• Journal of Periodontal and Implant Science
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• 제31권4호
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• pp.833-846
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• 2001

Recently, use of natural medicine is getting more attention, and some of them are believed to be effective in　the treatment of periodontitis. Among them, the seeds of safflower(Carthamus tinctrorius L.) have been proven to be effective through its use in bone diseases such as fracture and osteoporosis. During the last few years, studies using the seeds of safflower gown in Korea have been active, and it has been reported that safflower seed extract increase the proliferation and the alkaline phosphatase(ALP) activity of human periodontal ligament fibroblast(hPDLF), osteoblast, and that they promote the mineralization process. In animal studies, when safflower seed extract were administered orally new bone formation was promoted. Recently, in an effort to find out the most effective osteogenic components, among many components of the safflower seed, various safflower seed fraction extracts were obtained by multistep extraction of the safflower components using various solvents. Among these, saf-M-W fraction extracted by methanol and water was most effective in increasing osteogenic potential of osteoblasts. In this study, the effect of safflower seed fraction extract, saf-M-W, on the growth and differentiation of hPDLF and MC3T3-E1 cell was investigated. The toxicity of saf-M-W on both cells was measured using M'IT(3-(4,5dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide) test, and ALP activity was measured using the colorimetric assay of hPDLF. In addition, in MC3T3-El cells, the expression of ALP, bone sialoprotein(BSP) mRNA was observed using Northern blot, and the mineralized nodule formation Was observed using von Kossa stain and phase-contrast microscope. 1. In concentrations below $10{\mu}g/ml$, saf-M-W didn't show any toxicity on hPDLF and MC3T3-El cell. 2. The change in saf-M-W concentration had no effect on the ALP activity of hPDLF. 3. In MC3T-E1 cells, mRNA expressions of ALP and BSP were greater in the experimental group treated with $10{\mu}g/ml$ concentration of saf-M-W compared with the control group. 4. In MC3T3-El cells, abundance of mineralized nodules were formed in the experimental group treated with $10{\mu}g/ml$ Concentration of saf-M-W, while no mineralized nodule was formed in the control group. These results suggest that safflower seed fraction extract, saf-M-W. didn't show any toxicity on hPDLF and MC3T3-E1 cell at concentrations below $10{\mu}g/ml$ and effectively enhanced the differentiation and osteogenic potential of MC3T3-El cell.

• Restorative Dentistry and Endodontics
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• 제36권1호
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• pp.26-36
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• 2011

연구목적: 본 연구는 in vitro 상에서 치수, 치은, 치주인대 세포를 neuropeptide (substance P, Calcitonin gene related peptides (CGRP))및 inflammatory cytokine (TNF-$\alpha$)으로 자극 시 matrix metalloproteinase (MMPs)의 생성 및 발현을 관찰한 것으로 치아와 치아 주변 조직에 염증이 존재할 경우 neuropeptide 및 inflammatory cytokine과 치아 경조직 혹은 치조골의 remodeling에 중요한 역할을 하는 matrix metalloproteinase (MMPs)와의 관계를 규명하고자 하였다. 연구 재료 및 방법: 시편으로는 우식이 없는 건전한 제3대구치(n = 10)를 사용하였으며, 발거 후 즉시 Phosphate buffered saline (PBS)에 보관하고 치아에서 치은과 치주인대 조직을 채취하였다. 치아를 종축으로 절단하고 치수 조직을 채취하여 조각으로 분리한 후 시편을 PBS에서 세 번 세척하였다. Plate에 치수, 치은, 치주인대 시편 조각을 위치시켜 Dulbecco's Modified Eagle Medium (DMEM)을 첨가하여 세포를 배양하였다. 치수, 치은, 치주인대 세포를 culture dish에서 confluence에 도달할 때 까지 배양하여 Fetal Bovine Serum (FBS)가 포함되지 않은 배지로 교환하여, $37^{\circ}C$에서 24시간 동안 배양한 후 Phosphate buffered saline (PBS)로 1회 세척하고 Substance P ($10^{-8}\;M$, $10^{-5}\;M$)가 포함된 배양액과 Mock (배양액만 포함됨)으로 4시간, 24시간동안 자극하였다. CGRP ($10^{-6}\;M$)을 함유한 배양액 및 TNF-$\alpha$(2 ng/mL)를 포함한 배양액으로 각각 24시간동안 세포를 자극하였다. 각각 다른 농도의 TNF-$\alpha$(2 ng/mL, 10 ng/mL, 100 ng/mL)를 포함한 배양액으로 24시간 동안 세포를 자극 한 후 RNase protection assay 및 Enzyme linked immunosorbent assay를 시행하였다. 결과: SP와 CGRP는 치수, 치은, 치주인대 세포의 MMPs발현에 관여 하지 않았다. TNF-$\alpha$로 24시간 자극 시 치수, 치은, 치주인대 세포에서 MMP-1,-12, -13의 발현을 증가시켰다. 반면, TNF-$\alpha$는 치수, 치은, 치주인대 세포들에서 TIMP-3의 발현을 감소시켰다. 서로 다른 농도의 TNF-$\alpha$(2 ng/mL, 10 ng/mL, 100 ng/mL)로 24시간 자극 시 MMP-1과 MMP-13의 발현이 증가하였다. 결론: 치아와 치아 주변 조직에 염증이 존재 시 TNF-$\alpha$가 증가함에 따라 치수, 치은, 치주인대로부터의 치아의 경조직 혹은 치조골의 remodeling에 관여하는 matrix metalloproteinase (MMPs)가 중요한 역할을 하는 것으로 사료된다.

