the synthetic bone morphogenetic protein 2 (BMP-2)derived peptide on the osteogenic induction in the marrow mesenchymal stem cells (MSCs）and to evaluate the osteoinductivity and dosedependence of the BMP-2 derived peptide in vitro. Methods MSCs of 4-week old Wistar rats were separated and cultured. In the 3rd passage, the conditional culture medium was changed, in which the BMP-2-derived peptide in the following doses was added: 300,200, 100, 50, and 0 μg/ml, respectively (Groups A-E). The activity of alkaline phosphatase (ALP）and the amount of calciumdeposition were meassured at 5，10，15 and 20 days during the culture with the conditional culture medium. The real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was performed to measure the mRNA expressions of collagen type Ⅰ, osteopontin (OPN), and osteocalcin（OCN）and to measure the osteoinductivity of the BMP-2-derived peptide in the different concentrations.Results Under the inverted phase contrast microscope, MSCs cultured in the conditional culture medium for 3-4 days were changed in shape, from long fusiform to short fusiform or polygon. As the concentration of the BMP-2-derived peptide increased, the time for MSCs to change into the osteoblasts decreased. There was a significantly greater level of the ALP activity and amount of the calcium deposition in Groups A and B than in the other groups（Plt;0.05）. However,there was no significant difference between Group A and Group B （Pgt;0.05）. Theresult of FQPCR showed that after MSCs were cultured in the different doses of theconditional culture medium for 14 days, the mRNA expressions of collagen type Ⅰ, OPN andOCN were at higher levels. An increasing order in the level of the cycle threshold (Ct) was found in the following groups: Agt;Bgt;Cgt;D. Almost no expression was found in Group E. The Ct levels were significantly greater in Groups A and B thanin Groups C and D（Plt;0.05）. However, there was no significant difference between Group A and Group B （Pgt;0.05）.ConclusionThe BMP-2-derived peptide can greatly promote differentiation of MSCs into the osteoblasts, the promotion of osteogenesis has a dosedependent pattern, and the best inducing dosage is 200 μg/ml.
Objective To explore the expressions of bone morphogenetic protein 2 (BMP-2) and runt-related transcription facotr 2 (Runx2) and microarchitecture of trabecular bone periacetabula in adult patients with developmental dysplasia of the hip (DDH). Methods Between March and September 2008, the trabecular bone periacetabulum was collected from 8 patients with DDH who were scheduled for total hip arthroplasty (aged 37-55 years, 3 males and 5 females, trial group) and from 8 patients with avascular necrosis of the femoral head (Ficat stage II) who were scheduled for hip resurfacing arthroplasty (aged 36-55 years, 3 males and 5 females, control group). The expressions of BMP-2 and Runx2 in the trabecular bone were determined by real-time quantitative PCR, and the microarchitecture was observed by micro-CT and the following parameters were determined: bone volume/total volume (BV/TV), connectivity density (Conn.Dens), trabecular number (Tb. N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and structure model index (SMI). Results The expressions of BMP-2 and Runx2 were significantly lower in trial group than in control group (P lt; 0.05). The micro-CT showed sparse trabecular bone in trial group and dense trabecular bone in control group. BV/TV and Tb.N in trial group were significantly lower than those in control group, and SMI and Tb.Sp in trial group were significantly higher than those in control group (P lt; 0.05); there was no significant difference in Conn.Dens and Tb.Th between 2 groups (P gt; 0.05). Conclusion The trabecular bone is in a low metabolism condition and its microarchitecture is tendency to be osteoporosis trabecualr bone in adult patients with DDH. It may be related with the acetabular component loosening after total hip arthroplasty.
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.
Objective To investigate the effects of bone morphogenetic protein 2 (BMP-2) on the chondrogenic differentiation of human Achilles tendon-derived stem cells (hATDSCs) in vitro. Methods Achilles tendon was harvested from a voluntary donor with acute Achilles tendon rupture. And nucleated cells were obtained by digesting with collagenase and were cultured to the 3rd passage. The flow cytometry was used to measure the immunophenotyping; and Oil red O staining, alizarin red staining, and Safranin O/fast green staining were used to identify the adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation, respectively. The hATDSCs pellet was cultured in complete culture medium with (experimental group) or without recombinant human BMP-2 (rhBMP-2) (control grup) for 3 weeks. Chondrogenic differentiation of hATDSCs was evaluated by HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II; and the mRNA expressions of SOX9, collagen type II, and Aggrecan were detected by real-time fluorescence quantitative PCR. Results Primary hATDSCs cultured in vitro showed clonal growth; after cell passage, homogeneous spindle fibroblast-like cells were seen. The cells were positive for CD44, CD90, and CD105, while negative for CD34, CD45, and CD146. The results were positive for Oil red O staining at 3 weeks after adipogenic differentiation, for alizarin red staining at 4 weeks after osteogenic differentiation, and for Safranin O/fast green staining at 3 weeks after chondrogenic differentiation. After hATDSCs were induced with rhBMP-2 for 3 weeks, pellets formed in the experimental group, and the size of pellets was significantly larger than that in the control group; the results of HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II were all positive. The results of real-time fluorescence quantitative PCR showed that the mRNA expressions of SOX9, collagen type II, and Aggrecan in the experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion BMP-2 can promote proteoglycan deposition and induce chondrogenic differentiation of hATDSCs in vitro. The effect of BMP-2 on hATDSCs might provide a possible explanation for histopathological changes of tendinopathy.