• Journal of Periodontal and Implant Science
• /
• 제39권sup2호
• /
• pp.279-286
• /
• 2009

Purpose: Gene therapy (ex vivo) has recently been used as a means of delivering bone morphogenetic proteins (BMPs) to sites of tissue regeneration. In the present study, we investigated the effect of co-transduction of adenoviruses expressing BMP-2 and BMP-7 on osteogenesisof C2C12 cells in vitro. Methods: A replication-defective human adenovirus 5 (Ad5) containing a cDNA for BMPs in the E1 region of the virus (Ad5BMP-2 and Ad5BMP-7) was constructed by in vivo homologous recombination. Functional activity of Ad5BMP-2 and Ad5BMP-7 were evaluated in mouse stromal cells (W20-17cells). C2C12 cells are transduced with various MOI (multiplicity of infection) of Ad5BMP-2 and Ad5BMP-7 to assess most effective and stable titer. Based on this result, C2C12 cells were transduced with Ad5BMP-2 and Ad5BMP-7 alone or by combination. BMPs expression, alkaline phosphatase (ALPase) activity, cell proliferation, and mineralization were assessed. Results: Ad5BMP-2 and Ad5BMP-7 are successfully transduced to W20-17 cells, and secreted BMPs stimulated cell differentiation. Also, C2C12 cells transduced with Ad5BMPs showed expression of BMPs and increased ALPaseactivity. In all groups, cell proliferation was observed over times. At 7days, cells co-transduced with Ad5BMP-2 and Ad5BMP-7 showed lower proliferation than the others. C2C12 cells co-transduced with Ad5BMP-2 and Ad5BMP-7 had greater ALPaseactivity than that would be predicted if effect of individual Ad5BMPs were additive. Little mineralized nodule formation was detected in cells transduced with individual Ad5BMPs. In contrast, Ad5BMP-2 and Ad5BMP-7 combination stimulated mineralization after culturing for 10 days in mineralizing medium. Conclusions: Present study demonstrated that adenoviruses expressing BMPs gene successfully produced BMPs protein and these BMPs stimulated cells to be differentiated into osteoblastic cells. In addition, the osteogenic activity of Ad5BMPs can be synergistically increased by co-transduction of cells with Ad5BMP-2 and Ad5BMP-7.

• 한국한의학연구원논문집
• /
• 제13권1호통권19호
• /
• pp.139-145
• /
• 2007

Emodin is one of the main active components of a traditional Korean medicine isolated from the root and rhizomes of Rheum palmatum L. In this study, of 222 natural compounds to evaluate the anabolic activities, emodin activated bone morphogenetic protein (BMP)-2 promoter in the differentiation process of mouse osteoblastic MC3T3-E1 cells. Emodin was shown to significantly stimulate the activity and expression of alkaline phosphatase, an earlyphase marker of osteoblastic differentiation, on the differentiation day 7, and induce the osteopontin mRNA expression from the differentiation day 14. In addition, low concentration (up to 5 M) of emodin dramatically promoted the induction of mineralization in MC3T3-E1 subclone 4 cells. The stimulatory effect of emodin on the osteoblast differentiation/mineralization could be associated with its potential to stimulate the BMP-2 gene expression. Although further studies are needed to determine the precise mechanism, this study suggests that the use of herbal medicine containing natural compounds with anabolic activity such as emodin could have a beneficial effect on bone health.

• International Journal of Oral Biology
• /
• 제34권2호
• /
• pp.115-121
• /
• 2009

Bone morphogenetic protein (BMP) 2 is a potent osteogenic factor. Although both Smad1/5 and mitogenactivated protein kinases (MAPKs) are activated by BMP2, the hierarchical relationship between them is unclear. In this study, we examined if BMP2-stimulated MAPK activation is regulated by Smad1/5 or vice versa. When C2C12 cells were treated with BMP2, the activation of extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun-N-terminal kinase was evident within 5 min. The knockdown of both Smad1 and Smad5 by small interfering RNA did not affect the activation of these MAPKs. In addition, neither the overexpression of Smad1 nor Smad5 induced ERK activation. When ERK activation was induced by constitutively active MEK1 expression, the protein level and activation of Smad1 increased. Furthermore, the inhibition of constitutively active BMP receptor type IB-induced ERK activation significantly suppressed Smad1 activation. These results indicate that Smad1/5 activation is not necessary for BMP2-induced MAPK activation and also that ERK positively regulates Smad1 activation.

• 생명과학회지
• /
• 제10권2호
• /
• pp.218-227
• /
• 2000

Bone morphogenetic protein 1 (BMP-1) is part of a complex capable of inducing ectopic bone formation in mammals. Studies on TGF-β1 processing and Drosophila dorsal-ventral patterning have focused attention on BMP-1 as important in mediating the biological activity of this bone inducing complex. Herein, the bacterial expression, refolding, purification, and initial characterization of the BMP-1 proteolytic domain (BPD) are described. A semi-quantitative fluorescence-based thin layer chromatography assay was developed to assist in rapidly screening for optimal renaturation conditions. According to a preliminary screen for optimal conditions for the refolding of BPD , a detectable proteolytic activity against a high turnover substrate for astacin, a homologous protease from crayfish was observed. The conditions identified have allowed the expression of sufficient amounts of BPD for the characterization of the protein. Its proteolytic activity exhibits the same cleavage specificity as astacin against seven substrates that were previously synthesized for studying astacin. Furthermore, this activity is inhibited by the metal chelator 1,10-phenanthroline but not by its analogue 1,7-phenanthroline. The collagenase inhibitor Pro-Leu-Gly hydroxamate was found to inhibit both astacin and BPD activity. The results presented in this paper argue that BMP-1 does in fact possess an intrinsic proteolytic activity.

