• International Journal of Oral Biology
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• 제44권4호
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• pp.165-172
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• 2019

Berberine has been used clinically for more than a decade to treat various diseases, has been shown to be effective in osteoblast differentiation, and is a potential treatment option for osteoporosis. However, compared with existing osteoporosis drugs, berberine is somewhat less effective. This study aimed to identify a new compound with efficacy superior to that of berberine. The osteogenic activities of various berberine derivatives were evaluated via cell differentiation in C2C12 preosteoblast cell lines. Alkaline phosphatase (ALP) staining assay and structure-activity relationship demonstrated that compound 2b had a potent osteogenic effect. Furthermore, compound 2b dose dependently increased ALP activity and showed no toxicity at the effective concentration, indicating its efficacy. Additionally, compound 2b upregulated BMP2-induced transcriptional activity in a promoter activity assay using ALP, BSP, and OC promoters.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제31권6호
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• pp.461-467
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• 2005

Background. 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, succinimidyl ester mixed (CFSE) is the fluorescent labelling agent of living cells and used to trace the cells in vivo after transplatnation of various cells. The CFSE labelled cells can maintain fluorescence for up to 7 days after labelling. The MC3T3-E1 cell line (MC3T3) has been used for many studies about osteoblast, which is well known as a mouse preosteoblast. So the CFSE would be used to trace the transplanted MC3T3. However there are few reports about CFSE labelling of MC3T3. This study is aimed to know about adequate concenturation and incubation time of CFSE to MC3T3. Materials and methods. The MC3T3 was incubated in a humidified atmosphere of 95% air with 5% $CO_2$ at $37^{\circ}C$ using ${\alpha}$-minimal essential medium (${alpha}$-MEM) containing10% FBS and gentamycin. Ten mM CFSE solution in dimethylsulphoxide (DMSO: 1%) was diluted with phosphate buffered saline (PBS) and final concentration of culture medium was, respectively, 5, 10, 15, 20, 25 and 30 ${{\mu}M$. Then the MC3T3 was incubated with CFSE in a humidified atmosphere of 95% air with 5% $CO_2$ at $37^{\circ}C$ for 5, 10, 15, 20, 25, 30, 35, 40 and 45 minutes in each concentration. The fluorescence of CFSE labelled cells was analysed with a inverted fluorescence microscope. The duration of cell labelling was also studied. Trypan blue dye exclusion test was done for cell viability. Results. For concentration between 5 and 10 ${\mu}M$, CFSE did not significantly label the MC3T3 in vitro. The destruction of MC3T3 was observed at the concentration of 20 ${\mu}M$. In the concentration of 15 ${\mu}M$, the best labelling was obtained at an incubation period between 15 and 30 minutes. The MC3T3 labelled with an incubation period of 15 minutes at 15 ${\mu}M$ was still fluorescent 7 days after CFSE labelling. The mean cell viability was 95.93%. Conclusion. These results suggests an incubation period of 15 minutes at 15 ${\mu}M$ of CFSE provides best labelling of MC3T3 in vitro.

• 한국정밀공학회지
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• 제27권2호
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• pp.153-159
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• 2010

Recently, it has been reported that mechanical stimulation takes a role in improving cell growth. Also, became generally known that skeletal system as bone or cartilage tissues take influence of compression loading. In this study, we fabricated a custom-made bioreactor and analyzed that conditions of compressive loading would influence cell growth. To compare the effect of intermittent compressive loading on cell-encapsulated agarose scaffold, we cultured preosteoblast cell (MC3T3-E1 cells) statically and dynamically. And dynamic culture conditions were produced by changing parameters such as the iteration time and interval delay time. Also, cellencapsulated agarose scaffold were subjected to 10 % dynamic compressive strain at 1㎐ frequency for 7 days. After cell culture, cell proliferation was assessed with PI stain assay for fluorescence images and flow cytometry (FACS).

