박테리아 셀룰로오스 기반 기능성 나노복합체의 제조와 특성 및 응용 연구

학위논문 (박사)-- 서울대학교 대학원 : 바이오시스템·소재학부 바이오소재전공, 2016. 8. 현진호.본 논문에서는 적합한 합성전략을 통해 박테리아 셀룰로오스에 무기나노입자와 바이오폴리머를 도입한 나노복합체를 제작하였고, 그 응용가능성을 평가해 보았다. 첫 번째로 자성나노입자, 실리콘나노입자, 금나노입자를 각각 박테리아 셀룰로오스와 복합화하였다. 자성 박테리아셀룰로오스 복합체는 자성나노입자가 함유된 배지에 박테리아를 배양하는 생합성 방법을 통하여 제조하였고, 아닐린 모노머를 도입하여 전도성 고분자인 폴리아닐린을 중합하였다. 전자기 차폐막으로의 응용가능성을 파악하기 위해 자성과 전기전도성 측정 실험을 수행하였고, 합성된 복합체는 전도성과 자성을 모두 가졌다. 실리콘나노입자가 함유된 박테리아 셀룰로오스 나노복합체는 안정적으로 분산된 실리콘나노입자 분산용액에 박테리아셀룰로오스를 담침시키는 방법으로 제조하였다. 실리콘 나노입자들은 셀룰로오스 나노섬유에 균일하게 도입되었다. 이 후, 폴리아닐린을 중합하여 복합체에 전도성을 부여하였다. 완성된 복합체는 반복적인 구부림 시험에서 일정한 전기 전도성을 나타내었다. 이를 통하여 유연성 전극으로 응용이 가능함을 확인하였다. 금나노입자-박테리아셀룰로오스 복합체는 in situ 합성법을 통해 제조되었다. 셀룰로오스 나노섬유는 합성 과정에서 지지체이자 환원제로서 역할을 하였다. 제조된 복합체의 구조적 변형을 통하여 분석물질의 표면 증강 라만 산란 신호를 크게 증가시킬 수 있었다. 두 번째로, TEMPO/NaBr/NaClO 산화 시스템을 통해 개질된 박테리아셀룰로오스와 바이오폴리머를 이용한 복합체를 제작하였다. 수용액에 잘 분산된 산화 박테리아셀룰로오스를 알지네이트와 섞고, 칼슘이온에 의한 이온가교를 유도하여 하이드로젤 형태로 제조하였다. 형성된 복합체는 기계적∙화학적 저항성이 증가하였고, 세포 담지체로서의 응용가능성이 평가되었다. 수용액상에서 양전하를 갖는 엘라스틴 유래 폴리펩타이드와 음전하를 갖는 산화 박테리아 셀룰로오스를 이용하여 온도민감성 하이드로젤 복합체를 제조하였다. 온도가 증가함에 따라 엘라스틴 유래 폴리펩타이드가 물리적 가교제로 작용을 하여 하이드로젤이 형성되고, 온도가 낮아짐에 따라 젤-졸 전이가 일어났다. 이 후 세포 실험을 통하여 하이드로젤 내부에서 세포를 안정적으로 성장시킬 수 있었다. 위와 같은 적합한 합성전략을 통한 다양한 박테리아셀룰로오스 기반 나노복합체 연구들은 소재로서의 박테리아 셀룰로오스에 대한 이해를 넓혀주고, 다양한 분야로의 응용 가능성을 높여 줄 수 있을 것으로 기대한다.Bacterial cellulose (BC)-based nanocomposites incorporated with inorganic nanoparticles and biopolymers were fabricated by employing suitable synthesis strategies, and their potential applications were investigated. Firstly, a series of BC nanocomposites containing inorganic nanoparticles were synthesized using appropriate methods. BC magnetite nanocomposites (MNP-BC) were biosynthesized by incubating bacteria in a medium containing magnetic nanoparticles (MNPs). Subsequently, polyaniline (PANi) was synthesized on the MNPs-BC nanocomposites by carrying out oxidative polymerization of aniline. Magnetic and electrical measurements confirmed that the MNP-PANI-BC exhibited the potential to be used as an electromagnetic shielding material. Silicon nanoparticles (SiNPs)-BC nanocomposites were prepared by dipping BC in the SiNPs dispersion. SiNPs were uniformly attached to the BC pellicle surfaces along the nanofibers. The conductive PANi-Si-BC composite exhibited stable conductivity under repetitive bending stress, confirming its potential for flexible anode application. Gold nanoparticles (AuNPs)-BC nanocomposites were prepared by employing the in situ polymerization of AuNPs on the BC fibers, which could act as a template and an immobilized reducing agent. The surface-enhanced Raman scattering signals corresponding to molecules to be detected on the AuNPs-BC nanocomposite were significantly enhanced due to the spatial deformation of the composite. Secondly, a series of BC nanocomposites containing biopolymers were synthesized with TEMPO-oxidized bacterial cellulose (TOBC). TOBC fibers were obtained using a TEMPO/NaBr/NaClO system at pH 10 and room temperature. The fibrillated TOBCs mixed with alginate were cross-linked in the presence of Ca2+ solution to form hydrogel composites. Alginate/TOBC hydrogel composites exhibited improved mechanical and chemical resistance, indicating that the hydrogel could be used for cell encapsulation applications. Elastin-like polypeptide (ELP)-BC composites were synthesized, which could be used as a thermosensitive hydrogel for cell encapsulation applications. Positively charged ELP was used as a polymeric cross-linker for conjugating with negatively charged cellulose nanofibers. Hydrogel formation was triggered by increasing the temperature, and the hydrogel was converted to the liquid