• 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.

• Journal of Periodontal and Implant Science
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• v.41 no.4
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• pp.167-175
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• 2011

Purpose: Periodontal ligament (PDL) cell differentiation into osteoblasts is important in bone formation. Bone formation is a complex biological process and involves several tightly regulated gene expression patterns of bone-related proteins. The expression patterns of bone related proteins are regulated in a temporal manner both in vivo and in vitro. The aim of this study was to observe the gene expression profile in PDL cell proliferation, differentiation, and mineralization in vitro. Methods: PDL cells were grown until confluence, which were then designated as day 0, and nodule formation was induced by the addition of 50 ${\mu}g$/mL ascorbic acid, 10 mM ${\beta}$-glycerophosphate, and 100 nM dexamethasone to the medium. The dishes were stained with Alizarin Red S on days 1, 7, 14, and 21. Real-time polymerase chain reaction was performed for the detection of various genes on days 0, 1, 7, 14, and 21. Results: On day 0 with a confluent monolayer, in the active proliferative stage, c-myc gene expression was observed at its maximal level. On day 7 with a multilayer, alkaline phosphatase, bone morphogenetic protein (BMP)-2, and BMP-4 gene expression had increased and this was followed by maximal expression of osteocalcin on day 14 with the initiation of nodule mineralization. In relationship to apoptosis, c-fos gene expression peaked on day 21 and was characterized by the post-mineralization stage. Here, various genes were regulated in a temporal manner during PDL fibroblast proliferation, extracellular matrix maturation, and mineralization. The gene expression pattern was similar. Conclusions: We can speculate that the gene expression pattern occurs during PDL cell proliferation, differentiation, and mineralization. On the basis of these results, it might be possible to understand the various factors that influence PDL cell proliferation, extracellular matrix maturation, and mineralization with regard to gene expression patterns.

• Molecules and Cells
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• v.22 no.3
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• pp.353-359
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• 2006

The bone morphogenetic protein (BMP) family has been implicated in control of cartilage development. Here, we demonstrate that BMP-2 promotes chondrogenesis by activating p38 mitogen-activated protein kinase (MAPK), which in turn downregulates $Wnt-7a/{\beta}$-catenin signaling responsible for proteasomal degradation of Sox9. Exposure of mesenchymal cells to BMP-2 resulted in upregulation of Sox9 protein and a concomitant decrease in the level of ${\beta}$-catenin protein and Wnt-7a signaling. In agreement with this, the interaction of Sox9 with ${\beta}$-catenin was inhibited in the presence of BMP-2. Inhibition of the p38 MAPK pathway using a dominant negative mutant led to sustained Wnt-7a signaling and decreased Sox9 expression, with consequent inhibition of precartilage condensation and chondrogenic differentiation. Moreover, overexpression of ${\beta}$-catenin caused degradation of Sox9 via the ubiquitin/26S proteasome pathway. Our results collectively indicate that the increase in Sox9 protein resulting from downregulation of ${\beta}$-catenin/Wnt-7a signaling is mediated by p38 MAPK during BMP-2 induced chondrogenesis in chick wing bud mesenchymal cells.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.34 no.1
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• pp.1-11
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• 2012

