Methods for Evaluation of the Chemotactic Migration of the Cells in 3D Biomimetic Matrix

Cell migration plays a critical role in various biological processes such as embryogenesis, metastasis of tumor cells and wound healing. Therefore, the migratory behavior of the cells has been researched with various influencing factors including chemoattractants. Most researches have done with 2 dimensional (2D) structured stratums to investigate the cell migration, however, this study suggests three different methods for in vitro studies of cell migratory behavior which are performed in biomimetic 3D structural environment by using collagen matrix. In the firstly introduced method, wound model was built up with a silicon insert and the chemoattractant, PDGF-bb was applied. Then, the movement of the cells to the wound area was observed. It was designed for examination of the potential of wound healing effect of certain agents. Other two methods are to study chemotactic migration of the cells in the 3D matrix in the presence of the chemoattractant. The orientation of the cell migration was examined by the under-agarose cell migration assay as the cells were seeded between the control collagen gel and PDGF-bb containing collagen gel. The cells located in the middle of the migrating chamber were affected by the components in both sides and the direction of their movement was determined. The last method was also performed in a 3D collagen matrix and the cell suspension was directly contacted to the chemoattractant. Therefore, the movement behavior of the cells was influenced by the agent. These protocols have different characteristics to evaluate the migration of the cells, and thus an appropriate method would be chosen for various studies of migration.ope

Migration of Human Dermal Fibroblast is Affected by the Diameter of the Electrospun PLGA Fiber

Cell migration is an essential activity of the cells in various biological phenomena such as embryonic development, wound healing of damaged tissue, capillary vascularization in angiogenesis and migration of leukocytes to kill the bacteria around the wound site. The properties of nanofibrous surface enhancing cell adhesion, proliferation, migration and differentiation are necessary for application in tissue engineering. Recently, fabricated scaffolds at the nanometer scale are very similar to the architecture of natural human tissue, because of the development of nanofibers. In this study, we observed different cell migration behaviors on PLGA nanofibers with different diameters. 0.4 μm and 1.4 μm PLGA fibers were fabricated by electrospinning. Adhesion of neonatal human dermal fibroblasts (nHDFs) on the PLGA scaffolds was quantified by MTT assay. Real time observation system was used to analyze the migration of nHDF on the 0.4 μm and 1.4 μm PLGA scaffolds. There are no significant differences in cell attachment between 0.4 μm and 1.4 μm PLGA nanofibers. However, the migration was affected by the thickness of the PLGA fiber. The cells were migrated along with the 0.4 μm PLGA fiber but did not cross 1.4 μm PLGA fiber. In this research, it would be evaluated that different diameter of electrospun PLGA fiber effect on the cell migration and proliferation, and it could be applied for the development of the fibrous scaffold in tissue engineering.ope

Stimulated Migration and Penetration of Vascular Endothelial Cells Into Poly (L-lactic acid) Scaffolds Under Flow Conditions

BACKGROUND: The initial procedure of the development of engineered tissues is cell seeding into three-dimensional polymer scaffolds. However, it is hard to make the cells invade into scaffold due to the characteristic of pore and material. Electrospun poly (L-lactic acid) scaffold and flow perfusion system were used to overcome these seeding problems. RESULTS: Before starting the experiment, we set up the parallel plate chamber system to observe endothelial cell migration under flow condition. In individual cell migration model, human umbilical endothelial cells started to migrate in the direction of flow at 8 dyne/cm(2) and we observed the cytoskeleton alignment at 8 dyne/cm(2). This study has demonstrated the possibility to evaluate and analyze cell migration using the parallel plate chamber system and we may predict in vivo cell migration under flow condition based on these results. Also the flow perfusion system was established for the effective cell seeding into at three dimensional scaffolds. Moreover, shear stress induced by flow can enhance cell migration into PLLA scaffold that is in the form of cotton. CONCLUSIONS: Result indicated that cell penetration was achieved under flow condition better and more than under static condition throughout the matrix.ope