phase by decreasing the temperature.Chapter 1. Introduction 1 Chapter 2. Literature survey 7 2.1. Cellulose nanofibers 8 2.1.1. Cellulose 8 2.1.2. Processes of cellulose nanofibers preparation 12 2.1.3. Three types of cellulose nanofibers 12 2.1.4. Surface modification of cellulose nanofibers 20 2.2. Bacterial cellulose (BC) 23 2.2.1. General information of BC 23 2.2.2. Biosynthesis process of BC 25 2.2.3. Factors affecting the production capacity of BC 28 2.2.4. Productivity of BC 30 2.2.5. Characteristic differences between the plant cellulose and BC 34 2.2.6. Applications of BC 38 Chapter 3. Electromagnetic BC nanocomposite using magnetite nanoclusters and polyaniline 44 3.1. Introduction 45 3.2. Materials and method 48 3.2.1. Preparation of highly dispersive magnetite nanoparticles (MNPs) 48 3.2.2. Biosynthesis of MNP-incorporated BC (MNP-BC) nanocomposites 49 3.2.3. Polymerization of polyaniline (PANi) on the MNP-BC (PANi-MNP-BC) nanocomposites 49 3.2.4. Characterizations of nanocomposites 50 3.3. Results and discussion 51 3.3.1. Enhancement of the colloidal stability of MNP solutions 51 3.3.2. Biosynthesis of MNP-BC nanocomposites 54 3.3.3. Synthesis of PANi-MNP-BC nanocomposites 58 3.3.4. Electromagnetic properties of nanocomposites 64 3.4. Summary 66 Chapter 4. Flexible conductive BC combined with silicon nanoparticles and polyaniline 67 4.1. Introduction 68 4.2. Materials and method 71 4.2.1. Preparation of the silicon nanoparticle-embedded BC (Si-BC) nanocomposites 71 4.2.2. Polyaniline (PANi) polymerization with BC (PANi-BC) and Si-BC nanocomposites (PANi-Si-BC) 71 4.2.3. Characterization of nanocomposites 72 4.3. Results and discussion 74 4.3.1. Preparation of the Si-BC nanocomposites 74 4.3.2. Synthesis of PANi-Si-BC nanocomposites 80 4.3.3. Conductivity of nanocomposite under bending stress 92 4.4. Summary 94 Chapter 5. Surface-enhanced Raman scattering sensor based on a BC hydrogel 95 5.1. Introduction 96 5.2. Materials and method 99 5.2.1. In situ synthesis of gold nanoparticles (AuNPs)-BC 99 5.2.2. Characterization of AuNPs-BC nanocomposites 99 5.2.3. SERS experiments 100 5.3. Results and discussion 100 5.3.1. In situ synthesis of AuNPs-BC hydrogel 100 5.3.2. SERS measurement using undeformed AuNPs-BC 105 5.3.3. SERS measurement using deformed AuNPs-BC 110 5.3.4. Detection of molecules with weak affinity to Au surfaces 114 5.4. Summary 116 Chapter 6. Oxidized BC/alginate hydrogel for cell encapsulation 117 6.1. Introduction 118 6.2. Materials and method 121 6.2.1. Preparation of TEMPO-mediated oxidized BC (TOBC) 121 6.2.2. Preparation of alginate/TOBC beads 121 6.2.3. Characterization of alginate/TOBC beads 122 6.2.4. Viability and proliferation of encapsulated cells 123 6.3. Results and discussion 123 6.3.1. Preparation of alginate/TOBC beads 123 6.3.2. Mechanical and chemical stability of alginate/TOBC 131 6.3.3. Permeability of alginate/TOBC beads 135 6.3.4. Viability and proliferation of encapsulated cells 139 6.4. Summary 142 Chapter 7. Thermoresponsive hybrid hydrogel of oxidized BC using a elastin like polypeptide 143 7.1. Introduction 144 7.2. Materials and method 147 7.2.1. Elastin like polypeptide (ELP) synthesis 147 7.2.2. Sol-gel transition 147 7.2.3. Characterization of ELP/TOBC 147 7.2.4. Cell viability and proliferation 149 7.3. Results and discussion 151 7.3.1. Gel formation of ELP/TOBC 151 7.3.2. Rheological analysis of ELP/TOBC hydrogel 154 7.3.3. Morphological analysis of ELP/TOBC hydrogel 157 7.3.4. Mechanism of TOBC/ELP hydrogel formation 160 7.3.5. Viability and proliferation of cells 166 7.4. Summary 168 Chapter 8. Conclusions 169 References 173 초록 204Docto

