• 생명과학회지
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• 제29권10호
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• pp.1104-1110
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• 2019

최근 노령인구의 증가로 고령화 사회로 접어들면서 골다공증과 같은 골 대사 질환이 사회적 문제로 대두되고 있다. 파골세포는 골 흡수 역할을 하는 세포이며 골 흡수가 강하게 일어나는 경우 골다공증이 유발된다. 그러나 현재 사용 중인 골 흡수 억제제는 장기간 사용 시 부작용이 발생할 수 있어, 파골세포 분화 억제에 효과가 있는 새로운 소재 개발이 필요한 실정이다. 따라서 본 연구에서는 풀무치 에탄올 추출물을 대상으로 RANKL에 의해 유도된 파골세포 분화에 대한 억제 효능 확인 및 그 기작을 구명하고자 하였다. RAW264.7 파골세포에서 풀무치 추출물의 독성 및 증식 효과를 확인하기 위하여 MTS assay를 진행하였고 $2,000{\mu}g/ml$ 농도까지 세포 독성은 확인되지 않았다. 풀무치 추출물이 파골세포의 분화에 미치는 영향을 확인하기 위해 3일 동안 RAW264.7 세포에 파골세포 분화 촉진제인 RANKL을 단독 처리 및 풀무치 추출물과 함께 처리한 후 TRAP 활성을 비교하였다. 그 결과 RANKL에 의해 증가한 파골세포 분화가 풀무치 추출물 처리에 의해 농도 의존적으로 감소하는 것을 확인하였다. 파골세포 분화와 관련된 유전자(TRAP, RANK, NFATc1 및 CK)의 발현량 변화를 확인한 결과, RANKL 처리 시에 증가한 유전자 발현량이 풀무치 추출물 처리에 의해 현저하게 감소하는 것을 확인하였고, NFATc1, c-Src와 같은 분화 관련 단백질 발현량 또한 풀무치 추출물 처리에 의해 감소하였다. 풀무치 에탄올 추출물은 $NF-{\kappa}B$, ERK 및 JNK 신호전달에 영향을 미쳐 그 결과로 파골세포 분화 억제 효과가 나타나는 것으로 판단된다. 이러한 결과로 보아 풀무치 에탄올 추출물은 골 흡수를 억제하는 역할을 함으로써 골다공증 예방 및 치료를 위한 새로운 기능성 소재로 사용 가능성이 있음을 확인하였다.

• 대한치과교정학회지
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• 제25권6호
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• pp.715-722
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• 1995

Orthodontic tooth movement in response to orthodontic force results from actions of osteoclasts and osteeoblasts in the cell level. Convincing evidence has now been provided to support the view that osteoclasts are derived from mononuclear cells that originate in the bone marrow or other hematopoietic organs and they migrate to the bones via vascular routes. Nitric oxide(NO), which accounts for the biological properties of endothelium-derived relaxing factor(EDRF), is the endogenous stimulator of soluble guanylate cylase. The discovery of the formation of nitric oxide(NO) from L-arginine in mammalian tissues and its biological roles has, in the last 7 years, thrown new light onto many areas of research. Data from experiments in vitro showed that N-metyl-L-arginine(L-NMA) and L-nitro-L- arginine(L-NAME) are competitive inhibitors of nitric oxide synthase. This study suggest that the multinucleated cells in our culture have characteristics of osteoclasts and that the potential bone cell activity of nitric oxide in vitro may be mediated in part by stimulation of marrow mononuclear cells to form osteoclast-like cells. Bone marrow cells were obtaineed from tibia of 19-days old chick embryo. After sacrifice, tibia was quickly dissected and the bone were then split to expose the medullary bone. The cells were attached for 4 hours and the nonadherent cells were collected. Marrow cells weere cultured in 96-well plate in medium 199. To examine the number of TRAP-positive multinucleated cells(MNCs), $10^{-8}\;M\;Vit=D_3$ and various concentration of L-NMA and L-NAME weere added at the beginning of cultures and with each medium change. After 7 days of culture. tartrate-resistant acid phosphatase(TRAP) staining was performed for microscopic evaluation. Cells haying more than three nuclei per cell were counted as MNCs. The obsrved results were as follows;1. 1,25-dihydroxyvitamine $D_3$ stimulated the osteoclast-like multinucleated cells in cultures of chick embryo bone marrow. 2. Nitric oxide synthase inhibitors(NOSI ; N-NMA, N-NAME) stimulated the osteoclast-like cells in cultures of chick embry bone marrow. 3. 1,25-dihydroxyvitamine$D_3$ and nitric oxide synthase inhibitors did not appear to have additive effect on the generation of TRAP-positive MNCs. These results suggest that nitric oxide synthase inhibitors may stimulate the osteoclast-like multinucleated cell formation and fusion in cultures of chick bone marrow.