• Journal of Periodontal and Implant Science
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• 제36권3호
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• pp.767-782
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• 2006

이 연구의 목적은 DNA microarray 분석법을 이용하여 건강한 사람치주인대섬유모세포, 건강한 사람치은섬유모세포, 복제노화된 사람치은섬유모세포, 염증성 사람치주인대 섬유모 세포의 유전자 발현 형태를 상호비교하고자 하였다. 환자의 동의하에 충치, 치주염이 없이 교정발치된 치아의 치주인대세포를 배양하여 건강한 치주인대섬유모세포로, 만성치주염으로 발거된 치아에서 채취하여 배양한 세포를 염증성 치주인대섬유모세포로 선정하였다. 구강에서 채취한 치은결체조직에서 배양한 사람치은섬유모세포를 일차 배양한 후 계대배양을 통해 복제 노화를 유도하였다. $-198^{\circ}C$의 액화질소에 저장되어 있던 2, 4, 8, 15, 16세대 세포를 실험에 이용하였다. 위의 모든 세포들은 60 mm 배양접시에서 세포들이 80-90%의 밀생이 될 때까지 5% $CO_2$, $37^{\circ}C$, 100% 습도의 배양기에서 2일 간격으로 10% FBS가 함유된 DMEM 세포 배양액을 교체하면서 세포를 배양하였다. Trizol Reagent (Invitrogen, USA)를 이용하여 제조회사의 지시에 따라 total RNA를 추출하였다. 18S RNA와 28S RNA를 확인한 후 DNA microarray 분석을 실시하였다. 4배수 이상의 변화양상을 비교시 상호 유전자 발현의 차이를 나타내었다. 건강한 사람치은섬유모세포(2세대)와 노화된 치은섬유모세포에서 가장 높은 발현변화를 나타낸 반면 DMC1 dosage suppressor of mck1 homolog, meiosis-specific homologous recombination,은 건강한 치은섬유모세포에서 가장 높게 나타났다. 염증성 치은인대섬유모세포와 건강한 치주인대 섬유모세포를 비교시, Regucalcin은 염증성 치주인대섬유모세포에서 가장 높게 나타났고, Vascular cell adhesion molecule 1도 두 번째로 높게 나타났다. 건강한 치주인대섬유모 세포와 건강한 치은섬유모세포를 비교시, IL-11과 periostin이 치주인대섬유모세포에서 높은 발현을 나타낸 반면, Prostaglandin D2 synthase 21kDa과 Thioredoxin interacting protein은 치은섬유모세포에서 높은 발현을 나타내었다. 염증성 치주인대섬유모세포와 노화된 치은섬유모세포(15세대 이상)를 비교시 149개의 유전자가 유사한 발현 수준을 나타내었다. 이 연구는 노화, 염증, 세포 형태에 따라서 유전자 수준에서 가장 높거나 높은 수준 변화를 보이는 유전자가 다를 수 있음을 나타낸다. 향후, 치주염 환자들에서, 노염, 염증, 세포 특이성에 관한 유전자 표시지를 이용하여 진단하거나 치료에 응용하기 위해서는 더 많은 연구가 필요하리라 사료된다.

