• Journal of Periodontal and Implant Science
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• 제42권3호
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• pp.73-80
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• 2012

Purpose: The purpose of this study was to investigate the effects of the immunosuppressants FK506 and cyclosporin A (CsA) on the osteogenic differentiation of rat mesenchymal stem cells (MSCs). Methods: The effect of FK506 and CsA on rat MSCs was assessed in vitro. The MTT assay was used to determine the deleterious effect of immunosuppressants on stem cell proliferation at 1, 3, and 7 days. Alkaline phosphatase (ALP) activity was analyzed on days 3, 7, and 14. Alizarin red S staining was done on day 21 to check mineralization nodule formation. Real-time polymerase chain reaction (RT-PCR) was also performed to detect the expressions of bone tissue-specific genes on days 1 and 7. Results: Cell proliferation was promoted more in the FK506 groups than the control or CsA groups on days 3 and 7. The FK506 groups showed increased ALP activity compared to the other groups during the experimental period. The ALP activity of the CsA groups did not differ from the control group in any of the assessments. Mineralization nodule formation was most prominent in the FK506 groups at 21 days. RT-PCR results of the FK506 groups showed that several bone-related genes-osteopontin, osteonectin, and type I collagen (Col-I)-were expressed more than the control in the beginning, but the intensity of expression decreased over time. Runx2 and Dlx5 gene expression were up-regulated on day 7. The effects of 50 nM CsA on osteonectin and Col-I were similar to those of the FK506 groups, but in the 500 nM CsA group, most of the genes were less expressed compared to the control. Conclusions: These results suggest that FK506 enhances the osteoblastic differentiation of rat MSCs. Therefore, FK506 might have a beneficial effect on bone regeneration when immunosuppressants are needed in xenogenic or allogenic stem cell transplantation to treat bone defects.

• 생명과학회지
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• 제14권6호
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• pp.967-974
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• 2004

골조직은 골아세포, 파골세포, 골세포 등으로 구성되며, 골개조시 여러 인자가 세포증식, 분화, 활성화 및 골대사 조절에 관여한다. 이때 조골세포의 활성은 골형성에 중요하므로, 본 연구에서는 MC3T3-E1 조골세포주를 이용하여 식용자원인 복분자의 조골세포의 증식과 분화활성에 미치는 영향을 조사하였다. 복분자 추출물이 조골세포의 성장에 미치는 영향을 MTT 검색법으로 조사한 결과, 복분자 추출물 $10\;{\mu}g/mL$ 처리시 대조군과 비교하여 $142\%$ 증가하여 조골세포에 대해 높은 성장률을 보였다. 복분자 추출물이 ALP 효소 활성에 미치는 영향을 3일 간격으로 배지교환 및 시료처리를 하면서 27일 동안 배양시간에 따른 변화를 측정하여 조사하였다. 그 결과, 농도 1, 10, $100\;{\mu}g/mL$ 처리시 $100\;{\mu}g/mL$을 제외한 나머지 분획물들이 시간이 지남에 따라 ALP 활성을 증가시켰고, $10\;{\mu}g/mL$ 농도의 추출물은 27일째에 대조군에 비해 약 2.6배 이상, 양성대조군에 비해 약 1.5배 이상 ALP 활성을 증가시켰다. 복분자 추출물은 다시 ALP 효소 염색법과 Alizarin Red 염색으로 조골세포의 ALP활성유도, 분화와 석회화 형성능을 재확인하였으며 골기질 유전자의 발현의 변화도 확인하였다. 따라서 종래의 골질환에 좋다고 알려진 식품인 복분자 추출물이 양성대조군에 비해 빠르게 세포 증식과 분화를 유도하고 있어 앞으로 복분자에 대한 좀 더 깊은 분자생물학 수준 등의 구체적인 연구들과 기작연구가 지속적으로 이루어져야 할 것이라 추측된다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제27권5호
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• pp.453-456
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• 2001