In situ recruitment of human bone marrow-derived mesenchymal stem cells using chemokines for articular cartilage regeneration

Dept. of Medical Science/박사Articular cartilage has a limited capacity for self-regeneration after injury. Bone marrow-derived mesenchymal stem cells (BMSCs) are good sources of repair since they can migrate directly to the injury site and differentiate to articular chondrocytes. To repair articular cartilage injury, various surgical techniques such as subchondral drilling and autologous chondrocytes transplantation are widely used. However, these techniques have severe problems including the fibro-cartilage formation, two-step surgery, limited donor sites for osteochondral autograft and loss-of-chondrogenic potency of ex vivo expanded chondrocytes. Chondral defects do not completely heal due to lack of chondrocytes at the defect site and the insufficient migration of surrounding chondrocytes and BMSCs to the injury site. Therefore, enhancing migration of a large number of BMSCs, chondrogenic progenitor cells, to the injury site is considered to be an effective strategy for cartilage regeneration. The primary purpose of this study is to screen the chemokine receptors and their ligands inducing the chemotaxis of BMSCs and the second is to investigate the chemotaxis of BMSCs in vivo toward selected chemokines which may give rise to a complete regeneration of articular cartilage.BMSCs isolated from human bone marrow were grown in either presence or absence of pro-inflammatory cytokines, such as IL-1β and TNF-α. RT-PCR was performed to evaluate the expression of nineteen chemokine receptors. To evaluate changes in the expression of chemokine receptors by pro-inflammatory cytokines, a reverse dot-blot assay was performed. It was shown that CCR2, CCR4, CCR6, CXCR1, and CXCR2 were expressed in normal BMSCs and increased significantly upon treatment with pro-inflammatory cytokines. To evaluate the effect of chemokines on BMSCs, the ligands of screened chemokine receptors were selected using the “Protein-knowledge database” and other reports. After screening the meaningful chemokine receptors and ligands, MTT assay for cell proliferation and cytotoxicity was performed for MCP-1, MIP-3α, IL-8, and SDF-1α.To determine the effect of chemokine on cell movement, two-dimensional migration assay was performed using wound healing and live cell tracking techniques. The wound healing capacity was increased by MIP-3α and IL-8 more than that by MCP-1 or SDF-1α. These four chemokines significantly increased the migrating mean velocity and total migrated distance of BMSCs. Especially, MIP-3α was most potent in healing capacity among chemokines. To investigate the chemotactic effect of chemokines on BMSCs, in vitro chemotaxis assay was performed. As a result, IL-8 and MIP-3α significantly enhanced the chemotaxis of BMSCs compared to MCP-1, SDF-1α and control. Collectively, IL-8 and MIP-3α were finally selected as candidates for the recruitment of BMSCs. To examine whether chemokine candidates enhance osteogenesis and chondrogenesis of BMSCs, cell differentiation assay was performed. These candidates did not induce ALP expression and calcium deposition in osteogenic differentiated BMSCs. The expression of chondrogenic markers, such as collagen fibers, aggrecan and type II collagen, were also not affected by chondrogenic differentiation with IL-8 and MIP-3α. To evaluate chemotactic effect of either IL-8 or MIP-3α, in vitro/in vivo chemotaxis assay using human BMSCs, chemokines-containing PLGA scaffolds, and nude mice was performed with live in vivo imaging system. In nude mice, the in situ recruitment of human BMSCs toward transplanted either IL-8 or MIP-3α with scaffolds was significantly induced. According to the results of histological analysis, fibroblastic cells recruited into chemokines-containing scaffolds were identified as human BMSCs which were injected into tail vein of mice. In addition, the subcutaneous tissue formation was enhanced by recruited human BMSCs without inflammatory reaction.Conclusively, IL-8 and MIP-3α were significantly enhanced tissue formation by chemotaxis of BMSCs without inflammation. Therefore, this study suggests that either IL-8 or MIP-3α can be useful candidates for regeneration of damaged articular cartilage.prohibitio

(The) effect of TGF-β signaling pathway by integrin signaling pathway in articular chondrocytes

