• BMB Reports
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• v.49 no.3
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• pp.179-184
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• 2016

Bone morphogenetic protein 9 (BMP9) is a potent inducer of osteogenic differentiation of mesenchymal stem cells. The Wnt antagonist Dickkopf-1 (Dkk1) is involved in skeletal development and bone remodeling. Here, we investigated the role of Dkk1 in BMP9-induced osteogenic differentiation of MSCs. We found that overexpression of BMP9 induced Dkk1 expression in a dose-dependent manner, which was reduced by the P38 inhibitor SB203580 but not the ERK inhibitor PD98059. Moreover, Dkk1 dramatically decreased not only BMP9-induced alkaline phosphatase (ALP) activity but also the expression of osteocalcin (OCN) and osteopontin (OPN) and matrix mineralization of C3H10T1/2 cells. Furthermore, exogenous Dkk1 expression inhibited Wnt/β-catenin signaling induced by BMP9. Our findings indicate that Dkk1 negatively regulates BMP9-induced osteogenic differentiation through inhibition of the Wnt/β-catenin pathway and it could be used to optimize the therapeutic use of BMP9 and for bone tissue engineering.

• Journal of Ginseng Research
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• v.42 no.2
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• pp.199-207
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• 2018

Background: Ginseng is a well-known traditional Chinese medicine that has been widely used in a range of therapeutic and healthcare applications in East Asian countries. Microbial transformation is regarded as an effective and useful technology in modification of nature products for finding new chemical derivatives with potent bioactivities. In this study, three minor derivatives of ginsenoside compound K were isolated and the inducing effects in the Wingless-type MMTV integration site (Wnt) signaling pathway were also investigated. Methods: New compounds were purified from scale-up fermentation of ginsenoside Rb1 by Paecilomyces bainier sp. 229 through repeated silica gel column chromatography and high pressure liquid chromatography. Their structures were determined based on spectral data and X-ray diffraction. The inductive activities of these compounds on the Wnt signaling pathway were conducted on MC3T3-E1 cells by quantitative real-time polymerase chain reaction analysis. Results: The structures of a known 3-keto derivative and two new dehydrogenated metabolites were elucidated. The crystal structure of the 3-keto derivative was reported for the first time and its conformation was compared with that of ginsenoside compound K. The inductive effects of these compounds on osteogenic differentiation by activating the Wnt/b-catenin signaling pathway were explained for the first time. Conclusion: This study may provide a new insight into the metabolic pathway of ginsenoside by microbial transformation. In addition, the results might provide a reasonable explanation for the activity of ginseng in treating osteoporosis and supply good monomer ginsenoside resources for nutraceutical or pharmaceutical development.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.1
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• pp.7-15
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• 2010

Complex human tissues harbor stem cells and precursor cells, which are responsible for tissue development or repair. Recently, dental tissues such as dental pulp, periodontal ligament (PDL), dental follicle have been identified as easily accessible sources of undifferentiated cells. These tissues contain mesenchymal stem cells that can be differentiate into bone, cartilage, fat or muscle by exposing them to specific growth conditions. In this study, the authors procured the stem cell from pulp, PDL, and dental follicle and differentiate them into osteoblast and examine the bone induction capacity. Dental pulp stem cell (DPSC), periodontal ligament stem cell (PDLSC), and dental follicle precursor cell (DFPC) were obtained from human 3rd molar and cultured. Each cell was analyzed for presence of stem cell by fluorescence activated cell sorter (FACs) against CD44, CD105 and CD34, CD45. Each stem cell was cultured, expanded and grown in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix. Osteogenic pathway was checked by alizarin red staining, alkaline phosphatase (ALP) activity test and RT-PCR for ALP and osteocalcin (OCN) gene expression. According to results from FACs, mesenchymal stem cell existed in pulp, PDL, and dental follicle. As culturing with bone differentiation medium, stem cells were differentiated to osteoblast like cell. Compare with stem cell from pulp, PDL and dental follicle-originated stem cell has more osteogenic effect and it was assumed that the character of donor cell was able to affect on differential potency of stem cell. From this article, we are able to verify the pulp, PDL, and dental follicle from extracted tooth, and these can be a source of osteoblast and stem cell for tissue engineering.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.34 no.4
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• pp.412-418
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• 2008

