• 한국식품영양과학회지
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• 제34권9호
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• pp.1367-1374
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• 2005

사람의 정상 골모세포 FOB에서 아미노산 수송계 L의 발현 및 이들 수송계 L을 통한 아미노산 수송특성을 밝히기 위하여, FOB 세포에서 RT-PCR, western blot 분석 및 아미노산 uptake 실험 등을 수행하여 다음과 같은 결과들을 얻었다. FOB 세포에서 아미노산 수송계 L의 두 아형인 LAT1, LAT2및 그들의 보조인자 4F2hc의 발현을 확인할 수 있었다. FOB 세포에서 $_{L}-leucine$의 수송은 $Na^+$-비의존적이었다. FOB 세포에서 $_{L}-leucine$의 수송은 아미노산 수송계 L의 선택적 억제제인 BCH에 의해 완전히 차단되었다. FOB 세포에서 여러 아미노산들에 의한 L-leucine수송억제 실험결과는 Xenopus oocyte에서 시행되어 보고되어진 LAT1과 LAT2 수송억제 실험 결과의 특성을 모두 포함하였다. 본 연구의 결과로 사람의 정상 골모세포주인 FOB에서 는 세포성장 및 증식을 위한 중성 아미노산의 수송에 아미노산 수송계 L의 두 아형인 LAT1과 LAT2가 중요한 역할을 하고 있음을 확인할 수 있었다.

• Journal of Dental Anesthesia and Pain Medicine
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• 제16권4호
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• pp.263-271
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• 2016

Background: Bone injury is common in many clinical situations, such as surgery or trauma. During surgery, excessive reactive oxygen species (ROS) production decreases the quality and quantity of osteoblasts. Remifentanil decreases ROS production, reducing oxidative stress and the inflammatory response. We investigated remifentanil's protective effects against $H_2O_2$-induced oxidative stress in osteoblasts. Methods: To investigate the effect of remifentanil on human fetal osteoblast (hFOB) cells, the cells were incubated with 1 ng/ml of remifentanil for 2 h before exposure to $H_2O_2$. For induction of oxidative stress, hFOB cells were then treated with $200{\mu}M$ $H_2O_2$ for 2 h. To evaluate the effect on autophagy, a separate group of cells were incubated with 1 mM 3-methyladenine (3-MA) before treatment with remifentanil and $H_2O_2$. Cell viability and apoptotic cell death were determined via MTT assay and Hoechst staining, respectively. Mineralized matrix formation was visualized using alizarin red S staining. Western blot analysis was used to determine the expression levels of bone-related genes. Results: Cell viability and mineralized matrix formation increased on remifentanil pretreatment before exposure to $H_2O_2$-induced oxidative stress. As determined via western blot analysis, remifentanil pretreatment increased the expression of bone-related genes (Col I, BMP-2, osterix, and $TGF-{\beta}$). However, pretreatment with 3-MA before exposure to remifentanil and $H_2O_2$ inhibited remifentanil's protective effects on hFOB cells during oxidative stress. Conclusions: We showed that remifentanil prevents oxidative damage in hFOB cells via a mechanism that may be highly related to autophagy. Further clinical studies are required to investigate its potential as a therapeutic agent.

• Journal of Periodontal and Implant Science
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• 제34권2호
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• pp.317-332
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• 2004

Osteoblasts from alveolar bone may have an important role in the bone regeneration for periodontium, but their culture and characterization are not determined yet. The purpose of this study was to investigate the biological characteristics of primary explant cultured osteoblasts(PECO) from alveolar bone. Osteoblasts were isolated and cultured from alveolar socket of extracted tooth in children. To compare the characteristics, osteoblasts and gingival fibroblasts were cultured with DMEM at $37^{\circ}C$, 5% $CO_2$, l00% humidity incubator, and human fetal osteoblasts cell line(hFOB1) were cultured with DMEM at $34^{\circ}C$, 5%, $CO_2$ 100% humidity incubator. To characterize the isolated bone cells, morphologic change, cell proliferation and differentiation were measured. Morphology of PECO was small round body or cuboidal shape on inverted microscope and was similar with hFOB1. PECO became polygonal shape with stellate and had an amorphous shape at 9th passage in culture. PECO had significantly higher activity than that of gingival fibroblasts and hFOB1 in alkaline phosphatase activity. The expression of osteocalcin and bone sialoprotein in PECO was notably increased when compared with hFOB1 and gingival fibroblasts. These result indicated that PECO from alveolar bone in children has an obvious characteristics of osteoblast, maybe applied for the regeneration of bone.

