• Journal of Periodontal and Implant Science
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• v.35 no.2
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• pp.289-298
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• 2005

Bone morphogenetic protein-7(BMP-7), a member of the transforming growth factor superfamily, stimulates osteoblast differentiation and bone formation. There are lots of evidences supporting a direct participation of periodontal ligament(PDL) cells on periodontal tissue regeneration. The purpose of this study was to evaluate the effect of recombinant human(rh) BMP-7 on primary rat PDL cells in vitro, with special focus on the ability of bone formation. The PDL cells were cultured with rhBMP-7 at the concentration of 0, 10, 25, 50, 100 and 200ng/ml for MTT assay. We evaluated the alkaline phosphatase activity at 3 and 5 days of incubation and the ability to produce mineralized nodules of rat PDL cells at 14 days of cell culture in concentration of 0, 10, 25, 50 and 100ng/ml. The cell activity was not reduced in cells treated with BMP-7 at $10{\sim}100ng/ml$, whereas the cell activity was reduced in the concentration of 200ng/ml than the control at day 1 and 3(p<0.01). At 3 and 5 day, alkaline phosphatase activity was significantly increased in cells treated with BMP-7 at 50ng/ml and 100ng/ml(p<0.05). The area of mineralized bone nodule was greater in cells treated with BMP-7 at 50 and 100 ng/ml than the control(p<0.01). These results suggest that rhBMP-7 stimulate rat PDL cells to differentiate toward osteoblast phenotype and secretion of the extracellular matrix of rat PDL cells.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.29 no.6
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• pp.467-473
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• 2007

The most important factor for successful implantation is osseointegration between the implant and bone. The expression of bone morphogenetic proteins (BMPs) inducing bone formation would differ after maxillary sinus elevation. And within the same graft material. the expression of BMPs would change with time after graft. The aim of this study was to compare the relative expressions of BMP4 and BMP6 using real-time RT-PCR when maxillary sinus elevation was performed using deproteinated bovine bone powder (DBBP) as the graft material or absorbable gelatin sponge (AGS) as the filler without any graft material. Fifteen rabbits, each weighing between 3.0 to 3.5 Kg, were divided randomly into 5 groups of 3 animals each based on their time of sacrifice 0, 3, 5, 7 and 9 days). After exposure of the maxillary sinus bilaterally, bone graft was performed in the right maxillary sinus using DBBP ($BBP^{(R)}$ Oct Inc., Cheonan, Korea) and only AGS ($Gelfoam^{(R)}$ Pharmacia & Upjohn Company, Kalamazoo, MI, U.S.A) was placed into the left without any graft material. Each group of rabbits was sacrificed at 1, 3, 5, 7, or 9 days after operation and all specimens were harvested. And the following results were obtained using real-time RT-PCR from isolated total RNA of the samples. 1. The expression of BMP4 increased at postoperative 1 and 3 days in both DBBP group and AGS group. In AGS group. it decreased at postoperative 5 days. increased again at postoperative 7 days, and decreased at postoperative 9 days. In DBBP group, it increased until postoperative 7 days and decreased at postoperative 9 days. Although the expression of BMP4 was higher in DBBP group compared with AGS group, it was not statistically significant (p>0.05). 2. The expression of BMP6 increased at postoperative 1 and 3 days in both DBBP group and AGS group. In AGS group, it decreased at postoperative 5 days, increased again at postoperative 7 days, and decreased at postoperative 9 days. In DBBP group, it increased until postoperative 7 days and decreased at postoperative 9 days. Although the expression of BMP6 was higher in AGS group compared with DBBP group, it was not statistically significant (p>0.05). 3. There was no statistically significant difference in BMP expression in both groups during same period of time. It' s probably because DBBP and AGS both functioned as a space retainer so that the BMP expression in blood clot seemed to be similar. 4. Thus, DBBP would not offer many benefits for early bone regeneration compared with AGS. The expression of BMP in early bone formation seems to be more influenced by physical carrier rather than the graft type.

• Journal of Microbiology and Biotechnology
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• v.27 no.7
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• pp.1281-1287
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• 2017

Bone morphogenetic protein-4 (BMP-4) is considered to have therapeutic potential for various diseases, including cancers; however, the high expression of biologically active recombinant human BMP-4 (rhBMP-4) needed for its manufacture for therapeutic purposes has yet to be established. In the current study, we established a recombinant Chinese hamster ovary (rCHO) cell line overexpressing rhBMP-4 as well as a production process using 7.5-l bioreactor (5 L working volume). The expression of the mature rhBMP-4 was significantly enhanced by recombinant furin expression. The combination of a chemically defined medium and a nutrient supplement solution for high expression of rhBMP-4 was selected and used for bioreactor cultures. The 11-day fed-batch cultures of the established rhBMP-4-expressing rCHO cells in the 7.5-L bioreactor produced approximately 32 mg/l of rhBMP-4. The mature rhBMP-4 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 55% and a protein purity greater than 95%. The N-terminal amino acid sequences and N-linked glycosylation of the purified rhBMP-4 were confirmed by N-terminal sequencing and de-N-glycosylation analysis, respectively. The mature purified rhBMP-4 has been proved to be functionally active, with an effective dose concentration of $EC_{50}$ of 2.93 ng/ml.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.27 no.4
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• pp.307-314
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• 2005

