• Journal of Periodontal and Implant Science
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• v.26 no.1
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• pp.317-333
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• 1996

The main goal of periodontal regeneration is to be achieved by epithelial exclusion, periodontal ligament cell activation or alveolar bone regeneration. The purpose of this study was to investigate on the physico- chemical and biological characteristics of biodegradable chitosan beads. Chitosan beads were fabricated by ionic gelation with sodium tripolyphosphate and they had the size in 300um diameter. As therapeutic agent, flurbiprofen was incorporated into the beads by 10, 20% loading contents. The release of drugs from the chitosan beads was measured in vitro. Also, biological activity tests of flurbiprofen loaded chitosan beads including cytotoxicity test, ihhibition of $IL-1{\beta}$ production, suppression to $PGE_2$ production, collagenase inhibition test, the ability of total protein synthesis, and tissue response were evaluated. The amount of flurbiprofen released from chitosan was 33-50% during 7 days. Minimal cytotoxicity was observed in chitosan beads. Flurbiprofen released from chitosan beads significantly suppressed the $IL-1{\beta}$ production of monocyte, $PGE_2$ production and markedly inhibited collagenase activity. Meanwhile, flurbiprofen released from this system showed increased ability for protein synthesis. Throughout 4 -week implantation period, no significant inflammatory cell infiltrated around chitosan bead and also fibroblast like cell types at the beads - tissue interface were revealed with gradual degradation of implanted chitosan beads. From these results, it was suggested that flurbiprofen loaded chitosan beads can be effectively useful for biocompatible local delivery system in periodontal regeneration.

• The korean journal of orthodontics
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• v.27 no.3 s.62
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• pp.503-513
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• 1997

This study was undertaken to investigate the effect of oxygen tension on the activity and function of the cells derived from human periodontal ligament by measuring cell activity, total protein synthesis, collagen synthesis, $IL-1{\beta},\;IL-6,\;TNF-{\alpha}$ Human periodontal ligament fibroblasts were collected from premolars extracted for orthodontic treatment and incubated in the environment of $37^{\circ}C,\;5\%\;CO_2,\100\%$ humidity. After the fifth to sixth passage they were used for the experiment. Gaspack system to which $0.2{\mu}m$ Millipore filter was attached was connected to mixed-gas tanks. The mixed gases were composed of $10\%\;O_2,\;5\%\;CO_2,\;85\%\;N_2$ in hyoxic group or $90\%\;O_2,\;5\%\;CO_2,\;5\%\;N_2$ in hyperoxic group and $5\%\;CO_2,\;95\%$ air for control. After incubation in $37^{\circ}C$ for 2, 4, 6 days, cell activity was determined by tetrazolium(MTT) assay and total protein synthesis was assayed using sulforhodamine B(SRB). And measurement of 4-hydroxyproline was performed to assess collagen synthesis md $IL-1{\beta},\;IL-6,\;and\;TNF-{\alpha}$ were measured by enzymeimmunoassay. The results were as follows. 1. The cell activity and total protein synthesis in hypoxic group were a little higher than or almost the same with those in control group. 2. In hyperoxic group, the cell activity was lower than that in control group and total protein synthesis was decreased. 3. Collagen synthesis was significantly decreased initially in both hypoxic and hyperoxic group and increased nearly to the level of control group as the duration of cell incubation was longer 4. As a result of enzymeimmunoassay, the amount of cytokines was $IL-6,\;TNF-{\alpha}\;and\;IL-1{\beta}$ in order. 5. $IL-6,\;TNF-{\alpha}\;and\;IL-1{\beta}$ were increased more rapidly in both hypoxic and hyperoxic group than in control group as the duration of cell incubation was longer. 6. There were more $IL-6\;and\;TNF-{\alpha}$ in hyperoxic group than in control group after 6 days, and there were more $IL-6\;and\;TNF-{\alpha}$ after 6 days than after 2 or 4 days in hyperoxic group. These results suggested that oxygen tension might modulate the production of extracellular matrix and cytokines in the cells derived from human periodontal ligament.

