• Journal of Biomedical Engineering Research
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• v.40 no.5
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• pp.143-150
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• 2019

To evaluate the toxicity of ophthalmic drug, the Draize test and Bovine Corneal Opacity and Permeability (BCOP) test commonly used. In Draize test, experimental animals were under stress and pain due to long-term exposure of drug. In addition, regarding physiological functions, animal model is not perfectly reflected a human eye condition. Although some models such as $EpiOcular^{TM}$, HCE model, LabCyte Cornea-Model, and MCTT $HCE^{TM}$ were already presented advanced cornea ex-vivo model to replace animal test. In this sense, cornea tissue structure mimicked ex-vivo toxicity model was fabricated in this study. The corneal epithelial cells (CECs) and keratocytes (CKs) isolated from rabbit eyeball were seeded on non-patterned silk film (n-pSF) and patterned silk film (pSF) at $32,500cells/cm^2$ and $6,500cells/cm^2$. Sequentially, n-pSF and pSF were stacked to mimic a multi-layered stroma structure. The thickness of films was about $15.63{\mu}m$ and the distance of patterns was about $3{\mu}m$. H&E stain was performed to confirm the cell proliferation on silk film. F-actin of CKs was also stained with Phalloidin to observe the cytoskeletal alignment along with patterns of the pSF. In the results, CECs and CKs were shown the good cell attachment on the n-pSF and pSFs. Proliferated cells expressed the specific phenotype of cornea epithelium and stroma. In conclusion, we successfully established the ex-vivo cornea toxicity model to replace the eye irritation tests. In further study, we will set up the human ex-vivo cornea toxicity model and then will evaluate the drug screening efficacy.

• Journal of Microbiology and Biotechnology
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• v.33 no.3
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• pp.329-338
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• 2023

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that produces attaching and effacing lesions on the large intestine and causes hemorrhagic colitis. It is primarily transmitted through the consumption of contaminated meat or fresh produce. Similar to other bacterial pathogens, antibiotic resistance is of concern for EHEC. Furthermore, since the production of Shiga toxin by this pathogen is enhanced after antibiotic treatment, alternative agents that control EHEC are necessary. This study aimed to discover alternative treatments that target virulence factors and reduce EHEC toxicity. The locus of enterocyte effacement (LEE) is essential for EHEC attachment to host cells and virulence, and most of the LEE genes are positively regulated by the transcriptional regulator, Ler. GrlA protein, a transcriptional activator of ler, is thus a potential target for virulence inhibitors of EHEC. To identify the GrlA inhibitors, an in vivo high-throughput screening (HTS) system consisting of a GrlA-expressing plasmid and a reporter plasmid was constructed. Since the reporter luminescence gene was fused to the ler promoter, the bioluminescence would decrease if inhibitors affected the GrlA. By screening 8,201 compounds from the Korea Chemical Bank, we identified a novel GrlA inhibitor named Grlactin [3-[(2,4-dichlorophenoxy)methyl]-4-(3-methylbut-2-en-1-yl)-4,5-dihydro-1,2,4-oxadiazol-5-one], which suppresses the expression of LEE genes. Grlactin significantly diminished the adhesion of EHEC strain EDL933 to human epithelial cells without inhibiting bacterial growth. These findings suggest that the developed screening system was effective at identifying GrlA inhibitors, and Grlactin has potential for use as a novel anti-adhesion agent for EHEC while reducing the incidence of resistance.

• Journal of Periodontal and Implant Science
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• v.32 no.2
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• pp.429-455
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• 2002

There are numerous kind of materials and techniques to regenerate the periodontal tissue which has been lost due to destructive periodontal disease, including bone graft material. Many bone graft materials have been reported and among these materials, synthetic material has been developed fin the long time because of its sufficient supply economically. Calcium sulfate which was evaluated as including much calcium, has been used in the clinical field. In the dental field calcium sulfate has been used as bone graft material and Kim reported that improved bone formation and more amount of new attachment after grafting calcium sulfate. but, because calcium sulfate has the problem that it generates the heat in setting period and resolves fast, we need to evaluate the effect of the improved calcium sulfate on periodontal tissue. The present study evaluates the effect of paste type calcium sulfate on the epithelial migration, alveolar bone regeneration, cementum formation and gingival connective tissue attachment in intrabony defect in dogs. Four millimeter deep and four millimeter wide 3-wall defects were surgically created in the mesial or distal aspects of premolars or molars. the test group received paste-type calcium sulfate with a flap procedure and the control group underwent flap procedure only. Histologic analysis after 8 weeks of healing revealed the following results : 1. The length of epithelial growth(the distance from CEJ to the apical end of JE) was 0.52${\pm}$0.26mm in the control and 0.56${\pm}$0.25mm in the test group. there was no statistically significant difference between the two groups. 2. The length of connective tissue adhesion was 1.74${\pm}$1.06mm in the control and 1.28${\pm}$0.57mm in the test group. there was no statistically significant difference between the two groups. 3. The length of new bone was 2.01${\pm}$0.95mm in the control and 2.62${\pm}$0.81mm in the test group. there was no statistically significant difference between the two groups. 4. The length of new cementum was 1.86${\pm}$0.80mm in the control and 2.77${\pm}$ 0.86mm in the test group. there was a statistically significant difference between the two groups.(P<0.01) These results suggest that the use of paste type calcium sulfate in 3-wall intrabony defects has significant effect on new cementum formation , but doesn't have any significant effect on the prevention of junctional epithelium migration and new bone formation. Finally, the paste type calcium sulfate that is used in this study is suggested to be the material that can have a significant effect on the periodontal healing, if its biocompatibility is improved.

