• 대한통합의학회지
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• 제8권3호
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• pp.53-62
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• 2020

Purpose :Periodontal ligament stem cells maintain tissue homeostasis in periodontal ligament. The purpose of this study was to determine the characteristics of periodontal ligament stem cells isolated from premolar teeth and observe protective effects against oxidative damage caused by Triethylene glycol dimethacrylate (TEGDMA) following treatment with N-acetylsysteine amide (NACA) drug known as enzymatic antioxidants. Methods : Primary periodontal ligament stem cell (PDSC) culture was performed from simply extracted human premolar of orthodontic patients. The characteristics of the primary cultured PDSCs was analyzed using the FACS system. PDSCs was incubated with TEGDMA and NACA. The cell proliferation and survival was determined using WST-1 assay. Collected data were analyzed using SPSS Window 20. Results : Primary cultured PDSCs grow on the floor and develop rapidly in a cluster form from up to 14 days. The morphology of PDSCs showed the spindle-shaped cells and grew directionally. FACS analysis, In addition, positive expression of visible cells were observed in mesenchymal stem cell biomarkers. PDLSCs cell viability was significantly decreased at high concentration in both 3 and 6 hours after TEGDMA treatment. We observed a decrease in the number of cells as well as a morphological change of PDLSCs. Antioxidative effect was notable since the death of PDLSC death was significantly inhibited compared to the control group at 24 and 48 hours after NACA treatment. Conclusion : Therefore, based on the results of this study, further research should be encouraged considering the development of clinical treatment methods using various antioxidants as well as regenerative engineering techniques utilizing periodontal ligament stem cells.

• Journal of Periodontal and Implant Science
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• 제25권3호
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• pp.539-556
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• 1995

Periodontal therapeutic modalities should be re-establishing and regenerating the periodontal tissue previously lost to the disease. To achieve periodontal regeneration, periodontal ligament cells must selective migrate to the deneded root surface, attached and proliferated it. Local pH concentration is one of the most factors that periodontal regeneration. The aims of this study were to examine on biological effects of pH to the human periodontal ligament cells in vitro, especially on the cell morphology, attachment, activity, vitality and viability. Human periodontal ligament cells were cultured from extracted tooth for non-periodontal reason. Immediately after extraction, any soft tissue adhering to the cervical parts of the roots was carefully removed with a sterile curette. To produce different pH levels in the media, Eagle's MEM was adjusted from pH 6.6 to 8.2 in 0.2 intervals with 1 M NaOH and 1 N HCl. After cultivation, Then, Periodontal ligament cells were cultured at pH ranging from 6.6-8.2. attachment assay was done at 1, 2 day incubation and activity assay was done at 1, 2, 3 day incubation. The experiments were evaluated by scaning electron microscopic techniques (HITACHIX-650 Scaning Electron Microanalyzer, Tokyo, Japan), MTT assay, and the cultured periodontal ligament cells were fixed in neutral formalin for 24 hours and immunohistochemically processed by PCNA for proliferating ability. The surviving cells in the medium showed slightly increased volume and widening intercellular distances at low concentration of pH than control group (pH 7.4), and apparently shrinkage at high concentration of pH than control group (pH 7.4). The results of the statistical analysis from the experiment on attachment, vitality and viability were as follows. Attachment of periodontal ligament cells at 1st and 2nd day, similar attachment rate of low concentration pH compared with control value (pH 7.4). But above pH 8.0, attachment rate were statistically significant decrease from control value(P<0.05). Periodontal ligament cell's activities were maximum at pH 7.6 by MTT assay. Similar with control value at low concentration of pH. But, the activities were statistically significant decrease at high concentraration of pH(P<0.05). Cellular proliferating rate (PCNA index) were statistically significant decrease from control value at low and high concentration of pH(p<0.05). This results suggested that hjgh concentration pH, in other words, alkali pH was cytotoxic effects on human periodontal ligament cells in vitro.

• International Journal of Oral Biology
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• 제33권4호
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• pp.131-135
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• 2008

Substance P (SP) is known to be expressed in the nerve fibers of dental pulp and periodontal tissues. It was recently reported that SP expression increased in response to orthodontic force. In the present study, we investigated the effect of SP on expression of mineralization markers and heme oxygenase-1 (HO-1) in human immortalized periodontal ligament (IPDL) cells. Cell viability was measured using a 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay. The expression of mineralization markers, including alkaline phosphatase (ALP), osteonectin (ON) and bone sialoprotein (BSP), and heme oxygenase-1 (HO-1) was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. SP did not significantly change human IPDL cell viability, with the exception of the 24 hour treatment group. Treatment of human IPDL cells with $10^{-10}$ to $10^{-4}M$ SP upregulated mineralization marker and HO-1 expression in a time- and concentration-dependent manner. Our results suggest that SP may modulate osteoblastic cell differentiation of human IPDL cells through a mechanism involving HO-1 expression.