Bone morphogenetic proteins (BMPs) in combination with stem cells gain more significance for their use in bone tissue engineering. The mesenchymal stem cell can be differentiated into osteoblast by the treatment of BMP. The aim of this study is to characterize the osteogenic differentiation process of adult stem cells derived from buccal fat pad according to BMP-2 within culture media and decide the appropriate concentration of BMP-2 to facilitate osteogenesis. The authors procured the stem cell from buccal fat pad and analyzed for presence of stem cell by flow cytomety against CD-34, CD-105 and STRO-1. The buccal fat derived stem cells (BFDC) were treated by application of the different concentration with BMP-2 of 0, 10, 50, 100 and 200ng/ml, respectively. And their ability to differentiate into osteogenic pathway were checked by alkaline phosphatase(ALP) staining, Alizarin red staining and RT-PCR for osteocalcin(OC) gene expression at 7, 14 and 21day of culture. Flow cytometric analysis and biochemical assays demonstrated that BFDC might be a distinguished stem cells, and mineralization was accompanied in proportion to BMP-2 concentration. However, with 100ng/ml concentration of BMP-2, the BFDC demonstrated most efficient staining pattern of ALP and Alizarin red. The feasibility of the osteogenic differentiation in the group of both 50ng/ml and 200ng/ml of BMP-2 showed similar activity and relatively weaker than that of 100ng/ml. These results suggest that the BMP-2 stimulate osteogenesis by BFDC effectively and that bone induction might be controlled through negative regulatory feedback in higher concentration.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.28 no.6
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• pp.511-519
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• 2006

Autogenous bone grafts have been considered the gold standard for maxillofacial bony defects. However, this procedure could entail a complicated surgical procedure as well as potential donor site morbidity. Possibly the best solution for bone-defect regeneration is a tissue engineering approach, i.e. the use of a combination of a suitable scaffold with osteogenic cells. A major source of osteogenic cells is the bone marrow. Bone marrow-derived mesenchymal stem cells are multipotent and have the ability to differentiate into osteoblastic, chondrocytic, and adipocytic lineage cells. However, the isolation of cells from bone marrow has someproblems when used in clinical setting. Bone marrow aspiration is sometimes potentially more invasive and painful procedure and carries of a risk of morbidity and infection. A minimally invasive, easily accessible alternative would be cells derived from periosteum. The periosteum also contains multipotent cells that have the potential to differentiate into osteoblasts and chondrocytes. In the present study, we evaluated the osteogenic activity and mineralization of cultured human periosteal-derived cells. Periosteal explants were harvested from mandibule during surgical extraction of lower impacted third molar. The periosteal cells were cultured in the osteogenic inductive medium consisting of DMEM supplemented with 10% fetal calf serum, 50g/ml L-ascorbic acid 2-phosphate, 10 nmol dexamethasone and 10 mM -glycerophosphate for 42 days. Periosteal-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 14 of culture period, then decreased in intensity during the culture period. ALP mRNA expression increased up to day 14 with a decrease thereafter. Osteocalcin mRNA expression appeared at day 7 in culture, after that its expression continuously increased in a time-dependent manner up to the entire duration of culture. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. In conclusion, our study showed that cultured human periosteal-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix. As the periosteal-derived cells, easily harvested from intraoral procedure such as surgical extraction of impacted third molar, has the excellent potential of osteogenic capacity, tissue-engineered bone using periosteal-derived cells could be the best choice in reconstruction of maxillofacial bony defects.

• BMB Reports
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• v.42 no.7
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• pp.427-432
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• 2009

Orthodontic tooth movement results from the combinational process of both bone resorption and formation in the compressive and tension sides, respectively. However, the genes responsible for new bone formation in tension sides have not been determined. In this study, we used DNA microarray and real-time RT-PCR to identify genes in human periodontal ligament (PDL) cells that undergo significant changes in expression in response to static tensional forces (2 or 12 hours). The genes found were alkaline phospatase (ALP), matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), and several collagen genes. Furthermore, an ELISA evaluating the expression of VEGF, type IV collagen and MMP-2 found levels significantly increased after 24 and 72 hours (P < 0.05). ALP activity was also increased after 24 hours (P < 0.05). Collectively, we found the genes up-regulated in our study by the static tensional force are related to osteogenic processes such as matrix synthesis and angiogenesis.

• The Journal of the Korean dental association
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• v.22 no.1 s.176
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• pp.37-48
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• 1984

Clinical assessment of bone-graft healing in the maxillofacial region is generally limited to clinical evaluation, radiographs, and biopsy. Sequential interpretation of osseous repair, more sensitive than with conventional radiography is possible with a non-invasive, non-destructive radionuclide method. Technetium-99m radionuclide bone scan was used in the evaluation of the progress of osteogenic activity in autologous iliac bone graft and freeze-dried bone allograft of dog's mandible. Bone scan was performed at 1wk, 2wk, 4wk, 6wk, and 8wk after grafting. In autologous graft the activity ratio for the graft bone remained greater than that of the host since 2자 after grafting; however, in lyophilized allograft the activity ratio for graft bone was greater than that of the host at 6자 after grafting.