• Journal of Periodontal and Implant Science
• /
• 제29권4호
• /
• pp.765-785
• /
• 1999

Bone morphogenetic protein-2/4 (BMP-2/4) are members of Transforming Growth $Factor-{\beta}\;(TGF-{\beta})$ superfamily and they may differentiate the osteoprogenitor cell and induce formation of cartilage and bone in vivo. This study was performed to investigate the effects of bone morphogenetic protein-2/4 on the characteristics of rat periodontal ligament cells(RPDL) and rat calvaria cells(RCV). In the control group, the cells were cultured alone with Dulbeco's Modified Eagle's Medium contained with 20% fetal bovine serum, $100{\mu}/ml$ penicillin, $100{\mu}/ml$ streptomycin. In the experimental groups, recombinant human bone morphogenetic protein-2/4 (25ng, 100ng, 250ng/ml) were added into the above culture condition. And then each group was characterized by examing the cell proliferation at 1, 2, 3, 5, 7th day, the amount of total protein synthesis and alkaline phosphatase activity at 2, 5, 7th day. And also, the calcified nodule was examed. The results were as follows ; 1 . Both RCV and RPDL cells in both control and experimental groups proliferated during the entire experimental period, but there is no stastically significant difference according to the BMP-2/4 concentration. 2 . Amount of total protein synthesis of both cells in both groups was steadily increased until 5th day, but all experimental groups were significantly different from the control group at 7th day. 3. Alkaline phosphatase activity of both cells in both groups was increased during the entire experiment period. In RCV cells, the experimental group treated with 100ng/ml and 250ng/ml BMP-2/4 were significantly different from the control group at 7th day. In RPDL cells, the experimental group treated with 100ng/ml and 250ng/ml BMP-2/4 were significantly different from the control group at 5th day, and all experimental groups were significantly different from the control group at 7th day. 4. In the both of the cultured Rat Periodontal ligament and calvaria cell treated with BMP-2/4 to compared with control group, it revealed more rapid cell polarization, cell aggregation and hyperchromatic stained on HE agent, and even though only 1 day treated with BMP-2/4 both RPDL and RCV showed more rapid cell reaction than control group. More sensivitve cell reaction of RCV were observed than RPDL in this experiment. From the above results, we could conclude that BMP-2/4 influenced the induction, proliferation and differentiation of bone forming cells

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• 제47권6호
• /
• pp.454-464
• /
• 2021

Objectives: This study aimed to investigate the in vitro osteoinductivity of the combination of bone morphogenetic protein-2 (BMP-2) and nanohydroxyapatite (nHAp) and the in vivo effects of implants coated with nHAp/BMP-2. Materials and Methods: To evaluate the in vitro efficacy of nHAp/BMP-2 on bone formation, bone marrow-derived mesenchymal stem cells (BM-MSCs) were seeded onto titanium disks coated with collagen (Col), Col/nHAp, or Col/nHAp/BMP-2. Protein levels were determined by a biochemical assay and reverse transcriptase-polymerase chain reaction. Stem cell differentiation was analyzed by flow cytometry. For in vivo studies with mice, Col, Col/nHAp, and Col/nHAp/BMP-2 were injected in subcutaneous pockets. Titanium implants or implants coated with Col/nHAp/BMP-2 were placed bilaterally on rabbit tibias and evaluated for 4 weeks. Results: In the in vitro study, BM-MSCs on Col/nHAp/BMP-2 showed reduced levels of CD73, CD90, and CD105 and increased levels of glycosaminoglycan, osteopontin, and alkaline phosphatase activity. After 4 weeks, the Col/nHAp/BMP-2 implant showed greater bone formation than the control (P=0.07), while no differences were observed in bone implant contact and removal torque. Conclusion: These results suggest that a combination of BMP-2 and an nHAp carrier would activate osseointegration on dental implant surfaces.

• 대한치과의사협회지
• /
• 제54권4호
• /
• pp.274-283
• /
• 2016

Bisphosphonates are widely used mainly for the treatment of osteoporosis and bone metastasis of malignancy. Since the first report of MRONJ, there have been many studies associated, however the pathogenesis of MRONJ is not yet clear. Medication-related osteonecrosis of the jaws (MRONJ) is a serious complication associated with long-term medication therapy. It is characterized by exposed necrotic bonein the jaw, which has persisted for more than 8weeks despite continuous treatment by dentist. The mechanism of development of MRONJ is still unclear and there is no definitive standard treatment for MRONJ. The purpose of this study is to investigate the jaw bone destruction mechanism of accumulated bisphosphonates, so that we can develop therapeutic method to repair the defect and stop the destruction process. The authors performed simultaneous application of PRF(Platelet rich fibrin) and BMP-2(Bone morphogenetic protein-2) to stimulate not only soft tissue healing but also osseous regeneration. Our case series demonstrate that simultaneous application of platelet rich fibrin and bone morphogenetic protein-2 can be a treatment of choice for MRONJ.