• Journal of Periodontal and Implant Science
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• v.29 no.2
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• pp.311-326
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• 1999

This study was investigated to observe the effect of Treponema denticola(TDC) and Treponema lecithinolyticum(TLC) on cultured human periodontal ligament cells. Several experiments were performed including MTT test for the inhibition effect of cell proliferation, LDH test for the cytotoxicity , gelatin zymography for the gelatinase activation and observation of cell morphology change using the phase-contrast microscopy. The results were as follows. 1. The effect of concentration on cell proliferation with time showed an inhibitory effect at high concentration $(150{\mu}g/well)$ for TLC and at low concentration( $9.4{\mu}gwell$ ) for TDC. 2. The effect of time on cell proliferation with concentration showed an inhibitory effect at $150{\mu}g/well$ on 2-day incubation for TLC and at $9.4{\mu}g/well$ on 2-day incubation for TDC. 3. The effect of heat-treated TDC and TLC on the inhibition of cell proliferation showed the difference in the heat-treated group compared to the non-heat treated group for TDC, whereas no difference was found for TLC. 4. The morphological changes which were observed from the phase-contrast microscopy showed the difference in the test group compared to the control group. The loss of spindle-like appearance, cell-to-cell detachment and inhibition of cell proliferation were observed. 5. There was no difference of the cytotoxicity effect between the test group and the control group in the LDH test. 6. The active form of progelatinase A with molecular weight 72kDa was activated in both TDC and TLC on the gelatin zymography. Regarding to the above results, TDC and TLC have an effect on periodontal ligament cells by playing an inhibitory role in cell proliferation and appears to activate progelatinase A which degrades type IV collagen.

• Restorative Dentistry and Endodontics
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• v.31 no.5
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• pp.398-408
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• 2006

The purpose of this study was to verify the usefulness of MTT analysis as a tool of measurement of the periodontal ligament cell viability from the extracted rat molar. A total of 80 Sprague-Dawley white female rat of 4 week-old with a body weight of 100 grams were used. The maxillary left and right, first and second molars were extracted under Ketamine anesthesia. Twenty-four teeth of each group (divided as five groups depending upon the time-lapse after extraction such as Immediate, 10, 20, 40 and 60 minutes) were immersed in $200{\mu}l$ of MTT solution (0.5 mg/ml) and processed for optical density measurements. Another 10 teeth of each group were treated as same as above and sectioned at $10{\mu}m$ for microscopic examination. All measurements values were divided by the value of hematoxylin-eosin staining which represented the volume of each corresponding samples. Immediate and 10 minute groups showed highest MTT values followed by 20, 40, and 60 minutes consecutively. Statistical significance (p<0.05) existed between all groups except in immediate versus 10 minute groups and 40 versus 60 minutes. Histological findings also showed similar findings with MTT results in crystal shape and crystal numbers between the experimental groups. These data indicate that in vivo MTT analysis nay be of value for evaluation of the periodontal ligament cell viability without time- consuming cell culturing processes.

• Journal of Periodontal and Implant Science
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• v.27 no.4
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• pp.883-908
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• 1997

Bone remodeling is characterized by the coupling of osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The process is tightly regualted at the local level by an incompletely known netwotk of peptide and non-peptide fators. Nitric oxide(NO), synthesized by nitric oxide synthetase(NOS) from L-arginine, is becoming recognized as an important bioregualtory molecule in a variety of tissue, but little is known about its possible role in periodontal tissue. The purpose of this study is to investigate the expression of nitric oxide synthetase(NOS) in inflamed gingiva and the effects of cytokine on the expression of NOS protein. The expression of NOS in gingival tissue was evaluated by immunohistochemical staining for $NOS_1$, $NOS_2$, $NOS_3$. The effect of cytokine on the expression of NOS in human periodontal ligament cells and osteoblast-like HOS cells by western blot analysis. Further, we studied that NO functions in periodontal ligament cells as a regulatory molecule. PDL cells incubated with NOS inhibitor and donor. The protein expression, type I collagen & non-collagenous protein, nitrate production and cell proliferation were evaluated The results were as follows. 1. $NOS_1$, $NOS_2$, $NOS_3$ was rarely distributed in healthy gingiva, but stronger stained in gingival epithelium, endothelial cells, and mononuclear cells of inflammed gingiva. 2. The cytokine stimulated $NOS_1$, and $NOS_3$ protein were not inducing or inhibitory effect to compared with control in PDL and HOS cells. 3.Incubation of cells with combination of $TNF-{\alpha}$, $IFN-{\gamma}$, LPS result in a time dependant increase in $NOS_2$ expression, reaching a maximal level after 24 hours of stimulation. 4. The osteonectin protein inhibitory effect of NMA, inhibitor of NOS, was reversed by Larginine in dose dependant manner. 5. NMA decreased cell poliferation and nitrate production, but the inhibitory efffect of NMA was also prevented by the NO donor, sodium nitropruiside. These results suggest that exogenously synthesized NO was playing a stimulating effect on cell proliferation or on non-collagenous protein expression. Therefore NO have an important role in mediation of localized bone destruction associated inflammatory bone disease such as periodontitis.

