• Journal of Microbiology and Biotechnology
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• v.32 no.2
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• pp.238-247
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• 2022

Since the skin covers most surfaces of the body, it is susceptible to damage, which can be fatal depending on the degree of injury to the skin because it defends against external attack and protects internal structures. Various types of artificial skin are being studied for transplantation to repair damaged skin, and recently, the production of replaceable skin using three-dimensional (3D) bioprinting technology has also been investigated. In this study, skin tissue was produced using a 3D bioprinter with human skin cell lines and cells extracted from mouse skin, and the printing conditions were optimized. Gelatin was used as a bioink, and fibrinogen and alginate were used for tissue hardening after printing. Printed skin tissue maintained a survival rate of 90% or more when cultured for 14 days. Culture conditions were established using 8 mM calcium chloride treatment and the skin tissue was exposed to air to optimize epidermal cell differentiation. The skin tissue was cultured for 14 days after differentiation induction by this optimized culture method, and immunofluorescent staining was performed using epidermal cell differentiation markers to investigate whether the epidermal cells had differentiated. After differentiation, loricrin, which is normally found in terminally differentiated epidermal cells, was observed in the cells at the tip of the epidermal layer, and cytokeratin 14 was expressed in the lower cells of the epidermis layer. Collectively, this study may provide optimized conditions for bioprinting and keratinization for three-dimensional skin production.

• Journal of Periodontal and Implant Science
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• v.52 no.3
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• pp.242-257
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• 2022

Purpose: This study investigated periodontal ligament (PDL) restoration in osseointegrated implants using stem cells. Methods: Commercial pure titanium and zirconium oxide (zirconia) were coated with beta-tricalcium phosphate (β-TCP) using a long-pulse Nd:YAG laser (1,064 nm). Isolated bone marrow mesenchymal cells (BMMSCs) from rabbit tibia and femur, isolated PDL stem cells (PDLSCs) from the lower right incisor, and co-cultured BMMSCs and PDLSCs were tested for periostin markers using an immunofluorescent assay. Implants with 3D-engineered tissue were implanted into the lower right central incisors after extraction from rabbits. Forty implants (Ti or zirconia) were subdivided according to the duration of implantation (healing period: 45 or 90 days). Each subgroup (20 implants) was subdivided into 4 groups (without cells, PDLSC sheets, BMMSC sheets, and co-culture cell sheets). All groups underwent histological testing involving haematoxylin and eosin staining and immunohistochemistry, stereoscopic analysis to measure the PDL width, and field emission scanning electron microscopy (FESEM). The natural lower central incisors were used as controls. Results: The BMMSCs co-cultured with PDLSCs generated a well-formed PDL tissue that exhibited positive periostin expression. Histological analysis showed that the implantation of coated (Ti and zirconia) dental implants without a cell sheet resulted in a well-osseointegrated implant at both healing intervals, which was confirmed with FESEM analysis and negative periostin expression. The mesenchymal tissue structured from PDLSCs only or co-cultured (BMMSCs and PDLSCs) could form a natural periodontal tissue with no significant difference between Ti and zirconia implants, consequently forming a biohybrid dental implant. Green fluorescence for periostin was clearly detected around the biohybrid implants after 45 and 90 days. FESEM showed the invasion of PDL-like fibres perpendicular to the cementum of the bio-hybrid implants. Conclusions: β-TCP-coated (Ti and zirconia) implants generated periodontal tissue and formed biohybrid implants when mesenchymal-tissue-layered cell sheets were isolated from PDLSCs alone or co-cultured BMMSCs and PDLSCs.

• The Korea Journal of Herbology
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• v.28 no.6
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• pp.145-153
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• 2013

Objectives : Neuroinflammation is characterized by microglial activation and the expression of major inflammatory mediators. The present study investigated the inhibitory effect of ginsenoside Rg1 ($GRg_1$), a principle active ingredient in Panax ginseng, on pro-inflammatory cytokines and microglial activation induced by systemic lipopolysaccharide (LPS) treatment in the mouse brain tissue. Methods : Varying doses of $GRg_1$ was orally administered (10, 20, and 30 mg/kg) 1 h before the LPS injection (3 mg/kg, intraperitoneally). The mRNA expression of pro-inflammatory cytokines in the brain tissue was measured using the quantitative real-time PCR method at 4 h after the LPS injection, Microglial activation was evaluated using western blotting and immunohistochemistry against ionized calcium binding adaptor molecule 1 (Iba1) in the brain tissue. Cyclooxigenase-2 (COX-2) expressions also observed using western blotting and immunohistochemistry at 4 h after the LPS injection, In addition, double-immunofluorescent labeling of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) and COX-2 with microglia and neurons was processed in the brain tissue. Results : $GRg_1$ (30 mg/kg) significantly attenuated the upregulation of TNF-${\alpha}$, interleukin (IL)-$1{\beta}$ and IL-6 mRNA in the brain tissue at 4 h after LPS injection. Morphological activation and Iba1 protein expression of microglia induced by systemic LPS injection were reduced by the $GRg_1$ (30 mg/kg) treatment. Upregulation of COX-2 protein expression in the brain tissue was also attenuated by the $GRg_1$ (30 mg/kg) treatment. Conclusion : The results suggest that $GRg_1$ is effective in the early stage of neuroinflammation which causes neurodegenerative diseases.

