• Journal of Pharmaceutical Investigation
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• v.31 no.4
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• pp.241-256
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• 2001

Alginate microspheres, containing fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) or green fluorescent protein (GFP) were prepared and used as a model drug to develop the oral vaccine delivery system. The alginate microspheres were coated with poly-L-lysine or chitosan. Two methods, w/o-emulsion and spray, were used to prepare alginate microspheres. To optimize preparation conditions, effects of several factors on the particle size and particle morphology of microsphere, and loading efficiency of model antigen were investigated. In both preparation methods, the particle size and the loading efficiency were enhanced when the concentration of sodium alginate increased. In the w/o-emulsion preparation method, as the concentration of Span 80 was increased from 0.5% to 2%, the particle size was decreased, but the loading efficiency was increased. The higher the emulsification speed was, the smaller the particle size and loading efficiency were. The concentration of calcium chloride did not show any effect on the particle size and loading efficiency. In the spray preparation method, the particle size was increased as the nozzle pressure $(from\;1\;kgf/m^2\;to\;3\;kgf/m^2)$ and spray rate was raised. Increasing calcium chloride concentration (<7%) decreased the particle size, in contrast to no effect of calcium chloride concentration on the w/o-emulsion preparation method. Alginate microspheres prepared by two methods were different in the particle size and loading efficiency, the particle size of microspheres prepared by the spray method was about $2-6\;{\mu}m$, larger than that prepared by the w/o emulsion method $(about\;2{\mu}m)$, and the loading efficiency was also higher with spray method. Furthermore, drying process for the microspheres prepared by the spray was simpler and easier, compared with the w/o emulsion preparation. Therefore, the spray method was chosen to prepare alginate microspheres for further experiments. Release pattern of FITC-BSA in alginate microspheres was evaluated in simulated intestinal fluid and PBS (phosphate buffered saline). Dissolution rate of FITC-BSA from alginate/chitosan microsphere was lower than that from alginate microsphere and alginate/poly-L-lysine microsphere. By confocal laser scanning microscope, it was revealed that alginate/FITC-poly-L-lysine microspheres were present in close apposition epithelium of the Peyer's patches of rabbits following inoculation into lumen of intestine, which proved that microspheres could be taken up by Peyer's patch. In conclusion, it is suggested that alginate microsphere prepared by spray method, showing a particle size of & $10\;{\mu}m$ and a high loading efficiency, can be used as a model drug for the development of oral vaccine delivery system.

• Applied Chemistry for Engineering
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• v.19 no.2
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• pp.145-150
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• 2008

In this work we have studied the antifouling properties of the hydrophobic sol-gel modified sensing membrane and its optical properties for sensor application. E. coli JM109, B. cereus 318 and P. pastoris X-33 were cultivated in confocal cultivation dishes with glass surface, respectively. The glass surface was coated with the hydrophobic sol-gels prepared by the dimethoxy-dimethyl-silane (DiMe-DMOS) and tetramethyl-orthosilicate (TMOS). After cultivation, microorganisms adhered on the surface coated with sol-gels and glass surface were dyed by gram-staining method and the numbers of microorganisms were analyzed based on the image data of the scanning electronic microscope (SEM). A great number of microorganisms, about $2{\sim}3{\times}10^4/mm^2$, was adhered on the glass surfaces which no hydrophobic sol-gels were coated. But, the antifouling effect of the hydrophobic sol-gels was large, that microorganisms of less than $200{\sim}300/mm^2$ were adhered on the coated glass surface. The performance of the sensing membranes for detection of pH and dissolved oxygen was enhanced by recoating the light insulation layer prepared with the mixture of the hydrophobic sol-gel and graphite particles.

