• 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.36 no.2
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• pp.375-383
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• 2006

사람치주인대섬유모세포(human periodontal ligament fibroblast, PDLF)의 기능 손상과 클로르헥시딘(Chlorhexidine, CHX)의 세포독성에 관한 분자적인 기전은 최근까지도 불명확하다. 이 연구의 목적은 PDLF에 의한 골결절 형성에 있어서 CHX의 효과를 평가하고, 치주수술후에 치주병원균의 최소억제농도(minimal inhibitory concentration, MIC)를 평가하고자 하였다. CHX의 세포독성을 평가하기 위해서 MTT assay법을 실시하였다. CHX은 0.12%에서 0.00012%까지, 즉 10-1000배로 희석시킨 후 30, 60, 120초 동안 PDLF에 적용되었고, 석회화된 결절은 alizarin red 용엑에 염색되었다. 치주병원균에 대한 CHX의 MIC가 평가되었다. 이 연구 결과, 세포생존율 검사에서는, 단지 0.12% CHX 에 노출되었던 세포들만 세포 증식 소견을 다소 나타내었다. 모든 CHX 농도(0.12%-0.00012%)에서 PDLF에 의한 골결절 형성은 의미있는 감소를 나타내었다. 또한 치주병원균에 대한 CHX의 MIC는 0.0012%로 나타났다. PDLF의 골결절 형성에 영향을 주는 농도(0.00012%)는 세포독성을 나타내는 농도(0.12%)보다 더 낮은 농도를 보였고, 치주병원균의 최소억제에 필요한 농도는 0.0012%로 나타났다. 이런한 결과들은 통상적으로 상용되는CHX이 PDLF에 의한 골결절 형성에 있어서 영향을 미칠 수 있음을 시사하였다.

• Journal of Periodontal and Implant Science
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• v.23 no.1
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• pp.77-96
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• 1993

This in vitro study was undertaken to observe whether citric acid application aids the attachment and proliferation of human periodontal ligament cells to the root surfaces of periodontally diseased teeth. The roots were prepared so that the comparison could be made among the control healthy root surface, citric acid demineralized and non-demineralized root planted surfaces. Prior to the cell attachment experiment, each groups were prepared for scanning electron microscopic (SEM) examinations of root surface morphology, All specimens were fixed with phosphate buffered glutaraldehydes, postfixed with phosphate buffered osmium tetraoxide and stained with phosphate buffered tannic acid. dehydrated in ethanol, critical point dried, sputter coated with gold and examined under the SEM. In the cell attachement experiment, human cultured periodontal ligament cells at concentration to $4.5{\times}\;10^4\;cells/ml$ were seeded in each culture well which contained prepared roots and incubated for 30min 1, 2, 6, 12 and 24 hours at 37, 5% $CO_2$air incubator. Than the specimens were prepared for SEM examination using, the same methods as described above. In the cell proliferation experiment, $5{\times}\;10^4\;cells/ml$ cells were seeded incubated with the specimens for 6 hours. Then, all of the specimens were moved into fresh culture well and incubated for 24, 48, and 72 hours. The cell counting was done after trypsinization, under light microscope. The results were as follows. When viewed the surface morphology prior to the cell attachment, the non acid treated root planed surface displayed scaling striation and occasional bacteria and calculus. The citric acid treated specimens displayed little debris on the surface and funnel shaped orifices of dentinal tubules. There were no apparent differences in the morphology of cells attached to the control and experiment groups. However, in initial attachement, there was a slight more enhanced appearance in attachment in citric acid treated groups than other root surfaces. After 6 hours of incubation, most of the cells initiated the alteration of cell morphology from ovoid to spindle shapes. After 24 hours of incubation, most of the cells displayed proliferated appearance and connected with each other via numerous processes. In the cell proliferation experiments, there were statistically significant increased number of cells in citic acid treated groups than other groups.

• 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.31 no.1
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• pp.109-122
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• 2001

Gingival fibroblasts(GF) and periodontal ligament fibroblasts(PDLF) are the major cellular components of periodontal soft connective tissues, but the precise molecular biological differences between these cells are not yet known. In the present study, we investigated the expression of S100A4, S100A2 calcium-binding protein and osteoblast-specific factor 2(OSF-2, Periostin) mRNA in GF and PDLF in vitro through the process of reverse transcription-polymerase chain reaction(RT-PCR) and Northern blot analysis in each. Human GF and PDLF were isolated from the gingival connective tissue and the middle third of freshly extracted healthy third molars. They were cultured in Dulbecco's Modified Eagle Medium(DMEM) containing 10% fetal bovine serum and cells in the third passage were used in the experiments. After extracting total RNA from cultured cells, RT-PCR and Northern analysis were performed using S100A4-, S100A2- and Periostin-specific oligonucleotide primers and subcloned cDNA probes in each. In PT-PCR and Northern analysis, the expression of S100A4 and Periostin mRNA in GF was slightly detectable. Interestingly, the expression of S100A4 and periostin mRNA in PDLF was much higher than that in GF. On the other hand, S100A2 mPNA was highly expressed in both GF and PDLF. Since there was a marked difference of S100A4 and Periostin expression between GF and PDLF in vitro, these data suggest that S100A4 and periostin could be used as a useful marker for distinguishing cultured gingival fibroblasts and periodontal ligament cells.

• 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.