Purpose: The purpose of this study was to evaluate the bone regeneration capacity of silk fibroin (SF) when combined with beta tricalcium phosphate (${\beta}$-tricalcium phosphate [TCP]) and rh-bone morphogenetic protein (BMP) in vivo by micro-computed tomography (CT), soft x-ray, and histological analysis. Methods: A total of 56 critical size defects formed by a trephine bur made on 28 adult female Spague-Dawley rats were used for this study and the defect size was 5.0 mm in diameter. The defects were transplanted with (1) no graft material (raw defect), (2) autogenous bone, (3) SF ($10{\mu}g$), (4) SF-BMP ($10{\mu}g$, $0.8{\mu}g$ each), and (5) SF+${\beta}$-TCP ($10{\mu}g$). At 4 and 8 weeks after operation, the experimental animals were sacrificed. Samples were evaluated with soft x-ray, histological examinations and 3-dimensional micro-CT analysis. Results: In the 3-dimensional micro-CT evaluation, bone volume and bone surface data were higher in the SF-BMP ($12.8{\pm}1.5$, $138.6{\pm}45.0$ each) (P<0.05) and SF-TCP ($12.3{\pm}1.5$, $144.9{\pm}30.9$ each) group than in the SF group ($6.1{\pm}3.3$, $77.2{\pm}37.3$ each) (P<0.05), except for the autogenous group ($15.0{\pm}3.0$, $190.7{\pm}41.4$ each) at 4 weeks. At 8 weeks, SF-BMP ($16.8{\pm}3.5$, $173.9{\pm}34.2$ each) still revealed higher (P<0.05) bone volum and surface, but SF-TCP ($11.3{\pm}1.5$, $1132.9{\pm}52.1$ each) (P=0.5, P=0.2) revealed the same or lower amount compared with the SF group ($13.8{\pm}2.7$, $127.5{\pm}44.8$ each). The % of bone area determined by radiodensity was higher in the SF-TCP ($31.4{\pm}9.1%$) and SF-BMP ($36.2{\pm}16.2%$) groups than in the SF ($19.0{\pm}10.4$) group at the period of 4 weeks. Also, in the histological evaluation, the SF-BMP group revealed lower inflammation reaction, lower foreign body reaction and higher bone healing than the SF group at postoperative 4 weeks and 8 weeks. The SF-TCP group revealed lower inflammation at 4 weeks, but accordingly, as the TCP membrane was absorbed, inflammatory and foreign body reaction are increased at 8 weeks. Conclusion: The current study provides evidence that the silk fibrin can be used as an effective grafted material for tissue engineering bone generation through a combination of growth factor or surface treatment.

• International Journal of Oral Biology
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• v.32 no.3
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• pp.85-91
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• 2007

Dlx3 is a homeodomain protein and is known to playa role in development and differentiation of many tissues. Deletion of four base pairs in DLX3 (NT3198) is causally related to tricho-dento-osseous (TDO) syndrome (OMIM # 190320), a genetic disorder manifested by taurodontism, hair abnormalities, and increased bone density in the cranium. Although the observed defects of TDO syndrome involves bone, little is known about the role of Dlx3 in bone remodeling process. In this study, we examined the effect of wild type DLX3 (wtDlx3) expression on osteoclast differentiation and compared it with that of 4-BP DEL DLX3 (TDO mtDlx3). To examine whether Dlx3 is expressed during RANKL-induced osteoclast differentiation, RAW264.7 cells were cultured in the presence of receptor activator of nuclear factor-B ligand (RANKL). Dlx3 protein level increased slightly after RANKL treatment for 1 day and peaked when the fusion of prefusion osteoclasts actively progressed. When wtDlx3 and TDO mtDlx3 were overexpressed in RAW264.7 cells, they enhanced RANKL-induced osteoclastogenesis and the expression of osteoclast differentiation marker genes such as calcitonin receptor, vitronectin receptor and cathepsin K. Since osteoclast differentiation is critically regulated by the balance between RANKL and osteoprotegerin (OPG), we examined the effect of Dlx3 overexpression on expression of RANKL and OPG in C2C12 cells in the presence of bone morphogenetic protein 2. Overexpression of wtDlx3 enhanced RANKL mRNA expression while slightly suppressed OPG expression. However, TDO mtDlx3 did not exert significant effects. This result suggests that inability of TDO mtDlx3 to regulate expression of RANKL and OPG may contribute to increased bone density in TDO syndrome patients. Taken together, it is suggested that Dlx3 playa role as a positive regulator of osteoclast differentiation via up-regulation of osteoclast differentiation-associated genes in osteoclasts, as well as via increasing the ratio of RANKL to OPG in osteoblastic cells.

