• Korean Chemical Engineering Research
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• v.43 no.1
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• pp.103-109
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• 2005

Isolation and primary culture technique of rabbit oral keratinocytes, and the study for effect of environmental factors on the cell growth were carried out in T75-flask. $1.92{\pm}0.59{\times}10^6$ viable cells were isolated by trypsin enzymatic digestion method from $0.25cm^2$ biopsy of rabbit oral mucosa. Primary culture with 10 mL of K-SFM containing 50 mg/L BPE, $5.0{\mu}g/L$ EGF and 0.15 mM $Ca^{2+}$ showed confluence after 8 days and doubling time was 2.54 days. Effect of medium types, medium volume and supplement types on the cell growth was investigated after the cultured keratinocytes had been harvested from primary confluence. Serum addition showed adverse effect and the increase of serum concentration didn't have an effect on the cell growth. The increase of medium volume decreased the cell growth. The increase of calcium concentration increased the cell growth and 2.0 mM was optimum value. In conclusion, when rabbit oral keratinocytes was cultured in T75-flask, the most effective conditions was to use 10 mL of K-SFM containing 50 mg/L BPE, $5.0{\mu}g/L$ EGF and 2.0 mM $Ca^{2+}$, and doubling time was 1.32 days. This study can provide the useful informations to develop a process and design a bioreactor for the culture of keratinocytes in human body like skin and cornea, as well as mucosa.

• The Korea Journal of Herbology
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• v.26 no.2
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• pp.75-81
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• 2011

Objectives : This study aims at examining the anti-inflammatory effects of Scutellariae Radix extract. Methods : Scutellariae Radix was hot water extracted to make the samples(SR) for the experiment. Their effects were examined on the increase of cell viability in mouse macrophage Raw 264.7 cells, the creation of nitric oxide(NO) in lipopolysaccharide(LPS)-induced Raw 264.7 cells, and the creation of cytokines of interleukin(IL)-$1{\beta}$ and others. Results : The results of the experiment are as follows. 1. The MTT assay was carried out to check the cellular toxicity of the water extract of Scutellariae Radix. The results were found no significant toxicity caused to macrophages by the water extract of Scutellariae Radix. 2. The water extract of Scutellariae Radix significantly restricted the increase of NO in the LPS-induced macrophages after 24-hour culture. 3. The water extract of Scutellariae Radix significantly restricted the creation of IL-6, IL-10, IL-12p40, IL-17, interferon-inducible protein(IP)-10, keratinocyte-derived chemokine(KC), and vascular endothelial growth factor(VEGF) in the LPS-induced macrophages at the concentration of $25{\mu}g/mL$ or higher. Conclusion : The samples(SR) of hot water extract of Scutellariae Radix caused no significant cellular toxicity to macrophages and significantly restricted the creation of NO, IL-6, IL-10, IL-12p40, IL-17, IP-10, KC, and VEGF in the LPS-induced macrophages at $25{\mu}g/mL$ or higher, thus demonstrating significant anti-inflammatory effects.

• Archives of Plastic Surgery
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• v.33 no.5
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• pp.601-605
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• 2006

Purpose: Large skin defect by various causes, should be covered by autologous skin graft. But, the donor site of autologous skin graft is limited and leaves permanent donor scar and contracture. There have been our trial to engineer artificial skin using allogenic dermis (AlloDerm) with basement membrane. Methods: Dermal and epidermal layer were separated by immersing in dipase solution for 30 minutes, and the separated layers were treated with 0.05% trypsin for 10 minutes. And then each layer was cultivated to fibroblasts and keratinocytes on a culture medium. Fibroblasts were first penetrated into basement membrane of allogenic dermis facing down, then allogenic dermis was flipped over to face up and keratinocytes were transplanted to allogenic dermis. Results: Observing artificial skin fabricated in vitro, we found following: 1) The artificial skin opened in air for 5 days formed epidermal layer. In dermal layer, fibroblast was distributed evenly among all. 2) The artificial skin opened in air for 30 days formed thicker and thicker, and it formed basement membrane, spinous and granular layers. PAS stain to confirm existence of basement membrane showed positive reaction. 3) Cytokeratin 10 stain to confirm the formation of epidermal layer showed positive reaction. 4) The formation of thick keratin, lamellar body and desmosome similar to human skin were observed in result of an electron micrograph. Conclusion: As a result of research, the structure seen in normal skin such as rete ridge, is found in reproduced artificial skin. This type of artificial skin can be used as a useful model for investigating skin disease and for clinical application also.

