• 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.

• International Journal of Industrial Entomology and Biomaterials
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• v.32 no.2
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• pp.49-53
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• 2016

Bone morphogenetic proteins (BMPs) are essential growth factors for bone formation, skeletal development and bone regeneration. The BMP-2/7 heterodimer is known to have remarkable effects on osteogenic induction that are even stronger than the BMP-2 or BMP-7 homodimers. We designed a recombinant human BMP-2/7 (rhBMP-2/7) heterodimer protein with four glycine residues between BMP-2 and BMP-7 protein to facilitate free bond rotation of domains. The Baculovirus Expression Vector System (BEVS) is routinely used to produce recombinant proteins in the milligram scale. In this study, the BEVS was used to express the rhBMP-2/7 protein whrer the recombinant baculovirus was recovered in the host Sf9 cells. To confirm the biological activity of rhBMP-2/7 protein secreted from the BEVS as an osteogenic differentiation and induction factor, we measured the BMP-induced ALP activity. rhBMP-2/7 could be used as an alternative to BMPs to overcome limitations like short half-life and requirement for high concentrations. Furthermore, rhBMP-2/7 may be an efficient tool for various application studies such as bone regeneration and skeletal development.

• Journal of Periodontal and Implant Science
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• v.46 no.5
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• pp.350-359
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• 2016

Purpose: The purpose of the present study was to evaluate the effectiveness of a minimal concentration of bone morphogenetic protein-2 (BMP-2) in terms of quantitative and qualitative analyses of newly formed bone in a rabbit maxillary sinus model. Methods: In 7 rabbits, sinus windows were prepared bilaterally. Biphasic calcium phosphate (BCP) loaded with 0.05 mg/mL BMP-2 was grafted into one sinus (the BMP group) and saline-soaked BCP was placed into the other (the control group) in each animal. The animals were allowed an 8-week healing period before being sacrificed. Specimens including the augmented area and surrounding tissues were then removed and evaluated both radiographically and histologically. Results: There was a difference in the mineralization of new bone between the groups. In the BMP group, the greater part of the new bone consisted of mature lamellar bone with an evident trabecular pattern, whereas the control group showed mostly woven bone, consisting only partially of lamellar bone. Histometrically, the area of new bone was significantly greater ($4.55{\pm}1.35mm^2$ vs. $2.99{\pm}0.86mm^2$) in the BMP group than in the control group (P<0.05); however, the total augmentation volumes were not significantly different between the groups. Conclusions: Within the limitations of this study, it can be suggested that a minimal concentration of BMP-2 (0.05 mg/mL) had an osteoinductive effect with accelerated mineralization in a rabbit sinus model using a BCP carrier.

• Journal of Periodontal and Implant Science
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• v.32 no.3
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• pp.501-521
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• 2002

The purpose of this study is to evaluate the influences of the bone morphogenetic protein (BMP) on the healing of periodontal ligament and alveolar bone after replantation of tooth, and to examine the possibility of its clinical application. 45 Sprague Dawley rats weighted about 100 gram were divided into 3 experimental groups by different dose of BMP. All the upper right and left 1st molar were extracted after 5 days feeding of 0.4% ${\beta}$-aminopropionitrile, and right molar were used as experimental group and left molar were used as control group. The root surface of experimental molar were treated with 25,50 and l00ng/ml of human recombinant Bone morphogenetic protein-4 (rh-BMP-4) with micropipet, and 1M Sodium hypochloride were used on control root surface. All the experimental animals were sacrificed as 1, 2, 4, 7 and 14 days after autoreplantation of upper 1st molar into their own position. The maxilla were disected included both side of 1st molar. The collected tissue were processed from demineralization to paraffin embeding as usual procedure, and the specimens were prepared with Hematoxylin-Eosin stain for the light microscopic evaluation. The results of this study were as follows : 1. There was no significant differences between control and experimental site on 1 and 2 days after replantation of tooth. In the case of 4th days, the evidence of tissue regeneration were observed on experimental site to compare the controls. New osteoid were revealed on high concentration of BMP at 7 days after replantation, and it became more obvious at 14 days, 2. The effect of the rh-BMP-4 coated on root surface was revealed slight influences for the prolifertion of cells of periodontium and tissue regeneration as dose-dependent pattern. 3. Bony ankylosis was observed between alveolar bone and root surface due to the remarkable amount of osteoid formation on the 14 days after replantation of root. In the conclusion, it was suggested that topical application of the rhBMP-4 on the root surface has influence on the periodontal ligament and alveolar bone. The application method of BMP on the root should be designed with calculation of proper concentration.

• Molecules and Cells
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• v.38 no.3
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• pp.267-272
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• 2015

Nacre seashell is a natural osteoinductive biomaterial with strong effects on osteoprogenitors, osteoblasts, and osteoclasts during bone tissue formation and morphogenesis. Although nacre has shown, in one study, to induce bridging of new bone across large non-union bone defects in 8 individual human patients, there have been no succeeding human surgical studies to confirm this outstanding potency. But the molecular mechanisms associated with nacre osteoinduction and the influence on bone marrow-derived mesenchymal stem cells (BMSC's), skeletal stem cells or bone marrow stromal cells remain elusive. In this study we highlight the phenotypic and biochemical effects of Pinctada maxima nacre chips and the global nacre soluble protein matrix (SPM) on primary human bone marrow-derived stromal cells (hBMSCs) in vitro. In static co-culture with nacre chips, the hBMSCs secreted Alkaline phosphatase (ALP) at levels that exceeded bone morphogenetic protein (rhBMP-2) treatment. Concentrated preparation of SPM applied to Stro-1 selected hBMSC's led to rapid ALP secretions, at concentrations exceeding the untreated controls even in osteogenic conditions. Within 21 days the same population of Stro-1 selected hBMSCs proliferated and secreted collagens I-IV, indicating the premature onset of an osteoblast phenotype. The same SPM was found to promote unselected hBMSC differentiation with osteocalcin detected at 7 days, and proliferation increased at 7 days in a dose-dependent manner. In conclusion, nacre particles and nacre SPM induced the early stages of human bone cell differentiation, indicating that they may be promising soluble factors with osteoinductive capacity in primary human bone cell progenitors such as, hBMSC's.

