Mechanism of Youguiwan Regulating BMP-2/Smad Signaling Pathway to Promote Bone Formation in Postmenopausal Rats with Osteoporosis / 中国实验方剂学杂志

ObjectiveTo observe the effects of Youguiwan on bone metabolism and bone morphogenetic protein-2 （BMP-2）/Smad signaling pathway in ovaries-removed rats with osteoporosis and study the mechanism of Youguiwan in the prevention and treatment of osteoporosis. MethodA postmenopausal rat model of osteoporosis was prepared by bilateral ovariectomy. The 40 female SD rats were randomly divided into five groups， including sham operation group， model group， alendronate sodium group （0.1 mg·kg-1）， and high-dose and low-dose （5.36 and 2.68 g·kg-1） groups of Youguiwan. The drug was given seven days after modeling， once a day for 12 weeks. After the treatment， the changes in femur tissue structure were observed by micro-CT， including bone mineral density （BMD）， bone volume/total volume （BV/TV）， trabecular number （Tb.N）， trabecular thickness （Tb.Th）， bone surface/bone volume （BS/BV）， and trabecular separation （Tb.Sp）. Ossification was observed by saffrane-solid green staining， and serum levels of bone metabolism markers， including bone alkaline phosphatase （BALP）， osteocalcin （BGP）， type Ⅰ procollagen amino terminal propeptide （PINP）， and tartrate-resistant acid phosphatase 5b （TRACP-5b）， were determined by enzyme-linked immunosorbent assay （ELISA）. The protein and mRNA expression levels of Runx2， BMP-2， and Smad1 in rat femur were detected by Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the sham operation group， bone trabecula in the model group was sparse. BMD， BV/TV， Tb.N， and Tb.Th were decreased （P<0.05， P<0.01）. BS/BV （P<0.05） and Tb.Sp were increased. The content of BGP， BALP， PINP， and TRACP-5b in serum was significantly increased （P<0.01）. The mRNA and protein expressions of Runx2， BMP-2， and Smad1 in rat femur were significantly decreased （P<0.05， P<0.01）. Compared with the model group， the number of bone trabeculae in the high-dose and low-dose groups of Youguiwan was increased， and the bone microstructure was improved. BMD， BV/TV， Tb.N， and Tb.Th were increased significantly （P<0.05， P<0.01）， and BS/BV and Tb.Sp were increased. The content of bone metabolic markers decreased （P<0.05， P<0.01）. ConclusionYouguiwan has certain preventive and therapeutic effects on postmenopausal osteoporosis， and its mechanism may be related to promoting bone formation by regulating the BMP-2/Smad signaling pathway.

Comparative study of the effects of intramedullary nail fixation and minimally invasive percutaneous plate internal fixation technique on platelet activation and serum transforming growth factor-β1(TGF-β) 1 and bone morphogenetic protein-2 (BMP-2) in patients with tibial and fibular fracture / 中国骨伤

OBJECTIVE@#To investigate the effect of intramedullary nail fixation (IMN) and minimally invasive percutaneous plate internal fixation (MIPPO) techniques on tibiofibular fractures and their effect on platelet activation and serum transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2).@*METHODS@#Total of 105 patients with tibiofibular fractures from February 2019 to February 2020 were selected and divided into 53 cases in the MIPPO group and 52 cases in the IMN group. There were 29 males and 24 females with an average age of (41.74±6.05) years old in MIPPO group;in IMN group, 31 males and 21 females with an average age of (40.59±5.26) years old. The perioperative surgical indexes, postoperative complications, ankle function recovery at 12 months postoperatively, platelet activation indexes at 3 and 7 days preoperatively and postoperatively, and serum TGF-β1 and BMP-2 levels at 4 and 8 weeks preoperatively and postoperatively were compared between the two groups.@*RESULTS@#The operating time and fracture healing time in the MIPPO group were shorter than those in the IMN group(P<0.05); Compared with the preoperative period, the levels of GMP-140, PAC-1, CD63, and CD61 increased in both groups at 3 and 7 days after surgery, but were lower in the MIPPO group than in the IMN group(P<0.05);the levels of serum TGF-β1 and BMP-2 increased in both groups at 4 and 8 weeks after surgery compared with the preoperative period, and the postoperative complication rate in the MIPPO group was lower than that in the IMN group(P<0.05);the difference was not statistically significant in the excellent rate of ankle function recovery at 12 months follow-up after surgery between two groups(P>0.05).@*CONCLUSION@#Both intramedullary nail fixation and MIPO technique for treatment of tibia and fibula fractures can improve ankle joint function, but the latter has the advantages of short operation time, fast fracture healing, fewer complications, and light platelet activation. Serum TGF-β1, BMP-2 level improves quickly.

