• Tissue Engineering and Regenerative Medicine
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• 제15권6호
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• pp.793-801
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• 2018

BACKGROUND: The aim of this study was to evaluate the combined effect of low-level laser treatment (LLLT) and recombinant human bone morphological protein-2 (rhBMP-2) applied to hypoxic-cultured MC3T3-E1 osteoblastic cells and to determine possible signaling pathways underlying differentiation and mineralization of osteoblasts under hypoxia. METHODS: MC3T3-E1 cells were cultured under 1% oxygen tension for 72 h. Cell cultures were divided into four groups: normoxia control, low-level laser (LLL) alone, rhBMP-2 combined with LLLT, and rhBMP-2 under hypoxia. Laser irradiation was applied at 0, 24, and 48 h. Cells were treated with rhBMP-2 at 50 ng/mL. Alkaline phosphatase activity was measured at 3, 7, and 14 days to evaluate osteoblastic differentiation. Cell mineralization was determined with Alizarin red S staining at 7 and 14 days. Western blot assays were performed to evaluate whether p38/protein kinase D (PKD) signaling was involved. RESULTS: The results indicate that LLLT and rhBMP-2 synergistically increased alkaline phosphatase (ALP) activity and mineralization. Western blot analyses showed that expression of type I collagen, runt-related transcription factor 2 (RUNX2), and Osterix (Osx), increased and expression of hypoxia-inducible factor 1-alpha ($HIF-1{\alpha}$), decreased more in the LLLT and rhBMP-2 combined group than in the rhBMP-2 or LLL alone groups. Moreover, LLLT and rhBMP-2 stimulated p38 phosphorylation and rhBMP-2 and LLLT increased Prkd1 phosphorylation. CONCLUSION: Combined treatment with rhBMP-2 and LLL induced differentiation and mineralization of hypoxic-cultured MC3T3-E1 osteoblasts by activating p38/PKD signaling in vitro.

• 치위생과학회지
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• 제21권4호
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• pp.243-250
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• 2021

Background: Endodontic sealers or their toxic components may become inflamed and lead to delayed wound healing when in direct contact with periapical tissues over an extended period. Moreover, an overfilled sealer can directly interact with adjacent tissues and may cause immediate necrosis or further resorption. Therefore, the treatment outcome conceivably depends on the endodontic sealer's biocompatibility and osteogenic potential. This study aimed to evaluate the cell viability and osteogenic effects of four different sealers in osteoblastic cells. Methods: AH Plus (resin-based sealer), Pulp Canal Sealer EWT (zinc oxide-eugenol sealer), BioRoot RCS (calcium silicate-based sealer), and Well-Root ST (MTA-based calcium silicate sealer) were mixed strictly according to the manufacturer's instructions, and dilutions of sealer extracts (1/2, 1/5 and 1/10) were determined. Cell viability was measured using the water-soluble tetrazolium-8 (WST-8) assay. Differentiation was assessed by alkaline phosphatase (ALP) activity and mineralized nodule formation by Alizarin Red S staining. Results: The cell viability of the extracts derived from the sealers excluding Well-Root ST was concentration dependent, with sealer extracts having the least viability at a 1/2 dilution. At sealer extract dilution of 1/10, the test groups showed the same survival rate as that control group, with the exception of BioRoot RCS. Among all experimental groups, BioRoot RCS showed the highest cell viability after 48 hours. The ALP activity was significantly higher in a concentration-dependent manner. Furthemore, all four materials promoted ALP activity and mineralized nodule formation compared to the control at 1/10 dilutions. Conclusion: This is the first study to highlight the differences in biological activity of these four materials. These results suggest that the composition of root canal sealers appears to alter the form of biocompatibility and osteoblastic differentiation.

• The Korean Journal of Physiology and Pharmacology
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• 제26권1호
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• pp.37-45
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• 2022

