• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.34 no.4
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• pp.405-411
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• 2008

Purpose : Cyclosporine A (CsA) is a versatile immunosuppresive agent used to prevent graft rejection syndrome and treat autoimmune disease. One of the major side effects associated with CsA is the abnormal gingival hyperplasia. The purpose of this study was to investigate the relationship between the mRNA expression of the MMP-1, TIMP-1, and $TGF-{\beta}_1$ and the concentration of CsA in cultured human gingival keratinocytes. Materials & Methods : Gingival keratocytes were obtained from gingival tissues of 4 healthy donors. The cultured gingival keratocytes were incubated with increasing concentrations of CsA (0-2000 ng/ml) for 24 hours and the expression of MMP-1, TIMP-1, and $TGF-{\beta}_1$ were determined by reverse transcription-polymerase chain reaction (RT-PCR). Results : The expressions of MMP-1 and $TGF-{\beta}_1$ were not significantly different according to the concentrations of CsA. The expression of TIMP-1 was significantly increased at the CsA concentration of 500 ng/ml. Conclusion : We concluded that the gingival hyperplasia induced by CsA was more related with TIMP-1 than MMP-1 or $TGF-{\beta}_1$ on gingival collagen metabolism in patients treated with CsA.

• International Journal of Oral Biology
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• v.46 no.1
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• pp.39-44
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• 2021

This study aimed to investigate whether neurotransmitter receptors in the nervous system were also expressed in oral keratinocytes. Expressions of various neurotransmitter receptor genes in immortalized mouse oral keratinocyte (IMOK) cells were examined by reverse transcriptase polymerase chain reaction. IMOK cells expressed calcitonin gene-related peptide (CGRP) receptor subunit genes Ramp1 and Ramp3 and glutamate receptor subunit genes Grina, Gria3, Grin1, Grin2a, and Grin2d. Moreover, IMOK cells expressed Adrb2 and Chrna5 that encode beta 2 adrenergic receptor and cholinergic receptor nicotinic alpha 5 for sympathetic and parasympathetic neurotransmitters, respectively. The expression of Bdkrb1 and Ptger4, which encode receptors for bradykinin and prostaglandin E2 involved in inflammatory responses, was also observed at low levels. Expressions of Ramp1 and Grina in the mouse gingival epithelium were also confirmed by immunohistochemistry. When the function of neurotransmitter receptors expressed on IMOK cells was tested by intracellular calcium response, CGRP, glutamate, and cholinergic receptors did not respond to their agonists, but the bradykinin receptor responded to bradykinin. Collectively, oral keratinocytes express several neurotransmitter receptors, suggesting the potential regulation of oral epithelial homeostasis by the nervous system.

• International Journal of Oral Biology
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• v.32 no.1
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• pp.35-43
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• 2007

Tooth loss in elderly is mainly caused by alveolar bone loss via severe periodontitis. Although the severity of periodontitis is known to be affected by age, the aging process or the genetic changes during the aging of periodontal tissue cells are not well characterized. In this study, we investigated the effect of in vitro aging on the change of gene expression pattern in periodontal fibroblasts. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDL) were obtained from two young patients and replicative senescence was induced by sequential subcultivation. When more than 90% cells were positively stained with senescence-associated ${\beta},-galactosidase$, those cells were regarded as aged cells. In aged GF and PDL, the level of phosphorylated retinoblastoma (RB) and $p16^{INK4A}$ protein was significantly decreased and increased, respectively. However, the protein level of p53 and p21, well known senescence-inducing genes, did not increase in aged GF and PDL. Although $p27^{Kip1}$ and $p15^{INK4B}$, another cyclin-dependent kinase inhibitors, were reported to be involved in replicative senescence of human cells, they were decreased in aged GF and PDL. Because senescent cells showed flattened and enlarged cell shape and are known to have increased focal adhesion, we examined the protein level of several integrins. Aged GF and PDL showed increased protein level of integrin ${\alpha}2$, ${\alpha}v$, and ${\beta}1$. When the gene expression profiles of actively proliferating young cells and aged cells were compared by cDNA microarray of 3,063 genes and were confirmed by reverse transcription-polymerase chain reaction, 7 genes and 15 genes were significantly and commonly increased and decreased, respectively, in aged GF and PDL. Among them, included are the genes that were known to be involved in the regulation of cell cycle, gene transcription, or integrin signaling. The change of gene expression pattern in GF and PDL was minimally similar to that of oral keratinocyte. These results suggest that $p16^{INK4A}/RB$ might be involved in replicative senescence of periodontal fibroblasts and the change of gene expression profile during aging process is cell type specific.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.28 no.4
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• pp.277-286
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• 2006

