• Journal of Periodontal and Implant Science
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• v.38 no.3
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• pp.543-550
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• 2008

Purpose: The purpose of this study was to investigate induction of cytokine expression in human gingival fibroblasts (HGFs) by whole cell and the components of T. forsythia. Material and Methods: After HGFs were treated with lipopolysaccharide (LPS), membrane protein isolated from T. forsythia or culture media of T. forsythia, the induction of interleukin (IL)-1, IL-6 and IL-8 was examined with real-time PCR and ELISA. Their induction ability of cytokines was compared with whole bacteria. Result: The expression of IL-6 and IL-8 was significantly induced in HGFs by whole bacteria and membrane protein. The expression of IL-$1{\beta}$ was induced by membrane protein of T. forsythia, not by whole bacteria. LPS and condition media of T. forsythia slightly activated HGFs. Conclusion: The membrane protein of T. forsythia could be one of virulence factors.

• Journal of Periodontal and Implant Science
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• v.32 no.2
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• pp.389-402
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• 2002

BMP can induce ectopic bone formation when implanted into sites such as rat muscle and can greatly enhance healing of bony defects when applied exogenously. In addition, BMP stimulated osteoblastic differentiation in vitro in various types of cells. The aim of this study was to investigate the effect of recombinant human bone morphogenetic protein(rhBMP-2) on the proliferation and osteoblastic differentiation of human periodontal ligament cells and gingival fibroblasts. The cell number and alkaline phosphatase activity were measured in 3 experimental groups of human periodontal ligament cells and gingival fibroblasts (control group, rhBMP-2 50ng/ml group, and rhBMP-2 100ng/ml group) at 1 and 2 weeks after culture. At the same time, total RNA of cultured cells were extracted and reverse trascription polymerase chain reaction(RT-PCR) was performed to determine the expression of mRNA of bone matrix protein. RhBMP-2 had no effect on the cell proliferation of human periodontal ligament cells and gingival fibroblasts. Alkaline phosphatase activity was elevated significantly by rhBMP-2 in both cells. And periodontal ligament cells showed significantly higher alkaline phosphatase activity than gingival fibroblasts. ${\beta}$-actin, type I collagen, alkaline phosphatase, BMP-2 mRNA were expressed in all of the samples. Osteopontin, osteocalcin mRNA were expressed in all periodontal ligament cell groups, and rhBMP-2 50ng/ml group, rhBMP-2 100ng/ml group of 2 week culture period of gingival fibroblasts. Bone sialoprotein mRNA was only expressed in rhBMP-2 50ng/ml group and rhBMP-2 100ng/ml group of 2-week culture period. These results suggest that rhBMP-2 stimulates osteoblastic differentiation in human periodontal ligament cells and gingival fibroblasts in vitro.

• Journal of the korean academy of Pediatric Dentistry
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• v.24 no.1
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• pp.204-219
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• 1997

The use of fluoride is one of the most effective methods for caries prevention. Fluoridation of public water supply has been recognized, for many years, as an effective way to reduce dental caries. The fluoride supplement has been recommended when the natural fluoride was unavailable or below the optimal range. However the mechanism of caries prevention by fluoride has not yet been clarified and it is well known that an overdose of fluoride results inacute and chronic toxicity, especially dental fluorosis. Fluoride mouthrinsing solution is widely used in dentistry due to its effectiveness in carrying anticariogenic action. Understanding the effects of fluoride mouthrinsing solution on human gingival fibroblasts will provide the safety rationale for its use during the caries preventive therapy. The purpose of this study was to evaluate the cytotoxic effect of fluoride mouthrinsing solution on the human gingival fibroblast in vitro. The human gingival fibroblasts were cultured from healthy gingiva on the extracted deciduous teeth of children. Cells were inoculated into a 24-well plate with $1{\times}10^4cells/well$ of medium at $37^{\circ}C$, 100% humidity, 5% $CO_2$ incubator for 24 hours. And the cells were counted by using the hemocytometer at each designed study. Human gingival fibroblasts were cultured in growth medium after one minute application range of 0.02%-0.2% NaF solution and 0.1% $SnF_2$ solution. The cells used in this study were between fifth to eighth passage number. The cell morphology was examined by inverted microscope and cell proliferation was measured by incorporating $[^3H]$-thymidine into DNA. DNA synthesis by human gingival fibroblasts was assessed by $[^3H]$-thymidine uptake assays while the cell activity was measured by MTT assay. Each concentrated fluoride mouthrinsing solution was estimated for its biocompatability with fibroblasts by the tissue culture technique. The results of this study were as follows : 1. It was observed that at 0.05%, 0.2% NaF mouthrinsing solution the cytoplasmic processes became globular. When 0.1% $SnF_2$ mouthrinsing solution was applied, the cytoplasmic process and cell morphology were disappeared. 2. DNA synthetic activity was reduced regardless of the concentration of the fluoride mouthrinsing solution. However, the result is statistically insignificant except 0.1% $SnF_2$ mouthrinsing solution(p<0.05). 3. Our results indicate that 0.02%, 0.05% concentrations of NaF mouthrinsing solution caused minimal cytotoxicity. But 0.2% NaF and 0.1% $SnF_2$ concentration were a significant difference between the cell activity in the experimental group and control group (p<0.05). 4. After appling 0.05% & 0.02% NaF fluoride mouthrinsing solution, cell activity was restored to the control groups level according to incubating time. The results suggest that direct exposure to fluoride solution inhibits gingival fibroblast activity. Therefore, for the most effective use of fluoride use, lowering the concentration of fluoride mouthrinsing is advisable because it maintains biocompatability and free ion in the oral fluid.