• Journal of Periodontal and Implant Science
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• v.39 no.2
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• pp.119-128
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• 2009

Purpose: The aim of this study was to evaluate the stemness of cells from canine dental tissues and bone marrow. Methods: Canine periodontal ligament stem cells (PDLSC), alveolar bone stem cells (ABSC) and bone marrow stem cells(BMSC) were isolated and cultured. Cell differentiations (osteogenic, adipogenic and chondrogenic) and surface antigens (CD146, STRO-1, CD44, CD90, CD45, CD34) were evaluated in vitro. The cells were transplanted into the subcutaneous space of nude mice to assess capacity for ectopic bone formation at 8 weeks after implantation. Results: PDLSC, ABSC and BMSC differentiated into osteoblasts, adipocytes and chondrocytes under defined condition. The cells expressed the mesenchymal stem cell markers differently. When transplanted into athymic nude mice, these three kinds of cells with hydroxyapatite /${\beta}$- tricalcium phosphate (HA/TCP) carrier showed ectopic bone formation. Conclusions: This study demonstrated that canine dental stem cells have stemness like bone marrow stem cells. Transplantation of these cells might be used as a therapeutic approach for dental stem cell-mediated periodontal tissue regeneration.

• Journal of Periodontal and Implant Science
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• v.25 no.3
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• pp.497-517
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• 1995

The cells associated with normal defense mechanism in inflammation release free oxygen radicals, hydroxy radicals, and various protease, all of which can damage the surrounding cells(fibroblasts) and matrix molecules(collagen). The objective of this study was to evaluate the effects of "scavenger" enzyme, superoxide dismutase(SOD). to periodontal ligament (PDL) cells. Human PDL cells were cultured from the teeth extracted for non-periodontal reason. Cultured PDL cells in vitro were treated with SOD and LPS according to dosage and culture times. Cellular activity was exaimed by Microtitration(MTT) assay. The quantitative expression of cellular proliferation by proliferating cell nuclear antigen(PCNA), collagen type I and fibronectin by indirect immunocytochemically stain in PDL cells were done. The results were as follows: 1. As only SOD treated group at 2 and 3 days, PDL cell activity was significantly increased at more than 150U(P<0.05). 2. When LPS(0.5, $5{\mu}g/m{\ell}$) and SOD(more than 150U) were added together, it was significantly increased than LPS only treated and control groups at 2 days(P<0.05). 3. When LPS($5{\mu}g/m{\ell}$) and SOD(150, 300U) were added together, PCNA index was significantly increased than LPS only treated and control groups at 2 and 3 days(P<0.05). 4. When LPS($5{\mu}g/m{\ell}$) and SOD(150U) were added together, collagen type I was significantly increased than LPS only treated and control groups at 3 days(P<0.05). 5.When LPS($5{\mu}g/m{\ell}$) and SOD(300U) were added together, fibronectin was significantly increased than LPS only treated and control groups at 3 days(P<0.05). On the above the results, the SOD in association with collagen type I, fibonectin, and PCNA may afford biological protection to oxy-radicals that were typically liberated during normal inflammatory response. Thus, the exogenous application of SOD may be effective in sthe treatment of the localized breakdown associated with chronic periodontal disease.

• Journal of Periodontal and Implant Science
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• v.26 no.2
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• pp.491-510
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• 1996

