• International Journal of Oral Biology
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• v.39 no.3
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• pp.145-151
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• 2014

During bone remodeling, there is requirement of differentiation of osteoblastic cells. Previously, we identified proteins differentially expressed in soft tissue during bone healing. Of these proteins, we focused the effect of LTF on differentiation of osteoblast. In order to analyze the osteogenic ability of LTF, we treated conditioned media collected from human LTF-stably transfected HEK293T cells into osteoblastic MC3T3-E1. The results showed that the activity and expression of alkaline phosphatase were increased in MC3T3-E1 cells treated with conditioned media containing LTF in dose- and time-dependent manner. At the same time, we observed the significant increase of the expression of osteoblastic genes, such as ALP, BSP, COL1A1, and OCN, and along with matrix mineralization genes, such as DMP1 and DMP2, in LTF conditioned media-treated groups. Moreover, the result of treating recombinant human LTF directly into osteoblastic MC3T3-E1 showed the same pattern of treating conditioned media containing LTF. Our study demonstrated that LTF constitutively enhances osteoblastic differentiation via induction of osteoblastic genes and activation of matrix mineralization in MC3T3-E1 cells.

• Journal of Periodontal and Implant Science
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• v.47 no.5
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• pp.273-291
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• 2017

Purpose: Although static magnetic fields (SMFs) have been used in dental prostheses and osseointegrated implants, their biological effects on osteoblastic and cementoblastic differentiation in cells involved in periodontal regeneration remain unknown. This study was undertaken to investigate the effects of SMFs (15 mT) on the osteoblastic and cementoblastic differentiation of human osteoblasts, periodontal ligament cells (PDLCs), and cementoblasts, and to explore the possible mechanisms underlying these effects. Methods: Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, mineralized nodule formation based on Alizarin red staining, calcium content, and the expression of marker mRNAs assessed by reverse transcription polymerase chain reaction (RT-PCR). Signaling pathways were analyzed by western blotting and immunocytochemistry. Results: The activities of the early marker ALP and the late markers matrix mineralization and calcium content, as well as osteoblast- and cementoblast-specific gene expression in osteoblasts, PDLCs, and cementoblasts were enhanced. SMFs upregulated the expression of Wnt proteins, and increased the phosphorylation of glycogen synthase $kinase-3{\beta}$ ($GSK-3{\beta}$) and total ${\beta}-catenin$ protein expression. Furthermore, p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), and nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) pathways were activated. Conclusions: SMF treatment enhanced osteoblastic and/or cementoblastic differentiation in osteoblasts, cementoblasts, and PDLCs. These findings provide a molecular basis for the beneficial osteogenic and/or cementogenic effect of SMFs, which could have potential in stimulating bone or cementum formation during bone regeneration and in patients with periodontal disease.

• BMB Reports
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• v.50 no.2
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• pp.103-108
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• 2017

Heme oxygenase (HO-1) catalyzes heme to carbon monoxide (CO), biliverdin/bilirubin, and iron and is known to prevent the pathogenesis of several human diseases. We assessed the beneficial effect of heme degradation products on osteoclastogenesis induced by receptor activator of NF-${\kappa}B$ ligand (RANKL). Treatment of RAW264.7 cells with CORM-2 (a CO donor) and bilirubin, but not with iron, decreased RANKL-induced osteoclastogenesis, with CORM-2 having a more potent anti-osteogenic effect. CORM-2 also inhibited RANKL-induced osteoclastogenesis and osteoclastic resorption activity in marrow-derived macrophages. Treatment with hemin, a HO-1 inducer, strongly inhibited RANKL-induced osteoclastogenesis in wild-type macrophages, but was ineffective in $HO-1^{+/-}$ cells. CORM-2 reduced RANKL-induced NFATc1 expression by inhibiting IKK-dependent NF-${\kappa}B$ activation and reactive oxygen species production. These results suggest that CO potently inhibits RANKL-induced osteoclastogenesis by inhibiting redox-sensitive NF-${\kappa}B$-mediated NFATc1 expression. Our findings indicate that HO-1/CO can act as an anti-resorption agent and reduce bone loss by blocking osteoclast differentiation.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.35 no.5
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• pp.304-309
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• 2009