• 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.34 no.1
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• pp.205-221
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• 2004

Periodontal ligament(PDL) cells have been known as playing an important roles in periodontal regeneration and gingival fibroblasts are also important to periodontal regeneration by forming connective tissue attachment. There were rare studies about the gene expression patterns of PDL cells and gingival fibroblasts, therefore in this study, we tried cDNA microarray-based gene expression monitoring to explain the functional differences of PDL cells and gingival fibroblasts in vivo and to confirm the characteristics of PDL cells. Total RNA were extracted from PDL cells and gingival fibroblasts of same person and same passages, and mRNA were isolated from the total RNA using Oligotex mRNA midi kit(Qiagen) and then fluorescent cDNA probe were prepared. And microarray hybridization were performed. The gene expression patterns of PDL cells and gingival fibroblasts were quite different. About 400 genes were expressed more highly in the PDL cells than gingival fibroblasts and about 300 genes were more highly expressed in the gingival fibroblasts than PDL cells. Compared growth factor- and growth factor receptor-related gene expression patterns of PDL cells with gingival fibroblasts, IGF-2, IGF-2 associated protein, nerve growth factor, placental bone morphogenic protein, neuron-specific growth- associated protein, FGF receptor, EGF receptor-related gene and PDGF receptor were more highly expressed in the PDL cells than gingival fibroblasts. The results of collagen gene expression patterns showed that collagen type I, type III, type VI and type VII were more highly expressed in the PDL cells than gingival fibroblasts, and in the gingival fibroblasts collagen type V, XII were more highly expressed than PDL cells. The results of osteoblast-related gene expression patterns showed that osteoblast specific cysteine-rich protein were more highly expressed in the PDL cells than gingival fibroblasts. The results of cytoskeletal proteins gene expression patterns showed that a-smooth muscle actin, actin binding protein, smooth muscle myosin heavy chain homolog and myosin light chain were more highly expressed in the PDL cells than gingival fibrobalsts, and ${\beta}-actin$, actin-capping protein(${\beta}$ subunit), actin- related protein Arp3(ARP) and myosin class I(myh-1c) were more highly expressed in the gingival fibroblasts than PDL cells. Osteoprotegerin/osteoclastogenesis inhibitory factor(OPG/OCIF) was more highly expressed in the PDL cells than gingival fibroblasts. According to the results of this study, PDL cells and gingival fibroblasts were quite different gene expression patterns though they are the fibroblast which have similar shape. Therefore PDL cells & gingival fibroblasts are heterogeneous populations which represent distinct characteristics. If more studies about genes that were differently expressed in each PDL cells & gingival fibroblasts would be performed in the future, it would be expected that the characteristics of PDL cells would be more clear.

• Journal of Periodontal and Implant Science
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• v.24 no.3
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• pp.661-670
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• 1994