• Applied Chemistry for Engineering
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• v.19 no.2
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• pp.222-227
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• 2008

In this work we have studied the antifouling properties of the hydrophobic sol-gel modified sensing membrane and its optical properties for sensor application. E. coli JM109, B. cereus 318 and P. pastoris X-33 were cultivated in confocal cultivation dishes with glass surface, respectively. The glass surface was coated with the hydrophobic sol-gels prepared by the dimethoxy-dimethyl-silane (DiMe-DMOS) and tetramethyl-orthosilicate (TMOS). After cultivation, microorganisms adhered on the surface coated with sol-gels and glass surface were dyed by gram-staining method and the numbers of microorganisms were analyzed based on the image data of the scanning electronic microscope (SEM). A great number of microorganisms, about $2{\sim}3{\times}10^4/mm^2$, was adhered on the glass surfaces which no hydrophobic sol-gels were coated. However, the antifouling effect of the hydrophobic sol-gels was large, that microorganisms of less than $200{\sim}300/mm^2$ were adhered on the coated glass surface. The performance of the sensing membranes for detection of pH and dissolved oxygen was enhanced by recoating the light insulation layer prepared with the mixture of the hydrophobic sol-gel and graphite particles.

• Korean Journal of Clinical Laboratory Science
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• v.52 no.1
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• pp.53-61
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• 2020

Cell migration is a central process for recovering from wounds triggered by physical distress besides embryogenesis and cancer metastasis. Wound healing assay is widely used as a fundamental research technique for investigation of two-dimensional cell migration in vitro. The most common approach for imitating physical wound in vitro is mechanical scratching on the surface of the confluent monolayer by using sharp materials. The iron metal pin with a suspension spring for fine adjustment of the orthogonal contact surface between the scratching point and the individual bottom of multi-well plate with planar curvatures were adopted for the creative invention of a 96-well plate wound maker. While classic tips drew diverse and zigzag scratching patterns on the confluent monolayer, our wound maker displayed synchronized linear wounds in the middle of each well of a 96-well plate that was seeded with several cell lines. Given that several types of multi-well plates commercially available are compatible with our lab-made wound maker for creating uniform scratches on the confluent monolayer for the collective cell migration in wound healing assay, it is certain that the application of this wound maker to the real-time wound healing assay in high content screening (HCS) is superior than utilization of typical polypropylene pipette tips.

• Korean Journal of Clinical Laboratory Science
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• v.52 no.1
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• pp.1-17
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• 2020

Three-dimensional microscopic approaches in histopathology display multiplex properties that present puzzling questions for specimens as related to their comprehensive volumetric information. This information includes spatial distribution of molecules, three-dimensional co-localization, structural formation and whole data set that cannot be determined by two-dimensional section slides due to the inevitable loss of spatial information. Advancement of optical instruments such as two-photon microscopy and high performance objectives with motorized correction collars have narrowed the gap between optical theories and the actual reality of deep tissue imaging. However, the benefits gained by a prolonged working distance, two-photon laser and optimized beam alignment are inevitably diminished because of the light scattering phenomenon that is deeply related to the refractive index mismatch between each cellular component and the surrounding medium. From the first approaches with simple crude refractive index matching techniques to the recent cutting-edge integrated tissue clearing methods, an achievement of transparency without morphological denaturation and eradication of natural and fixation-induced nonspecific autofluorescence out of real signal are key factors to determine the perfection of tissue clearing and the immunofluorescent staining for high contrast images. When performing integrated laboratory workflow of tissue for processing frozen and formalin-fixed tissues, clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in situ hybridization-compatible tissue hydrogel (CLARITY), an equipment-based tissue clearing method, is compatible with routine procedures in a histopathology laboratory.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.7
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• pp.857-867
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• 2017

This study was performed to encapsulate low molecular weight marine collagen and ${\gamma}$-aminobutyric acid (GABA)-producing lactic acid bacteria to inhibit degradation and improve survival rate during exposure to adverse conditions of the gastro-intestinal tract. Calcium-alginate method was used for the manufacture of a double-layered coated capsule. The inner core material was composed of collagen and lactic acid bacteria, and the coating materials were alginate and chitosan. The sizes and shapes of the double-coated capsule were affected mainly by centrifuge speed and pH. Manufactured capsules were observed with a scanning electron microscope and by confocal laser scanning microscopy to confirm the micromorphological changes of capsules and bacterial cells. As a result, double-layered coated capsules were not degraded at pH 1.2, whereas degradation occurred at pH 7.4. In addition, GABA and collagen were maintained in stable state at pH 1.2. Therefore, double-layered coated capsules developed in this study would not be degraded in the stomach and could be stably delivered to the small intestine to benefit intestinal and dermatic health.