Purpose: Fibrous dysplasia (FD) is a fibro-osseous disease associated with activating missense mutations of the gene encoding the $\alpha$-subunit of stimulatory G protein. FD may affect a single bone (called monostotic form) or multiple bones (called polyostotic form). The extent of lesions reflects the onset time of mutation. In this study, cells from monostotic FD in maxilla of a patient were isolated and cultured in vitro for characterization. Materials and Methods: The single cells were released from FD lesion which was surgical specimen from 15 years-old boy. These isolated cells were cultured in vitro and tested their proliferation activity with MTT assay. In osteogenic media, these cells underwent differentiation process comparing with its normal counterpart i.e. bone marrow stromal cells. The proliferated FD cells were detached and transplanted into the dordsal pocket of nude mouse and harvested in 6 weeks and 12 weeks. Results and Summary: FD cells have an increased proliferation rate and poor differentiation. As a result, cells isolated from FD lesion decreased differentiation into osteoblast and increased proliferation capacity. MTT assay presented that proliferation rate of FD cells were higher than control. However, the mineral induction capacity of FD was lesser than that of control. Monostotic FD cells make fewer amounts of bone ossicles and most of them are woven bone rather than lamellar bone in vivo transplantation. In transplanted FD cells, hematopoietic marrow were not seen in the marrow space and filled with the organized fibrous tissue. Therefore, they were recapitulated to the original histological features of FD lesion. Collectively, these results indicated that the FD cells were shown that the increased proliferation and decreased differentiation potential. These in vitro and in vivo system can be useful to test FD cell's fate and possible.

• Journal of Periodontal and Implant Science
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• v.34 no.4
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• pp.791-805
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• 2004

The purpose of this study was to evaluate the effects of DFPR on differentiation of mouse calvarial cell in vitro, to examine the possibility for periodontal regeneration. $10{\mu}g/ml$ of DFPR was used as experimental concentration. osteogenic medium only was assigned as control, Experimental 1 was supplemented with 10nM dexamethasone, Experimental 2 with $10{\mu}g/ml$ DFPR and Experimental 3 with l0nM dexamethasone + $10{\mu}g/ml$ DFPR. cellular activity was evaluated by MTT method at 8, 12, 16 days, expression of mRNA of ALP, osteopontin, osteocalcin, collagen type-l was detected by RT-PCR method at 4, 8, 12, 16 days of culture. extent of mineralization was observed by Von Kossa staining at 16 day of culture. The results are as follows 1)Any acceleration of differentiation was not observed at expression of differentiation marker, 2) Decrease in expression of extracelluar matrix and in bone nodule formation was observed The results suggested that DFPR have negative effect on the rate of differentiation on rat calvarial cell, decrease extracelluar matrix formation ,decrease bone nodule formation. Ongoing studies are necessary in order to determine effect of DFPR on periodontal regeneration.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.17 no.4
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• pp.337-349
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• 1995

Periosteum in general is described as a specialized fibrous membrane of mesenchymal origin consisting of two basis layers : outer fibrous layer consists of irregularly arranged dense connective-tissue with fibroblasts, and inner osteogenic or cambial layer is composed of more loosely arranged fibers, greater vascularity and flatted spindle-shaped pre-osteoblasts. This periosteum may serve in controlling bone growth, especially mandibular growth has been emphasized. But, the periosteum enwrapping the facial skeleton have been studied for many years leaving a controversy in opinion regarding the function of these structures. We evaluated the bone formation activity of te periosteum in allogeneic bone grafts which bones are made of freeze-dried preparation preoperatively. We made the calvarial bone defects, 5 ${\times}$ 7mm sized, amd grafted with allogeneic bone in rats, which a half of specimens has dissected the overlying periosteum and a rest intacted. After bone grafting, we evaluated the capacity ofbone formation of periosteum, 1, 2, 4, 6, 8 weeks postoperatively. There are subtle differences of bone formation during early healing period after demineralized allogeneic bone grafting between control groups with periosteum and experimental groups without periosteum.

• Journal of Periodontal and Implant Science
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• v.25 no.3
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• pp.623-634
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• 1995

Periodontitis is characterized by gingival inflammation and results in periodontal pocket formation with loss of the supporting alveolar bone and connective tissue around the teeth. Therapeutic modalities should therefore aim not only at eliminating the gingival inflammatory process and preventing the progression of periodontal disease but also at reestablishing and regenerating the periodontal tissue previously lost to the disease. To achieve periodontal regeneration, progenitor cells must migrate to the denuded root surface, attach to it, proliferate and mature into an organized and functional fibrous attachment apparatus. Likewise, progenitor bone cells must also migrate, proliferate, and mature in conjunction with the regenerating periodontal ligament. Significant advances have been made during the last decade in understanding the factors controlling the migration, attachment and proliferation of cells. A group of naturally occuring molecules known as polypeptide growth factors in conjunction with certain matrix proteins are key regulators of these biological events. Of these, the fibroblast growth factor(FGF), platelet-derived growth factor(PDGF) , insulin like growth factor(CIGFs), transforming growth factor(TGFs), epidermal growth factor(EGF) and bone morphogenetic growth factor(BMPs) apper to have an important role in periodontal wound healing. The purpose of this study was to determine the effects of BMP on periodontal ligament cells. Human periodontal ligament cells were cultured from extracted tooth for non-periodontal reason. Cultured periodontal ligament cells were treated with BMP. Cellular activities were determined by MTT(3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay and ALP(alkaline phosphatase) activity. The results were as follows ; Regardless of cultured time, cellular activities were stimulated by BMP. Also, BMP greatly increased alkaline phosphatase(ALP) in periodontal ligament cells. These results suggest that BMP not only have no cytotoxic effect on periodontal ligament cells, but also have osteogenic stimulatory effect on periodontal ligament cells.