• The korean journal of orthodontics
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• v.18 no.2
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• pp.311-327
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• 1988

Vascular changes in the periodontal ligament of the rat incisors following application of experimental orthodontic forces were examined by the India ink perfusion method. 57 rats were used for this experiment. The rats were divided into experimental group (54 rats) and control group (3 rats). 54 experimental rats were divided into group I (27 rats) and group II (27 rats). The right and left upper incisors of group. I and group II rats were separated distally with forces of 20gm, 70gm respectively. The vascular changes of periodontal ligament were observed histologically by means of light microscope after 1, 2 and 3 days of tooth movement and 1,3,5,8,14, and 21 days after removal of orthodontic force. The results were as follows; 1. After one day of tooth movement, occlusion of blood vessels, hyalinization of periodontal ligament and resorption of alveolar bone adjacent to the alveolar crest on pressure side were observed. Above the tissue changes on the pressure side of group II were more severe than those of group I. Especially, septal bone of group II was separated after 2 days of tooth movement. 2. In tension zones, periodontal space was widened and periodontal fibers were orientated in the direction of puil. The blood vessels of periodontal ligament were distended. New bone deposition was seen along the inner surface of the alveolus after 2 days of tooth movement. 3. After 3 days of tooth movement, deposition of new bone was seen along the periosteal surface of alveolar bone on pressure side, progressing with increasing after removal of orthodontic force. Remodelling of the new bone was occurred 5 days after removal of orthodontic force. 4. 3 days after removal of orthodontic force, invasion of blood vessels into the marginal periodontal ligament on pressure side was observed clearly and the vessels below the epithelial attachment were increased. 5. After removal of orthodontic force, hyalinized structures disappeared concomittantly with an invasion of blood vessels from the neighboring periodontal ligament. 14 days after removal of orthodontic force, the vessels in the periodontal ligament of group I were finished the vascular rearrangement. 21 days after removal of orthodontic force, the vessels in the periodontal ligament of group II were finished the vascular rearrangement.

• Parasites, Hosts and Diseases
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• v.36 no.4
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• pp.227-234
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• 1998

Actin and tropomyosin of Cryptosporidium muris were localized by immunogold labeling. Two kinds of antibodies for actin labeling were used. The polyclonal antibody to skeletal muscle (chicken back muscle) actin was labeled on the pellicle and cytoplasmic vacuoles of parasites. The feeder organelle has showed a small amount of polyclonal actin antibody labeling as well. Whereas the monoclonal antibody to smooth muscle (chicken gizzard muscle) actin was chiefly labeled on the filamentous cytoplasm of parasites. The apical portion of host gastric epithelial cell cytoplasm was also labeled by smooth muscle actin together. The polyclonal antibody to tropomyosin was much more labeled at C. muris than host cells, so it could be easily identified even with low magnification (${\times}2,000$). The tropomyosin was observed along the pellicle, cytoplasmic vacuoles, and around the nucleus also. The skeletal muscle type actin seems to play a role in various celluar functions with tropomyosin in C. muris; on the other hand, the smooth muscle type actin was located mainly on the filamentous cytoplasm and supported the parasites firm attachment to host cells. Tropomyosin on the pellicle was thought to be able to stimulate the host as a major antigen through continuous shedding out by the escape of sporozoites or merozoites from their mother cells.