• Journal of Korean Academy of Oral and Maxillofacial Radiology
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• v.29 no.1
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• pp.105-117
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• 1999

Purpose: Ionizing radiations have been reported as an apoptosis initiating stimulus in various cells and it has established that sustained elevations in [Ca/sup 2+/] can lead to DNA fragmentation by Ca/sup 2+/-dependent endonucleases, ultimately resulting in apoptotic cell death. The previous experiments have been reported by using primarily thymocytes and lymphocytes and the change of [Ca/sup 2+/] was measured only by minutes or hours respectively. We need to evaluate [Ca/sup 2+/] in both several minutes and hours after irradiation of radiation of radiation therapy and verify the apoptotic cells. Materials and Methods: We have measured [Ca/sup 2+/] in human gingival epitheloid cancer cell with 10Gy irradiation, at minutely intervals and hourly intervals using digitized video-intensified fluorescence microscopy and the fluorescent Ca/sup 2+/ indicator dye, fura-2. In order to find out that the transient rise in [Ca/sup 2+/] could induced apoptosis, cells were incubated for 1 hour at 37℃ with TdT enzyme, rinsed and resuspended containing fluorescence and observed under a confocal fluorescence microscope. MTT assay was done to determine cell activity and LDH assay was done to determine the amount of necrotic cells. Results: After irradiation, the transient and temporal increasing of [Ca/sup 2+/] in the KB cells was founded. Though, there was no change in the intracellular [Ca/sup 2+/] at 30 minutes and 2 hours after irradiation. We could detect of DNA fragmented cells at 4 hours after 10Gy irradiated cells. There were no significant differences between 4 hour, 1 day, 3 day cells. There were no significant differences in MTT and LDH assay between the irradiated group and the control group after 4 hours and 1 day. Though after 3 days there were differences in MTT and LDH assay between the irradiated group was significantly decreased than the control group, in LDH assay the number of necrotic cell death of the irradiated was higher than the control group. Conclusion: In KB cells there were incipient and temporal increasing of the [Ca/sup 2+/] with 10Gy irradiation and the apoptosis was founded from 4 hours later which was earlier than seeing of the change of the amount of the cellular ability and necrosis.

• The Journal of Korean Academy of Prosthodontics
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• v.44 no.3
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• pp.343-355
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• 2006

Statement of problem. The success of osseointegration can be enhanced with an implant that has improved surface characteristics. Anodic oxidation is one of the surface modifying method to achieve osseointegration. Voltage of anodic oxidation can change surface characteristics and cell activity Purpose. This study was performed to evaluate MG63 cell responses such as affinity, proliferation and to compare surface characteristics of anodic oxidized titanium in various voltage. Material and method. The disks for cell culture were fabricated from grade 3 commercially pure titanium,1 m in thickness and 12 mm in diameter. Surfaces of 4 different roughness were prepared. Group 1 had a machined surface, used as control. Group 2 was anodized under 220 V, group 3 was anodized under 300 V and group 4 was anodized under 320 V. The microtopography of specimens was observed by scanning electron microscope (JSM-840A, JEOL, Japan) and atomic force microscope(Autoprobe CP, Park Scientific Instrument, USA). The surface roughness was measured by confocal laser scanning microscope(Pascal, LSM5, Zeiss, Germany). The crystal structure of the titanium surface was analyzed with x-ray diffractometer(D8 advanced, Broker, Germany). MG63 osteoblast-like cells were cultured on these specimens. The cell morpholgy was observed by field emission electron microscope(Hitachi S-4700, Japan). The cell metabolic and proliferative activity was evaluated by MTT assay Results and conclusion. With in limitations of this in vitro study, the following conclusions were drawn. 1. In anodizing titanium surface, we could see pores which did not show in control group. In higher anodizing voltage, pore size was increased. 2. In anodizing titanium surface, we could see anatase. In higher anodizing voltage, thicker oxide layer increased crystallinity(anatase, anatase and rutile mixed). 3. MG63 cells showed more irregular, polarized and polygonal shape and developed more lamellipodi in anodizing group as voltage increased. 4. The activity of cells in MTT assay increased significantly in group 3 and 4 in comparison with group 1 and 2. However, there was no difference between group 3 and 4 at P<0.05. Proliferation of MG63 cells increased significantly in pore size($3-5.5{\mu}m$) of group 3 and 4 in comparison with in pore size($0.2-1{\mu}m$ ) of group 2.