In the present study, I investigated the effects of N-methyl-D-aspartate (NMDA), arachidonic acid (AA), and Nitric Oxide Synthase Inhibitor (NOS-I), alone or in combination, on the viability of cultured primary normal human oral keratinocytes (NHOK). Specifically, we examined whether AA and NOS-I could protect primary NHOK from glutamate cytotoxicity. The purpose of this study was therefore the preliminary study for the examination of the interaction between these agents and NHOK in order to elucidate the mechanisms by which epithelial growth and regeneration are regulated. NHOK were obtained from gingival tissue of 20 individuals aged 20 to 29, and third passage (P3) cells were used for this study. Cell viability was measured by the MTT assay. NMDA and NNA, a calcium dependent NOS inhibitor, induced an initial increase in cell number, which subsequently decreased by the $7^{th}$ day. Low concentration of AA ($0.5\;{\mu}M$ & $1\;{\mu}M$) induced an increase in cell number while high concentrations of AA ($5\;{\mu}M$ & $10\;{\mu}M$) induced a decrease in cell number. The decrease in cell number induced by NMDA at the $7^{th}$ day was abolished by the addition of low concentrations of AA ($0.5\;{\mu}M$ & $1\;{\mu}M$) or NOS inhibitors. Low concentrations of AA ($0.5\;{\mu}M$ & $1\;{\mu}M$) or NOS inhibitors may protect the NHOK from NMDA induced cytotoxicity. These reactions might be related to the NMDA receptor in the cell and the change of the intracellular calcium ion concentration.

• Journal of Korean society of Dental Hygiene
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• v.16 no.3
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• pp.471-477
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• 2016

Objectives: Fluoride is widely used in the prevention and control of dental caries. The purpose of this study is to examine the biological effects of Sodium fluoride on the proliferation of oral normal cell in vitro(MDPC-23, HaCaT, HGF-1 cells). Methods: The proliferation of normal cells and the cyto-skeletal change of normal cells were assessed by WST-1 assay and F-actin stain assay. The statistical significances of the resulting data were analyzed using SPSS(Window 12.0). Results: The sodium fluoride(0-12 mM) treatment decreased the cell viability in a dose and time dependent manner: HaCaT(6 h): $100{\pm}0$, $98{\pm}0.39$, $82{\pm}2.68$, $75{\pm}0.83$, $69{\pm}1$, $67{\pm}1.42%$(p<0.005); HaCaT(24 h): $100{\pm}0$, $98{\pm}1.85$, $54{\pm}0.64$, $43{\pm}0.4$, $38{\pm}0.32$, $36{\pm}0.13%$(p<0.006), MDPC-23(6 h): $100{\pm}0$, $93{\pm}1.48$, $85{\pm}0.28$, $82{\pm}1.58$, $79{\pm}1.48$, $76{\pm}1.93%$(p<0.009); MDPC-23(24 h): $100{\pm}0$, $91{\pm}1.26$, $58{\pm}0.65$, $49{\pm}1$, $44{\pm}0.74$, $2{\pm}0.05%$(p<0.005), HGF-1(6 h): $100{\pm}0$, $97{\pm}2.93$, $89{\pm}5$, $71{\pm}5.42$, $58{\pm}4.82$, $43{\pm}3.47%$(p<0.009); HGF-1(24 h): $100{\pm}0$, $97{\pm}2.05$, $73{\pm}1.73$, $22{\pm}1.61$, $14{\pm}1.73$, $7{\pm}0.85%$(p<0.005). Thus, changes in cell morphology and disruption of filamentous(F)-actin organization were observed in higher concentration. Conclusions: These results suggest that higher concentrations of fluoride lead to a reduce the number of cells and morphology change of normal cell.