• BMB Reports
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• v.48 no.11
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• pp.636-641
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• 2015

There are controversial findings regarding the roles of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway on bone metabolism under oxidative stress. We investigated how Nrf2/HO-1 pathway affects osteoblast differentiation of MC3T3-E1 cells in response to hydrogen peroxide (H₂O₂), N-acetyl cysteine (NAC), or both. Exposing the cells to H₂O₂ decreased the alkaline phosphatase activity, calcium accumulation, and expression of osteoblast markers, such as osteocalcin and runt-related transcription factor-2. In contrast, H₂O₂ treatment increased the expression of Nrf2 and HO-1 in the cells. Treatment with hemin, a chemical HO-1 inducer, mimicked the inhibitory effect of H₂O₂ on osteoblast differentiation by increasing the HO-1 expression and decreasing the osteogenic marker genes. Pretreatment with NAC restored all changes induced by H₂O₂ to near normal levels in the cells. Collectively, our findings suggest that H₂O₂-mediated activation of Nrf2/HO-1 pathway negatively regulates the osteoblast differentiation, which is inhibited by NAC.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.35 no.2
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• pp.120-124
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• 2009

The concept of biostimulation of wounds by low-level laser therapy(LLLT) is attracting considerable attention. Although its effect on whole tissues has been studied quite extensively, the biological and cellular mechanisms underlying LLLT have not been clarified. In an experimental radius fracture in rabbits, Tang and Chai reported that LLLT enhanced the activity of red blood cells, macrophages, fibroblasts, chondrocytes, and osteoclasts within the fracture area. The purpose of the present study was to evaluate the effect of LLLT with a GaAlAs diode laser device on bone healing in rabbit mandibular fractures. We use 12 rabbits for this study. All rabbits were fractured mandible angle area using saw in anesthetic condition. In control group(n=6), none treatment was performed at fracture site. In experimental group(n=6), LLLT with a GaAlAs diode laser was radiated at fracture site daily for 7 days. All rabbits were sacrificed at 6 weeks later from performed fracture day. We studied the immunohistochemical staining of CD34 and Vimentin and the histochemical analysis for calcium and phosphorus content. The results were as follows. 1. In the histological and immunohistological staining, after 6week, fibroblasts, osteogenic cells and collgen fibers were observed more in experimental group than in control group. 2. In the histochemical analysis, the amount of calcium and phosphorus contents of the experimental group were more than the control group. From the results obtained, we suggest that the bone healing is stimulated by low-level laser irradiation in bone fractures.

• Journal of Embryo Transfer
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• v.27 no.3
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• pp.175-181
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• 2012

The synovial tissues are a valuable MSCs source for cartilage tissue engineering because these cells are easily obtainable by the intra-articular biopsy during diagnosis. In this study, we isolated and characterized the canine MSCs derived from synovial fluid of female and male donors. Synovial fluid was flushed with saline solution from pre and post-puberty male (cM1-sMSC and cM2-sMSC) and female (cF1-sMSC and cF2-sMSC) dogs, and cells were isolated and cultured in advanced-DMEM (A-DMEM) supplemented with 10% FBS in a humidified 5% $CO_2$ atmosphere at $38.5^{\circ}C$. The cells were evaluated for the expression of the early transcriptional factors, such as Oct3/4, Nanog and Sox2 by RT-PCR. The cells were induced under conditions conductive for adipogenic, osteogenic, and chondrogenic lineages, then evaluated by specific staining (Oil red O, von Kossa, and Alcian Blue staining, respectively) and analyzed for lineage specific markers by RT-PCR. All cell types were positive for alkaline phosphatase (AP) activity and early transcriptional factors (Oct3/4 and Sox2) were also positively detected. However, Nanog were not positively detected in all cells. Further, these MSCs were observed to differentiate into mesenchymal lineages, such as adipocytes (Oil red O staining), osteocytes (von Kossa staining), and chondrocytes (Alcian Blue staining) by cell specific staining. Lineage-specific genes (osteocyte; osteonectin and Runx2, adipocytes; PRAR-${\gamma}2$, FABP and LEP, and chondrocytes; collagen type-2 and Sox9) were also detected in all cells. In this study, we successfully established synovial fluid derived mesenchymal stem cells from female and male dogs, and determined their basic biological properties and differentiation ability. These results suggested that synovial fluid is a valuable stem cell source for cartilage regeneration therapy, and it is easily accessible from osteoarthritic knee.