• Archives of Plastic Surgery
• /
• 제35권3호
• /
• pp.229-234
• /
• 2008

Purpose: There are some reports presenting that peripheral blood contain circulating hematopoietic cells as well as, in significantly smaller quantities, mesenchymal stem cells. The purposes of this study is to isolate and characterize circulating mesenchymal progenitor cells with osteogenic potential from human peripheral blood. Methods: Human buffycoat containing mononuclear cells was harvested from peripheral blood of normal persons and isolated using a density gradient centrifugation and serially subcultured in osteogenic media for 1-4 weeks. The proliferation capability, phase-contrast microscopy, transmission electron microscopy, immunophenotype FACS analysis, Alizarin red staining and RT-PCR assays for osteogenic differentiation potential were performed. Results: The phenotype of cultured cells changed from small round or cuboidal cells at passage 1 into large spindle-shaped fibroblastic morphology cells at passage 4. Surface marker expressed CD14, but did not express CD34, CD80, CD83. Strong positive staining was observed for Alizarin reds in osteogenic medium on day 14, Using RT-PCR, the mRNA levels of bone- specific genes, such as ALP, c-bfa-1 and osteocalcin were detected. Conclusion: A new subset of peripheral blood derived progenitor cells described here has the ability to proliferate and differentiate into osteogenic cell lineages in vitro, and to be candidate for regenerative therapy.

• 대한한의학방제학회지
• /
• 제25권4호
• /
• pp.527-536
• /
• 2017

Objectives : The present study, to confirm the osteoblast differentiation effects of Acer tegmentosum Maxim. (AT) extract. Methods : In this experiment, cell viability, Alizarin red S assay, and Alkaline phosphatase (ALP) activity with AT extract (50, $100{\mu}g/m{\ell}$). Also, we studied the expression of differentiation regulator with AT extract in primary calvarial osteoblasts cells (pOB). Results : As a result of AT treatment, we determined that AT extract stimulates ALP activity and alizarin red activities in the pOB cells for mineralization for 18 days. Moreover, these factors increasing osteogenic markers such as Runt-related transcription factor2 ($Run{\times}2$), osteocalcin (OC), osteopontin, osterix, smad1, smad5, activating transcription factor4 (ATF4) and collagen type I alpha 1. Conclusions : These results indicate that AT extract have effect on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of bone diseases.

• Restorative Dentistry and Endodontics
• /
• 제39권3호
• /
• pp.187-194
• /
• 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.

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

• The Journal of Advanced Prosthodontics
• /
• 제6권3호
• /
• pp.157-164
• /
• 2014

PURPOSE. This study focused on in vitro cell differentiation and surface characteristics in a magnesium coated titanium surface implanted on using a plasma ion source. MATERIALS AND METHODS. 40 commercially made pure titanium discs were prepared to produce Ti oxide machined surface (M) and Mg-incorporated Ti oxide machined surface (MM). Surface properties were analyzed using a scanning electron microscopy (SEM). On each surface, alkaline phosphatase (ALP) activity, alizarin red S staining for mineralization of MC3T3-E1 cells, and quantitative analysis of osteoblastic gene expression, were evaluated. Actin ring formation assay and gene expression analysis of TRAP and GAPDH performing RT-PCR were performed to characterize osteoclast differentiation on mouse bone marrow-derived macrophages (BMMs). RESULTS. MM showed similar surface morphology and surface roughness with M, but was slightly smoother after ion implantation at the micron scale. M was more hydrophobic than MM. No significant difference between surfaces on ALP activity at 7 and 14 days were observed. Real-time PCR analyses showed similar levels of mRNA expression of the osteoblast phenotype genes; osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and collagen 1 (Col 1) in cell grown on MM at 7, 14 and 21 days. Alizarin red S staining at 21 days showed no significant difference. BMMs differentiation increased in M and MM. Actin ring formation assay and gene expression analysis of TRAP showed osteoclast differentiation to be more active on MM. CONCLUSION. Both M and MM have a good effect on osteoblastic cell differentiation, but MM may speed the bone remodeling process by activating on osteoclast differentiation.