• Restorative Dentistry and Endodontics
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• 제20권2호
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• pp.744-757
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• 1995

Implantation of demineralized bone matrices was done into the amputated pulp in vivo and sequential reaction of the pulpal ectomesenchymal cells was observed. The bone matrices, obtained from cat long bone were crushed into below $700{\mu}m$, demineralized with 0.5N HCl and allografted into pulp of molar teeth. At seven days after implantation many undifferentiated mesenchymal cells aggregated near the matrices in the pulpal tissue. At fourteen days after implantation, the cells differentiated into preosteoblast-like cells which have secretory cell characteristics. At one or two months after implantation osteoid tissue was formed. The cells, which are located at the surface of the tissue, contained abundant dilated rough endoplasmic reticulum, Golgi apparatus and secretory granules in the cytoplasm. The matrix of the tissue has less collagen fibers than those in normal dentin. These results suggest that the interaction of pulpal mesenchymal cells with demineralized bone matrix can be a model which induces mineralization.

• BMB Reports
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• 제47권8호
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• pp.463-468
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• 2014

Osteoblasts are specialized mesenchymal cells that are responsible for bone formation. In this study, we examine the role of GATA4 in osteoblast differentiation. GATA4 was abundantly expressed in preosteoblast cells and gradually down-regulated during osteoblast differentiation. Overexpression of GATA4 in osteoblastic cells inhibited alkaline phosphatase activity and nodule formation in osteogenic conditioned cell culture system. In addition, overexpression of GATA4 attenuated expression of osteogenic marker genes, including Runx2, alkaline phosphatase, bone sialoprotein, and osteocalcin, all of which are important for osteoblast differentiation and function. Overexpression of GATA4 attenuated Runx2 promoter activity, whereas silencing of GATA4 increased Runx2 induction. We found that GATA4 interacted with Dlx5 and subsequently decreased Dlx5 binding activity to Runx2 promoter region. Our data suggest that GATA4 acts as a negative regulator in osteoblast differentiation by downregulation of Runx2.

• 한국식품영양과학회지
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• 제46권3호
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• pp.314-319
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• 2017

본 연구에서는 마가목 열매에서 추출한 chlorogenic acid의 유사체인 cryptochlorogenic acid(CCA)가 조골세포 분화에 미치는 영향에 대해서 알아보았다. 먼저 세포독성 여부를 확인하기 위해 MTT assay를 수행하였고 독성이 없다고 확인된 $5{\mu}M$의 농도에서 실험을 진행하였다. 그리고 조골세포로 분화할 수 있는 다분화능 세포인 C3H10T1/2와 조골세포인 MC3T3-E1에 CCA를 처리하여 표지 유전자인 Id1, Dlx5, Runx2의 발현을 확인하였다. 확인한 결과 표지유전자들의 발현이 대조군에 비교해서 증가한 것을 확인하였고, 그중 조골세포의 핵심 전사조절인자인 Runx2의 전사활성에 미치는 영향을 알아보기 위해 promoter assay를 수행하여 Runx2의 전사활성이 증가하는 것을 재확인하였다. 이러한 결과들을 토대로 CCA는 조골세포 분화를 촉진한다는 것을 알게 되었고, 골 질환 관련 제제로 CCA가 이용 가능할 수 있다고 생각된다.

• Biomaterials and Biomechanics in Bioengineering
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• 제1권2호
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• pp.81-93
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• 2014

An in vitro cell study evaluating cell adhesion to hydroxyapatite (HA) coated prosthetic Ti-6Al-4V alloy via laser treatment is presented in comparison with uncoated alloy. Based on our previous in vitro biocompatibility study, which demonstrated higher cell attachment and proliferation with MC3T3-E1 preosteoblast cells, the present investigation aims to reveal the effect of laser coating Ti alloy with HA on the adhesion strength of bone-forming cells against centrifugal forces. Remaining cells on different substrates after centrifugation were visualized using fluorescent staining. Semi-quantifications on the numbers of cells were conducted based on fluorescent images, which demonstrated higher numbers of cells retained on HA laser treated substrates post centrifugation. The results indicate potential increase in the normalized maximum force required to displace cells from HA coated surfaces versus uncoated control surface. The possible mechanisms that govern the enhancing effect were discussed, including surface roughness, chemistry, wettability, and protein adsorption. The improvement in cell adhesion through laser treatment with a biomimetic coating could be useful in reducing tissue damage at the prosthetic to bone junction and minimizing the loosening of prosthetics over time.