• 대한임상검사과학회지
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• 제51권3호
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• pp.379-385
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• 2019

골다공증은 호르몬의 변화와 무기질 감소에 의해 골밀도의 감소를 유발하여 골절의 위험을 높이는 질환이다. 최근 보고에 의하면, 간염바이러스 및 에이즈 치료제로 사용되고 있는 Adefovir dipivoxil (ADV)의 장기적 복용에서 골다공증 부작용이 유발할 수 있음이 보고 되고 있다. 이에 대한 연구수행을 위해 골모세포주 hFOB1.19와 혈관내피세포 HUVEC을 이용하여 ADV에 대한 생물학적 연관성을 평가하였다. 우선적으로 ADV를 농도별로 처리한 후 각 세포와 핵의 형태학적 분석을 위해 DAPI와 crystal violet 염색을 시행하였다. 또한 세포 증식에 대한 약물 효과를 평가하기 위하여 CCK-8분석과 골모세포에 대한 분화유도 및 억제 효과를 확인하기 위하여, ALP 염색을 진행하였다. 그 결과, ADV는 hFOB1.19 세포와 HUVEC 세포에서 농도 의존적으로 세포의 비대 현상을 유발하였고, 세포의 증식이 억제되었다. 이러한 원인들을 규명하기 위해 TGF-${\beta}$발현을 조사하였을 뿐만 아니라 이러한 발현 감소에 의한 생물학적 영향이 골모세포로부터 골세포로의 분화 과정에 관여하고 있음을 확인 할 수 있었다. 결론적으로, 본 연구에서는 ADV 약물이 골모세포와 혈관내피세포의 TGF-${\beta}$의 발현을 억제하여 핵의 크기 증가와 세포형태의 비대증을 유발하며, 세포의 증식억제 및 골모세포 분화능에 영향을 줌으로서 골다공증을 유발할 수 있는 가능성을 확인하였다. 이러한 결과는 ADV 복용에 따른 골다공증 발병 원인을 이해하기 위한 기초 연구 및 이를 이용한 임상영역에 활용 될 수 있을 것으로 사료된다.

• Journal of Periodontal and Implant Science
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• 제33권2호
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• pp.259-269
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• 2003

The ideal goal of periodontal therapy is the regeneration of periodontal tissue repair of function. Although is very difficult to attain the goal, recent advances in periodontal wound healing concepts encourage hope reaching it. Recently many efforts are concentrated on the regeneration potential of material used in traditional Korean medicine. Phlomidis Radix has been used for the treatment of blood stasis, bone fracture and osteoporosis in traditional Korean medicine. The purpose of this study is to examine effects of dichloromethane fraction Phlomidis Radix on Bone Formation in Human Fetal Osteoblasts. Human fetal osteoblastic cell line(hFOB1 1.19 ;American Type Culture Collection, Manassas, VA) were used and cells were cultured containing DMEM and dichloromethane fraction Phlomidis Radix(100 ng/ml , 1 ${\mu}$/ml, 10 ${\mu}$/ml) at 34$^{\circ}C$ with 5% $CO_2$ in 100% humidity. MTT was performed to examine the viability of the cell, and alkaline phosphatase activity was analyzed to examine the mineralization. Also bone calcification nodules were evaluated. The cellular activity of hFOB1 was increased in 100 ng/ml, 1 ${\mu}$/ml , 10 ${\mu}$/ml of dichloromethane fraction of Phlomidis Radix and especially significant increation was showed in 100 ng/ml of dichloromethane fraction of Phlomidis Radix at 6days (p <0.05). ALP level of hFOB1 was significantly increased in 100 ng/ml , 1 ${\mu}$/ml, 10 ${\mu}$/ml of dichloromethane fraction of Phlomidis Radix and especially more increation was showed in 10 ${\mu}$/ml of dichloromethane fraction of Phlomidis Radix (p <0,05). Calcification nodules of hFOB1 significantly increased in 10 ${\mu]$/ml of dichloromethane fraction of Phlomidis Radix at 21 days of incubation(p<0.05). The results indicate that dicholoromethane fraction of Phlomidis Radix has excellent effects on mineralization of hFOB1.