• Journal of Periodontal and Implant Science
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• v.45 no.3
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• pp.120-126
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• 2015

Purpose: With the significance of stable adhesion of alveolar bone and peri-implant soft tissue on the surface of titanium for successful dental implantation procedure, the purpose of this study was to apply microgrooves on the titanium surface and investigate their effects on peri-implant cells and tissues. Methods: Three types of commercially pure titanium discs were prepared; machined-surface discs (A), sandblasted, large-grit, acid-etched (SLA)-treated discs (B), SLA and microgroove-formed discs (C). After surface topography of the discs was examined by confocal laser scanning electron microscopy, water contact angle and surface energy were measured. Human gingival fibroblasts (hGFs) and murine osteoblastic cells (MC3T3-E1) were seeded onto the titanium discs for immunofluorescence assay of adhesion proteins. Commercially pure titanium implants with microgrooves on the coronal microthreads design were inserted into the edentulous mandible of beagle dogs. After 2 weeks and 6 weeks of implant insertion, the animal subjects were euthanized to confirm peri-implant tissue healing pattern in histologic specimens. Results: Group C presented the lowest water contact angle ($62.89{\pm}5.66{\theta}$), highest surface energy ($45{\pm}1.2mN/m$), and highest surface roughness ($Ra=22.351{\pm}2.766{\mu}m$). The expression of adhesion molecules of hGFs and MC3T30E1 cells was prominent in group C. Titanium implants with microgrooves on the coronal portion showed firm adhesion to peri-implant soft tissue. Conclusions: Microgrooves on the titanium surface promoted the adhesion of gingival fibroblasts and osteoblastic cells, as well as favorable peri-implant soft tissue sealing.

• Journal of Periodontal and Implant Science
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• v.43 no.2
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• pp.72-78
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• 2013

Purpose: The purpose of this study was to compare the phototoxic effects of blue light exposure on periodontal pathogens in both planktonic and biofilm cultures. Methods: Strains of Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis, in planktonic or biofilm states, were exposed to visible light at wavelengths of 400.520 nm. A quartz-tungsten-halogen lamp at a power density of $500mW/cm^2$ was used for the light source. Each sample was exposed to 15, 30, 60, 90, or 120 seconds of each bacterial strain in the planktonic or biofilm state. Confocal scanning laser microscopy (CSLM) was used to observe the distribution of live/dead bacterial cells in biofilms. After light exposure, the bacterial killing rates were calculated from colony forming unit (CFU) counts. Results: CLSM images that were obtained from biofilms showed a mixture of dead and live bacterial cells extending to a depth of $30-45{\mu}m$. Obvious differences in the live-to-dead bacterial cell ratio were found in P. gingivalis biofilm according to light exposure time. In the planktonic state, almost all bacteria were killed with 60 seconds of light exposure to F. nucleatum (99.1%) and with 15 seconds to P. gingivalis (100%). In the biofilm state, however, only the CFU of P. gingivalis demonstrated a decreasing tendency with increasing light exposure time, and there was a lower efficacy of phototoxicity to P. gingivalis as biofilm than in the planktonic state. Conclusions: Blue light exposure using a dental halogen curing unit is effective in reducing periodontal pathogens in the planktonic state. It is recommended that an adjunctive exogenous photosensitizer be used and that pathogens be exposed to visible light for clinical antimicrobial periodontal therapy.