The effects of BMP-2 expression in the related periodontal tissue on bone remolding of rapid tooth movement by resistance reduction and distration method / 实用口腔医学杂志

Objective:To investigate the effects of bone morphogenetic protein 2(BMP-2)expression in the related periodontal tissue on bone remodeling under different distracting force during rapid tooth movement by resistance reduction and distraction. Methods:1 2 Beagle dogs were randomly divided into 4 groups as follows:5 d distraction,1 5 d distraction,1 5 d distraction and 1 0 d retaining and 1 5 d distraction and 90 d retaining.4 4 were distalized.6 teeth in each group were randomly assigned to re-sistance and distracting method,resistance and conventional method and conventional method,and there were 2 teeth in each group.Moving teeth models were prepared regularly.BMP-2 expression was examined by immunohistochemistry.Results:The BMP-2 positive expression of the 3 groups of different distraction schedule showed similar distribution area,and it reached peak at the end of 1 5-day distration,but the group of resistance and distracting method showed the maximum peak(P 0.05).Conclusion:Resistance reduction with sustained strong distracting force can significantly increased the positive expression of BMP-2 and effectively accelerate new bone formation in periodontal tissue.

The expression of BMP-2 mRNA of rat osteoblast cell sheets cultured with ascorbic acid / 实用口腔医学杂志

Objective:To study the effects of ascorbic acid on BMP-2 mRNA expression of osteoblast cell sheets.Methods:Rat os-teoblasts were primaryly cultured and identified;osteoblast cell sheets were built by physical scraping method in vitro;the osteoblast cell sheets were cultured with 1 5,50 and 85 mg/L ascorbic acid for 1 and 2 weeks respectively,and the expression of BMP-2 mRNA of the cell sheets was detected by RT-qPCR.Results:The obtained cells were conformed to be osteoblasts.The osteoblast cell sheets could be rolled into tube in vitro.The expression of BMP-2 mRNA of osteoblast cell sheets in experiment group,whether in week one or week two was higher than that in control group,50 mg/L group showed the highest expression(first week P 0.05);the expression of any group in week two was higher than that in week one(P <0.05).Conclusion:Ascorbic acid may pro-mote the expression of BMP-2 mRNA in osteoblast cell sheets.

The effects of bone morphogenetic protein-2 and enamel matrix derivative on the bioactivity of mineral trioxide aggregate in MC3T3-E1cells

OBJECTIVES: The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. MATERIALS AND METHODS: MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). RESULTS: Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). CONCLUSIONS: These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.

The effects of nano-chitosan/bone morphogenetic protein gel on the repair of mandibular defect / 实用口腔医学杂志

Objective:To investigate the effects of Nano-chitosan rhBMP-2 Gel(NCS/BMP-2 Gel)on the repair of mandibular de-fect.Methods:NCS/BMP-2 Gel and NCS Gel were prepared and respectively injected into the subcutaneous space on both sides of dorsum of 9 rats.3 rats were respectively sacrificed 10,20 and 30 d after injection.The subcutaneous nodules were histologicaly ex-amined.Mandibular defect was made in 54 SD rats and the rats were divided randomly and into 3 groups(n=18):NCS/BMP-2 Gel was implanted into the defects in group 1,NCS Gel in group 2,no injection in group 3.Animals were sacrificed 3,6 and 9 weeks after transplantation.X-ray examination,pathologic observation were conducted.Results:Subcutaneous nodules were found in both sides of the rat dorsum.The residual mandibular defect area in study group 1 was apparently smaller than that in group 2 and 3(P<0.05). More new bone formation was observed in the gel injected area in group 1 than in group 2 and 3.Conclusion:Nano-CS/BMP-2 Gel is biocompatible and can accelerate the healing of mandibular defect.