The aim of the present study was to investigate the physiological role of nicotinamide phosphoribosyltransferase (NAMPT) associated with odontogenic differentiation during tooth development in mice. Mouse dental papilla cell-23 (MDPC-23) cells cultured in differentiation media were stimulated with the specific NAMPT inhibitor, FK866, and Visfatin (NAMPT) for up to 10 days. The cells were evaluated after 0, 4, 7, and 10 days. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The mineralization assay was performed by staining MDPC-23 cells with Alizarin Red S solution. After cultivation, MDPC-23 cells were harvested for quantitative PCR or Western blotting. Analysis of variance was performed using StatView 5.0 software (SAS Institute Inc., Cary, NC, USA). Statistical significance was set at p < 0.05. The expression of NAMPT increased during the differentiation of murine odontoblast-like MDPC-23 cells. Furthermore, the up-regulation of NAMPT promoted odontogenic differentiation and accelerated mineralization through an increase in representative odontoblastic biomarkers, such as dentin sialophosphoprotein, dentin matrix protein-1, and alkaline phosphatase in MDPC-23 cells. However, treatment of the cells with the NAMPT inhibitor, FK866, attenuated odontogenic differentiation, as evidenced by the suppression of odontoblastic biomarkers. These data indicate that NAMPT regulated odontoblastic differentiation through the regulation of odontoblastic biomarkers. The increase in NAMPT expression in odontoblasts was closely related to the formation of the extracellular matrix and dentin via the Runx signaling pathway. Therefore, these data suggest that NAMPT is a critical regulator of odontoblast differentiation during tooth development.

• Journal of Periodontal and Implant Science
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• 제53권1호
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• pp.54-68
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• 2023

Purpose: The purpose of this study was to determine whether metformin (MF) could alleviate the expresssion of reactive oxygen species (ROS) and improve the osteogenic ability of bone marrow mesenchymal stem cells derived from diabetic rats (drBMSCs) in vitro, and to evaluate the effect of MF on the ectopic osteogenesis of drBMSCs in a nude mouse model in vivo. Methods: BMSCs were extracted from normal and diabetic rats. In vitro, a cell viability assay (Cell Counting Kit-8), tests of alkaline phosphatase (ALP) activity, and western blot analysis were first used to determine the cell proliferation and osteogenic differentiation of drBMSCs that were subjected to treatment with different concentrations of MF (0, 50, 100, 200, 500 µM). The cells were then divided into 5 groups: (1) normal rat BMSCs (the BMSCs derived from normal rats group), (2) the drBMSCs group, (3) the drBMSCs + Mito-TEMPO (10 µM, ROS scavenger) group, (4) the drBMSCs + MF (200 µM) group, and (5) the drBMSCs + MF (200 µM) + H₂O₂ (50 µM, ROS activator) group. Intracellular ROS detection, a senescence-associated β-galactosidase assay, ALP staining, alizarin red staining, western blotting, and immunofluorescence assays were performed to determine the effects of MF on oxidative stress and osteogenic differentiation in drBMSCs. In vivo, the effect of MF on the ectopic osteogenesis of drBMSCs was evaluated in a nude mouse model. Results: MF effectively reduced ROS levels in drBMSCs. The cell proliferation, ALP activity, mineral deposition, and osteogenic-related protein expression of drBMSCs were demonstrably higher in the MF-treated group than in the non-MF-treated group. H₂O₂ inhibited the effects of MF. In addition, ectopic osteogenesis was significantly increased in drBMSCs treated with MF. Conclusions: MF promoted the proliferation and osteogenic differentiation of drBMSCs by inhibiting the oxidative stress induced by diabetes and enhenced the ectopic bone formation of drBMSCs in nude mice.

• Restorative Dentistry and Endodontics
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• 제48권2호
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• pp.18.1-18.10
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• 2023

Objectives: This study aimed to determine whether collagen triple helix repeat containing-1 (CTHRC1), which is involved in vascular remodeling and bone formation, can stimulate odontogenic differentiation and angiogenesis when administered to human dental pulp stem cells (hDPSCs). Materials and Methods: The viability of hDPSCs upon exposure to CTHRC1 was assessed with the WST-1 assay. CTHRC1 doses of 5, 10, and 20 ㎍/mL were administered to hDPSCs. Reverse-transcription polymerase reaction was used to detect dentin sialophosphoprotein, dentin matrix protein 1, vascular endothelial growth factor, and fibroblast growth factor 2. The formation of mineralization nodules was evaluated using Alizarin red. A scratch wound assay was conducted to evaluate the effect of CTHRC1 on cell migration. Data were analyzed using 1-way analysis of variance followed by the Tukey post hoc test. The threshold for statistical significance was set at p < 0.05. Results: CTHRC1 doses of 5, 10, and 20 ㎍/mL had no significant effect on the viability of hDPSCs. Mineralized nodules were formed and odontogenic markers were upregulated, indicating that CTHRC1 promoted odontogenic differentiation. Scratch wound assays demonstrated that CTHRC1 significantly enhanced the migration of hDPSCs. Conclusions: CTHRC1 promoted odontogenic differentiation and mineralization in hDPSCs.