• International Journal of Oral Biology
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• 제36권1호
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• pp.37-42
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• 2011

The working mechanism of bisphosphonate on bone cells is unclear despite its powerful inhibitory activity on bone resorption. The differentiation and activation of osteoclasts are essential for bone resorption and are controlled by the stimulatory RANKL and inhibitory OPG molecules. Teeth exhibit a range of movement patterns during their eruption to establish their form and function, which inevitably accompanies peripheral bone resorption. Hence, the mandible, which contains the teeth during their eruption processes, is a good model for revealing the inhibitory mechanism of bisphosphonate upon bone resorption. In the present study, RANKL and OPG expression were examined immunohistochemically in the mandible of rats with developing teeth after alendronate administration (2.5 mg/kg). The preeruptive mandibular first molars at postnatal days 3 to 10 showed the developing stages from bell to crown. No morphological changes in tooth formation were observed after alendronate administration. The number of osteoclasts in the alveolar bone around the developing teeth decreased markedly at postnatal days 3, 7 and 10 compared with the control group. RANKL induced strong positive immunohistochemical reactions in the dental follicles and stromal cells around the mandibular first molar. In particular, many osteoclasts with strongly positive reactions to RANKL appeared above the developing mandibular first molars at postnatal days 3 and 10. Immunohistochemical reactions with RANKL after alendronate administration were weaker than the control groups. However, the immunohistochemical reactivity to OPG was stronger after alendronate administration, at postnatal days 3 and 10. These results suggest that alendronate may decrease bone resorption by regulating the RANKL/OPG pathway in the process of osteoclast formation, resulting in a delay in tooth eruption.

• 동의생리병리학회지
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• 제27권4호
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• pp.431-436
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• 2013

Bone homeostasis is maintained by co-ordination of bone-resorbing osteoclasts and bone-forming osteoblasts. Imbalance between osteoclasts and osteoblasts leads to many bone diseases such as osteoporosis, rheumatoid arthritis. Taxillus chinensis is a herb that has been widely used to improve bone health. However, the effect and mechanism of Taxillus chinensis extract on osteoclast differentiation and bone resportion has been unknown. Thus, We investigated the effect of Taxillus chinensis on expression of receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL)-induced osteoclast differentiation and bone resorption. Also, the action of Taxillus chinensis on mechanisms relating to osteoclast differentiation was studied. In this results, we identified that Taxillus chinensis significantly inhibited RANKL-induced osteoclast differentiation and bone resportion. Moreover, Taxillus chinensis was suppressed the activation of NF-${\kappa}B$ in bone marrow macrophage treated RANKL and M-CSF. Taxillus chinensis was down-regulated the mRNA expression of c-Fos, nuclear factor of activated T-cells (NFAT)c1, osteoclast-associated receptor (OSCAR), tartrate-resistant acid phosphatase (TRAP). The cell adhesion-related molecules such as integrin ${\alpha}v$ and integrin ${\beta}3$, and the filamentous actin (F-actin) rings of mature osteoclasts-related molecules such as dendritic cell-specific transmembrane preotein (DC-STAMP) and cathepsin K are also suppressed. Taken together, these results indicated that Taxillus chinensis will be a good candidate to treat osteoclast-mediated bone diseases.

• 한국미생물·생명공학회지
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• 제44권4호
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• pp.557-562
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• 2016