• BMB Reports
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• v.56 no.3
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• pp.202-207
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• 2023

We investigated the neuroprotective effects of deca nano-graphene oxide (daNGO) against reactive oxygen species (ROS) and inflammation in the human neuroblastoma cell line SH-SY5Y and in the 6-hydroxydopamine (6-OHDA) induced Parkinsonian rat model. An MTT assay was performed to measure cell viability in vitro in the presence of 6-OHDA and/or daNGO. The intracellular ROS level was quantified using 2',7'-dichlorofluorescein diacetate. daNGO showed neuroprotective effects against 6-OHDA-induced toxicity and also displayed ROS scavenging properties. We then tested the protective effects of daNGO against 6-OHDA induced toxicity in a rat model. Stepping tests showed that the akinesia symptoms were improved in the daNGO group compared to the control group. Moreover, in an apomorphine-induced rotation test, the number of net contralateral rotations was decreased in the daNGO group compared to the control group. By immunofluorescent staining, the animals in the daNGO group had more tyrosine hydroxylase-positive cells than the controls. By anti-Iba1 staining, 6-OHDA induced microglial activation showed a significantly decrease in the daNGO group, indicating that the neuroprotective effects of graphene resulted from anti-inflammation. In conclusion, nano-graphene oxide has neuroprotective effects against the neurotoxin induced by 6-OHDA on dopaminergic neurons.

• Clinical and Experimental Pediatrics
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• v.51 no.10
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• pp.1112-1117
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• 2008

Purpose : We intended to observe cell death and apoptotic changes in neurons in organotypic hippocampal slice cultures following oxygen-glucose deprivation (OGD), using propidium iodide (PI) uptake, Fluoro-Jade (FJ) staining, TUNEL staining and immunofluorescent staining for caspase-3. Methods : The hippocampus of 7-day-old rats was cut into $350{\mu}m$ slices. The slices were cultured for 10 d (date in vitro, DIV 10) and and exposed to OGD for 60 min at DIV 10. They were then incubated for reperfusion under normoxic conditions for an additional 48 h. Fluorescence of PI uptake was observed at predetermined intervals, and the cell death percentage was recorded. At 24 h following OGD, the slices were Cryo-cut into $15{\mu}m$ thicknesses, and Fluoro-Jade staining, TUNEL staining, and immunofluorescence staining for caspase-3 were performed. Results : 1) PI uptake was restricted to the pyramidal cell layer and DG in the slices after OGD. The fluorescent intensities of PI increased from 6 to 48 h during the reperfusion stage. The cell death percentage significantly increased time-dependently in CA1 and DG following OGD (P<0.05). 2) At 24 h after OGD, many FJ positive cells were detected in CA1 and DG. Some neurons had distinct nuclei and processes while others had fragmented nuclei and disrupted processes in CA1. TUNEL and immunofluorescent staining for caspase-3 showed increased expression of TUNEL labeling and caspase-3 in CA1 and DG at 24 h after OGD. Conclusion : The numerous dead cells in the slice cultures after OGD tended to display apoptotic changes mediated by the activation of caspase-3.

• Pediatric Infection and Vaccine
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• v.8 no.1
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• pp.94-100
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• 2001