• Imaging Science in Dentistry
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• v.30 no.4
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• pp.275-279
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• 2000

Purpose: Radiation damage is produced and viable cell number is reduced. We need to know the type of cell death by the ionizing radiation and the amount and duration of cell cycle arrest. In this study, we want to identified the main cause of the cellular damage in the oral cancer cells and normal keratinocytes with clinically useful radiation dosage. Materials and Methods: Human gingival tissue specimens obtained from healthy volunteers were used for primary culture of the normal human oral keratinocytes (NHOK). Primary NHOK were prepared from separated epithelial tissue and maintained in keratinocyte growth medium containing 0.15 mM calcium and a supplementary growth factor bullet kit. Fadu and Hep-2 cell lines were obtained from KCLB. Cells were irradiated in a /sup 137/Cs γ-irradiator at the dose of 10 Gy. The dose rate was 5.38 Gy/min. The necrotic cell death was examined with Lactate Dehydrogenase (LDH) activity in the culture medium. Every 4 day after irradiation, LDH activities were read and compared control group. Cell cycle phase distribution and preG1-incidence after radiation were analyzed by flow cytometry using Propidium Iodine staining. Cell cycle analysis were carried out with a FAC Star plus flowcytometry (FACS, Becton Dickinson, USA) and DNA histograms were processed with CELLFIT software (Becton Dickinson, USA). Results: LDH activity increased in all of the experimental cells by the times. This pattern could be seen in the non-irradiated cells, and there was no difference between the non-irradiated cells and irradiated cells. We detected an induction of apoptosis after irradiation with a single dose of 10 Gy. The maximal rate of apoptosis ranged from 4.0% to 8.0% 4 days after irradiation. In all experimental cells, we detected G2/M arrest after irradiation with a single dose of 10 Gy. Yet there were differences in the number of G2/M arrested cells. The maximal rate of the G2/M ranges from 60.0% to 80.0% 24h after irradiation. There is no significant changes on the rate of the G0/G1 phase. Conclusion: Radiation sensitivity was not related with necrosis but cell cycle arrest and apoptosis. These data suggested that more arrested cell is correlated with more apoptosis.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.33 no.2
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• pp.124-130
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• 2007

In the present study, I investigated the effects of N-methyl-D-aspartate (NMDA), arachidonic acid (AA), and Nitric Oxide Synthase Inhibitor (NOSI), alone or in combination, on the proliferation of cultured primary normal human oral keratinocytes (NHOK). The purpose of this study was therefore the preliminary study for the examination of the interaction between these agents and NHOK in order to elucidate the mechanisms by which epithelial growth and regeneration are regulated. NHOK were obtained from gingival tissue of 20 individuals aged 20 to 29, and third passage (P3) cells were used for this study. The DNA synthesis was measured by the BrdU assay. Addition of low concentration of AA ($1{\mu}M$) and high concentration of AA with NMDA group (NMDA+AA $10{\mu}M$) made DNA synthesis rate increase significantly at the early stage. Adding NNA ($10{\mu}M$) affected DNA synthesis rate to increase significantly in 4 hours. At the early stage, DNA synthesis was significantly active in the NOS-I with NMDA groups than in the control and the NMDA-only group, while it didn't become statistically meaningful in 24 hours. AA $1{\mu}M$ and NNA $10{\mu}M$ may induce the proliferation of the NHOK independently and NOS-I may induce the proliferation of the NHOK with NMDA. These reactions might be related to the NMDA receptor in the cell and the change of the intracellular calcium ion concentration.