• Journal of Nutrition and Health
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• v.55 no.6
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• pp.617-629
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• 2022

Purpose: Osteoporosis is characterized by structural deterioration of the bone tissue because of the loss of osteoblastic activity or the increase in osteoclastic activity, resulting in bone fragility and an increased risk of fractures. Hederagenin (Hed) is a pentacyclic triterpenoid saponin isolated from Dipsaci Radix, the dried root of Dipsacus asper Wall. Dipsaci Radix has been used in Korean herbal medicine to treat bone fractures. In this study, we attempted to demonstrate the potential anti-osteoporotic effect of Hed by examining its effect on osteoblast differentiation in MC3T3-E1 cells. Methods: Osteoblastic MC3T3-E1 cells were cultured in 0, 1, and 10 ㎍/mL Hed for 3 and 7 days. The activity of alkaline phosphatase (ALP), bone nodule formation and level of expression of bone-related genes and proteins were measured in MC3T3-E1 cells exposed to Hed. The western blot test was used to detect the activation of the bone morphogenetic protein-2 (BMP2)/ Suppressor of Mothers against Decapentaplegic (SMAD)1 pathway. Results: Hed significantly increased the proliferation of MC3T3-E1 cells. Intracellular ALP activity was significantly increased in the 1 ㎍/mL Hed-treated group. Hed significantly increased the concentration of calcified nodules. Furthermore, Hed significantly upregulated the expression of genes and proteins associated with osteoblast proliferation and differentiation, such as Runt-related transcription factor 2 (Runx2), ALP, osteopontin (OPN), and type I procollagen (ProCOL1). Induction of osteoblast differentiation by Hed was associated with increased BMP2. In addition, Hed induced osteoblast differentiation by increasing the activity of SMAD1/5/8. These results suggest that Hed has the potential to prevent osteoporosis by promoting osteoblastogenesis in osteoblastic MC3T3-E1 cells via the modulation of the BMP2/SMAD1 pathway. Conclusion: The results presented in this study indicate that Hed isolated from Dipsaci Radix has the potential to be developed as a healthcare food and functional material possessing anti-osteoporosis effects.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.5
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• pp.366-374
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• 2010

Introduction: This study evaluated the capability of silk fibroin (SF) and recombinant human bone morphogenetic protein-2 loaded SF (SF-BMP) as a bone defect replacement matrix when grafted in a calvarial bone defect of rats in vivo. Materials and Methods: A total 70 calvarial critical size defects (5.0 mm in diameter) made on 35 adult female Sprague-Dawley rats were used in this study. The defects were transplanted with (1) rhBMP-2 loaded silk fibroin graft (SF-BMP: 0.8+$10\;{\mu}g$), (2) Silk fibroin (SF: $10\;{\mu}g$), and (3) no graft material (Raw). The samples were evaluated with soft x-rays, alkaline phosphatase activity, calcium/phosphate quantification, histological and histomorphometric analysis at postoperative 4 and 8 weeks. Results: The SF-BMP group ($48.86{\pm}14.92%$) had a significantly higher mean percentage bone area than the SF group ($24.96{\pm}11.01%$) at postoperative 4 weeks.(P<0.05) In addition, the SF-BMP group ($40.01{\pm}12.43%$) had a higher % bone area at postoperative 8 weeks than the SF group ($33.26{\pm}5.15%$). The mean ratio of gray scale levels to the host bone showed that the SF-BMP group ($0.67{\pm}0.08$) had a higher mean ratio level than the SF group ($0.61{\pm}0.09$) at postoperative 8 weeks. These differences were not statistically significant.(P=0.168 and P=0.243, respectively) The ratio of the calcium and phosphate contents of the SF-BMP ($0.93{\pm}0.22$) group was lower than that of the SF ($1.90{\pm}1.42$) group at postoperative 4 weeks. However, the SF-BMP group ($0.75{\pm}0.31$) had a higher Ca/$PO_4$ ratio than the SF ($0.68{\pm}0.04$) at postoperative 8 weeks. These differences were not statistically significant.(P=0.126 and P=0.627, respectively) For the bone-specific alkaline phosphatase (ALP) activity, which is recognized as a reliable indicator of the osteoblast function, the SF-BMP ($23.71{\pm}8.60\;U/L$) groups had a significantly higher value than the SF group ($12.65{\pm}6.47\;U/L$) at postoperative 4 weeks.(P<0.05) At postoperative 8 weeks, the SF-BMP ($21.65{\pm}10.02\;U/L$) group had a lower bone-specific ALP activity than the SF group ($16.72{\pm}7.35\;U/L$). This difference was not statistically significant.(P=0.263) For the histological evaluation, the SF-BMP group revealed less inflammation, lower foreign body reactions and higher bone healing than the SF group at postoperative 4 and 8 weeks. The SF group revealed more foreign body reactions at postoperative 4 weeks. However, this immunogenic reaction decreased and the remnant of grafted material was observed at postoperative 8 weeks. For histomorphometric analysis, the SF-BMP group had a significantly longer bone length to total length ratio than those of the SF group at postoperative 4 and 8 weeks.(P<0.05) Conclusion: The rhBMP-2 loaded silk fibroin graft revealed fewer immunoreactions and inflammation as well as more new bone formation than the pure silk fibroin graft. Therefore, silk fibroin may be a candidate scaffold for tissue engineered bone regeneration.