• Journal of Periodontal and Implant Science
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• v.27 no.2
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• pp.363-377
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• 1997

The main goal of periodontal therapy is the regeneration of periodontal tissue which has been lost due to destructive periodontal diseases. Although conventional forms of periodontal therapy show sound clinical results, the healing results in long junctional epithelium. There have been numerous materials and surgical techniques developed for new attachment and bone regeneration. Bone grafts can be catagorized into: autografts, allografts, xenografts and bone substitutes. Synthetic bone substitute materials include hydroxyapatite, tricalcium phosphate, calcium carbonate, and Plaster of Paris. Calcium sulfate has found its use in dental practice for the last 30 years. Recent animal studies suggest that periodontal regeneration in 3 wall intrabony defect may be enhanced by the presence of calcium sulfate. And it is well known that 2 wall & 1 wall defect have less osteogenic potential, So we need to study the effect of calcium sulfate in 1 wall intrabony defect in dogs. The present study evaluates the effects of calcium sulfate on the epithelial migration, alveolar bone regeneration and cementum formation in intrabony defects of dogs. Four millimeter-deep one-wall intrabony defects were surgically created in the mesial aspect of anterior teeth and mesial & distal aspects of premolars. The test group received calcium sulfate grafts with a flap procedure. The control underwent flap procedure only. Histologic analysis following 8 weeks of healing revealed the following results: 1. The lengths of junctional epithelium were: 2.52mm in the control, and 1.89mm in the test group. There was no statistical significance between the two groups. 2. Alveolar bone formation were: 0.61mm in the control, and 1.88mm in the test group. There was a statistically significant difference between the two groups (p<0.05). 3. Cementum formations were: l.lmm in the control, and 2.46mm in the test group. There was a statistically significant difference between the two groups (p<0.05). 4. The length of CT adhesion were: O.97mm in the control, and 0.17mm in the test group. There was no statistically significant differences between the two groups These results suggest that the use of calcium sulfate in intrabony defects has little effect on junctional epithelium migration, but has significant effects on new bone and new cementum formations.

• Journal of Periodontal and Implant Science
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• v.36 no.1
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• pp.27-37
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• 2006

Although the main purpose of periodontal treatment to regenerate is the complete regeneration of periodontal tissue due to periodontal disease, most of the treatment cannot meet such purpose because healing by long epithelial junction. Therefore, diverse materials of resorbable and non-resorbable have been used to regenerate the periodontal tissue. Due to high risk of exposure and necessity of secondary surgical procedure when using non-resorbable membrane, guided tissue regeneration using the resorbable membrane has gain popularity, recently. However, present resorbable membrane has the disadvantage of not having sufficient time to regenerate date to the difference of resorption rate according to surgical site. Meanwhile, other than the structure stability and facile manipulation, acellular dermal matrix has been reported to be a possible scaffold for cellular proliferation due to rapid revascularization and favorable physical properties for cellular attachment and proliferation. The purpose of this study is to estimate the influence of acellular dermal matrix on periodontal ligament, cementum and alveolar bone when acellular dermal matrix is implanted to 1-wall alveolar bone defect. 4 dogs of 12 to 16 month old irrelevant to sex , which below 15Kg of body weight, has been used in this study. ADM has been used for the material of guided tissue regeneration. The 3rd premolar of the lower jaw was extracted bilaterally and awaited for self-healing. subsequently buccal and lingual flap was elevated to form one wall intrabony defect with the depth and width of 4mm on the distal surface of 2nd premolar and the mesial surface of 4th premolar. After the removal of periodontal ligament by root planing. notch was formed on the basal position. Following the root surface treatment, while the control group had the flap sutured without any treatment on surgically induced intrabony defect. Following the root surface treatment, the flap of intrabony defect was sutured with the ADM inserted while the control group sutured without any insertion. The histologic specimen was observed after 4 and 8 weeks of treatment. The control group was partially regenerated by periodontal ligament, new cementum and new alveolar bone. the level of regeneration is not reached on the previous formed notch. but, experimental group was fully regenerated by functionally oriented periodontal ligament fiber. new cementum and new alveolar bone. In conclusion, we think that ADM seems to be used by scaffold for periodontal ligament cells and the matrix is expected to use on guided tissue regeneration.

• Polymer(Korea)
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• v.35 no.4
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• pp.289-295
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• 2011

Biomaterials for retinal tissue engineering must demonstrate several critical features for potential utility, including mechanical integrity, biocompatibility, and slow biodegradation. Silk film biomaterials were designed and characterized to meet these functional requirements. We prepared natural/synthetic hybrid silk/PLGA films using 0, 10, 20, 40, and 80 wt% of silk by a solvent evaporation method. MIT assay was used to confirm the number of cells attached on film at 1, 2, and 3 days, respectively. The morphology of cellular adhesion on films was also confirmed by scanning electron microscope (SEM). RT-PCR was conducted to confrrm mRNA expression of retinal pigment epithelitun (RPE) using RPE65 as a RPEs marker and the expression of cytokeratin were determined by immunofluorescence staining. We confirmed that the silk/PLGA film of 20~40 wt% silk was superior for the adhesion and proliferation of RPEs.

