• Journal of Periodontal and Implant Science
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• v.36 no.3
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• pp.757-765
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• 2006

Human periodontal ligament fibroblasts (hPDLF) are very important for curing the periodontal tissue because they can be differentiated into various cells. A tissue engineering approach using a cell-scaffold is essential for comprehending today's periodontal tissue regeneration procedure. This study examined the possibility of using an acellular dermal matrix as a scaffold for human periodontalligament fibroblast (hPDLF). The hPDLF was 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 with 5% $CO_2$. The acellular dermal matrix(ADM) was provided by the US tissue banks(USA). Second passage cells were used in this study. The hPDLF cells were cultured with the acellular dermal matrix for 2 days, and the dermal matrix cultured by the hPDLF was transferred to a new petri dish and used as the experimental group. The control group was cultured without the acellular dermal matrix, The control and experimental cells were cultured for six weeks. The hPDLF cultured on the acellular dermal matrix was observed by Transmission Electron microscopy (TEM). Electron micrography shows that the hPDLF was proliferated on the acellular dermal matrix. This study suggests that the acellular dermal matrix can be used as a scaffold for hPDLF.

• Journal of Periodontal and Implant Science
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• v.37 no.4
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• pp.655-669
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• 2007

Reactive oxygen species (ROS) have been implicated in the pathogenesis of various diseases. And vitamin C has shown a protective effect for the tissues. The aim of this study was to evaluate the effects of $H_2O_2$ and ascorbic acid on matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase (TIMP: TIMP-1, TIMP-2), Type 1 collagen, fibronectin, and PDLs22 level in human periodontal ligament fibroblasts (hPDLF) via reverse transcription-polymerase chain reaction (RT-PCR). hPDLF was obtained from a healthy periodontium and cultured in Dulbecco's modified Eagles's medium plus 10% fetal bone serum. The concentration of ascorbic acid in hPDLF was $50{\mu}g/ml$, and that of $H_2O_2$ in hPDLF was 0.03% and 0.00003%. Ascorbic acid only, $H_2O_2$ only and mixture of ascorbic acid and $H_2O_2$ were applied with hPDLF for 1-, 3-, and 30-min. respectively. The gene expression of MMP-1-, TIMP-1-, TIMP-2-, Type 1 collagen-, fibronectin-, and PDLs22-mRNA in hPDLF was analysed via RT-PCR. The results were as follows; 1. hPDLF in response to 30-min. incubation with 0.03% $H_2O_2$ did not show any gene expression. 2. In all the experimental groups, the gene expression of fibronectin mRNA showed the decreased tendency compared to control. 3. In all the experimental groups, the gene expression of TIMP-1 mRNA showed the tendency similar to control. 4. hPDLF in response to 30-min. incubation with 0.03% $H_2O_2$ and ascorbic acid increased mRNA induction for MMP-1. 5. In all the experimental groups, hPDLF increased mRNA induction for PDLs22, collagen type 1, and TIMP-2 compared to control. Within the limited experiments, $H_2O_2$ and ascorbic acid increased mRNA induction for PDLs22, collagen type 1, TIMP-2 in hPDLF. More research will be needed in order to confirm the relative importance of the different roles of ROS and antioxidants in hPDLF from a periodontal regeneration or repair standpoint.

• Journal of Periodontal and Implant Science
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• v.35 no.3
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• pp.623-634
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• 2005

Human periodontal ligament fibroblast(hPDLF) is very important to cure periodontal tissue because it can be diverged into various cells. This study examined the expression of MMP-1, TIMP-1, periodontal ligament specific PDLs22, Type I collagen, Fibronectin, TIMP-2, telomerase mRNA in a replicative senescence of hPDLF. The periodontal ligament tissue was obtained from periodontally healthy and non-carious human teeth extracted for orthodontic reasons at the Chosun University Hospital of Dentistry with the donors' informed consent. The hPDLF cells were cultured in a medium containing Dulbecco's modified Eagle medium(DMEM, Gibco BRL, USA) supplemented with 10% fetal bovine serum(FBS, Gibco BRL, USA) at 37C in humidified air with 5% $CO_2$. For the reverse transcription-polymerase chain reaction(RT-PCR) analysis, the total RNA of the 2, 4, 8, 16, 18, and 21 passage cells was extracted using a Trizol Reagent(Invitrogen, USA) in replicative hPDL cells. Two passage cells, i.e. young cells, served as the control, and ${\beta}-actin$ served as the internal control for RT-PCR The results of this study about cell morphology and gene expression according to aging of hPDLF using RT-PCR method are as follows: 1. The size of hPDLF was increased with aging and it was showed that the hPDLF was dying in the final passage. 2. PDLs22 mRNA was expressed in young hPDLF of the two, four, and six passage. 3. TIMP-1 mRNA was expressed in young hPDLF of the two and four passage. 4. There was a tendency that MMP-1 mRNA was weakly expressed over eighteen. 5. Type 1 collagen mRNA was expressed in almost all passages, but it was not expressed in the final passage. 6. Fibronectin mRNA was observed in all passages and it was weakly expressed in the final passage. 7. TIMP-2 and telomerase mRNA were not expressed in this study. Based on above results, it was observed that PDLs22, Type 1 collagen, Fibronectin, MMP-1. and TIMP-1 mRNA in hPDLF were expressed differently with aging. The study using the hPDLF that is collected from healthy patients and periodontitis patients needs in further study.

• Journal of Periodontal and Implant Science
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• v.38 no.4
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• pp.645-656
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• 2008

Purpose: To evaulate the effects of chlorhexidine and $H_2O_2$ on matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase(TIMP-1, TIMP-2), Type 1 collagen, fibronectin and UNCL expressions in human periodontal ligament fibroblasts (hPDLF). Materials and Methods: $1.2{\times}10^{-1}%$, $1.2{\times}10^{-2}%$ and $1.2{\times}10^{-3}%$ CHX and $3{\times}10^{-3}%$, $3{\times}10^{-4}%$ and $3{\times}10^{-5}%$ $H_2O_2$ and mixture of CHX and $H_2O_2$ were applied to hPDLF for 1 min and 30 min. The mRNA expressions of MMP-1, TIMP-1 and 2, Type 1 collagen, fibronectin and UNCL in hPDLF were analysed by RT-PCR. Results: The result were as follows: 1. The expression of UNCL mRNA was higher than that of other mRNAs. 2. $1.2{\times}10^{-3}%$ CHX increased mRNA expressions of hPDLF as application time increased. 3. $H_2O_2$ lower than $3{\times}10^{-3}%$ increased expression of UNCL mRNA, and did not decrease mRNA expression of hPDLF. 4. hPDLF treatment with $1.2{\times}10^{-1}%$ CHX (with or without $H_2O_2$) resulted in no gene expression. 5. hPDLF treatment with $1.2{\times}10^{-2}%$ CHX (with or without $H_2O_2$) for 30 minutes resulted in no gene expression. Conclusion: Because low concentration of CHX and $H_2O_2$ increased UNCL mRNA expression of hPDLF, low concentraction of CHX and $H_2O_2$ may have an antioxidative effect.

• Journal of Periodontal and Implant Science
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• v.31 no.4
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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.

• Journal of Periodontal and Implant Science
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• v.36 no.3
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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.

• Journal of Periodontal and Implant Science
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• v.37 no.1
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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.