치주질환은 만성염증성 질환으로 치조골소실을 일으켜 성인치아상실을 유발하는 요인 중 하나이다. 그람 음성세균인 Aggregatibacter actinomycetemcomitans와 Porphyromonas gingivalis는 치주질환환자의 병소에서 쉽게 동정된다. 지질다당체(Lipopolysaccharide; LPS)는 그람 음성세균의 핵심 독력인자로 알려져 있다. 이러한 세균과 LPS는 파골세포에 의한 골소실을 조절하는 요인 중 하나이다. 그러므로 본 연구에서는 동물모델을 활용하여 A. actinomycetemcomitans와 P. gingivalis의 의한 골소실 여부를 확인하고, 기전규명을 위하여 A. actinomycetemcomitans, P. gingivalis, A. actinomycetemcomitans와 P. gingivalis에서 분리한 LPS에 의한 파골세포분화 영향을 연구하였다. 열사멸한 A. actinomycetemcomitans (HKAa)와 열사멸한 P. gingivalis (HKPg)가 복강으로 투여된 쥐의 대퇴골은 대조군에 비해 감소된 골량을 보여주었다. 이러한 골소실의 증가가 파골세포분화 때문인지 확인하기 위해 파골세포분화를 연구한 결과, bone marrow-derived macrophage (BMM)의 RANKL-매개 파골세포분화를 감소시켰으나, committed osteoclast precursor의 파골세포분화를 유도함을 확인하였다. 세균에 의한 파골세포분화 결과와 동일하게 A. actinomycetemcomitans와 P. gingivalis에서 분리한 LPS 역시 RANKL-매개 파골세포분화는 감소시키고, committed osteoclast precursor의 파골세포분화를 유도하였다. 결과적으로 치주원인균인 A. actinomycetemcomitans와 P. gingivalis는 committed osteoclast precursor의 파골세포분화를 증가시키는데, 이 세균들의 LPS가 핵심 역할을 수행하는 것으로 판단되며 이를 통해 골 흡수를 유발함을 알 수 있었다.

• Journal of Dental Anesthesia and Pain Medicine
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• 제18권1호
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• pp.9-17
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• 2018

Background: The structure and function of bone tissue is maintained through a constant remodeling process, which is maintained by the balance between osteoblasts and osteoclasts. The failure of bone remodeling can lead to pathological conditions of bone structure and function. Remifentanil is currently used as a narcotic analgesic agent in general anesthesia and sedation. However, the effect of remifentanil on osteoclasts has not been studied. Therefore, we investigated the effect of remifentanil on pre-osteoclast (pre-OCs) differentiation and the mechanism of osteoclast differentiation in the absence of specific stimulus. Methods: Pre-OCs were obtained by culturing bone marrow-derived macrophages (BMMs) in osteoclastogenic medium for 2 days and then treated with various concentration of remifentanil. The mRNA expression of NFATc1 and c-fos was examined by using real-time PCR. We also examined the effect of remifentanil on the osteoclast-specific genes TRAP, cathepsin K, calcitonin receptor, and DC-STAMP. Finally, we examined the influence of remifentanil on the migration of pre-OCs by using the Boyden chamber assay. Results: Remifentanil increased pre-OC differentiation and osteoclast size, but did not affect the mRNA expression of NFATc1 and c-fos or significantly affect the expression of TRAP, cathepsin K, calcitonin receptor, and DC-STAMP. However, remifentanil increased the migration of pre-OCs. Conclusions: This study suggested that remifentanil promotes the differentiation of pre-OCs and induces maturation, such as increasing osteoclast size. In addition, the increase in osteoclast size was mediated by the enhancement of pre-OC migration and cell fusion.

• 치위생과학회지
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• 제23권4호
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• pp.282-295
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• 2023

Background: Secretory leukocyte protease inhibitor (SLPI) protects tissues from proteases and promotes cell proliferation and healing. SLPI also reduces periodontal inflammation and alveolar bone resorption by inhibiting proinflammatory cytokine expression in rat periodontal tissues and osteoblasts. However, little is known of the role of SLPI in the expression of osteoclast regulatory factors from osteoblasts, which are crucial for the interaction between osteoblasts and osteoclasts. Therefore, we aimed to determine the effects of SLPI on the regulation of osteoclasts and osteoblasts in LPS-treated alveolar bone and osteoblasts. Methods: Periodontitis was induced in rats using LPS. After each LPS injection, SLPI was injected into the same area. Immunohistochemical analysis was performed with antibodies against SLPI, RANKL, OPG, and Runx2 in the periodontal tissue. RT-PCR and western blotting were performed to determine the expression levels of SLPI, RANKL, OPG, and Runx2 in LPS- and SLPI/LPS-treated MC3T3-E1 cells. SLPI/LPS-treated MC3T3-E1 cells were also stained with Alizarin Red S. Results: Immunohistochemical analysis showed that the expression levels of SLPI, OPG, and Runx2 were higher while that of RANKL was lower in the LPS/SLPI group relative to those in the LPS group. The mRNA and protein expression of SLPI, OPG, and Runx2 was higher in SLPI/LPS/MC3T3-E1 cells than in LPS/MC3T3-E1 cells, and RANKL expression was lower. During differentiation, OPG and Runx2 protein levels were higher whereas RANKL levels were lower in SLPI/LPS/MC3T3-E1 than in LPS/MC3T3-E1 cells on days 0, 4, 7, and 10. In addition, mineralization and matrix deposition were higher in SLPI/LPS/MC3T3-E1 than in LPS/MC3T3-E1 on days 7 and 10. SLPI decreased RANKL expression in LPS-treated alveolar bone and osteoblasts but increased the expression of OPG and Runx2. Conclusion: SLPI can be considered as a regulatory molecule that indirectly regulates osteoclast activation via osteoblasts and promotes osteoblast differentiation.