인공혈관의 적절한 혈관 내피화를 위한 혈관 내피 세포의 이동 분석

Dept. of Medical Science/석사[한글] [영문]The main reason of restenosis that happened after bypass surgery, balloon angioplasty or stent placement is the damage of the endothelium, which generally forms the inner lining of the blood vessel walls. A rapid reendothelialization of the blood vessel walls is crucial for the prevention of thrombosis and intimal hyperplasia. Endothelial cell migration is critical and initiating the repair of injured vessels. Most of the researches on EC migration were completed under static conditions, but endothelial cells in vivo are under flow condition. Shear stress can induce many changes in endothelial cells, including alterations in cell shape, orientation, proliferation, and the reorganization of cytoskeleton etc. In this study, we set up the parallel plate chamber system to evaluate endothelial cell migration under flow condition. In wound closure model, the cells in the upstream part more migrated into the wound area under flow condition compared with in the absence of flow, while shear stress inhibited cell migration in the direction to the wound area in downstream part. In individual cell migration model, human umbilical endothelial cells started to migrate in the direction of flow at 8 dyne/cm2. We observed focal adhesions formation at the leading edge of the cell in the direction of flow and the alignment of actin cytoskeleton at 8 dyne/cm2. Also the flow perfusion system was estabilished for the effective cell seeding into a three dimensional scaffold. In this study, we found that under flow condition a better and more uniform cell distribution throughout the matrix was achieved than under static condition.This study has demonstrated the possiblity to evaluate and analyze cell migration using the parallel plate chamber system and we may predict in vivo cell migration under flow condition based on these results. It may give some help to design stent or artificial blood vessels.ope

Effect of high strength glass filler content in the system of MgO-CaO-Al2O3-SiO2 on hydrophilic addition

의과학과/박사[한글] 치과용 인상재는 치아 및 구강조직을 복제하는데 사용된다. 인상재의 경화 후 구강에서 제거할 때 치은 열구와 함몰부위에서 찢어지지 않아야 한다. 실리카는 치과용 인상재의 낮은 기계적 강도를 극복하기 위하여 필러로 광범위하게 사용되고 있다. 본 연구는 MgO-CaO-Al2O3-SiO2 체제의 고강도 필러를 제조하고 부가중합형 실리콘 인상재의 기질에 첨가하여 필러의 효과를 실험하였다. 실험군으로는 12.5MgO-17.5CaO-20Al2O3-50SiO2의 4성분계 글라스 필러를 35 wt%, 30 wt%, 25 wt%, 그리고 실란 처리한 필러를 25 wt% 첨가한 인상재를 제조하였고, 대조군으로는 Contrast(VOCO, 독일), Examix(GC, 일본), Express(3M/ESPE, 미국) 및 Perfect-F(한대케미칼, 한국) 등 4종의 라이트 바디(light body) 인상재를 선택하였다. 실험은 ASTM D624에 따라 찢김 강도, ISO 4823에 따라 점주도, 압축 변형률, 변형 회복률, 미세부 재현성, 선체적 변화율을 측정하였다. 그 결과 찢김 강도는 실험군이 대조군 보다 증가된 양상을 보였다. 필러의 조성 변화와 필러 함량의 증가는 찢김 강도를 증가시켰고, 점주도는 35 wt%를 제외하고 모두 IS0 규격에 만족하였다. 압축 변형률, 변형 회복률, 미세부 재현성, 선체적 변화율 등은 미세한 차이를 보였으나 실험군이나 대조군 모두 IS0 규격에 만족하였다. 그러므로 12.5MgO-17.5CaO-20Al2O3 -50SiO2 조성의 글라스는 25 wt%에서 가장 적절한 낮은 점도를 유지하면서 찢김강도를 증가시키므로 부가 중합형 실리콘 인상재의 라이트 바디에 유용한 필러로 기대된다. [영문]Dental impression materials are used to register or reproduce the form and relationship of the teeth and oral tissues. They should not be torn when removing from the mouth after its setting. Nowadays, silica is widely used as filler to overcome the low mechanical strength of the dental impression materials. The purpose of this study was to synthesize high strength glass in the system of MgO-CaO-Al2O3-SiO2 and to evaluate its usefulness after its mixing with addition silicone base. Commercial products, Contrast(VOCO, Germany), Examix(GC, Japan), Express(3M/ESPE, USA), and Perfect-F(Handae Chem., Korea) were selected as control groups. The glass filler of 12.5MgO-17.5CaO-20Al2O3-50SiO2 was admixed in each component of addition silicone impression material base at various contents of 35%(E1), 30%(E2), 25%(E3) and also silane-treated glass filler 25%(E4) in weight. Tear strength, consistency, strain in compression, recovery from deformation, detail reproduction and linear dimensional change were determined according to the standards of ISO 4823 and ASTM D624. Tear strength of E1 and E2 was increased significantly than that of the control group, and that of E3 and E4 was not statistically different from EP, the highest in control groups. Consistency was satisfied all groups the ISO standard except E1. Strain in compression, recovery from deformation, detail reproduction and linear dimensional change were satisfied the ISO standard either all experimental groups or control groups. Therefore, the glass in the system of 12.5MgO-17.5CaO-20Al2O3-50SiO2 is expected the useful filler in the light body of hydrophilic addition silicone impression materials because of its improved tear strength and low viscosity.ope