• Biomolecules & Therapeutics
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• 제31권5호
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• pp.515-525
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• 2023

The most common heart valve disorder is calcific aortic valve stenosis (CAVS), which is characterized by a narrowing of the aortic valve. Treatment with the drug molecule, in addition to surgical and transcatheter valve replacement, is the primary focus of researchers in this field. The purpose of this study is to determine whether niclosamide can reduce calcification in aortic valve interstitial cells (VICs). To induce calcification, cells were treated with a pro-calcifying medium (PCM). Different concentrations of niclosamide were added to the PCM-treated cells, and the level of calcification, mRNA, and protein expression of calcification markers was measured. Niclosamide inhibited aortic valve calcification as observed from reduced alizarin red s staining in niclosamide treated VICs and also decreased the mRNA and protein expressions of calcification-specific markers: runt-related transcription factor 2 and osteopontin. Niclosamide also reduced the formation of reactive oxygen species, NADPH oxidase activity and the expression of Nox2 and p22^phox. Furthermore, in calcified VICs, niclosamide inhibited the expression of β-catenin and phosphorylated glycogen synthase kinase (GSK-3β), as well as the phosphorylation of AKT and ERK. Taken together, our findings suggest that niclosamide may alleviate PCM-induced calcification, at least in part, by targeting oxidative stress mediated GSK-3β/β-catenin signaling pathway via inhibiting activation of AKT and ERK, and may be a potential treatment for CAVS.

• Advances in nano research
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• 제15권4호
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• pp.295-304
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• 2023

In this paper, a powerful photocatalyst based on carbon nanocomposite is developed in order to obtain a new material applicable in water treatment and especially for the discoloration of effluents used in the textile industry. For that, TiO₂-graphene nanocomposites have been successfully synthesized by a mixture of Functionalized Graphene Sheet (FGS) and tetrachlorotitanium complexes to form FGS-TiO₂ nanocomposite. In the presence of an anionic surfactant, we used a new chemical process to functionalize graphene sheets in order to make them an excellent medium for blocking and preventing the aggregation of TiO₂ nanoparticles. The components of these nanocomposites are characterized by means of X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), which confirms the successful formation of the FGS-TiO₂ nanocomposite. It was found that the TiO₂ nanoparticles were dispersed uniformly on the graphene plane which possesses better charge separation capability than pure TiO₂. The FGS-TiO₂ nanocomposites exhibited higher photocatalytic activity compared to pure TiO₂ for the removal of three dyes: such as Methylene Blue (MB), Bromophenol Blue (BB) and Alizarin Red-S (AR) in water. The removal process was fast and more efficient with FGS-TiO₂ nanocomposite in daylight (in the absence of UV irradiation) compared to pure TiO₂ nanoparticles without and under UV in all pH range.

• 분석과학
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• 제37권2호
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• pp.87-97
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• 2024

The appropriate intake of fluoride (F^-) is beneficial to human health; however, the over-consumption can result in various potentially harmful effects. This study compared different colorimetric reagents, i.e., aluminium-xylenol orange (Al-XO), zirconium-xylenol orange (Zr-XO), and zirconium-alizarin red S (Zr-ARS), for fluoride measurements by the UV-Vis, in terms of reaction mechanisms, method sensitivity, and interferences from aluminium and ferric ions. The colorimetric procedures were optimized, and the analytical methods were evaluated. The goodness of linearity (R² > 0.998) was obtained for all three assays within the concentration range of 1.0-20.0 mg/L fluoride in deionized water, in which the method sensitivity followed the descending order of Zr-XO > Al-XO > Zr-ARS. The Zr-XO was applied for determining the fluoride in different tea extracts in water (90 ℃ and 60-minute-brewing) and black tea demonstrated the highest fluoride content (3.0-3.6 mg/L). The effects of brewing time and temperature on the release of fluoride in the tea extracts were also investigated, indicating these are critical factors for the fluoride extraction. This study highlighted the application potentials of the UV-Vis measurement as a simple, convenient, and cheap analytical approach and discussed different colorimetric reagents used for fluoride determination in tea extracts in the context that the UV-Vis spectrophotometers are commonly equipped in most laboratories.