Purpose : Enterovirus is a common cause of aseptic meningitis and nonspecific febrile illness in young children. During the summer and fall months, enterovirus-infected young children are frequently admitted and evaluated to rule out bacterial sepsis and/or meningitis. The purpose of this study was to evaluate the relationship between nonpolio enterovirus infection and febrile illness in infants under 3 months of age during the summer, fall months by using a stool culture to identify the presence of enterovirus. Methods : Patients included febrile infants under 3 months of age admitted to Masan Fatima Hospital for sepsis evaluation from May 1999 to September 1999. Cultures were performed from stool and Cerebrospinal fluid samples and then were tested for enterovirus infection. Viral isolation and serotype identification were performed by cell culture and immunofluorescent testing. Enteroviruses not typed by immunofluorescent testing were confirmed by reverse transcription-polymerase chain reaction. Results : A total of 44 febrile infants were enrolled; of those, 20(45%) were positive for enterovirus. Two enterovirus culture-positive infants had concomitant urinary tract infection and one had Kawasaki disease. All infants infected with an enterovirus recovered without complications. Serotype of 20 enteroviruses were isolated from stool, 3 of echovirus type 9, 1 of echovirus type 11, 1 Coxsachievirus type B4, 15 of untyped enteroviruses. One untyped enterovirus was isolated in the CSF. Conclusion : Nonpolio enterovirus infections are associated with nonspecific febrile illnesses in infants under 3 months of age.

• Pediatric Infection and Vaccine
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• v.5 no.1
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• pp.104-114
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• 1998

Purpose : Enteroviruses are the most common cause of aseptic meningitis. The epidemics of aseptic meningitis in 1993 and 1996 were mostly caused by echovirus type 9. Identification of the causative virus of aseptic meningitis in epidemics, is very important not only for diagnosis but also for epidemiologic purpose. The purpose of this study was to identify the causative virus and investigate the relationship between aseptic meningitis, prevailed in Masan and surrounding areas in Kyoungsangnamdo in 1997, and its clinical manifestations. Methods : One hundred twenty eight cerebrospinal fluid(CSF) and 239 stool specimens were obtained from 239 patients(213 children and 26 adult patients) with aseptic meningitis were admitted to Masan Fatima Hospitals from March to October 1997. Viral isolation and serotype identification was performed by cell culture and immunofluorescent test. Enteroviruses not typed by immunofluorescent test was confirmed by reverse transcription-polymerase chain reaction(RT-PCR). Results : 1) The peak incidence was noted in June. 2) The age of 239 patients(pediatrics-213 cases, internal medicine-26 cases) that were diagnosed ranged from neonate to 35 years, the age of the patients of pediatrics ranged from neonate to 15years(mean 4.9 years), the age of the patients of internal medicine (above 16 years) ranged from 16 years to 35 years(mean 24.2 years). 3) Fifty-three(41.4%) of 128 CSF specimens were positive for enteroviruses, and 163(68.2%) of 239 stool specimens were positive for enteroviruses respectively. 4) Serotypes of 53 enteroviruses isolated from CSF were 16(30.2%) of echovirus type 30, 6(11.3%) of echovirus type 6, 1 of echovirus type 4, 4 of untyped echovirus, 1 of coxsackievirus type B5, and 24 isolates of untyped enteroviruses. Of 163 enterovirus isolated from stool were 72(44.2%) of echovirus type 30, 21(12.9%) of echovirus type 6, 1 of echovirus type 4, 17(10.4%) of undetermined subtyped echovirus, 1 of coxsackievirus type B5, 2 of A24, 3 of undetermined subtyped coxsackievirus type B, and 46 isolates of untyped enterovirus. Conclusion : There were epidemics of aseptic meningitis in the central areas of Kyoungsangnamdo from March to October 1997. The main causative organism was thought to be the echovirus type 30, and echovirus type 4, 6, coxsackievirus B5 and A24 were also thought to contribute to the epidemics.

• Radiation Oncology Journal
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• v.20 no.4
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• pp.367-374
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• 2002

Purpose : To investigate the growth inhibitory effects, and the underlying mechanism of human colon cancer cell (HT-29) death, induced by a new synthetic bile acid derivative (HS-1200). Materials and Methods : Human colon cancer cells (HT-29), in exponential growth phase, were treated with various concentrations of a new synthetic bile acid derivative (HS-1200). The growth inhibitory effects on HT-29 cells were examined using a frypan blue exclusion assay. The extent of apoptosis was determined using agarose gel electrophoresis, TUNEL assays and Hoechst staining. The apoptotic cell death was also confirmed by Western blotting of PARP, caspase-3 and DNA fragmentation factor (DFF) analysis. To investigate the involvement of mitochondria, we employed immunofluorescent staining of cytochrome c and mitochondrial membrane potential analyses. Results : The dose required for the half maximal inhibition $(IC_{50})$ of the HT-29 cell growth was $100\~150\;{\mu}M$ of HS-1200. Several changes, associated with the apoptosis of the HT-29 cells, were reveal by the agarose gel eletrophoresis, TUNEL assays and Hoechst staining, following their treatment with $100\;{\mu}M$ of HS-1200. HS-1200 treatment also induced caspase-3, PARP and DFF degradations, and the western blotting showed the processed caspase-3 p20, PARP p85 and DFF p30 and p11 cleaved products. Mitochondrial events were also demonstrated. The cytochrome c staining indicated that cytochrome c had been released from the mitochondria in the HS-1200 treated cells. The mitochondrial membrane potential $(\Delta\Psi_m)$ was also prominently decreased in the HS-1200 treated cells. Conclusion : These findings suggest that the HS-1200 - induced apoptosis of human colon cancer cells (HT-29) is mediated via caspase and mitochondrial pathways.