• International Journal of Oral Biology
• /
• 제38권3호
• /
• pp.111-119
• /
• 2013

Objective. To investigate the effects of the hypoxia inducible factor-1 (HIF-1) activation-mimicking agent cobalt chloride ($CoCl_2$) on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and elucidate the underlying molecular mechanisms. Study design. The dose and exposure periods for $CoCl_2$ in hMSCs were optimized by cell viability assays. After confirmation of $CoCl_2$-induced HIF-$1{\alpha}$ and vascular endothelial growth factor expression in these cells by RT-PCR, the effects of temporary preconditioning with $CoCl_2$ on hMSC osteogenic differentiation were evaluated by RT-PCR analysis of osteogenic gene expression, an alkaline phosphatase (ALP) activity assay and by alizarin red S staining. Results. Variable $CoCl_2$ dosages (up to $500{\mu}M$) and exposure times (up to 7 days) on hMSC had little effect on hMSC survival. After $CoCl_2$ treatment of hMSCs at $100{\mu}M$ for 24 or 48 hours, followed by culture in osteogenic differentiating media, several osteogenic markers such as Runx-2, osteocalcin and osteopontin, bone sialoprotein mRNA expression level were found to be up-regulated. Moreover, ALP activity was increased in these treated cells in which an accelerated osteogenic capacity was also verified by alizarin red S staining. Conclusions. The osteogenic differentiation potential of hMSCs could be preserved and even enhanced by $CoCl_2$ treatment.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• 제34권4호
• /
• pp.412-418
• /
• 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.

• 대한치과교정학회지
• /
• 제36권2호
• /
• pp.103-113
• /
• 2006

중년 이후의 여성의 경우 칼슘 섭취가 부족하거나, 인위적인 난소 적출 또는 폐경 등으로 인하여 골밀도가 감소하는 경향이 있다. 이 경우 교정 치료 시, 골내 고정원으로 마이크로스크류를 이용할 수 있는지를 알아보기 위하여, 생후 4개월 된 Sprague-Dawley계 백서를 실험군으로 난소 적출(Ovariectomy Screw, OS)군, 대조군으로 Sham operation 시행한(Sham operation Screw, SS) 군의 두 군으로 나누었다. OS군과 SS군 모두 상악 대구치 사이 구개골에 마이크로스크류를 식립하였고, NiTi coil spring을 이용하여, 상악 중절치 2개를 견인하였다. 각 군당 3일, 7일 후 7마리씩 희생시켰다. 희생 3일전 Alizarin red S를 주입하고, 상악골, 장골, 심장혈을 채득한 후, 심장혈을 성분분석하고, 상악골과 장골은 비탈회 레진표본을 제작하여, 편광현미경, 형광현미경으로 관찰하였다. 심장혈을 통한 성분 조사 결과 OS군, SS군 모두 7일째 alkaline phosphatase (ALP)의 농도가 높게 나타나 골개조의 가능성을 보였고, 편광현미경 사진에서 SO, SS군에서 모두 마이크로스크류의 압박측, 긴장측, 치아의 긴장측에서 3일 보다 7일에서 기존골에 비하여 골밀도가 낮은 영역이 적었고, 치아의 압박측에서는 골밀도가 낮은 영역이 증가하였다. 형광 현미경 관찰결과는 3일째 보다 7일째에 더 Alizarin red S로 침착된 골이 나타난 것으로 보아, 새로운 골의 형성이 있었음을 나타내었다. 골다공증이 유도된 백서에서 계속된 골의 흡수보다는 골개조의 가능성이 증가하고, 신생골의 형성이 증가함을 확인할 수 있었고, 이는 골다공증에서도 마이크로스크류의 식립으로 고정원 확보가 가능한 것으로 생각한다.

• Natural Product Sciences
• /
• 제6권1호
• /
• pp.40-43
• /
• 2000

The chemical investigation on the cell suspension culture of Morinda elliptica L. yielded eight anthraquinones, two of which, anthragallol-1,2-dimethyl ether (3) and purpurin-1-methyl ether (4), have not been isolated from the original plant. Other compounds isolated include nordamnacanthal (1), alizarin-1-methyl ether (2), rubiadin (5), soranjidiol (6), $lucidin-{\omega}-methyl$ ether (7), and morindone (8). The structures of anthraquinones were established based on spectral studies.

• 생약학회지
• /
• 제23권3호
• /
• pp.137-141
• /
• 1992

The cells of Rubia cordifolia var. pratensis were transformed by Agrobactrium tumefaciens strain 11157. Surface-sterilized young leaves and stems of the plants were cocultivated with bacterial suspensions. Crown galls induced from stems were cultured with variation of culturing conditions and compared with untransformed cells. The growth rates and production of anthraquinone pigments of cells were remarkably improved by transformation. Furthermore, hairy roots were induced by inoculation or cocultivation with Agrobacterium rhizogenes strains.