BMP2 expression impacts the growth and osteogenic differentiation of BMSCs in the co-culture system / 中华显微外科杂志

Objective To investigate the effects of BMP2 in the co-culture system of hBMSCs and hUVECs.Methods Based on the co-culture system of cells,a simple culture group (hBMSCs group),a combined culture group (BMSCs + hUVECs group) and a BMP2 silenced group (BMSCs + BMP2 silenced hUVECs) were established.On days 4,6,8,10,cells were counted for drawing cell growth curves.In addition,ALP and OC expression in BMSCs was detected in each group.Results At each time point,cell number in the BMSCs + hUVECs group were significantly greater than in the BMSCs group,while the BMP2 silenced group were in the median.ALP and OC expression in the BMSCs + hUVECs group were significantly greater than in the BMSCs group,while the BMP2 silenced group were in the median.Conclusion BMP2 plays an important role in the co-culture system,which can promote osteogenic differentiation and the growth of BMSCs.

Tissue transglutaminase (TG2) activity regulates osteoblast differentiation and mineralization in the SAOS-2 cell line

Tissue transglutaminase (type II, TG2) has long been postulated to directly promote skeletal matrix calcification and play an important role in ossification. However, limited information is available on the expression, function and modulating mechanism of TG2 during osteoblast differentiation and mineralization. To address these issues, we cultured the well-established human osteosarcoma cell line SAOS-2 with osteo-inductive conditioned medium and set up three time points (culture days 4, 7, and 14) to represent different stages of SAOS-2 differentiation. Osteoblast markers, mineralization, as well as TG2 expression and activity, were then assayed in each stage. Furthermore, we inhibited TG activity with cystamine and then checked SAOS-2 differentiation and mineralization in each stage. The results showed that during the progression of osteoblast differentiation SAOS-2 cells presented significantly high levels of osteocalcin (OC) mRNA, bone morphogenetic protein-2 (BMP-2) and collagen I, significantly high alkaline phosphatase (ALP) activity, and the increased formation of calcified matrix. With the same tendency, TG2 expression and activity were up-regulated. Furthermore, inhibition of TG activity resulted in a significant decrease of OC, collagen I, and BMP-2 mRNA and of ALP activity and mineralization. This study demonstrated that TG2 is involved in osteoblast differentiation and may play a role in the initiation and regulation of the mineralization processes. Moreover, the modulating effects of TG2 on osteoblasts may be related to BMP-2.

Effect of gene therapy on the expression of bone morphogenetic protein-2 during mandible distraction / 中华医学美学美容杂志

Objective To investigate the expression patterns of major bone growth factors and bone morphogenetic protein-2 (BMP2) in the distracted calluses following transfected gene during mandibular distraction osteogenesis in a rabbit model.Methods Bilateral mandibular osteotomies were performed in New-Zeland rabbits.After a latency of 3 days,the mandibles were elongated using distractors for 7 days.After the completion of distraction,the rabbits were randomly divided into 5 groups.Three animals each were sacrificed at the end of the delay phase,at 7,14,and 28 days after completion of distraction,respectively.The lengthened mandibles were harvested and processed for immunohistochemical detection of BMP2 expression,the mean optic densities and integral optical density of BMP2 positive cells were measured by computerized image analyzer.Results Elevated cellular expression of BMP2,in the distraction gap,was observed following mandibular distraction.BMP2 staining was mainly located in inflammatory cells,and the connective tissues arrounding the new bone.Their strongest expression was the 7th day,some of those growth factors expressed weakly or negatively.Conclusions Electroporation-mediated gene transfection can promote BMP2 expression effectively,which plays an important role in cell differentiation and proliferation during distraction osteo genesis.The BMP2 stimulates extracellular matrix synthesis,induces the proliferation and differentiation of fibroblasts and osteoblasts,which then promotes the new bone formation and repair.

Effects of cyclic mechanical stretch on BMP-2 expression in RA and OA fibroblast-like synoviocytes / 医用生物力学

Objective To explore the role of mechanical stimulation in synovium under different pathological conditions through studying the effects of cyclic mechanical stretch on the expression of bone morphogenetic protein 2 (BMP-2) in rheumatoid arthritis (RA) and osteoarthritis (OA) fibroblast-like synoviocytes (FLS). Method 6% and 0.5 Hz stretch generated by Flex cell 4000 tension systems was applied on normal, RA and OA FLS of human knee joint source under normal and inflammatory conditions for 2 h or 6 h, respectively. Results Cyclic mechanical stretch of 6%, 0.5 Hz had no significant effects on the expression of BMP-2 in normal, RA and OA FLS at 2 h, while in RA FLS it increased significantly at 6 h. Inflammatory cytokines (IL-1β) didn’t influence normal FLS at 2 h, but made BMP-2 mRNA significantly increased at 6 h. IL-1β increased BMP-2 mRNA of RA FLS significantly both at 2 h and 6 h. IL-1β increased BMP-2 mRNA of OA FLS significantly only at 2 h, but had no significant effect at 6 h. The co-effect of IL-1β and cyclic mechanical stretch induced the ascension of BMP-2 expression significantly in normal and RA FLS at 2 h, and in normal, RA and OA FLS at 6 h. Conclusions Response of BMP-2 mRNA to mechanic stimulation and IL-1β in normal, RA and OA FLS were different. Inflammation may play a more important role than mechanical stimulation in the pathogenesis of RA and OA. Synergetic effect in inflammation and mechanical stimulation were found in OA FLS at 6 h, which reveals that they may co-act in the occurrence and development of OA.