• 구강회복응용과학지
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• 제40권1호
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• pp.1-12
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• 2024

목적: 세 가지 칼슘 실리케이트 계열 실러가 흰쥐 하악골 조직에 미치는 영향을 조직학적으로 평가하고자 하였다. 연구 재료 및 방법: 흰쥐의 하악골에 와동을 형성하고 즉시 또는 2주 후 희생한 군, 와동에 CeraSeal (CS), AH Plus Bioceramic (AHB), One-Fil (OF) 실러를 각각 주입하여 2주 후 희생한 군으로 무작위 배정하였다. 모든 군을 조직 처리하고 현미경 하에서 와동 내외 평균 골 면적 분율(%)을 계산하고 파골세포를 계수하였고, 각 군의 결과를 비교하고 일원분산 분석 및 Tukey's test로 통계 분석하였다. 결과: 와동 형성 2주 후 희생한 대조군과 AHB 실러 주입 2주 후 희생한 군에서 와동 내 골조직 형성과 파골세포의 존재를 확인할 수 있었고, 다른 군들에 비해 유의하게 높은 와동 내 골 면적 분율(%)을 보여주었다. 다른 군들에서는 와동 내 어떠한 염증이나 이물 반응이 나타나지 않고 파골세포 또한 관찰되지 않았다. 결론: 실험에 사용된 칼슘 실리케이트 계열 실러는 흰쥐 하악골에 주입하였을 때 양호한 골조직 반응을 보였고, 특히 AHB에서 더 높은 와동 내 골조직 형성이 관찰되었다.

• Molecules and Cells
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• 제37권10호
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• pp.747-752
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• 2014

Protein kinase C (PKC) family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. However, the role of PKC in receptor activator of NF-${\kappa}B$ ligand (RANKL) signaling has remained elusive. We now demonstrate that $PKC{\beta}$ acts as a positive regulator which inactivates glycogen synthase kinase-$3{\beta}$ (GSK-$3{\beta}$) and promotes NFATc1 induction during RANKL-induced osteoclastogenesis. Among PKCs, $PKC{\beta}$ expression is increased by RANKL. Pharmacological inhibition of $PKC{\beta}$ decreased the formation of osteoclasts which was caused by the inhibition of NFATc1 induction. Importantly, the phosphorylation of GSK-$3{\beta}$ was decreased by $PKC{\beta}$ inhibition. Likewise, down-regulation of $PKC{\beta}$ by RNA interference suppressed osteoclast differentiation, NFATc1 induction, and GSK-$3{\beta}$ phosphorylation. The administration of PKC inhibitor to the RANKL-injected mouse calvaria efficiently protected RANKL-induced bone destruction. Thus, the $PKC{\beta}$ pathway, leading to GSK-$3{\beta}$ inactivation and NFATc1 induction, has a key role in the differentiation of osteoclasts. Our results also provide a further rationale for $PKC{\beta}$'s therapeutic targeting to treat inflammation-related bone diseases.

• BMB Reports
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• 제47권8호
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• pp.439-444
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• 2014

Inhibition of an increase of osteoclasts has become the most important treatment for osteoporosis. The CXCR4 antagonist, AMD3100, plays an important role in the mobilization of osteoclast precursors within bone marrow (BM). However, the actual therapeutic impact of AMD3100 in osteoporosis has not yet been ascertained. Here we demonstrate the therapeutic effect of AMD3100 in the treatment of ovariectomy-induced osteoporosis in mice. We found that treatment with AMD3100 resulted in direct induction of release of SDF-1 from BM to blood and mobilization of hematopoietic stem/progenitor cells (HSPCs) in an osteoporosis model. AMD3100 prevented bone density loss after ovariectomy by mobilization of HSPCs, suggesting a therapeutic strategy to reduce the number of osteoclasts on bone surfaces. These findings support the hypothesis that treatment with AMD3100 can result in efficient mobilization of HSPCs into blood through direct blockade of the SDF-1/CXCR4 interaction in BM and can be considered as a potential new therapeutic intervention for osteoporosis.