• Journal of Periodontal and Implant Science
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• v.30 no.2
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• pp.241-257
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• 2000

Autogenous periosteal grafts are an attractive alternative to existing barrier membrane materials since they meet the reqiurements of an ideal material. But no histological data are available on the effectiveness of periosteal membranes in the treatment of periodontal defects. The purpose of this study was to evaluate effect of autogenous periosteal graft on periodontal regeneration histologically. Class II furcation defects were surgically created on the second, third and the fourth premolars bilaterally in the mandibules of six mongrel dogs. The experimental sites were divided into three groups according to the treatment modalities; control group - surgical debridement only; Group I- autogenous periosteal membrane placement after surgical debridement; Group II-autogenous periosteal membrane placement after surgical debridement and bone grafting. The animals were sacrificed at 2, 4 and 12 weeks after periodontal surgery and the decalcified and undecalcified specimens were prepared for histological and histometrical analysis. Clinically all treated groups healed without significant problems. Under light microscope, at 2 weeks, control group showed significant apical epithelial migration and bone remodelling only below the notch area. But for the group I, II with autogenous periosteal graft, less apical migration of epithelium appeared and large amount of osteoid tissue showed above the notch area. Grafted periosteal membrane was indiscernable at 4 weeks, so periosteal membrane might be organized to surrounding tissues. Histometrically, at 4 and 12 weeks, all the test and control groups didn't show significant change of epithelial zone but new attachment level tended to be gained in the test groups than control group. These results suggest that autogenous periosteal grafts should be a good alternative for guided tissue regeneration.

• Journal of Periodontal and Implant Science
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• v.26 no.4
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• pp.949-965
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• 1996

The main goal of periodontal therapy is to restore the lost periodontal tissue and establish the attachment appratus. Current acceptable therapeutic techniques are included : removal of diseased soft tissue, demineralization of exposed root surface, using the barrier membrane for preventing the downgrowth of gingival epithelial cell, insertion of graft materials as a scaffolding action, and biological mediators for promoting the cell activity. The latest concept one among them has been studied which based on the knowledge of cellular biology of destructed tissue. Platelet-derived growth factor(PDGF) is one of the polypeptide growth factor which have been reported as a biological mediator to regulate activities of wound healing progress including cell proliferation, migration, and metabolism. The purposes of this study is to evaluate the influences of the PDGF as biological mediator to periodontal ligament and bone marrow cell. Both right and left maxillary first molar were extracted from rat which had treated with 0.4% ${\beta}-Aminopropionitril$ for 5 days, and feeded until designed date to sacrifice under anesthesisa. Periodontal ligament were removed from the extracted socket of the rat, and cultured with Dulbecco's Modified Essential Medium(DMEM) contained with 10% Fetal Bovine Serum, 100U/ml penicillin, $100{\mu}g/ml$ streptomycin, $0.5{\mu}g/ml$ amphotericin-B. Bone marrow cell were culture from bone marrow suspension with which washed out from femur with same medium. The study was performed to evaluate the effect of PDGF to periodontal ligament and bone cell, cell proliferation rate, total protein synthesis, and alkaline phosphatase activity of rat periodontal ligament(PDL) cell and bone stromal(RBS) cell in vitro. The effects of growth factors on both cells were measured at 3, 5th day after cell culture with (control group) or without growth factors(experimental group). The results were as follows: 1. The tendency of cell proliferation under the influence of PDGF showed more rapid proliferation pattern than control at 3 and 5 days after inoculation. 2. The activity of Alkaline phosphatase revealed 14, 80% increased respectively at 3, 5 days culture than control group. Measurements of ALPase levels indicated that PDL cells had significantly higher activity when compared with that of co-culture groups and GF only(P<0.05). And, ALPase activity in 10 days was higher than that of 7 days(P<0.05). 3. The tendency of formation of the mineralized nodule were observed dose-depend pattern of PDL cells. There was statistically significant difference among group l(PDL 100%), 2(PDL 70%:GF 30%), and 3(PDL 50%:GF 50%)(P<0.01). But, there was no difference among group 3, 4(PDL 30%:GF 70%), and 5(GF 100%). 4. Also, the number of nodule was greater in co-culture of PDL 70% and GF 30% than in culture of PDL 70%(P<0.05). From the above results, it is assumed that the PDGF on PDL cells and RMB cell culture. GF stimulates the cell growth, which is not that of PDL cells but GF. And, the activity of ALPase depends on the ratio of PDL cells, and ALPase may relate to the initial phase of nodule formation. Also, it is thought that the calcified nodule formation principally depends on PDL cells, is inhibited by GF, and affected by cell density. In conclusion, platelet-derived growth factor can promote rapid osteogenesis during early stage of periodontal tissue regeneration.