It is well known that the application of dressings after periodontal surgery have benefits to provide the comforts to patient and to promote the healing process with action of bleeding control and temporary stabilization for the operated mobile teeth. But until recently the relationship between periodontal dressings and cells which are composed of periodontium has not been clear. The purpose of this study was to evaluate the cytotoxic effect of soluble extracts from the four different kinds of periodontal dressings, two of them were eugenol type (K.H.pack, Wondrpak) and the others were non-eugenol type (Coe-pak, Periocare), on the human gingival fibroblasts in vitro. Human gingival fibroblasts were primarily cultured from gingiva around third molar during the extraction for preventive purposes. Extracts solution were prepared with culture medium by means of imersing the consistent size of periodontal dressing made from plastic mold. Cell were inoculated into the 24 well plate with $3\;{\times}\;10^4\;cells/well$ of medium at $37\;^{\circ}C$, 100% of humidity, 5% of $CO_2$, incubator for 24 hours. After discard of the supernatant of medium, those cells were cultured with original, 1/2, 1/5, 1/10 diluted soluble extract for 24, 48 and 72 hours, and counted the number of cells using the hemocytometer at each designed time and concentration. Also, the cytotoxic effect of soluble extract was measured by Wataha's MTT assay method. In briefly, cells were inoculated and cultured into 96 well culture plate with $2\;{\times}\;10^4\;cells/well$ for 24 hours. Soluble extracts were applied to cultured cells and incubated for 48 hours at same condition. $50\;{\mu}l$ of MTT solution and DMSO were added into each well for the detection of absorbance with ELISA reader. The measured data were calculated by value of colorimetric assay for survival rate. The results were as follows ; In the case of eugenol type of dressing, original, 1/2 and 1/5 diluted extracts of K.H.pack showed very low survival rate. And original extract of Wondrpak showed strong cytotoxic effect and 1/2 diluted extract showed moderate cytotoxic effect. In the case of Non-eugenol type of dressings, only original extract of Coe-pak revealed strong cytotoxic effect and Periocare had little cytotoxic effect. It is concluded that eugenol type of dressings showed more cytotoxic effect than non-eugenol types. This study suggest that use of non-eugenol dressings after periodontal surgery is recommended.

• Journal of Periodontal and Implant Science
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• v.25 no.2
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• pp.279-289
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• 1995

The most important object of periodontal treatment is the perfect regeneration of destructed periodontal tissue. The healing of periodontal lesion is affected by several cells & factors, which result in formation of long juntional epithelium, root resorption, bony ankylosis or connective tissue attachment. And ideal healing is enhanced by epithilial exclusion or periodontal ligament cell activation. In this investigation, I studied the effect of Zizyphus Fructus extract which enhances biologic activity& collagen synthesis, on the chemotaxis & cell nature. The cells were obtained from interdental area & middle third area of the freshly extracted teeth for the orthodontic purpose. And they were fully incubated in${\alpha}-MEM$ solution containing $100{\mu]g/ml$ penicillin & $100{\mu]g/ml$ streptomycin followed by 6 generation incubation. The test cells were collected by trypsin-EDTA & centrifuge in the fully incubated cells, counted by Hernacyotmeter, incbated $5{\times}10^5/ml$ cells for 24 hours, re-incubated 24 hours in media containing natural extract and photographed. The cells were incubated for 4 hours in 48 well microchemotaxis chamber bisecting upper & lower chamber by 8ug/m pore polycarbonate membrane coating 5mg/ml gelatin solution. The migrated cells in microscope were counted, which meaned cell chemotaxis activity. The study had shown that the morphology of cell was spindle-shaped as the control group, and the subextract test groups were not significantly different. In gingival fibroblasts, the chemotaxis effect of PDGF was statistically significant compared to control group. The Zizyphus Fructus extract was more or less enhanced chemotaxis effect and in $1{\mu}g/ml$ concentration the chemotaxis effect was slightly elevated compared with $10{\mu}g/ml$ concentration. But, among the subextracts, it was not significantly defferent. In PDL cells, the chemotaxis effect of PDGF in statistically significant, and the zizyphus Fructus extract had shown the enhanced effect. The effect was slightly higher in $1{\mu}g/ml$ concentration than 10g/ml concentration,and no significance among the subextracts.

• Journal of Periodontal and Implant Science
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• v.26 no.4
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• pp.841-858
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• 1996