Polydopamine-mediated immobilization of multiple bioactive molecules for the development of functional vascular graft materials

In this study, we introduced a simple method for polydopamine-mediated immobilization of dual bioactive factors for the preparation of functionalized vascular graft materials. Polydopamine was deposited on elastic and biodegradable poly(lactic acid-co-ɛ-caprolactone) (PLCL) films, and a cell adhesive RGD-containing peptide and basic fibroblast growth factor were subsequently immobilized by simple dipping. We used an enzyme-linked immunosorbent assay and fluorescamine assay to confirm that we had stably immobilized bioactive molecules on the polydopamine-coated PLCL film in a reaction time-dependent manner. When human umbilical vein endothelial cells (HUVEC) were cultured on the prepared substrates, the number of adherent cells and proliferation of HUVEC for up to 14 days were greatest on the film immobilized with dual factors. On the other hand, the film immobilized with RGD peptide exhibited the highest migration speed compared to the other groups. The expression of cluster of differentiation 31 and von Willebrand factor, which indicates maturation of endothelial cells, was highly stimulated in the dual factor-immobilized group, and passively adsorbed factors showed a negligible effect. The immobilization of bioactive molecules inspired by polydopamine was successful, and adhesion, migration, proliferation and differentiation of HUVEC were synergistically accelerated by the presence of multiple signaling factors. Collectively, our results have demonstrated that a simple coating with polydopamine enables the immobilization of multiple bioactive molecules for preparation of polymeric functionalized vascular graft materials.ope

Mussel-inspired immobilization of vascular endothelial growth factor (VEGF) for enhanced endothelialization of vascular grafts

Most polymeric vascular prosthetic materials have low patency rate for replacement of small diameter vessels (<5 mm), mainly due to failure to generate healthy endothelium. In this study, we present polydopamine-mediated immobilization of growth factors on the surface of polymeric materials as a versatile tool to modify surface characteristics of vascular grafts potentially for accelerated endothelialization. Polydopamine was deposited on the surface of biocompatible poly(L-lactide-co-ε-caprolactone) (PLCL) elastomer, on which vascular endothelial growth factor (VEGF) was subsequently immobilized by simple dipping. Surface characteristics and composition were investigated by using scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. Immobilization of VEGF on the polydopamine-deposited PLCL films was effective (19.8 ± 0.4 and 197.4 ± 19.7 ng/cm(2) for DPv20 and DPv200 films, respectively), and biotin-mediated labeling of immobilized VEGF revealed that the fluorescence intensity increased as a function of the concentration of VEGF solution. The effect of VEGF on adhesion of HUVECs was marginal, which may have been masked by polydopamine layer that also enhanced cell adhesion. However, VEGF-immobilized substrate significantly enhanced proliferation of HUVECs for over 7 days of in vitro culture and also improved their migration. In addition, immobilized VEGF supported robust cell to cell interactions with strong expression of CD 31 marker. The same process was effective for immobilization of basic fibroblast growth factor, demonstrating the robustness of polydopamine layer for secondary ligation of growth factors as a simple and novel surface modification strategy for vascular graft materials.ope

