• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.33 no.4
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• pp.322-330
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• 2007

Backgrounds: To overcome limited amount of autogenous mucosa for the reconstruction of various mucosal defect including oral mucosal defect, tissue engineered mucosa has been recently introduced. However, introduced conventional technique of tissue engineered mucosa still have serious pitfalls such as long fabrication time, fragility of the reconstructed mucosa, and complexity of the technique. Aim of the study: To examine whether the complex of preconfluent autologous keratinocytes and autologous PRP(Platelet rich plasma) can reconstruct oral mucosa on the muscular flap with easier and faster way compared to conventional mucosal tissue engineering technique. Materials and methods: One day before the operation, oral mucosa(3mm in diameter) were taken and treated for extraction of oral keratinocytes according to the routine manner. The day of operation, oral keratinocytes were prepared in the laboratory and then moved to the operating theater. Autologous PRP was also prepared and then mixed with oral keratinocytes just before grafting on the prepared muscular flap. After keratinocyte-PRP complex was seated, then a sterilized rubber sheet was placed on the graft and the elevated skin flap was replaced and sutured. Biopsies were proceeded at 3, 5, 7, 14 and 21 days. Tissue samples were evaluated clinically, histologically, and immunohistochemically. Results: All of the oral keratinocyte-PRP complexes were successfully grafted on the recipient sites(100%). On 3 days after the operation, 1-2 continuous epithelial layer and many inflammatory cells were observed. On 5 days after the operation, increase of layers of keratinocyte was observed with less inflammatory response. Thickness of the layers was gradually increased from 7 to 21 days after the operation. Cytokeratin confirms epithelium in every specimen. Conclusions: Preconfluent graft of autogenous oral keratinocytes mixed with autogenous PRP have successfully reconstructed myo-mucosal flap. This technique could be a useful alternative for oral mucosal reconstruction in the near future.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.27 no.6
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• pp.483-490
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• 2001

There were many controversies in the cause and progress of tumorigenesis. Recently, studies on the mutation of genes related to the tumor have extensively been performed due to development of molecular biology. Structural and morphological changes of chromosomes, which are related to the abnormal activation of oncogenes or inactivation of tumor suppression genes, transform the normal cells into the tumor cells. p53 and Rb are well known tumor suppressor genes, while oncogenes include c-myc, bcl-2 and ras, etc. When exposed to cell damaging agents, p53 inhibits cell growth by inducing transcription of p21. Especially p73, which is homo-logy of p53, frequently deleted in melanoma, neuroblastoma, colon cancer, and breast cancer, when over produced, p73 activates the transcription of p21, bax-1 and inhibits cell growth by inducing apoptosis. For study on mRNA expression of p21 and p73, normal oral keratinocytes, and cell lines of primary and metastatic oral squamous cell carcinoma were cultured and then electrophoresis and RT-PCR(reverse transcription-polymerase chain reaction) were performed. 1. The mRNA of p21 and p73 in normal oral keratinocyte expressed lower than that of primary squamous cell carcinoma. 2. The mRNA of p21 in metastatic oral squamous carcinoma cell lines was expressed as various patterns compared with that of normal oral keratinocyte. 3. In the metastatic oral squamous cell lines, the mRNA of HN8 expressed higher than that of HN12 or HN19. 4. The mRNA of p73 in primary oral squamous cell lines expressed 4-5 times higher than that of normal keratinocyte. 5. In metastatic oral squamous cell lines, there was no significant expression of p73 mRNA compared with that of normal oral keratinocyte. From the results obtained in this study, mRNA expression of p73 in primary oral squamous cell lines was remarkable, while mRNA expression of p21 and p73 in metastatic oral squamous cell lines were statistically insignificant.

• BMB Reports
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• v.51 no.4
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• pp.188-193
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• 2018

Caffeoylserotonin (CaS), one derivative of serotonin (5-HT), is a secondary metabolite produced in pepper fruits with strong antioxidant activities. In this study, we investigated the effect of CaS on proliferation and migration of human keratinocyte HaCaT cells compared to that of 5-HT. CaS enhanced keratinocyte proliferation even under serum deficient condition. This effect of CaS was mediated by serotonin 2B receptor (5-HT2BR) related to the cell proliferation effect of 5-HT. We also confirmed that both CaS and 5-HT induced G1 progression via 5-HT2BR/ERK pathway in HaCaT cells. However, Akt pathway was additionally involved in upregulated expression levels of cyclin D1 and cyclin E induced by CaS by activating 5-HT2BR. Moreover, CaS and 5-HT induced cell migration in HaCaT cells via 5-HT2BR. However, 5-HT regulated cell migration only through ERK/AP-1/MMP9 pathway while additional Akt/NF-${\kappa}B$/MMP9 pathway was involved in the cell migration effect of CaS. These results suggest that CaS can enhance keratinocyte proliferation and migration. It might have potential as a reagent beneficial for wound closing and cell regeneration.