Background: Autogenous alveolar bone cell transplantation may be suitable for tissue engineering for alveolar bone reconstruction. This study aimed to isolate human alveolar bone-derived cells (HABDCs) and to evaluate the ability of collagen gels to support HABDC proliferation and differentiation for human alveolar bone tissue engineering applications. Method: Cultures of primary HABDCs were established from alveolar bone chips obtained from 10 persons undergoing tooth extraction. These cells were expanded in vitro until passage 3 and used for the in vitro characterization of HABDCs and the in vitro analysis of collagen gels for alveolar bone tissue engineering. Results: Of the 10 attempts made to obtain HABDC cultures, eight were successful. HABDCs expressed the osteoblastic phenotype characterized by alkaline phosphatase activity, osteocalcin expression and the mineralization of the extracellular matrix in vitro. When seeded on collagen gels, HABDCs penetrated into the collagen gel matrices and proliferated inside the gels. Significantly, when HABDCs were embedded into the gels, collagen fibers and mineralization were produced within the gels. Conclusion: This study demonstrates the feasibility of using cultured HABDCs and collagen gels for human alveolar bone tissue engineering applications.

• Journal of Periodontal and Implant Science
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• 제47권5호
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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.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제39권4호
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• pp.150-155
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• 2013

Objectives: The objective of this study is to determine the adequate concentration and to evaluate the osteogenic potential of simvastatin in human maxillary sinus membrane-derived stem cells (hSMSC). Materials and Methods: Mesenchymal stem cells derived from the human maxillary sinus membrane were treated with various concentrations of simvastatin. The adequate concentration of simvastatin for osteogenic induction was determined using bone morphogenetic protein (BMP-2). The efficacy of osteogenic differentiation of simavastatin was verified using osteocalcin mRNA, and the mineralization efficacy of hSMSCs and simvastatin treatment was compared with alkaline phosphatase and von Kossa staining. Results: Expression of BMP-2 mRNA and protein was observed after three days and was dependent on the concentration of simvastatin. Expression of osteocalcin mRNA was observed after three days in the $1.0{\mu}M$ simvastatin-treated group. Mineralization was observed after three days in the simvastatin-treated group. Conclusion: These results suggest that simvastatin induces the osteogenic potential of mesenchymal stem cells derived from the human maxillary sinus membrane mucosa.

• Imaging Science in Dentistry
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• 제38권4호
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• pp.195-202
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• 2008

Purpose : To characterize the effects of 2-deoxy-D-glucose (2-DG) and quercetin (QCT) on gene expression of osteonectin (ON) and osteopontin (OP) in irradiated MC3T3-El cells. Materials and Methods : When MC3T3-El osteoblastic cells had reached 70-80% confluence, cultures were transferred to a differentiating medium supplemented with 5 mM 2-DG or 10 ${\mu}M$ QCT and then irradiated with 2, 4, 6, and 8 Gy. At various times after irradiation, the cells were analyzed for the expression of bone mineralization genes such as ON and OP. Results : The mRNA expression of both ON and OP was increased according to the culture time in the differentiation medium, and the increase of the genes peaked at 14 days after the differentiation induction. In the case of OP, the increase of mRNA expression was maintained to 28 days after the differentiation, while the mRNA level of ON was reduced to the basal level at the same time. Irradiation adding 2-DG showed a significant peak value in the expression pattern of ON at 4 Gy 7 days after irradiation. Irradiation adding QCT increased the mRNA expression of ON and OP in a dose-dependant manner, but irradiation adding 2-DG did not show any differences between the control and experiments 14 days after irradiation. Irradiation adding QCT increased significantly the expression patterns of ON 21 days after irradiation. Conclusion : The results showed that QCT acted as a radiosensitizer in the gene expression of ON and OP during differentiation of the late stage of irradiated MC3T3-E1 osteoblastic cells in vitro. (Korean J Oral Maxillofac Radiol 2008; 38: 195-202)

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제37권3호
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• pp.214-224
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• 2011