Enhanced cellular responses of vascular endothelial cells on poly-γ-glutamic acid/PU composite film treated with microwave-induced plasma at atmospheric pressure

Poly-γ-glutamic acid (γ-PGA), which is produced by microbial fermentation, is a biodegradable, hydrophilic and non-toxic biomaterial. γ-PGA has many carboxyl groups that makes it a polyanionic biopolymer with swelling ability, biocompatibility and anticoagulant activity. On the other hand, few studies have examined the effect of γ-PGA on the cellular activity of human umbilical vein endothelial cells (HUVECs). The present study evaluated the effects of γ-PGA and fabricated γ-PGA/PU composite films on the attachment and proliferation of HUVECs after treatment with microwave plasma at atmospheric pressure. The results confirmed that γ-PGA is capable of increasing the proliferation and differentiation of HUVECs to form capillary tubes with enhanced alignment and organization. Moreover, the microwave plasma modified γ-PGA/PU composite film was more hydrophilic and the surface roughness was enhanced. In addition, the attachment and proliferation of the HUVECs were increased by the plasma treatment. These results suggest that γ-PGA and surface modified γ-PGA/PU composite can be applied as bioactive and biocompatible materials in vascular tissue engineering but further testing of γ-PGA and γ-PGA/PU composite films will be needed to confirm the potential effects for use in vascular applicationsope

Promoted cell and material interaction on atmospheric pressure plasma treated titanium

Surface carbon contamination is a natural phenomenon. However, it interferes with cell–biomaterial interaction. In order to eliminate the interference, atmospheric pressure plasma treatment was employed. Dielectric barrier discharge treatment of titanium surface for less than 10 min turned titanium super-hydrophilic. Adsorption of fibronectin which is the major cell adhesive protein increased after plasma treatment. Cell attachment parameters of osteoblast cells such as population, cell area, perimeter, Feret's diameter and cytoskeleton development were also enhanced. Cell proliferation increased on the plasma treated titanium. In conclusion, dielectric barrier discharge type atmospheric pressure plasma system is effective to modify titanium surface and the modified titanium promotes cell and material interactions. --------------------------------------------------------------------------------ope

Relation of smoking and periodontal status among 30s-50s Adults in Metropolitan Area

Objectives: Smoking is well known risk factor of various disease and cause of periodontal disease. So the aim of this study was to investigate the relationship between smoking and periodontal status. Methods: This study conducted a survey for the 15 1 adults aged 30 to 59 from Oct. 1,2006 to Mar. 30,2007 and the 1st examination through a clinical periodontal index and a radiograph to analyze the periodontal status in smoking status. For the inspection of periodontal status, t-test and one-way analysis of variance was conducted. Results: In terms of the number of quadrants possessing periodontal pocket, the groups of smokers(3.34) and former smokers(3.38) were higher than the non-smokers group(2.72) and the amount of loss for interproximal bone was noticeably more abundant in the groups of smokers(67.23) and former smokers(81.97) than in the non-smoker group(49.44). There were no differences in smoking characteristics between the smoking terms, but there were statistically significant differences in the amount of smoking between the group who smoked less than 20 cigarettes(56.24) and that smoked more than 21 cigarettes(97.45) and the group who smoked less than 10 cigarettes(49.44) while drinking alcohol and that smoked more than 11 cigarettes(87.56) while drinking. Conclusions: Smoking is not related to 0' Leary Plaque Index, Loe & Silness index and number of missing teeth; however, it has been found that the impact of smoking was conspicuous in the improvement effects of periodontal status as well as the number of quadrants possessing periodontal pocket, amount of loss for interproximal bone