• 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.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.39 no.2
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• pp.63-70
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• 2013

Objectives: The purpose of this study was to investigate the wound healing effect of primary cultured oral mucosal keratinocytes (OMKs) and to assess their roles in skin wounds. Materials and Methods: OMK labeled with BromodeoxyUridine were scattered onto $1.5{\times}1.5$ cm skin defects of adult female nude mice (OMK group, n=15). For the control, culture media were placed on the wound (control group, n=15). Mice in both groups were sacrificed at three days (n=5), one week (n=5), and two weeks (n=5), and histomorphometric and immunoblot analyses with keratinocyte growth factor (KGF), interleukin (IL)-6, and IL-$1{\alpha}$ antibody were performed for the biopsied wound specimen. To verify the effect of the cytokine, rhIL-$1{\alpha}$ was applied instead of OMK transplantation, and the OMK and control groups were compared with regard to re-epithelialization. Results: Histomorphometric analyses demonstrated faster re-epithelialization in the graft group than in the control group at the third day, first week, and second week. Newly forming epithelium showed maintenance of the histological character of the skin epithelium. The graft group showed superior expression of KGF, IL-6, and IL-$1{\alpha}$ protein, compared with the control group. Similar faster re-epithelialization was observed after treatment with rhIL-$1{\alpha}$ instead of OMK transplantation. Conclusion: We successfully confirmed that the graft of primary cultured OMKs promoted regeneration of skin defects. The mechanism of accelerated wound healing by primary cultured OMKs was attributed to inducement of cytokine expression as required for re-epithelialization.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.31 no.2
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• pp.143-149
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• 2005

Objects : With the advancement of tissue engineering techniques, the effort to develop bioartificial mucosa have been actively delivered. The problem we met with this technique is the lack of mechanical strength between kerationocyte layer and dermal layer, where in the normal skin and mucosa, they are tightly bound with rete ridge structure. The purpose of this study is to understand the 2D and 3D structure of rete ridge of mucosa and skin paddle for rendering more biomimetic structure to the artificial mucosa. Materials and Methods : Oral mucosa and skin from the patients who received the oral surgery and maxillofacial reconstruction were harvested. The epidermis was separated from the dermis after treating with dispase for 12-16 hours. H&E staining was performed for 2D(dimensional) structure study and confocal LASER and SEM study were performed for 3D structure. Mean height(Sc) and arithmetic mean deviation(Sa) of all surface height were calculated. Results : The average height of rete ridge of skin flap was between $67.14{\mu}m$ and $194.55{\mu}m$. That of oral mucosa was between $146.26{\mu}m$ and $167.51{\mu}m$. Pressure bearing area and attached gingiva of oral mucosa showed deeper rete ridges. Conclusion : To obtain the adequate strength of artificially cultured keratinocyte skin and mucosa flap, it is necessary to imitate the original skin and mucosa structure, especially rete ridge. Through this study, 2D and 3D rete ridge structure of normal mucosa and skin was obtained. These results can be used as basis for substrate morphology for keratinocytes culture.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.27 no.6
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• pp.510-522
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• 2005

To assess the clinical applicability of bio-artificial mucosa which was made with autologous oral keratinocytes and human acellular dermal matrix, the formation of basement membrane and stratification of oral keratinocytes were evaluated. Six New Zealand white rabbits (around 2kg in weight) were anesthetized and its buccal mucosa was harvested (1.0 $\times$ 0.5cm size). Oral keratinicytes were extracted and cultured primarily with the feeder layer of pretreated NIH J2 3T3 fibroblast. These confluent cells were innoculated on the human acellular dermal matrix and cultured in multiple layer by air-rafting method. After 3, 5, 7, 10, 14 days of culture, each cultured bio-artificial mucosa was investigated the number of epthelial layer of by H&E stain and toluidine blue stain. The immuhohistochemical methods were used to evaluate the cell division capacity, the formation of basement membrane, and it's property of specific cells (PCNA, cytokeratin 14, laminin). Transmission electromicroscopy was used for the attachment between cells and matrix with the number of hemidesmosome. In result, the numbers of layer of stratified growth of oral keratinocyte cultured on the human acellular dermal matrix and the number of hemidesomal attachment between epithelial cells and human acellular dermal matrix were similar to the layers of normal oral mucosa after 10 days of culture. The cell division rate, basement membrane formation and proliferation rate increased as culture period increased. With these results, bio-artificial mucosa with autologous oral epithelial cells cultured on the acellular dermal matrix had clinically adaptable properties after 10 days' culture and this new bio-artificial mucosa model with relatively short culture time can be expected clinical applicability.