This study was performed to evaluate the effects of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) on the characteristics of beagle dog's periodontal ligament (BPD) cells and bone marrow (BBM) cells which have the important role on the early stage of periodontal tissue regeneration in vitro. In control group, the cells ($1.5{\times}10^5$cells/ml) were cultured alone with Dulbecco's Modified Eagle's Medium contained with 10% fetal bovine serum, $50{\mu]g/ml$ ascorbic acid, and 10mM/ml ${\beta}-glycerophosphate$. In experimental groups, growth factors, PDGF or EGF(10ng/ml), were added into the above culture condition. And then each group was characterized by examining the cell proliferation rate, amount of total protein synthesis, alkaline phosphatase activity at 1, 5, 9, 13, 17th day after seeding of cells into the culture wells. The results were as follows: 1. Both BPD and BBM cells in PDGF-treated group proliferated more rapidly than non-treated cells. This finding also was observed in EGF-treated group but it was not as prominent as that of PDGF-treated group. The proliferation rates of both cells showed the time-dependent pattern during experimental periods in all three groups. 2. Amount of total protein synthesis was more increased in PDGF-treated group than in control group. But no significant difference between EGF-treated group and control group was observed throughout experimental periods even though the tendency of amount of protein synthesis was time-dependent pattern. 3. Alkaline phosphatase activity also more increased in PDGF-treated group than control group. But slight decrease tendency was seen in both cells of EGF-treated group. From the above results, PDGF appeared to enhance the proliferation and cellular activities including amount of total protein synthesis and alkaline phosphatase activity of BPD and BBM cell, but EGF did not show notable effects. The optimal application of these growth factors was thought to be useful as the adjunctive means in periodontal regeneration procedures.

• Journal of dental hygiene science
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• v.19 no.4
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• pp.254-260
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• 2019

Background: The primary aims of periodontal disease treatment is to remove dental plaque and calculus, the main causes of tooth loss, and restore periodontal tissue destroyed by inflammation. Periodontal disease treatment should also help maintain the alveolar bone, alleviate inflammation, and promote periodontal ligament cell proliferation, which is essential for tissue regeneration. Conventional antibiotics and anti-inflammatories have adverse side effects, especially during long-term use, so there is a need for adjunct treatment agents derived from natural products. The purpose of this study was to investigate whether the herbal flavone baicalein has the osteogenic activity under inflammatory conditions, and assess the involvement of osteoblast immediate early response 3 (IER3) expression. Methods: Human osteoblastic MG-63 cells were cultured with the pro-inflammatory cytokines tumor necrosis factor α and interleukin 1β in the presence and absence of baicalein. Proliferation was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and expression of IER3 mRNA was assessed using real-time polymerase chain reaction. The expression of IER3 protein levels and activation of associated signal transduction pathways were assessed using western blotting. Results: Baicalein increased IER3 mRNA and protein expression synergistically. In addition, baicalein reversed the suppression of cell proliferation, and the downregulation of osteogenic transcription factor runt-related transcription factor 2 and osterix induced by pro-inflammatory cytokines. Baicalein also upregulated the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK 1/2). The upregulation of IER3 by pro-inflammatory cytokines was blocked by pretreatment with inhibitors of AKT, p38, JNK, and ERK 1/2. Conclusion: Baicalein mitigates the deleterious responses of osteoblasts to pro-inflammatory cytokines. Further, IER3 enhanced the effect of baicalein via activation of AKT, p38, JNK, and ERK pathways.

• Journal of Periodontal and Implant Science
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• v.50 no.5
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• pp.291-302
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• 2020

Purpose: The objective of this study was to investigate whether phelligridin D could reduce glucose-induced oxidative stress, attenuate the resulting inflammatory response, and restore the function of human periodontal ligament cells (HPDLCs). Methods: Primary HPDLCs were isolated from healthy human teeth and cultured. To investigate the effect of phelligridin D on glucose-induced oxidative stress, HPDLCs were treated with phelligridin D, various concentrations of glucose, and glucose oxidase. Glucose-induced oxidative stress, inflammatory molecules, osteoblast differentiation, and mineralization of the HPDLCs were measured by hydrogen peroxide (H₂O₂) generation, cellular viability, alkaline phosphatase (ALP) activity, alizarin red staining, and western blot analyses. Results: Glucose-induced oxidative stress led to increased production of H₂O₂, with negative impacts on cellular viability, ALP activity, and calcium deposition in HPDLCs. Furthermore, HPDLCs under glucose-induced oxidative stress showed induction of inflammatory molecules (intercellular adhesion molecule-1, vascular cell adhesion protein-1, tumor necrosis factor-alpha, interleukin-1-beta) and disturbances of osteogenic differentiation (bone morphogenetic protein-2, and -7, runt-related transcription factor-2), cementogenesis (cementum protein-1), and autophagy-related molecules (autophagy related 5, light chain 3 I/II, beclin-1). Phelligridin D restored all these molecules and maintained the function of HPDLCs even under glucose-induced oxidative stress. Conclusions: This study suggests that phelligridin D reduces the inflammation that results from glucose-induced oxidative stress and restores the function of HPDLCs (e.g., osteoblast differentiation) by upregulating autophagy.