After dental implant are planted into their bony site among the various growth factors associated with bone formation. BMP is expressed in the bone surrounding the implant fixture. By taking a close look at BMP2, BMP4 which are growth factors that take put in bone formation, its histologic features and radiographic bone healing patterns we would like to examine the mechanism of osseointegration. We randomly used 8 male and female house rabbit amd used diameter 5 mm height spiral shaped implants(Ostem, Korea) for animal use handled as a resorbable blast machined(RBM) surface and machined surface. 2group were formed and each group had RBM surface and machined surface implant or a simple bone cavity. After 3, 7, 14 and 28 days post surgery 2 objects were sacrificed from each group and histologic specimens were acquired. RT-PCR analysis was conducted and after H&E staining the extent of osseointegration was measured applying a histologic feature and histomorphometric analysis program. Quanitity one -4.41(Bio-Rad, USA) was used after scanning the PCR product image of the growth factors manifested in each group. According to the histomorphometric features the RBM, Machined surface group showed increased contact between bone and implant surface at 3, 7, 14 and 28 days after surgery. The BMP2 level increased in both experiment groups but remained unchanged in the contrast group. BMP4 levels stayed steady after the early post implantation period for RBM but showed decreased in the machined surface group and contrast group. The amount of contact between bone and implant surface increased with the passage of time. BMP2, BMP4 were expressed in both experimental group and contrast group. These growth factors play a role in osseointegration of implant.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.4
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• pp.317-326
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• 2006

Purpose : The aim of this study was to evaluate the effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) injected on the rate of new-bone formation for distraction osteogenesis on dogs. Materials & Methods : Twelve adult dogs were randomly selected into two groups of six dogs on each. Unilateral osteotomies were performed on the body of the mandible and an intraoral distractor was mounted to the mandible on dogs. One group was treated with injection of rhBMP-7 and the other group served as the control. RhBMP-7 was administered on the day of surgery by single injection into the medullary bone at the osteotomy gap. Distraction was performed five days after osteotomy as a rate of 0.5 mm twice per day for 10 days. The animals were then sacrificed at 2, 4, and 8 weeks after completion of the distraction. Two dogs in each group, totaling four dogs, were killed at 2 weeks, 4 weeks, and 8 weeks after completion of distraction, respectively. The lengthened mandibles were harvested and processed for radiographic and histological examinations. In addition, immunohistochemical examination using osteocalcin expression was studied. Results : Radiographs showed accelerated regenerate ossification with maturation of new bone in the rhBMP-7 group comparing with the control group at the 4 weeks of the consolidation. There was no significant difference in the radiographic findings at the 2 weeks and 8 weeks of the consolidation period. Histological findings demonstrated increased bone healing pattern in the rhBMP-7-treated group during all observation period. The expression of osteocalcin immunoreactivity was hardly detected in the normal mandible of dog, but the expression was detected in all experimental rhBMP-7 treated specimens. There were also significant increasing in number of positive immunostaining cells and staining intensity of osteocalcin expression in the rhBMP-7 treated group compared with those of the control group on 2-weeks and 4-weeks. There was a significant decreasing in staining intenstiy of all both two groups on 8 weeks of consolidation period, but significant differences of immunostaining was not seen in two groups. Conclusions : A single injection of rhBMP-7 at the time of osteotomy may stimulate the rate of regenerate ossification and increase callus maturation during distraction osteogenesis. In addition, it may shorten the distraction osteogenesis procedure and decrease the prevalence of complications associated with mandibular distraction osteogenesis.

• The Journal of Korean Academy of Prosthodontics
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• v.50 no.1
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• pp.44-52
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• 2012

Purpose: In this paper we tried to evaluate the most appropriate surface for rhBMP-2 coating among 4 rough titanium surfaces. Materials and methods: We used machined surface as a control group and anodized, RBM and SLA surfaces as test groups. We coated rhBMP-2 on the 4 surfaces and with uncoated surfaces for each case, we cultured human mesenchymal stem cells on all 8 surfaces. 24 hours after we measured the stem cell' attachment with SEM, and on 3rd, 7th, and 14th days, we checked the cell proliferation and differentiation by using MTT and ALP activity assay. And on the 7th day after the culture, we performed RT-PCR assay to determine whether the expression levels of Type I collagen, osteocalcin, osteopontin were changed. Results: We observed with SEM that 4 rhBMP-2 coated surfaces exhibited wider and tighter cell attachment and more cell process spreading than uncoated surfaces. The anodized rhBMP-2 surface caused robustest effects. In MTT assay we could not find any meaningful difference. In ALP assay there was a significant increase (P<.05) in the ALP activity of anodized rhBMP-2 coated surface compared with that of the control (3rd and 14th days) and with that of the RBM rhBMP-2 coated surface (14th day). In RT-PCR assay there was increased expressions in the anodized rhBMP-2 coated surface for osteocalcin, and osteopontin. Conclusion: We found that the anodized rhBMP-2 coated surface were most prominent stem cell attachment and differentiation in compared to control and Machined rhBMP-2 coated, RBM rhBMP-2 coated surface.