• Restorative Dentistry and Endodontics
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• 제31권3호
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• pp.153-160
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• 2006

본 연구는 치수조직, 치은, 치주인대로부터 배양된 조직을 SP (Substance P)로 4시간 SP, CGRP (Calcitonin Gene-related Peptide) , Tumor necrosis factor-$\alpha$ (TNF-$\alpha$)로 8시간 자극 후 RNase Protection Assay를 시행하고, IL-8의 분비량을 측정해 다음 결과를 얻었다. 1. IL-8 mRNA는 모든세포에서 발현됐다. 2. IL-8 mRNA 발현은 SP ($10^{-5}M$)와 SP ($10^{-8}M$)로 4시간 자극 시 증가되지 않았다. 3. IL-8 mRNA 발현은 SP ($10^{-4}M$)와 CGRP ($10^{-6}M$)로 8시간 자극 시 증가되지 않았다. 4. TNF-$\alpha$ (2 ng/ml) 자극 시, IL-8 mRNA 발현이 증가됐다. 5. 치은 세포를 CGRP ($10^{-6}M$)로 8시간 자극 시, IL-8 분비량이 증가했다 (p<0.05). 6. 치주인대 세포를 SP ($10^{-4}M$)로 8시간 자극 시 IL-8 분비량이 증가했다 (p<0.05).

• Journal of Periodontal and Implant Science
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• 제29권4호
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• pp.803-823
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• 1999

In order to reveal immunopathogenesis of periodontal tissue destruction, it is important to clarify the molecular mechanism of trafficking and retention of activated leukocytes, including monocytes/macrophages. Gingival fibroblasts may be involved in the regulation of inflammatory cell accumulation in the extravascular periodontal connective tissues via cytokine production and surface expression of adhesion molecules. In this study, it was investigated the molecular basis for the adhesive interactions between monocytes and fibroblasts such as peri-odontal ligament fibroblast(PDLF), human gingival fibroblast(HGF), and human dermal fibroblast(HDF). First, it was examined the evidence whether monocyte-fibroblast cell contact may cause signal transduction in fibroblasts. Being directly in contact with fixed human monocyte cell line THP-1, or U937, upregulation of IL-6 production, $TNF-{\alpha}$ mRNA expression and increased cell proliferation could be seen for fibroblasts. IL-6 production induced by monocyte- fibroblast coculture were further increased when fibroblasts had been pretreated with $IFN-{\gamma}$ or $IL-1{\beta}$ , and monocytes with LPS. Next, it was examined the expression of ICAM-1 which has been known to be involved in accumulation and activation of leukocytes in inflammatory diseases such as periodontitis. ICAM-1 was upregulated up to 10-fold on PDLF, HGF, and HDF by exposure to $IFN-{\gamma}$ or $IL-1{\beta}$. Furthermore, anti-ICAM-1 monoclonal antibody clearly blocked cocultureinduced IL-6 production by fibroblasts, suggesting that $ICAM-1/{\beta}_2$integrin pathway is involved in periodontal fibroblastmonocyte interaction. Overall, these findings provide evidence that periodontal fibroblasts could be involved in the accumulation and retention of monocytes/macrophages in periodontal inflammatory lesion at least in part by ICAM-1 expression. In addition, periodontal fibroblast-monocyte interaction could cause activation signals in fibroblasts intracellularly which result in cytokine production and cell proliferation. Thus, periodontal fibroblasts are speculated to play an important role in immunoregulation and tissue destruction in chronic periodontal diseases by interaction with monocytes/macrophages.

• Journal of Periodontal and Implant Science
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• 제30권4호
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• pp.895-913
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• 2000