• Journal of Periodontal and Implant Science
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• v.35 no.2
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• pp.345-357
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• 2005

Prostaglandin plays a significant role in the local control of bone metabolism associated with periodontal disease. ${\Delta}^{12}-PGJ_2$ is a natural $PGD_2$ metabolite that is formed in vivo in the presence of plasma. It is known for ${\Delta}^{12}-PGJ_2$ to stimulate calcification in osteoblastic cells. Bone morphogenetic protein(BMP) stimulated osteoblastic differentiation in various types of cells and greatly enhanced healing of bony defects. The purpose of this study was to evaluate the effect of rhEMP-2 on ${\Delta}^{12}-PGJ_2$ induced osteoblastic differentiation and mineralization in vitro. A human osteosarcoma cells line Saos-2 were cultured. In the test groups, 10-7M of ${\Delta}^{12}-PGJ_2$ or mixture of 10-8M of ${\Delta}^{12}-PGJ_2$ and 100ng/ml of rhBMP-2 or 100ng/ml of rhEMP-2 were added to culture media. After 1 day, 2 days and 4 days of culture period, the cell number was measured. Alkaline phosphatase activity was measure at 3 days. Reverse transcription polymerase chain reaction(RT-PCR) was performed to determine the expression of mRNA of bone matrix protein at 8 hours, 1 day and 7 days. The ability to produce mineralized nodules in rat osteoblasts(MC3T3-E1) was evaluated at 21 days. The results were as follows : 1. rhEMP-2 or mixture of rhBMP-2 and ${\Delta}^{12}-PGJ_2$ inhibited cell proliferation of human osteosarcoma cells. 2. rhEMP-2 or mixture of rhBMP-2 and ${\Delta}^{12}-PGJ_2$ stimulated alkaline phosphatase activity significantly higher than ${\Delta}^{12}-PGJ_2$ alone. 3. rhBMP-2 or mixture of rhEMP-2 and ${\Delta}^{12}-PGJ_2$ stimulated mineralization compared to ${\Delta}^{12}-PGJ_2$ alone. 4. mRNA of alkaline phosphatase, BMP-2, cbfa 1, Type I collagen were detected in the group treated with ${\Delta}^{12}-PGJ_2$/rhBMP-2, rhBMP-2 alone, ${\Delta}^{12}-PGJ_2$ alone. These results show that mixture of ${\Delta}^{12}-PGJ_2$ and rhBMP-2 causes more bone formation than ${\Delta}^{12}-PGJ_2$ alone while the bone formation effects of mixture of ${\Delta}^{12}-PGJ_2$ and rhBMP-2 are less than those of rhBMP-2 alone. Further researches would be necessary to clarify the interactions of these agents.