• Molecules and Cells
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• 제26권4호
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• pp.380-386
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• 2008

During embryonic and cancer development, the Hedgehog family of proteins, including Sonic Hedgehog, play an important role by relieving the inhibition of Smo by Ptc, thus activating the Smo signaling cascade. Recently, a purine compound, purmorphamine, has been reported to target the Hedgehog signaling pathway by interacting with Smo. Interestingly, both Sonic Hedgehog and purmorphamine were found to promote the osteogenic differentiation of mouse chondroprogenitor cells. However, there is insufficient information as to how the activation of this seemingly unrelated signaling pathway, either by Sonic Hedgehog or purmorphamine, contributes to osteogenesis. Using alkaline phosphatase assays, we screened 125 purmorphamine derivatives from the Korea Chemical Bank for effects on the differentiation of preosteoblast C2C12 cells. Here, we report that two purine derivatives modulate ALP activity as well as the expression of genes whose expression is known or suggested to be involved in osteogenesis.

• Journal of Dental Anesthesia and Pain Medicine
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• 제19권2호
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• pp.91-99
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• 2019

Background: The imbalance between osteoblasts and osteoclasts can lead to pathological conditions such as osteoporosis. It has been reported that opioid adversely affect the skeletal system, but it is inconsistent. Remifentanil is currently used as an adjuvant analgesic drug in general anesthesia and sedation. The aim of the present study was to investigate the effect of remifentanil on the osteoblast differentiation and mechanism involved in this effect. Methods: The C2C12 cells (mouse pluripotent mesenchymal cell line) were used as preosteoblast. Osteoblastic differentiation potency was determined by alkaline phosphatase (ALP) staining. C2C12 cell migration by remifentanil was evaluated using Boyden chamber migration assay. The expression of Runx2 and osterix was evaluated by RT-PCT and western blot analysis to investigate the mechanism involved in remifentanil-mediated osteoblast differentiation. Results: ALP staining showed that remifentanil increased significantly osteoblast differentiation. In Boyden chamber migration assay, C2C12 cell migration was increased by remifentanil. RT-PCR and western blot analysis showed that the expression of Runx2 and osterix was upregulated by remifentanil. Conclusions: We demonstrated that remifentanil increased osteoblast differentiation in vitro by upregulation of Runx2 and osterix expression. Therefore, remifentanil has the potential for assisting with bone formation and bone healing.

• 한국재료학회:학술대회논문집
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• 한국재료학회 2009년도 추계학술발표대회
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• pp.41.1-41.1
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• 2009

Aerosol Deposition (AD) is anovel way to fabricate bioactive ceramic coatings in biomedical implants and prostheses applications. In the present work, silicon-substituted hydroxyapatite (HA) coatings on commercially pure titanium were prepared by aerosol deposition using Si-HA powders. The incorporation of silicon in the HA lattice is known to improve the bioactivity of the HA, makingsilicon-substitute HA an attractive alternative to pure HA in biomedical applications. Si-HA powders with the chemical formula $Ca_{10}(PO_4)_6-x(SiO_4)x(OH)_2-x$, having silicon contents up to x=0.5 (1.4 wt%), were synthesized by solid-state reaction of $Ca_2P_2O_7$, $CaCO_3$, and $SiO_2$. The Si-HA powders were characterized by X-ray diffraction (XRD), X-ray fluorescence spectrometry (XRF), and Fourier transform infrared spectroscopy(FT-IR). The corresponding coatings were also analyzed by XRD, scanning electron microscopy (SEM), and electron probe microanalyzer (EPMA). The results revealed that a single-phase Si-HA was obtained without any secondary phases such as $\alpha$- or $\beta$-tricalcium phosphate (TCP) for both the powders and the coatings.The Si-HA coating was about $5\;{\mu}m$ thick, had a densemicrostructure with no cracks or pores. In addition, the proliferation and alkaline phosphatase (ALP) activity of MC3T3-E1 preosteoblast cells grown on the Si-HA coatings were significantly higher than those on the bare Ti and pure HA coating. These results revealed the stimulatory effects induced by siliconsubstitution on the cellular response to the HA coating.