• Imaging Science in Dentistry
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• v.33 no.2
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• pp.97-105
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• 2003

Purpose : To evaluate the effect of all-trans-retinoic acid (ATRA) on the radiosensitivity of normal human oral keratinocyte (NHOK). Materials and methods: Relative cell survival fraction including SF2 (survival fraction at 2 Gy) was calculated on the basis of colony formation assay. Data were fitted to the linear-quadratic model to establish the survival curve and calculate α and β values. Using flow cytometry at 1, 2, 3, 4, and 5 days after exposure to 2 and 10 Gy irradiation, cell cycle arrest and apoptosis were analysed. To understand the molecular mechanism of the radiosensitization of ATRA on NHOK, proteins related with apoptosis and cell cycle arrest were investigated by Western blot analysis. Results: Treatment with ATRA resulted in a significant decrease of SF2 value for NHOK from 0.63 to 0.27, and increased α and β value, indicating that ATRA increased radiosensitivity of NHOK. ATRA increased LDH significantly, but increasing irradiation dose decreased LDH, suggesting that the radiosensitizing effect of ATRA is not directly related with increasing cell necrosis by ATRA. ATRA did not induce appotosis but increased G2 arrest after 10 Gy irradiation, implying that the increased radiosensitivity of NHOK may be due to a decrease in mitosis casued by increasing G2 arrest. ATRA inhibited the reduction of p53 at 3 days after l0Gy irradiation and increased p21 at 1 day after 10 Gy irradiation. Further study is required to determine the precise relationship between this effect and the radiosensitizing effect of A TRA. Conclusion: These results suggested that ATRA increase radiosensitivity by inhibiting mitosis caused by increasing G2 arrest.

• Journal of The Korean Dental Society of Anesthesiology
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• v.14 no.2
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• pp.101-106
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• 2014

Background: Remifentanil, an ultra-short-acting mu-opioid receptor agonist, is unique from other opioids because of its esterase-based metabolism, minimal accumulation, and very rapid onset and offset of clinical action. Remifentanil can prevent the inflammatory response and can suppress inducible nitric oxide synthase expression in a septic mouse model. However, the effects of remifentanil on human keratinocyte and autophagy have yet to be fully elucidated during hypoxia-reoxygenation. Here we investigated whether remifentanil confers protective effect against hypoxia-reoxygenation in human keratinocyte and, if so, whether autophagy mediates this effect. Methods: The human keratinocytes were cultured under 1% oxygen tension. The cells were gassed with 94% $N_2$, and 5% $CO_2$ and incubated for 24 h at $37^{\circ}C$. To determine whether the administration of affects human keratinocytes hypoxia-reoxygenation injury, cells were then exposed to various concentrations of remifentanil (0.01, 0.1, 0.5 and 1 ng/ml) for 2 h. After remifentanil treatment, to simulate reoxygenation and recovery, the cells were reoxygenated for 12 h at $37^{\circ}C$. Control group did not receive remifentanil treatment. Normoxia group did not receive hypoxia and remifentanil treatment for 36 h. 3-MA group was treated 3-methyladenine (3-MA) for 1h before remifentanil treatment. Cell viability was measured using a quantitative colorimetric assay with MTT, showing the mitochondrial activity of living cells. Cells were stained with fluorescence and analyzed with Western blot analysis to find out any relations with activation of autophagy. Results: Prominent accumulation of autophagic specific staining MDC was observed around the nuclei in RPT group HaCaT cells. Similarly, AO staining, red fluorescent spots appeared in RPT group HaCaT cells, while the Normoxia, control and 3-MA groups showed mainly green cytoplasmic fluorescence. We here examined activation of autophagy related protein under H/R-induced cells by Western blotting analysis. Atg5, Beclin-1, LC3-II (microtubule-associated protein 1 light chain 3 form II) and p62 was elevated in RPT group cells. But they were decreased when autophagy was suppressed by 3-MA (Fig. 5). Conclusions: Although the findings of this study are limited to an in vitro interpretation, we suggest that remifentanil may have a beneficial effect in the recovery of wound from hypoxia-reoxygenation injury.

• Journal of the Korea Convergence Society
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• v.10 no.4
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• pp.81-89
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• 2019

This study was analyzed about the relationship between culture microenvironment and cell differentiation of HPV 16 E6/E7-transfected immortalized oral keratinocyte(IHOK). By the alteration of culture environment, IHOK-EF and IHOK-EFKGM were obtained, and the modulation of cell properties was observed by cell proliferation assay, immunofluorescence, microarray, and quantitative real-time PCR analysis. IHOK-EF losed the properties of epithelial cells and obtained the properties of mesenchymal cells, and in the result of microarray analysis, genes related to the inhibition of differentiation such as IL6, TWIST1, and ID2 were highly expressed in IHOK-EF. When the culture environment was recovered to initial environment, these changes were recovered partially, presenting the return of genes involved in the inhibition of differentiation such as IL6, and ID2, particularly. This study will contribute to understand adjustment aspect for cell surviving according to the change of culture microenvironment in the study for determining the cell characteristic, and facilitate therapeutic approach for human disease by applying surviving study according to the change of cancer microenvironment.