It is becoming increasingly clear that human gingival fibroblasts(HGF) may play a role in regulating immune responsiveness in inflammatory periodontal lesions. Stimulation of HGF with locally-secreted T cell cytokine $IFN_{\gamma}$ induces human leukocyte antigen class II(HLA II) expression on HGF, which is one of the characteristic feature of professional antigen presenting cells(pAPC). However, $IFN_{\gamma}$-treated HGF and other nonprofessional antigen presenting cells(npAPC) are known to be ineffective or less effective antigen presenter to resting T cells. This study, therefore, was undertaken in an effort to elucidate the differences in expression of cell surface molecules between npAPC in periodontal tissues, such as HGF and periodontal ligament fibroblasts(PDLF), and pAPC such as monocytes/macrophages. Using flow cytometry, the levels of cell surface expression of HLA-D, ICAM-1, LFA-3, and B7-1, which are involved in antigen presentation, were determined in HGF, PDLF and human myelomonocytic cell line THP-1. $IFN_{\gamma}$ clearly induced HLA-D expression on both of fibroblasts and monocytes dose dependently. However, expression level on monocytes were 4 to 5 times higher than that on fibroblasts, and induction rate was faster in monocytes than in fibroblasts. The levels of ICAM-1 expression on fibroblasts and monocytes were enhanced by $IFN_{\gamma}$ in a dose dependent manner. On the other hand, the expression of LFA-3 molecule, which could be detected in fibroblasts and monocytes without cytokine stimulation, was no more enhanced by addition of $IFN_{\gamma}$. B7-1, important costimulatory molecule in T cell activation and proliferation, was not detected on both of fibroblasts and monocytes even when stimulated with $IFN_{\gamma}$, except on monocytes fully differentiated by pretreatment of PMA and treated by $IFN_{\gamma}$. These results suggest that delayed expression of HLA-D and absence of B7-1 on $IFN_{\gamma}$ - treated fibroblasts may at least in part be involved in the ineffectiveness of fibroblasts as primary APC. And it is postulated that although periodontal fibroblasts may not serve as primary APC in normal periodontium, sustained expression of HLA II on ubiquitous fibroblasts in inflammatory lesions may perpetuate immune responses and produce chronic inflammation and tissue injury.

• International Journal of Oral Biology
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• 제41권4호
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• pp.163-173
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• 2016

Flavonoid myricetin, usually found in tea and medicinal plants, has antioxidant and anti-inflammatory effects. Our objectives in this study were to verify the effects of myricetin on periodontal ligament fibroblasts (PDLFs) under inflammatory conditions and to observe its effects on osteoclast generation and on cytokine expression in RAW264.7 cells. To determine the effects of myricetin on PDLFs, we examined the expression and activity of proteolytic enzymes, including MMP-1, MMP-2, and MMP-8, which all play an important role in chronic periodontitis. We observed the effects of myricetin on intracellular signal transduction to verify the molecular mechanism involved. By measuring the formation of TRAP-positive multinucleated cells and the expression and activity of MMP-8, we were able to assess the effects of myricetin on osteoclast generation. In addition, by measuring the secretion of IL-6 and NO, we could evaluate the effects of myricetin on inflammatory mediators. We found that Myricetin had no effect on the viability of the PDLFs in the presence of inflammation, but it did decrease both the expression of MMP-1 and MMP-8 and the enzyme activity of MMP-2 and MMP-8 in these fibroblasts. Myricetin also decreased the lipopolysaccharide-stimulated phosphorylation of JNK, p38 signaling, IKKB, AKT, and p65RelA in the PDLFs. In the RAW264.7 cells, myricetin inhibited both the expression and the activity of MMP-8. Furthermore, Myricetin not only suppressed the generation of LPS-stimulated osteoclasts, but it also slightly inhibited LPS-stimulated degradation of IkB and decreased the release of LPS-induced IL-6 and NO. These findings suggest that myricetin alleviates the tissue-destructive processes that occur during periodontal inflammation.

• Journal of Periodontal and Implant Science
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• 제37권1호
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• pp.45-51
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• 2007

This study evaluated the possibility of the 3-dimensional attachment of human periodontal ligament fibroblasts to a periodntally involved root surface after an EDTA treatment in vitro. The human PDL fibroblasts were isolated from the middle third of the root of periodontally healthy teeth extracted for orthodontic reasons. The cells were cultured in a medium containing Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at $37^{\circ}C$ in humidified air containing 5% $CO_2$. Eight single-rooted teeth were obtained from patients diagnosed with periodotitis. After scaling and root planing, four teeth were etched with 24% ethylenediaminetetracetic acid (EDTA) for two minutes (Experimental group). The other four teeth were not treated with EDTA and were used as the control group. The human PDL fibroblasts were placed in the total root surface and cultured for 4 weeks. The teeth were fixed in 2.5% glutaraldehyde in PBS before preparation for the scanning electron microscopy (SEM) examination. The human PDL fibroblasts showed a healthy morphology on the root surfaces treated with EDTA (Experimental group) and a relatively unhealthy appearance on the treated root surfaces (Control group). This suggests that EDTA favorably affects the 3-dimensional attachment of human PDL fibroblasts cultured on the root surfaces. which may play an important role in periodontal healing and regeneration.