• Restorative Dentistry and Endodontics
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• v.39 no.3
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• pp.187-194
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• 2014

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.

• Tissue Engineering and Regenerative Medicine
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• v.15 no.6
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• pp.793-801
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• 2018

BACKGROUND: The aim of this study was to evaluate the combined effect of low-level laser treatment (LLLT) and recombinant human bone morphological protein-2 (rhBMP-2) applied to hypoxic-cultured MC3T3-E1 osteoblastic cells and to determine possible signaling pathways underlying differentiation and mineralization of osteoblasts under hypoxia. METHODS: MC3T3-E1 cells were cultured under 1% oxygen tension for 72 h. Cell cultures were divided into four groups: normoxia control, low-level laser (LLL) alone, rhBMP-2 combined with LLLT, and rhBMP-2 under hypoxia. Laser irradiation was applied at 0, 24, and 48 h. Cells were treated with rhBMP-2 at 50 ng/mL. Alkaline phosphatase activity was measured at 3, 7, and 14 days to evaluate osteoblastic differentiation. Cell mineralization was determined with Alizarin red S staining at 7 and 14 days. Western blot assays were performed to evaluate whether p38/protein kinase D (PKD) signaling was involved. RESULTS: The results indicate that LLLT and rhBMP-2 synergistically increased alkaline phosphatase (ALP) activity and mineralization. Western blot analyses showed that expression of type I collagen, runt-related transcription factor 2 (RUNX2), and Osterix (Osx), increased and expression of hypoxia-inducible factor 1-alpha ($HIF-1{\alpha}$), decreased more in the LLLT and rhBMP-2 combined group than in the rhBMP-2 or LLL alone groups. Moreover, LLLT and rhBMP-2 stimulated p38 phosphorylation and rhBMP-2 and LLLT increased Prkd1 phosphorylation. CONCLUSION: Combined treatment with rhBMP-2 and LLL induced differentiation and mineralization of hypoxic-cultured MC3T3-E1 osteoblasts by activating p38/PKD signaling in vitro.

• Restorative Dentistry and Endodontics
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• v.32 no.5
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• pp.403-410
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• 2007

The purpose of this study was to investigate whether rhBMP-2 (BMP2) could induce synergistic effect with $Pro-Root^{(R)}$ MTA (MTA) in pulpotomized teeth in the rats. Healthy upper first molars from thirty-two, 10 weeks old, Sprague-Dawley rats were used for this investigation. The molars were exposed with round bur, and light pressure was applied with sterilized cotton to control hemorrhage. 1.2 grams of MTA cement was placed in right first molars as a control group. In left first molars, $1\;{\mu}g$ of BMP2 was additionally placed on exposed pulps with MTA. All cavities were back-filled with light-cured glass-ionomer cements. The rats were sacrificed after 2 weeks and 7 weeks, respectively. Then histologic sections were made and assessed by light microscopy. Data were statistically analyzed via student t-test with SPSSWIN 12.0 program (p < 0.05). Inflammation observed in 2 weeks groups were severe compared to the 7 weeks groups. But the differences were not statistically significant. BMP2-addition groups had less inflammation than MTA groups in both periods, though these differences were also not statistically significant. In conclusion, the combination of BMP2 and MTA showed no differences with MTA only for pulpotomy of rat teeth.

• BMB Reports
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• v.44 no.7
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• pp.446-451
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• 2011

The conversion of fibroblasts into osteoblasts requires the activation of key signaling pathways, including the BMP pathway. Although Runx2 is known to be a component of the BMP pathway, the combination of Runx2 and BMP2 has not yet been examined with respect to the conversion of fibroblasts into osteoblasts. Here, human ligamentum flavum (LF) fibroblast-like cells from six patients were tested for their conversion into osteoblasts using adenoviruses expressing Runx2 or BMP2. The forced expression of Runx2 or BMP2 in primary cultured LF cells resulted in a variety of proliferation and differentiation behaviors. Combined treatment of BMP2 plus Runx2 resulted in better osteoblastic differentiation than treatment with either component alone. These results indicate that the Runx2 and BMP2 pathways possess both common and independent target genes. Collectively, Runx2 plus BMP2 mediated efficient conversion of fibroblast-like LF cells into osteoblast-like cells, suggesting the possible use of these components for clinical applications such as spinal fusion.