• Journal of Periodontal and Implant Science
• /
• 제38권1호
• /
• pp.41-50
• /
• 2008

Purpose: Bone morphogenetic protein-2(BMP-2) has been shown to possess significant osteoinducitve potential. There have been attempts to overcome a limitation of mass production, and economical efficiency of BMP. The aim of this study was to produce recombinant human BMP-2(rhBMP-2) from E. coli in a large scale and evaluate its biological activity. Materials and Methods: The E.coli strain BL21(DE3) was used as a host for rhBMP-2 production. Dimerized rhBMP-2 was purified by affinity chromatography using Heparin column. To determine the physicochemical properties of the rhBMP-2 expressed in E. coli, we examined the HPLC profile and performed Western blot analysis. The effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and representing morphological change using C2C12 cell. Results: E. coli was genetically engineered to produce rhBMP-2 in a non-active aggregated form. We have established a method which involves refolding and purifying a folded rhBMP-2 dimer from non-active aggregates. The purified rhBMP-2 homodimer was characterized by SDS-PAGE as molecular weight of about 28kDa and eluted at 34% acetonitrile, 13.27 min(retention time) in the HPLC profile and detected at Western blot. The purified rhBMP-2 dimer stimulated ALP activity and induced the transformation from myogenic differentiation to osteogenic differentiation. Conclusion: rhBMP-2 was produced in E. coli using genetic engineering. The purified rhBMP-2 dimer stimulated ALP activity and induced the osteogenic differentiation of C2C12 cells.

• Journal of Periodontal and Implant Science
• /
• 제53권6호
• /
• pp.429-443
• /
• 2023

Purpose: The aim of this study was to determine 1) the bone-regenerative effect of porcine bone block materials with or without collagen matrix incorporation, 2) the effect of a collagen barrier, and 3) the effect of adding recombinant human bone morphogenetic protein-2 (rhBMP-2) to the experimental groups. Methods: Four treatment modalities were applied to rabbit calvaria: 1) deproteinized bovine bone mineral blocks (DBBM), 2) porcine bone blocks with collagen matrix incorporation (PBC), 3) porcine bone blocks alone without collagen matrix incorporation (PB), and 4) PBC blocks covered by a collagen membrane (PBC+M). The experiments were repeated with the addition of rhBMP-2. The animals were sacrificed after either 2 or 12 weeks of healing. Micro-computed tomography (micro-CT), histologic, and histomorphometric analyses were performed. Results: Micro-CT indicated adequate volume stability in all block materials. Histologically, the addition of rhBMP-2 increased the amount of newly formed bone (NB) in all the blocks. At 2 weeks, minimal differences were noted among the NB of groups with or without rhBMP-2. At 12 weeks, the PBC+M group with rhBMP-2 presented the greatest NB (P<0.05 vs. the DBBM group with rhBMP-2), and the PBC and PB groups had greater NB than the DBBM group (P>0.05 without rhBMP-2, P<0.05 with rhBMP-2). Conclusions: The addition of rhBMP-2 enhanced NB formation in vertical augmentation using bone blocks, and a collagen barrier may augment the effect of rhBMP-2.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• 제36권2호
• /
• pp.37-49
• /
• 2014

Purpose: This study compares the bone formation ability of tricalcium phosphate (TCP) with and without recombinant human bone morphogenetic protein-2 (rhBMP-2) and assesses TCP as a carrier of rhBMP-2. Methods: Bilateral round defects (diameter: 8.0 mm) were formed in the cranium of eight New Zealand white rabbits. The defects were grafted with TCP only (control group) or with rhBMP-2-coated TCP (experimental group). The animals were sacrificed at 1st week, 2nd week, 4th week, and 8th week postoperatively; two rabbits sacrificed each time. The skulls were harvested and subjected to radiographic and histological examination. Results: Radiologic evaluation showed faster bone remodeling in the experimental group than in the control group. Histologic evaluation (H&E, Masson's trichrome stain) showed rapid bone formation, remodeling and calcification in the 1st and 2nd week in the experimental group. Immunohistochemical evaluation showed higher expression rate of osteoprotegerin, receptor activator of nuclear factor ${\kappa}B$ ligand, and receptor activator of nuclear factor ${\kappa}B$ in the experimental group at the 1st and 2nd week than in the control group. Conclusion: rhBMP-2 coated TCP resulted in rapid bone formation, remodeling, and calcification due to rhBMP-2's osteogenic effect. TCP performed properly as a carrier for rhBMP-2. Thus, the use of an rhBMP-2 coating on TCP had a synergic effect on bone healing and, especially, bone remodeling and maturation.