Objective: This study examined the potential of the in vitro osteogenesis of microtopographically modified surfaces, RBM (resorbable blasting media) surfaces, which generate hydroxyapatite grit-blasting. Methods: RBM surfaces were modified hydroxyapatite grit-blasting to produce microtopographically modified surfaces and the surface morphology, roughness or elements were examined. To investigate the potential of the in vitro osteogenesis, the osteoblastic cell adhesion, proliferation, and differentiation were examined using the human osteoblast-like cell line, MG-63 cells. Osteoblastic cell proliferation was examined as a function of time. In addition, osteoblastic cell differentiation was verified using four different methods of an ALP activity assay, a mineralization assay using alizarin red-s staining, and gene expression of osteoblastic differentiation marker using RT-PCR or ELISA. Results: Osteoblastic cell adhesion, proliferation and ALP activity was elevated on the RBM surfaces compared to the machined group. The cells exhibited a high level of gene expression of the osteoblastic differentiation makers (osteonectin, type I collagen, Runx-2, osterix). imilar data was represented in the ELISA produced similar results in that the RBM surface increased the level of osteocalcin, osteopontin, TGF-beta1 and PGE2 secretion, which was known to stimulate the osteogenesis. Moreover, alizarin red-s staining revealed significantly more mineralized nodules on the RBM surfaces than the machined discs. Conclusion: RBM surfaces modified with hydroxyapatite grit-blasting stimulate the in vitro osteogenesis of MG-63 cells and may accelerate bone formation and increase bone-implant contact.

• Bulletin of the Korean Chemical Society
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• 제31권4호
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• pp.929-933
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• 2010

A new compound, 4-methoxyl 5-hydroxymethyl benzoic 3-O-$\beta$-D-glucopyranoside (1), has been isolated from the leaves and stems of Acer mandshuricum, along with nine known compounds (2-10). Their structures were determined by a variety of spectroscopic analyses. The effect of compounds 1-10 on the function of osteoblastic MC3T3-E1 cells was examined by determining alkaline phosphatase (ALP) activity, collagen synthesis, and mineralization. Compound 1 significantly increased the function of osteoblastic MC3T3-E1 cells; $5.0\;{\mu}M$ of 1 increased ALP activity, collagen synthesis, and mineralization of MC3T3-E1 cells to 114.7, 119.5, and 108.2% (P < 0.05) of the basal value, respectively. In addition, compounds 2-10 also potently increased the function of osteoblastic MC3T3-E1 cells.

• The Korean Journal of Ecology
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• 제28권4호
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• pp.181-188
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• 2005

Methods used to study carbon sequestration by soil aggregates have often excluded the concentric spatial variability and other dynamic processes that contribute to resource accessibility and solute transport within aggregates. We investigated the spatial gradients of carbon (C) and nitrogen (N) from the exterior to interior layers within macroaggregates, $6.3\sim9.5$ mm, sampled from conventional tillage (CT) and no tillage (NT) sites of a Hoytville silt clay loam. Spatial gradients in C accumulation within macroaggregates were related to the differences in C dynamics by determining the sizes and the turnover rates of fast C and slow C pools in the concentric layers of aggregates. Aggregate exteriors contained more labile C and were characterized by greater C mineralization rates than their interiors in both management systems. In contrast, C in the interior layers of aggregates was more resistant in both systems. These results indicated the spatial differentiation of C dynamics within macroaggregates, i.e., exterior layers as a reactive site and interior layers as a protective site. Greater total C distribution in the exterior layers of NT aggregates indicated more influx of C from the macropores in interaggregate space than C. mineralization (net gain of C), whereas lower C distribution within the exterior layers of CT aggregates indicated net loss of C by greater C mineralization than C influx. We found total C increased approximately 1.6-fold by the conversion of CT soils to NT management systems for a period of 36 years. Differences in total accumulation and the spatial distribution of C within aggregates affected by management were attributed to the differences in aggregate stability and pore networks controlling the spatial heterogeneities of resource availability and microbial activity within aggregates.

• BMB Reports
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• 제49권3호
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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.