• Restorative Dentistry and Endodontics
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• 제39권1호
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• pp.39-44
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• 2014

Objectives: The purpose of this study was to evaluate in vitro cytotoxicity of the pozzolan cement and other root-end filling materials using human periodontal ligament cell. Materials and Methods: Endocem (Maruchi), white ProRoot MTA (Dentsply), white Angelus MTA (Angelus), and Super EBA (Bosworth Co.) were tested after set completely in an incubator at $37^{\circ}C$ for 7 days, Endocem was tested in two ways: 1) immediately after mixing (fresh specimens) and 2) after setting completely like other experimental materials. The methods for assessment included light microscopic examination, cell counting and WST-1 assay on human periodontal ligament cell. Results: In the results of microscopic examination and cell counting, Super EBA showed significantly lower viable cell than any other groups (p < 0.05). As the results of WST-1 assay, compared with untreated control group, there was no significant cell viability of the Endocem group. However, the fresh mixed Endocem group had significantly less cell viability. The cells exposed to ProRoot MTA and Angelus MTA showed the highest viability, whereas the cells exposed to Super EBA displayed the lowest viability (p < 0.05). Conclusions: The cytotoxicity of the pozzolan cement (Endocem) was comparable with ProRoot MTA and Angelus MTA. Considering the difficult manipulation and long setting time of ProRoot MTA and Angelus MTA, Endocem can be used as the alternative of retrofilling material.

• Journal of Periodontal and Implant Science
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• 제43권4호
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• pp.168-176
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• 2013

Purpose: The purpose of the current study was to examine the effect of dexamethasone (Dex) at various concentrations on the apoptosis and mineralization of human periodontal ligament (hPDL) cells. Methods: hPDL cells were obtained from the mid-third of premolars extracted for orthodontic reasons, and a primary culture of hPDL cells was prepared using an explant technique. Groups of cells were divided according to the concentration of Dex (0, 1, 10, 100, and 1,000 nM). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed for evaluation of cellular viability, and alkaline phosphatase activity was examined for osteogenic differentiation of hPDL cells. Alizarin Red S staining was performed for observation of mineralization, and real-time polymerase chain reaction was performed for the evaluation of related genes. Results: Increasing the Dex concentration was found to reduce cellular viability, with an increase in alkaline phosphatase activity and mineralization. Within the range of Dex concentrations tested in this study, 100 nM of Dex was found to promote the most vigorous differentiation and mineralization of hPDL cells. Dex-induced osteogenic differentiation and mineralization was accompanied by an increase in the level of osteogenic and apoptosis-related genes and a reduction in the level of antiapoptotic genes. The decrease in hPDL cellular viability by glucocorticoid may be explained in part by the increased prevalence of cell apoptosis, as demonstrated by BAX expression and decreased expression of the antiapoptotic gene, Bcl-2. Conclusions: An increase in hPDL cell differentiation rather than cellular viability at an early stage is likely to be a key factor in glucocorticoid induced mineralization. In addition, apoptosis might play an important role in Dex-induced tissue regeneration; however, further study is needed for investigation of the precise mechanism.

• 대한치과교정학회지
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• 제36권6호
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• pp.465-473
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• 2006

교정적 치아이동과 밀접한 관련이 있는 치주인대세포에서, 산화질소가 세포의 증식과 분화에 미치는 영향을 알아보고자 하였다. 인간 치주인대세포를 분주한 후에 산화질소의 donor인 SNP의 농도에 따라 실험군을 구분하고, 세포 활성 염기성 인산분해효소 활성, Western blot 분석을 통한 osteonectin과 bone sialoprotein의 발현 정도를 측정하였다. 0.2 mM 이하의 저농도 SNP를 처리한 실험군에서 대조군과 비교하여 세포 활성, 염기성 인산분해 효소의 활성, 그리고 osteonectin과 bone sialoprotein의 발현이 유의하게 증가하였다. 그러나 1 mM 이상의 고농도 SNP를 처리한 실험군에서는 오히려 감소하였다. 산화질소는 인간 치주인대세포에서 저농도는 세포의 증식과 분화에 촉진 효과를, 고농도는 억제 효과를 보이는 biphasic effect를 갖는다.