Comparison of Osteogenic Potential in vivo between Autologous Bone Marrow Mesenchymal Stem Cell Implanting and Fibrin Sealant Combined with Recombined Human Bone Morphogenetic Protein-2 Grafting / 中国康复理论与实践

@#Objective To investigate the osteogenic potential for size-critical bone defect of fibrin sealant combined with recombined human bone morphogenetic protein-2 (rhBMP-2) grafting and varied autologous bone marrow mesenchymal stem cells (BMSCs) implanting in vivo. Methods BMSCs were cultured and induced with osteogenic supplement (OS) medium. BMSCs with and without OS induction were collected and percutaneously autologous injected respectively into the 15 mm bone defect of experimental rabbit model. The grafts were BMSCs, osteo-induced BMSCs, BMSCs and osteo-induced BMSCs, BMP combined with fibrin sealant, 0.9% NaCl solution. Osteogenesis at the defect area was assessed with regular radiography, histology and biomechanics. Results The FS/BMP group and the BMSCs+osteo-induced BMSCs group achieved complete bone healing with medullary cavity united, with the most new bone formation and the maximal load among those groups. Conclusion The osteogenic potential of both osteo-induced BMSCs combined with BMSCs and FS/BMP are similar, which are superior to that of BMSCs or osteo-induced BMSCs along.

Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells / 药物分析学报

Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42.8±6.1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP 2 in mesenchymal stem cells / 西安交通大学学报·英文版

Objective: To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods: Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometers analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results: Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42. 8 ± 6. 1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12 K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion: Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

Effect of exogenous transforming growth factor-beta 2(TGF-?_2) and bone morphogenetic protein 2(BMP_2) and allografts bone on radius defects healing of experimental rabbits / 中国矫形外科杂志

[Objective]To investigate the effect of exogenous TGF-?2and BMP2 and allografts bone on radius defects.[Method]The model of adult rabbit radius defects was made and treated with TGF-?2 and BMP2 respectively or in combination and allografts bone in the radius defects.A group:BMP2 and allografts bone.B group:autogenous bone.C group:TGF-?2 and allografts bone.D group:BMP2 and TGF-?2 and allografts bone.Fracture healing was evaluated in different time by radiograph,biomechanical tests,measurement of the bone mineral density and calcium content in callus.[Result]The results of B group excelledthose of A,C,D group in aspects of calcium content in callus,the ability of radius defects and biomechanical strength of bone(P

Expression of Bone Morphogenetic Protein-2 in Gunshot Fracture Healing Detected with Real Time Fluorescence Quantitative Reverse Transcript Polymerase Chain Reaction / 中国康复理论与实践

@#ObjectiveTo detect the levels of bone morphogenetic protein-2(BMP-2) mRNA in gunshot fracture healing of the rabbits.MethodsA model of primary treating gunshot fracture of the rabbit with external fixator and the real time fluorescence quantitative reverse transcription polymerase chain reaction(FQ-RT-PCR) technology with SYBR Green I were established.The levels of BMP-2 mRNA 1,2,3 and 4 weeks after operation were detected with FQ-RT-PCR respectively.ResultsIn the control group,BMP-2 mRNA of the bone tissue began to rise at the first week after fracture and the peak of mRNA expression appeared at the second week.The expression returned to normal at the forth week.In the experimental group,the BMP-2 mRNA rose more slowly that it arrived peak stage at the third week and was significantly lower in experimental group than that in control group at the same phases.ConclusionReal time FQ-RT-PCR is a good method for measuring the levels of BMP-2 mRNA during gunshot fracture healing.The mRNA expression rose more slowly and was significantly lower in gunshot fracture than in common fracture at the same phases.