• Archives of Plastic Surgery
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• v.33 no.5
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• pp.587-591
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• 2006

Purpose: Since Rheinwald and Green laid the foundation of epidermal cell culture technology in 1975, many clinicians and scientists have attempted to prove the effectiveness of cultured epidermal autologous(CEA) or homogenetic(CEH) grafts in the wound healing process. In contrast to CEA which cultured from a patient's skin on demand, Cultured Epidermal Homograft(CEH) can be readily available to use on cleaned wounds. In this study, we conducted a controlled clinical trial in order to confirm the effectiveness of CEH in treating partial-thickness 2nd degree burn wounds. Methods: From July 2003 to January 2004 at Hangang Sacred Heart Hospital, we performed a clinical trial in which 35 patients who suffered from 2nd degree burns were enrolled. Wounds were randomly divided into two parts, control and test sites. Test sites were treated with allogeneic keratinocyte sheets ($Kaloderm^{(R)}$, Tegoscience Inc.), a CEH commercialized in Korea. Results: All wounds healed completely without any major complication. The complete healing took $8.3{\pm}2.8$($mean{\pm}S.D.$) days in the test sites as opposed to $11.7{\pm}3.3days$ in the control sites. Conclusion: Based on these results, we concluded that CEH accelerates re-epithelialization of partial thickness burn wounds and CEH can be an safe alternative to skin grafts for 2nd degree burns.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.30 no.4
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• pp.308-315
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• 2004

Aim: The aim of this study was to investigate the mechanism of promoted skin wound healing in skin defects with primary cultured oral mucosal keratinocytes. Materials and methods: Thirty adult female nude mice weighing $20{\pm}2g$ were used for the experiment. Primary cultured and suspended oral mucosal keratinocytes, labeled with BrdU, were scattered onto $1.5cm{\times}1.5cm$ sized full thickness skin defects in the experimental group(N=15), and no grafts were placed the control group(N=15). They were sacrificed at 3 days, 1 week and 2 weeks after the treatment respectively. Histological examination of each wounds were performed to review the healing progress on measuring the length from the wound margin to regenerating epithelial front. The role of keratinocytes were assessed by double immunohistochemical staining with Anti-BrdU and Anti-cytokeratin AE1/3. Results: In the experimental group the wound was completely covered with regenerating epithelia in 2 weeks, but partially regenerated in the control group. The immunohistochemical studies unexpectedly reveal that most of regenerating epithelial cells were induced from marginal epithelium of the margin, not from the scattered keratinocytes. Conclusion: We could successfully confirm that graft of primary cultured oral mucosal keratinocytes promotes the regeneration of skin defects.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.27 no.1
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• pp.17-27
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• 2001

A cell-based assay system for monitoring NF-$textsc{k}$B activity was developed to determine the influence of activated NF-$textsc{k}$B in human HaCaT cells. The pNF-$textsc{k}$B-SEAP-NPT plasmid that permits expression of the secreted alkaline phosphatase (SEAP) reported gene in response to the NF-$textsc{k}$B activity and contains neomycin phosphotransferase (NPT) gene for the geneticin resistance in host cells was constructed and transfected into human keratinocyte cell line HaCaT. Human HaCaT transfectant cells secreted the SEAP enzyme into the culture medium in a time-dependent manner until 72h. NF-$textsc{k}$B activities were measured in the SEAP reporter gene assay using a fluorescent detection method. The treatment of HaCaT cell transfectants with known antioxidants [e.g., N-acetyl-L-cysteine and vitamin C] showed inhibition of NF-$textsc{k}$B activity in a time-and concentration-dependent manner. The phorbol 12-myristate 13-acetate (PMA) known as a stimulator of NF-$textsc{k}$B expression demonstrated that it increased NF-$textsc{k}$B activity in a time- and concentration-dependent manner. This assay system could be used to determine the quantitative measurement of NF-$textsc{k}$B activity in the human skin and allow the screening of anti-inflammatory agents from various synthetic chemicals and natural products for dermatological purpose. Abbrevitions used: NF-$textsc{k}$B, nuclear factor kappa B; I-$textsc{k}$B, Inhibitory kappa B; SEAP, secreted alkaline phosphatase; NPT, neomycin phosphotransferease; PCR, polymerase chain reaction: dNTP, deoxynucleoside triphosphates; DMEM, dulbecco’s modified eagle medium; FBS, fetal bovine serum; PBs, phosphate-buffered saline; MUP, 4-methylumbellifery phosphate; NAC, N-acetyl-L-cysteine; DMSO, dimethyl sulfoxide; PMA, phorbol 12-myristate 13-acetate.