• Journal of Periodontal and Implant Science
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• v.35 no.3
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• pp.747-760
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• 2005

Periodontal therapy has dealt primarily with attempts at arresting progression of disease. however, more recent techniques have focused on regenerating the periodontal ligament having the capacity to regenerate the periodontium. Recombinant human bone morphogenetic protein-7(rhBMP-7) can differentiate the osteoprogenitor cells and induce bone formation. The purpose of this study was to evaluate the effect of BMP-7 on rat periodontal ligament cells differentiation, in vitro. In the control group, cells was cultured with DMEM media. In the experimental groups, cells were cultured with rhBMP-7 in concentration of 10, 25, 50 and 100 ng/ml. Each group was characterized by examining alkaline phosphatase activity at 3 and 5 days of culture and the ability to produce mineralized nodules of rat calvarial cells at 14 days of culture. Synthesis of type I collagen(COL-I), osteocalcin(OCN), and bone sialoprotein(BSP) was evaluated by RT-PCR at 7 days of culture. Activation of Smad proteins and p38 MAP kinase was determined by western blot analysis of the cell lysates. Alkaline phosphatase activity was significantly increased in the concentration of BMP-7 50 ng/ml and 100 ng/ml compared to the control(p<0.05). The mineralized bone nodule formation was greater with addition of 50 ng/ml and 100 ng/ml BMP-7 than the control(p<0.01). In 7 days' culture, the expressions of COL-I, BSP, and OCN was increased by BMP-7 in concentration of 10 $ng/ml{\sim}100$ ng/ml. In western blot analysis, BMP-7 treated culture cells expressed Smad 1,5,8 in dose-dependent manner, whereas BMP-7 did not activate phosphorylated form of p38 MAP kinase. These result suggested that BMP-7 stimulate rat periodontal ligament cells to differentiate toward osteoblast phenotype and increase bone matrix production by activation of BMP-Smad pathway.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.7
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• pp.3527-3531
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• 2016

Recombinant human bone morphogenetic protein-2 (rhBMP-2 ), a member of the TGF-${\beta}$ family, has been used widely in recent years to regenerate defects of the maxillary and mandible bones. Such defects are sometimes caused by resection of oral squamous cell carcinoma (OSCC) yet the biologic effects of rhBMP-2 on these carcinomas are not fully clear. The objective of this study was to determine histologically whether rhBMP-2 produces adverse effects on angiogenesis during induction of OSCC, a biologic process critical for tumor formation in an experimental model in the buccal pouch of golden Syrian hamsters. Buccal cavities were exposed to painting with 0.5% DMBA in liquid paraffin three times a week for 14 weeks, then biopsies were taken. Division was into 2 groups: a study group of 10 hamsters receiving $0.25{\mu}g/ml$ of rhBMP-2 in the $3^{rd}$ and $6^{th}$ weeks; and a control group of 10 hamsters which did not receive any additional treatment. VEGF expression and microvessel density were measured but no differences were noted between the two groups. According to this study, rh-BMP-2 does not stimulate angiogenesis during induction of OCSSs.

• 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.

• Journal of Life Science
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• v.22 no.4
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• pp.456-465
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• 2012

Heterodimeric recombinant human bone morphogenetic proteins (rhBMPs) are powerful tools for bone tissue engineering. However, BMPs have several important limitations in their application to bone regeneration. BMPs have a short half-life and must be used in high concentrations, which may be cost-inefficient. To overcome these problems, we established a stable cell line that expressed the fusion protein comprised of recombinant human BMP2/7 heterodimer protein and PTD (rhBMP2/7-PTD). This stable cell line enabled high process yields by continuously expressing rhBMP2/7-PTD products at high levels throughout cultivation. This synthesized BMP7 was fused to a BMP2 protein with four glycine residues (to allow free bond rotation of the domains) and PTD. To demonstrate that the rhBMP2/7-PTD protein that was secreted from an rhBMP2/7-PTD-expressing stable cell line exhibited biological activity consistent with its role as an osteogenic differentiation induction growth factor, we evaluated BMP-induced ALP activity. Our results suggest that this cell line may be a powerful and efficient tool for applications such as bone tissue regeneration.

• International Journal of Industrial Entomology and Biomaterials
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• v.35 no.1
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• pp.7-13
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• 2017

In this study, changes in gene expression after administration of silk fibroin fragments ($size{\approx}30kDa$) were evaluated in MG63 cells using a cDNA microarray assay. In addition, the level of alkaline phosphatase (ALP) activity and cellular proliferation in the group administered moderately sized silk fibroin fragments ($size{\approx}30kDa$) (MSF) were compared to those in the group administered smaller silk fibroin fragments (size < 1 kDa) (SSF). The results of the cDNA microarray assay show increased expression of genes that are related to the cell cycle and inflammation. ALP, bone morphogenetic protein-7, bone morphogenetic protein receptor type IA, and runt-related transcription factor 2 exhibited significantly lower expression compared to control cells (fold ratio < 0.5). Relative ALP activity of the $100{\mu}g/mL$ MSF group was significantly lower than that of the SSF group (P < 0.05). Thus, the MSF group showed increased expression of genes associated with cellular proliferation and inflammation but decreased expression of genes associated with osteogenesis.