• Journal of Periodontal and Implant Science
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• 제40권3호
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• pp.111-118
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• 2010

Purpose: The aim of this study was to investigate whether vitrification in the cryopreservation of periodontal ligament (PDL) cells could be useful for tooth banking. Methods: In step 1, primary cultured human PDL cells were cryopreserved in 100% conventional cryopreservation media and 100% vitrification media (ESF40 media) in different temperatures for 2 weeks. In step 2, a series of modified vitrification formulae named T1 (75% vitrification media + 25% F-media), T2 (50% vitrification media + 50% F-media) and T3 (25% vitrification media + 75% F-media) were used to store PDL cells for 2 weeks and 4 weeks in liquid nitrogen. MTT assay was performed to examine the viability of PDL cells. Results: Maximum cell viability was achieved in cells stored in 100% conventional cryopreservation media at $-196^{\circ}C$ (positive control group) in step 1. Compared to the positive control group, viability of the cells stored in 100% vitrification media was very low as 10% in all test conditions. In step 2, as the percentage of vitrification media decreased, the cell viability increased in cells stored for 2 weeks. In 4-week storage of cells in step 2, higher cell viability was observed in the T2 group than the other vitrification formulae while the positive control group had the highest viability. There was no statistically significant difference in the cell viability of 2-week and 4-week stored cells in the T2 group. Conclusions: These observations indicate 100% vitrification media is not successful in PDL cell cryopreservation. Conventional cryopreservation media is currently the most appropriate media type for this purpose while T2 media would be interesting to test for long-term storage of PDL cells.

• Journal of Korean Dental Science
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• 제8권2호
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• pp.57-64
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• 2015

Purpose: This study was conducted to determine cell viability and differentiation capability of human periodontal ligament (PDL) cells and to elucidate the effects of cryopreservation on the activity of human third molar PDL cells by comparing PDL cells with and without cryopreservation. Materials and Methods: Human PDL fibroblasts obtained from immature third molars were cultured and divided into two groups. The experimental group was cryopreserved with a slow freezing rate of $0.5^{\circ}C/min$ from $4^{\circ}C$ to $-35^{\circ}C$ followed by plunging in liquid nitrogen at $-196^{\circ}C$ and cultured after fast thawing. The control group was cultured without cryopreservation. Cell viability, growth capacity and morphology were evaluated in both groups. Bivariate statistics were used to compare 2 groups and linear mixed model analysis was used to investigate the growth trends difference over time. Result: Cell viability and growth capacity were not significantly different between the 2 groups (P>0.05). Cultured cell of both groups showed fibroblast-like in appearance, and there were no significant differences in morphology between 2 groups. The mixed model analysis revealed no significant difference of growth capacity between 2 groups over time (${\beta}=-0.0009$; P=0.138). Conclusion: This study demonstrates that cryopreservation under control does not affect the biological properties of PDL cells, supporting the feasibility of autotransplantation of cryopreserved impacted third molars.

• 한국산학기술학회논문지
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• 제20권6호
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• pp.95-103
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• 2019

대표적인 치주질환인 치주염은 출혈, 통증 및 치아 손실을 초래하며, 산화적 스트레스는 치주염의 주요 원인으로 알려져 있다. 본 연구는 지방조직 유래 무막줄기세포추출물의 $H_2O_2$ 유도 산화적 손상에 대한 치주염 보호 효과를 확인하고자, 치주인대 섬유모세포(human periodontal ligament fibroblasts; HPLF)를 이용하여 세포 생존율, 염증 및 세포 사멸 관련 단백질 발현을 측정하였다. $H_2O_2$로 산화적 스트레스를 유도한 HPLF 세포에 무막줄기세포추출물 처리 시, $H_2O_2$만을 처리한 control군에 비해 유의적으로 세포 생존율이 증가함을 통해 산화적 손상에 대한 세포 보호 효과를 확인하였다. 또한, 무막줄기세포추출물은 nuclear factor kappa light chain enhancer of activated B cells, inducible nitric oxide synthase 및 interleukin-6와 같은 염증 관련 단백질 발현을 감소시켜 $H_2O_2$로 유도된 염증반응 보호 효과를 확인할 수 있었다. 뿐만 아니라, 무막줄기세포추출물 처리 군은 caspase-9, -3, poly (ADP-ribose) polymerase 단백질 발현 감소와 B-cell lymphoma 2 (Bcl-2)-associated X protein/Bcl-2 비율을 저하시켜 $H_2O_2$ 유도 산화적 손상에 대한 세포사멸 보호 효과를 보였다. 따라서 지방조직 유래 무막줄기세포추출물은 $H_2O_2$ 유도 산화적 손상에 대한 HPLF 세포의 염증반응 및 세포사멸을 저해함으로써 치주염으로부터 보호 효과가 있어, 치주질환 치료용 소재로써의 활용 가능성이 있을 것으로 기대된다.

• 대한치과보존학회:학술대회논문집
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• 대한치과보존학회 2007년도 Autumn Scientific Meeting(the 128th) of Korean Academy of Comservative Dentistry
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• pp.641-641
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• 2007