• Pediatric Infection and Vaccine
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• v.9 no.1
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• pp.67-73
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• 2002

Purpose : Despite of the appropriate measles vaccination programs, epidemics occur every 2~3 years and especially occurred in large group in late of 2000 and early of 2001. To evaluate the effect of the vaccination, needs for revaccination and to determine the optimal age for revaccination, we examined measles specific IgG and IgM in mealses patients and investigated different antibody appearance according to vaccination history. Methods : Anti-measles antibodies were checked in sera of 201 patients(male : 117, female : 84) that are responsible for Criteria for Disease Control among 298 patients that are suspicious of measles including inpatients and outpatients in Wonju Christian Hospital from June in 2000 to June in 2001. They were checked by immunofluorescent assay. Then we classified them according to sex, month, distribution of age due to vaccination and appearance of measles antibody. Results : The ratio of male and female was 1.4 : 1. The maximum incidence was 38 cases(18.9%) in May in 2001. Incidence was increased from November in 2000 to January in 2001 and decreased in February and March in 2001. Thereafter it was increased from April in 2001 again and decreased from June. There were 93 cases(46.3%) in vaccinated group and 108 cases(53.7%) in unvaccinated group. In the distribution according to age in vaccinated group, there were 54 cases(58.1%) in more than 10 years old, 15 cases(16.0%) between 7 and 10 years old, 12 cases(12.9%) between 15 months and 3 years old, 6 cases (6.5%) between 4 and 6 years old and 6 cases(6.5%) between 6 months and 14 months old. In the distribution according to age in unvaccinated group, there were 88 cases(81.5%) between 6 months and 14 months old, 9 cases(8.3%) between 15 months and 3 years old, 7 cases(6.5%) less than 6 months old, 3 cases(2.8%) more than 10 years old and 1 case(0.9%) between 7 and 10 years old. In the distribution of measles specific IgG and IgM, 78 cas (87.6%) were IgG(+), IgM(+) and 11 cases(12.4%) are IgG(+), IgM(-) in vaccinated group. In unvaccinated group, there were 69 cases(63.9%) of IgG(+), IgM(+) and 39 cases (36.1%) of IgG(-), IgM(+). Con c lu s i on s : We thought that measles incidence was peaked between 6 months and 14 months old in unvaccinated group because of maximum decrement of transplacental matenal antibody and was peaked in more than 10 years old in vaccinated group because of maximum decrement of measles specific IgG. We think that measles revaccination as well as vaccination and especially optimal age for revaccination is very important to prevent measles successfully.

• Journal of Life Science
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• v.21 no.10
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• pp.1385-1392
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• 2011

It is well known that both insulin-like growth factor-I and suppressor of cytokine signaling-3 (SOCS-3) are known to modulate various aspects of physiology in skeletal muscle cells. Furthermore, although SOCS-3 expression is related to insulin resistance in non-skeletal muscle cells and is known to interact with insulin-like growth factor-I receptor, the effect of IGF-I on SOCS-3 gene expression in skeletal muscle cells is presently unknown. C2C12 myotubes were treated with different concentrations (0-200 ng/ml) of IGF-I or for various periods of time (3-72 hr). Immunofluorescent staining image revealed that IGF-I induced SOCS-3 protein expression in a dose-dependent manner. Western blot data also showed that SOCS-3 proteins were induced by IGF-I (200 ng/ml) in C2C12 myotubes in a time-dependent manner. The level of SOCS-3 mRNA was also significantly increased after 3hr of IGF-I (10-100 ng/ml) treatment. However, the levels of SOCS-3 mRNA were significantly decreased after 24 and 48 hr of IGF-I (10-100 ng/ml) treatment compared to the control. In conclusion, SOCS-3 protein is induced by IGF-I treatment in C2C12 skeletal muscle cells and this induction is regulated pretranslationally. The modulating effect of IGF-I on SOCS-3 expression may be an important regulator of gene expression in skeletal muscle cells.