• BMB Reports
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• v.55 no.11
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• pp.565-570
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• 2022

Pulmonary arterial hypertension (PAH) is a progressive and devastating disease whose pathogenesis is associated with a phenotypic switch of pulmonary arterial vascular smooth muscle cells (PASMCs). Bone morphogenetic protein (BMP) signaling and potassium two pore domain channel subfamily K member 3 (KCNK3) play crucial roles in PAH pathogenesis. However, the relationship between BMP signaling and KCNK3 expression in the PASMC phenotypic switching process has not been studied. In this study, we explored the effect of BMPs on KCNK3 expression and the role of KCNK3 in the BMP-mediated PASMC phenotypic switch. Expression levels of BMP receptor 2 (BMPR2) and KCNK3 were downregulated in PASMCs of rats with PAH compared to those in normal controls, implying a possible association between BMP/BMPR2 signaling and KCNK3 expression in the pulmonary vasculature. Treatment with BMP2, BMP4, and BMP7 significantly increased KCNK3 expression in primary human PASMCs (HPASMCs). BMPR2 knockdown and treatment with Smad1/5 signaling inhibitor substantially abrogated the BMP-induced increase in KCNK3 expression, suggesting that KCNK3 expression in HPASMCs is regulated by the canonical BMP-BMPR2-Smad1/5 signaling pathway. Furthermore, KCNK3 knockdown and treatment with a KCNK3 channel blocker completely blocked BMP-mediated anti-proliferation and expression of contractile marker genes in HPAMSCs, suggesting that the expression and functional activity of KCNK3 are required for BMP-mediated acquisition of the quiescent PASMC phenotype. Overall, our findings show a crosstalk between BMP signaling and KCNK3 in regulating the PASMC phenotype, wherein BMPs upregulate KCNK3 expression and KCNK3 then mediates BMP-induced phenotypic switching of PASMCs. Our results indicate that the dysfunction and/or downregulation of BMPR2 and KCNK3 observed in PAH work together to induce aberrant changes in the PASMC phenotype, providing insights into the complex molecular pathogenesis of PAH.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.8
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• pp.1066-1073
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• 2017

Objective: Growth-related traits are important economic traits in the swine industry. However, the genetic mechanism of growth-related traits is little known. The aim of this study was to screen the candidate genes and molecular markers associated with body dimension and body weight traits in pigs. Methods: A genome-wide association study (GWAS) on body dimension and body weight traits was performed in a White $Duroc{\times}Erhualian$ $F_2$ intercross by the illumina PorcineSNP60K Beadchip. A mixed linear model was used to assess the association between single nucleotide polymorphisms (SNPs) and the phenotypes. Results: In total, 611 and 79 SNPs were identified significantly associated with body dimension traits and body weight respectively. All SNPs but 62 were located into 23 genomic regions (quantitative trait loci, QTLs) on 14 autosomal and X chromosomes in Sus scrofa Build 10.2 assembly. Out of the 23 QTLs with the suggestive significance level ($5{\times}10^{-4}$), three QTLs exceeded the genome-wide significance threshold ($1.15{\times}10^{-6}$). Except the one on Sus scrofa chromosome (SSC) 7 which was reported previously all the QTLs are novel. In addition, we identified 5 promising candidate genes, including cell division cycle 7 for abdominal circumference, pleiomorphic adenoma gene 1 and neuropeptides B/W receptor 1 for both body weight and cannon bone circumference on SSC4, phosphoenolpyruvate carboxykinase 1, and bone morphogenetic protein 7 for hip circumference on SSC17. Conclusion: The results have not only demonstrated a number of potential genes/loci associated with the growth-related traits in pigs, but also laid a foundation for studying the genes' role and further identifying causative variants underlying these loci.