• Journal of Physiology & Pathology in Korean Medicine
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• v.24 no.4
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• pp.586-591
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• 2010

Chemokine and Growth Factor are major mediumtors of immuno-inflammatory pathway. The purpose of this study is to investigate whether productions of Chemokine and Growth Factor in lipopolysaccharide (LPS)-induced mouse macrophage RAW 264.7 cells are modulated by Gallic acid (GA), which is easily founded in tannin-containing natural materials such as red wine, green tea, grape juice, and Corni Fructus. Productions of Chemokine and Growth Factor were analyzed by High-throughput Multiplex Bead based Assay with Bio-plex Suspension Array System based on $xMAP^{(R)}$ (multi-analyte profiling beads) technology. At first, cell culture supernatant was obtained after treatment with LPS and GA for 24 hour. Then, the antibody-conjugated beads were added and incubated for 30 minutes. After incubation, detection antibody was added and incubated for 30 minutes. And Strepavidin-conjugated Phycoerythrin (SAPE) was added. After incubation for 30 minutes, the level of SAPE fluorescence was analyzed on Bio-plex Suspension Array System. Based on fluorescence intensity, concentrations of Chemokine and Growth Factor were determined. The results of the experiment are as follows. GA significantly inhibited the production of interferon-inducible protein (IP)-10, keratinocyte-derived chemokine(KC), and vascular endothelial growth factor (VEGF) in LPS-induced RAW 264.7 cells at the concentration of 25, 50, 100, 200 uM (p<0.05). GA significantly inhibited the production of monocyte chemoattractant protein-1(MCP-1) and macrophage-colony stimulating factor(M-CSF) in LPS-induced RAW 264.7 cells at the concentration of 50, 100, 200 uM (p<0.05). GA diminished the production of granulocyte macrophage-colony stimulating factor (GM-CSF) in LPS-induced RAW 264.7 cells. But GA did not show the inhibitory effect on the production of leukemia inhibitory factor (LIP) and macrophage inflammatory protein (MIP)-2 in LPS-induced RAW 264.7 cells. These results suggest that GA has the immuno-modulating activity related with its inhibitory effects on the production of IP-10, KC, MCP-1, VEGF, and M-CSF in LPS-induced macrophages.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.24 no.3
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• pp.134-145
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• 1998

Facial hyperpigmentation in women, which is considered to be a serious cosmetic disability and a cause of mental distress, requires proper management. Melanocyte dendricity is a crucial factor affecting epidermal pigmentation. We found that BQ-788, the endothelin-B (ETB) receptor antagonist, blocks the formation of multi-dendricity which is induced by cocultured keratinocytes. Melanocytes in vivo show numerous dendrites which are in close contact with multiple keratinocytes, forming the epidermal-melanin unit. While melanocytes transfer their melanosomes into the neighboring keratinocytes via dendrites, keratinocytes secrete many growth factors and cytokines that influence viability, morphology, and melanin formation of melanocytes. Endothelin-1 (ET-1), prostaglandin E2(PGE2), and leukotriene-C4 (LT-C4) have been suggested as the candidates for increasing dendricity. Other reports suggested that ET-1 has stimulatory effects on proliferation and melanin formation of melanocytes in vitro. In the present study, using type-specific ET receptor antagonists, we observed how the morphology of melanocytes could be modulated in a coculture system. In addition, the roles of ET-1 for morphology and proliferation on melanocytes were evaluated in different culture media. We suggest that ET-1 increases dendricity and proliferation of melanocytes, and that its dendrite-inducing effect and mitogenic effect are regulated independently.

• Proceedings of the SCSK Conference
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• 2003.09a
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• pp.700-718
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• 2003

Fructan, a polysaccharide existing in plants or produced by microorganisms, is a sugar polymer of fructose with $\beta$-2,6 linkages. In this study, we investigated some cosmeceutical properties of Fructan such as moisturizing effect, cell proliferation effect, anti-inflammation effect and cell cytotoxicity. Zymomonas mobilis, a microorganism producing Fructan, was cultured in a medium containing 10％ sucrose and 2％ yeast extract as main components for 24 hours at 37$^{\circ}C$ and pH 7. Fructan was obtained by precipitation from the cultured medium by adding alcohol (alcohol ratio of 1:3) after removing the enzyme by centrifuging. Fructan exhibited almost same moisturizing effect as hyaluronic acid and cell proliferation effect on human fibroblast and keratinocyte as well. Moreover, on cell proliferation test on bio-artificial skin constructed by 3-dimensional(3-D) culture after inducing primary skin inflammation with 0.5％ sodium lauryl sulfate (SLS), the 3-D artificial skin treated with 0.01 mg/ml, 0.05mg/ml of Fructan exhibited higher cell proliferation than the 3-D artificial skin treated with SLS only. On anti-inflammation test on 3-D artificial skin evaluated by measuring secreted quantity of interleukin-1$\alpha$ (IL-1$\alpha$) which is a pre-inflammatory mediator induced by SLS, the quantity of IL-1$\alpha$on the 3-D artificial skin treated with 0.01 mg/ml, 0.05mg/ml of Fructan was less than the one on the 3-D artificial skin treated with SLS only. As a result of these studies, Fructan has anti-inflammation effect against inflammatory reaction by a skin irritant as well as cell proliferation effect in bio-artificial skin. Fructan was also evaluated as a safe material without any toxicity in safety tests using fibroblasts and animals.

• 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.