Epidermal growth factor(EGF) is one of polypeptide growth factors. EGF has been reported as a biological mediator which regulates activities of wound healing process including the cell proliferation, migration and metabolism. The purposes of this study is to evaluate the effects of EGF on the human periodontal ligament cells and human gingival fibroblast cells that promote regeneration of periodntal tissue. The mitogenic effects of epidermal growth factor on human periodontal ligament cells and human gingival fibroblasts were evaluated by determining the incorporation of 5-Bromo-2'-deoxy-uridine into DNA of the cells in a dose dependent manner. The prepared cells were the primary cultured gingival fibroblast and periodontal ligament cells from humans, the fourth or sixth subpassages were used in the experiments. Cells were seeded in DMEM containing 10% FBS. 1, 10, 50, 100, $200{\eta}g/ml$ and epidermal growth factor were added to the quiescent cells for 24 hours, 48 hours and 72 hours. They were labeled with $10\{mu}l/200{\mu}l$ 5-Bromo-2'-deoxy-uridine for the last 6 hours of each culture. The results of the five determinants were presented as mean and S.D.. The results were as follows : The DNA synthetic activity of human gingival fibroblasts were increased dose dependently by epidermal growth factor at 24 hours, 48 hours and 72 hours. The mitogenic effects were similar at the 24 and 48 hours of epidermal growth factor, but the DNA synthetic activity of human gingival fibroblasts generally decreased at 72 hours. The DNA synthetic activity of human periodontal ligament cells were increased dose dependently by epidermal growth factor at 24 hours but the DNA synthetic activity decreased at $200{\eta}g/ml$ of each hour. Generally the maximum mitogenic effects were observed at the 48 hours application of epidermal growth factor. The DNA synthetic activity of human periodontal ligament cells generally decreased lower at 24, 72 hours than at 48 hours the application of epidermal growth factor. In the comparison of DNA synthetic activity between human gingival fibroblasts and human periodontal ligament cells, human periodontal ligament cells had slightly higher proliferation activity than human gingival fibroblasts for a longer time at the high dosage of the epidermal growth factor. In conclusion, epidermal growth factor have important roles in the stimulation of DNA synthesis in human periodontal ligament cells and human gingival fibroblasts, and thus may be useful for clinical applications in periodontal regenerative procedures.

• Journal of Periodontal and Implant Science
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• v.35 no.1
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• pp.87-98
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• 2005

The purpose of present study was to investigate the effects of physiologically active compound (SD62-122) from Phlomidis Radix on the cell cycle progression and its molecular mechanism in human gingival fibroblasts(HGFs). For this purpose, fibroblasts were isolated and cultured from excisioned gingiva during crown lengthening procedure in healthy adult. The following parameter were evaluated that there are cell number counting, MIT assay, cell cycle progression, western blot analysis. The cell number and MIT assay of primary cultured fibroblast was not increased at 2 days but significant increased compare to negative control at 3days(p<0.05). S phase was increased and G1 phase decreased in both $10^{-8}M$ and $10^{-9}M$ of SD62-122 in cell cycle analysis. The cell cycle regulation protein levels of Cyclin $D_1$, Cyclin E, cdk 2, cdk 4 and cdk 6 were increased compare to control in both $10^{-8}M$ and $10^{-9}M$ of SD62-122. The protein levels of p21 and p53 were decreased compare to control, but the level of pRb was not changed compare to control in $10^{-9}M$ of SD2-122. These results suggested that physiologically active compound (SD62-122) isolated from Phlomidis Radix increases the cell proliferation and cell cycle progression in HGFs, which is linked to increased cell cycle regulation protein levels of Cyclin $D_1$, Cyclin E, cdk 2, cdk 4 and cdk 6, and decreased the levels of p21, p53.

• Journal of Periodontal and Implant Science
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• v.27 no.4
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• pp.909-922
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• 1997

The purpose of this study was to evaluate the effect of high glucose on prostaglandin E2 production in human gingival fibroblasts and periodontal ligament cells in vitro. In control group, the cells($5{\times}10^4\;cells/ml$) were cultured with Dulbecco's Modified Eagle's Medium contained with 10% fetal bovine serum, 45mg/dl glucose. In experimental groups, glucose was added to the above culture condition at the final glucose concentrations of 100mg/dl(Test group 1), 200mg/dl (Test group 2) and 400mg/dl (Test group 3). Then each group was tested for the cell proliferation rate, protein levels, and prostaglandin E2 production at $\frac{1}{2}$, 1, 2, 5 days. The results were as follows : 1. As glucose concentration increased, cell proliferation rate decreased significantly at 1, 2, 5 days in human gingival fibroblasts and periodontal ligament cells(P<0.01). 2. In human gingival fibroblasts, test group 2 and 3 showed significantly decreased protein levels as compared to control group at 5 days (P<0.01). 3. In human periodontal ligament cells, as glucose concentration increased, protein levels decreased significantly at 2 days and 5 days(P<0.01). 4. Prostaglandin $E_2$ production in human gingival fibroblasts and human periodontal ligament cells significantly increased as glucose concentration increased(P<0.01). results at 5 days showed obvious difference as compared to those at 2 days. From the above results, high glucose appeared to affect cellular activities including cell proliferation rate, protein levels and enhance prostaglandin $E_2$ production. It was assumed that prostaglandin E2 production by high glucose enhances inflammatory reaction and has a toxic effect on human gingival fibroblasts and human periodontal ligament cells. This study suggests that periodontal disease in diabetic patient is related to prostaglandin $E_2$ production.