• 대한치과보철학회지
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• 제53권1호
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• pp.9-18
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• 2015

목적: 본 연구는 Tricalcium phosphate 입자를 사용한 모재분사 후 양극산화처리를 한 임플란트 표면의 특성을 분석하고, 골모유사세포의 반응을 평가하고자 하였다. 재료 및 방법: 직경 10 mm, 두께 3.0 mm 크기의 Grade IV 타이타늄 디스크를 시편으로 사용하였으며, 양극산화처리(ASD)군, 모재 분사 후 양극산화(RBM/ASD)군, 대조군(machined surface)으로 나누어 표면처리하였다. 표면처리 후 FE-SEM, 에너지분산분광기와 주사전자현미경을 사용하여 표면특성을 평가하였다. 세포의 부착을 평가하기 위해 골모유사세포를 이용해 crystal violet assay를 통해 세포부착을 평가하고, 세포 형태는 공초점 레이저 현미경을 사용하여 관찰하였다. 세포증식을 평가하기 위해 XTT 시험을, 세포분화는 역전사 중합효소연쇄반응을 사용하였으며 침착된 칼슘의 양을 측정하기 위해 Alizarin red S stain 을 이용하였다. 비교분석은 one-way ANOVA (SPSS version 18.0)로 유의수준 5%에서 검정하였다. 결과: ASD군과 RBM/ASD군에서, 분화구 모양의 표면 형상이 나타났으며, 대조군과 비교하여 산소와 인산 이온이 관찰되었다. 단위면적당 거칠기는 대조군에서 $0.08{\pm}0.04{\mu}m$, ASD군에서 $0.52{\pm}0.14{\mu}m$, RBM/ASD군에서 $1.45{\pm}0.25{\mu}m$를 보였다. 세포반응실험에서, ASD군과 RBM/ASD군이 대조군에 비해 세포의 부착정도가 높았으며 대조군이 세포증식에서 가장 높은 값을 보였다(P<.05). RT-PCR 실험에서, RBM/ASD군이 다른 군들보다 높은 ALP를 보였다(P<.05). ASD군과 비교했을 때 RBM/ASD군은 세포부착과 증식 정도에서 큰 값을 보였다(P<.05). 결론: 본 연구의 한계내에서 모재분사 후 양극산화 처리한 티타늄 표면 처리 방식이 단순 양극산화 처리한 군이나 대조군보다 골모유사세포의 반응에 효과적인 방법임을 확인하였다.

• 대한소아치과학회지
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• 제27권2호
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• pp.309-317
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• 2000

본 실험은 bisphosphonate가 조골세포 분화 및 파골세포 분화에 미치는 영향을 알아보고자, etidronate와 alendronate를 조골세포에 투여하여 조골세포 전사인자인 Cbfa1, 조골세포 표시 인자의 발현, 석회화된 골결절 형성을 평가하였다. Bisphosphonate가 조골세포의 석회화된 골결절 형성에 미치는 영향을 평가하기 위하여 배양액에 $10^{-6},\;10^{-5},\;10^{-4}M$의 etidronate 및 $10^{-8},\;10^{-7},\;10^{-6}M$의 alendronate를 첨가하였으며, 배양 15일 후에 alizarin red로 염색하여 관찰하였다. 또 조골세포의 분화에 미치는 bisphosphonate의 영향을 평가하고자 백서 두개관에서 얻은 조골세포에 etidronate $10^{-6},\;10^{-5},\;10^{-4}M$ 및 alendronate $10^{-6}$ M을 투여하고 배양 8일 후 총RNA를 수집하였고, 전기영동 및 Northern blot hybridization하여 Cbfa1, alkaline phosphatase, type I collagen, osteopontin, osteocalcin의 발현을 조사하였다. 이상의 실험결과 다음과 같은 결론을 얻었다. 1. Etidronate는 농도 의존적으로 골결절 석회화를 억제하였으나, alendronate는 골석회화를 억제하지 않았다. 2. Etidronate는 Cbfa1의 발현을 농도 의존적으로 억제하였으나, alendronate는 오히려 촉진하였다. 3. Etidronate는 type I collagen, osteocalcin 및 osteopontin의 발현을 농도 의존적으로 억제하였으나, alendronate는 오히려 증가시켰다. 4. Alkaline phosphatase의 발현은 사용된 etidronate와 alendronate에 의해 영향 받지 않았다. 이상의 결과에서 etidironate는 조골세포의 전사인자인 Cbfa1의 발현을 억제하며, 이에 의하여 조골세포의 분화표지인자인 type I collagen, osteopontin 및 osteocalcin의 합성이 억제되고, 결과적으로 석회화된 골결절의 형성을 억제하는 것으로 사료된다.