• Journal of Life Science
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• v.27 no.5
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• pp.501-508
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• 2017

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that play important roles in a variety of cellular functions. Among BMP family members, BMP2 efficiently promotes osteoblast differentiation through Smad-mediated runt-related transcription factor 2 (Runx2) expression. Several recent studies suggest that BMPs are associated with clock genes, in particular Bmal1. Bmal1 protein heterodimerizes with Clock protein and then induces period 1 (Per1) expression. However, the role of Per1 on osteoblast differentiation remains unclear. In this study, we investigated whether Per1 is involved in osteoblast differentiation. MC3T3-E1 cells were treated with BMP2 for induction of osteoblastic differentiation. Osteogenic maker gene and Per1 mRNA expression were measured using real-time PCR. Interestingly, BMP2 treatment induced Per1 mRNA expression in MC3T3-E1 cells. To further investigate the function of Per1 on osteoblast differentiation, MC3T3-E1 cells were transiently transfected with pCMV-Per1. Per1 overexpression increased Runx2 mRNA and protein levels. Also, mRNA expression and promoter activity of osteocalcin were upregulated by Per1 overexpression. To investigate the effect of interaction between Per1 and osteogenic condition, MC3T3-E1 cells were cultured in osteogenic medium containing ascorbic acid and ${\beta}$-glycerophosphate. Osteogenic medium-induced ALP staining level and mineralization were synergistically increased by overexpression of Per1. Taken together, these results demonstrate that Per1 is a positive regulator of osteoblast differentiation.

• Journal of Acupuncture Research
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• v.23 no.5
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• pp.69-77
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• 2006

Background : Mesenchymal stem cells (MSCs) are present in most of the tissue matrix, taking part in their regeneration when injury or damage occurs. The aim of this study was to investigate the presence of cells with pluripotential characteristics in human subchondral bone and the capacity of these cells to differentiate to osteoblast. Methods : Human subchondral bone were digested with collagenase. Isolated cells were cultured with a-MEM, 15% FBS, 10-8M dexamethasone and 50 ng/mL ascoric acid. Cells from 0 day(isolated cells), 7 day (first subculture) and 14 days (third subculture) were used to carry out phenotypic characterization experiments flowcytometry analysis with 11 monoclonal antibodies) and osteogenic differentiation experiments. Osteogenic differentiation of cells was assessment by quantification of bone extracellular matrix components by following analysis: alkaline phosphatase(ALP) stains to detect ALP activity, RT-PCR and western blot to detect osteocalcin (OCN), osteopontin (OPN) and type I collagen(Col I), and Alizarin red stains to detect calcium deposition. Results : Flowcytometry analyses showed that in our population more than 98% of cells were positive for MSC markers: SH-2(CD105, 99%), CD29 (95%), CD73 (95%). Cells were negative for hematopoietic markers (CD11b, CD34, and CD45). Furthermore, cells showed positive stain to multipotent markers such as CDl17 (c-kit) (15.1%), and CD166 (74.9%), and cell adhesion molecules such as CD54 (78.1%) and CD106 (63.5%). The osteogenic specific marker analyses showed that the culture of these cells for 7 and 14 days stimulates ALP, OCN, OPN and Col I synthesis by RT-PCR and Western blot analysis. Also, after 14 days in the culture of MSCs induces mineralization by Arizarin red stain. Conclusion : In this work, we demonstrated a new and efficient method for osteoblastic differentiation of human subchondral bone stem cells. As MSCs takes part in reparative processes of adult tissues, these cells could play an important role in osteogenesis.

• Polymer(Korea)
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• v.29 no.5
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• pp.501-507
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• 2005

Small intestinal submucosa (SIS) had been widely used as a biomaterial without immune rejection responses. SIS sponges prepared by crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). SIS powders dissolved in $3\%(v/v)$ acetic acid aqueous solution for 48hrs and freeze-dried. EDC solution ($H_2O$ : ethanol = 5 : 95) as a crosslink agent was used in concentration of 100mM. In vitro, rat-BMSCs seeded in SIS sponges and induced the osteogenesis for 28 days. We have characterized the osteogenic potential of rat-BMSCs in SIS sponges by alkaline phosphatase activity(ALP), n assay, SEM and RT-PCR for osteogenic phenotype. In SEM, all morphology of SIS sponges was regular and showed interconnected pore structure. By RT-PCR analysis, we observed type I collagen expression. These results demonstrate osteogenic differentiation of rat-BMSCs. In conclusion, we confirmed that the morphology of surface, cross-section, and side of SIS sponges were highly porous with good interconnections between each pores, which can support the surface of cell growth, proliferation, and differentiation. This result indicates that SIS sponge is useful for osteogenesis of BMSCs.