• Journal of Dental Anesthesia and Pain Medicine
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• 제16권4호
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• pp.295-302
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• 2016

Background: Reactive oxygen species play critical roles in homeostasis and cell signaling. Dexmedetomidine, a specific agonist of the ${\alpha}2$-adrenoceptor, has been commonly used for sedation, and it has been reported to have a protective effect against oxidative stress. In this study, we investigated whether dexmedetomidine has a protective effect against $H_2O_2$-induced oxidative stress and the mechanism of $H_2O_2$-induced cell death in normal human fetal osteoblast (hFOB) cells. Methods: Cells were divided into three groups: control group-cells were incubated in normoxia without dexmedetomidine, hydrogen peroxide ($H_2O_2$) group-cells were exposed to $H_2O_2$ ($200{\mu}M$) for 2 h, and Dex/$H_2O_2$ group-cells were pretreated with dexmedetomidine ($5{\mu}M$) for 2 h then exposed to $H_2O_2$ ($200{\mu}M$) for 2 h. Cell viability and apoptosis were evaluated. Osteoblast maturation was determined by assaying bone nodular mineralization. Expression levels of bone-related proteins were determined by western blot. Results: Cell viability was significantly decreased in the $H_2O_2$ group compared with the control group, and this effect was improved by dexmedetomidine. The Hoechst 33342 and Annexin-V FITC/PI staining revealed that dexmedetomidine effectively decreased $H_2O_2$-induced hFOB cell apoptosis. Dexmedetomidine enhanced the mineralization of hFOB cells when compared to the $H_2O_2$ group. In western blot analysis, bone-related protein was increased in the Dex/$H_2O_2$ group. Conclusions: We demonstrated the potential therapeutic value of dexmedetomidine in $H_2O_2$-induced oxidative stress by inhibiting apoptosis and enhancing osteoblast activity. Additionally, the current investigation could be evidence to support the antioxidant potential of dexmedetomidine in vitro.

• Journal of Dental Anesthesia and Pain Medicine
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• 제16권1호
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• pp.39-47
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• 2016

Background: Oxidative stress occurs during the aging process and other conditions such as bone fracture, bone diseases, and osteoporosis, but the role of oxidative stress in bone remodeling is unknown. Propofol exerts antioxidant effects, but the mechanisms of propofol preconditioning on oxidative stress have not been fully explained. Therefore, the aim of this study was to evaluate the protective effects of propofol against $H_2O_2$-induced oxidative stress on a human fetal osteoblast (hFOB) cell line via activation of autophagy. Methods: Cells were randomly divided into the following groups: control cells were incubated in normoxia (5% $CO_2$, 21% $O_2$, and 74% $N_2$) without propofol. Hydrogen peroxide ($H_2O_2$) group cells were exposed to $H_2O_2\;(200{\mu}M)$ for 2 h, propofol preconditioning (PPC)/$H_2O_2$ group cells were pretreated with propofol then exposed to $H_2O_2$, 3-methyladenine (3-MA)/PPC/$H_2O_2$ cells were pretreated with 3-MA (1 mM) and propofol, then were exposed to $H_2O_2$. Cell viability and apoptosis were evaluated. Osteoblast maturation was determined by assaying bone nodular mineralization. Expression levels of bone related proteins were determined by western blot. Results: Cell viability and bone nodular mineralization were decreased significantly by $H_2O_2$, and this effect was rescued by propofol preconditioning. Propofol preconditioning effectively decreased $H_2O_2$-induced hFOB cell apoptosis. However, pretreatment with 3-MA inhibited the protective effect of propofol. In western blot analysis, propofol preconditioning increased protein levels of collagen type I, BMP-2, osterix, and TGF-${\beta}1$. Conclusions: This study suggests that propofol preconditioning has a protective effect on $H_2O_2$-induced hFOB cell death, which is mediated by autophagy activation.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제40권6호
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• pp.291-296
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• 2014

Objectives: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a side effect of bisphophonate therapy that has been reported in recent years. Osteoclastic inactivity by bisphosphonate is the known cause of BRONJ. Bone morphogenetic protein-2 (BMP-2) plays an important role in the development of bone. Recombinant human BMP-2 (rhBMP-2) is potentially useful as an activation factor for bone repair. We hypothesized that rhBMP-2 would enhance the osteoclast-osteoblast interaction related to bone remodeling. Materials and Methods: Human fetal osteoblast cells (hFOB 1.19) were treated with $100{\mu}M$ alendronate, and 100 ng/mL rhBMP-2 was added. Cells were incubated for a further 48 hours, and cell viability was measured using an MTT assay. Expression of the three cytokines from osteoblasts, receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL), osteoprotegerin (OPG), and macrophage colony-stimulating factor (M-CSF), were analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results: Cell viability was decreased to $82.75%{\pm}1.00%$ by alendronate and then increased to $110.43%{\pm}1.35%$ after treatment with rhBMP-2 (P<0.05, respectively). OPG, RANKL, and M-CSF expression were all decreased by alendronate treatment. RANKL and M-CSF expression were increased, but OPG was not significantly affected by rhBMP-2. Conclusion: rhBMP2 does not affect OPG gene expression in hFOB, but it may increase RANKL and M-CSF gene expression.

• The Korean Journal of Physiology and Pharmacology
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• 제21권6호
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• pp.599-607
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• 2017

Most normal cells express L-type amino acid transporter 2 (LAT2). However, L-type amino acid transporter 1 (LAT1) is highly expressed in many tumor cells and presumed to support their increased growth and proliferation. This study examined the effects of JPH203, a selective LAT1 inhibitor, on cell growth and its mechanism for cell death in Saos2 human osteosarcoma cells. FOB human osteoblastic cells and Saos2 cells expressed LAT1 and LAT2 together with their associating protein 4F2 heavy chain, but the expression of LAT2 in the Saos2 cells was especially weak. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, potently inhibited L-leucine uptake in Saos2 cells. As expected, the intrinsic ability of JPH203 to inhibit L-leucine uptake was far more efficient than that of BCH in Saos2 cells. Likewise, JPH203 and BCH inhibited Saos2 cell growth with JPH203 being superior to BCH in this regard. Furthermore, JPH203 increased apoptosis rates and formed DNA ladder in Saos2 cells. Moreover, JPH203 activated the mitochondria-dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bad, Bax, and Bak, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. These results suggest that the inhibition of LAT1 activity via JPH203, which may act as a potential novel anti-cancer agent, leads to apoptosis mediated by the mitochondria-dependent intrinsic apoptotic signaling pathway by inducing the intracellular depletion of neutral amino acids essential for cell growth in Saos2 human osteosarcoma cells.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제38권
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• pp.48.1-48.8
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• 2016

Background: This study investigates the effect of alendronate-treated osteoblasts, as well as the effect of low-level laser therapy (LLLT) on the alendronate-treated osteoblasts. Bisphosphonate decreases the osteoblastic activity. Various treatment modalities are used to enhance the bisphosphonate-treated osteoblasts; however, there were no cell culture studies conducted using a low-level laser. Methods: Human fetal osteoblastic (hFOB 1.19) cells were treated with $50{\mu}M$ alendronate. Then, they were irradiated with a $1.2J/cm^2$ low-level Ga-Al-As laser (${\lambda}=808{\pm}3nm$, 80 mW, and 80 mA; spot size, $1 cm^2$; NDLux, Seoul, Korea). The cell survivability was measured with the MTT assay. The three cytokines of osteoblasts, receptor activator of nuclear factor ${\kappa}B$ ligand (RANKL), osteoprotegerin (OPG), and macrophage colony-stimulating factor (M-CSF) were analyzed. Results: In the cells treated with alendronate at concentrations of $50{\mu}M$ and higher, cell survivability significantly decreased after 48 h (p < 0.05). After the applications of low-level laser on alendronate-treated cells, cell survivability significantly increased at 72 h (p < 0.05). The expressions of OPG, RANKL, and M-CSF have decreased via the alendronate. The RANKL and M-CSF expressions have increased, but the OPG was not significantly affected by the LLLT. Conclusions: The LLLT does not affect the OPG expression in the hFOB cell line, but it may increase the RANKL and M-CSF expressions, thereby resulting in positive effects on osteoclastogenesis and bone remodeling.