의과학과/석사[한글]관절 연골세포의 증식 및 생존을 위해서 인테그린과 제 2형 교원질과 같은 세포외 기질간의 상호작용이 필수적으로 요구된다. 그러나 아직까지 제 2형 교원질과 특이적인 인테그린 신호전달경로의 상호작용의 기능에 대해 알려진 바가 거의 없다. TGF-β1은 연골세포의 특성을 유지하고 증식 및 세포외기질 합성을 촉진한다고 알려져 있다. 최근에는 연골세포에서 TGF-β1 신호전달경로는 다른 성장인자의 신호전달경로와 신호교차반응을 일으키는 것으로 밝혀지고 있다. 따라서 관절 연골세포에 제 2형 교원질 및 TGF-β1의 자극을 각각 또는 동시에 가한 후, 이로 인한 세포내 신호전달 단백의 활성화 변화를 분석함으로써, 인테그린 신호전달경로와 TGF-β1 신호전달경로의 신호교차반응의 기전을 밝히고자 하였다.외부자극에 의한 인테그린과 TGF-β1의 하위 신호전달경로를 분석하기 위해 제 2형 교원질을 세포 배양 용기에 코팅하였고, TGF-β1은 세포 배양액에 첨가하는 방법으로 가했으며, 각 자극 또는 두 가지 자극을 함께 처리한 조건에서 연골세포를 배양하였다. 세포내 총 단백질을 추출하여 western blot기법을 이용하여 신호전달 단백의 활성 변화를 분석한 결과, 제 2형 교원질 자극을 가한 군에서의 세포부착이 제 2형 교원질 자극을 가하지 않은 군에 비해 빨리 이루어졌으며, 제 2형 교원질 자극을 가한 군에서 SMAD 2와 SMAD 3의 발현이 관찰되었다. 특히 두 자극을 동시에 가한 군에서는 SMAD 2와 SMAD 3의 활성에 대한 시너지효과를 관찰하였다. TGF-β1 처리군에서 FAK의 타이로신 925번째 부위의 인산화가 확인 되었고, 동시 자극군에서 시너지효과를 확인하였다. FAK의 하위 신호전달경로인 extracellular signal-regulated protein kinase 1/2 (ERK 1/2)와 p38의 활성에는 유의한 변화가 없었다. 따라서 관절 연골세포에서 두 경로사이의 신호교차반응이 존재하며 이에, SMAD 2 와 SMAD 3 및 FAK (Y925) 가 관여할 것으로 사료된다. [영문]It is established that articular chondrocytes require integrin mediated interactions with extracellular matrix (ECM), such as type II collagen, a major matrix component of articular cartilage, to proliferate and survive. However, the functional interaction between type II collagen and specific integrin signaling pathway has not been identified. Transforming growth factor-beta 1 (TGF)-β1 seems to play a crucial role in maintaining chondrogenic properties, proliferation and stimulates ECM synthesis in chondrocytes. Recently, signal cross-talking between TGF-β1 and other growth factors in chondrocytes was reported. Therefore, in this study, the signal cross-talk between integrin signaling cascade and TGF-β1 signaling cascade in articular chondrocytes was identified, through the analysis the altered activation of intracellular signal transmitting proteins after the treatment of type II collagen and TGF-β1 each or both stimuli into cells.To analyze integrin or TGF-β1 mediated signaling pathways from extracellular stimuli, type II collagen was coated on the cell culture plate and TGF-β1 was injected into cell culture media. Chondrocy-tes were cultured in the conditioned media with each or both stimuli. As a result of altered activation of signaling proteins with western blotting technique of whole cell lysates, more rapid attachment of cells was observed in the type II collagen coated group than non-treated group. The phosphorylated smad 2 and 3 were expressed in the type II collagen coated group and synergistically up-regulated expression in the co-treated group. The phosphorylated FAK (Y925) was activated by TGF-β1 treatment and synergistically up-regulated by both stimuli. But any meaningfully changed phosphorylation of extracellular signal-regul-ated protein kinase (ERK) 1/2 and p38, as known downstream molecules of FAK cascade, was not observed. This result means that SMAD 2, SMAD 3 and Y925 phosphorylation site of FAK are involved in this signal cross-talking in articular chondrocytes.ope

In Situ Recruitment of Human Bone Marrow-Derived Mesenchymal Stem Cells Using Chemokines for Articular Cartilage Regeneration

Bone marrow-derived mesenchymal stem cells (BMSCs) are a good cell source for regeneration of cartilage as they can migrate directly to the site of cartilage injury and differentiate into articular chondrocytes. Articular cartilage defects do not heal completely due to the lack of chondrocytes or BMSCs at the site of injury. In this study, the chemotaxis of BMSCs toward chemokines, which may give rise to a complete regeneration of the articular cartilage, was investigated. CCR2, CCR4, CCR6, CXCR1, and CXCR2 were expressed in normal BMSCs and were increased significantly upon treatment with proinflammatory cytokines. BMSC migration was increased by MIP-3α and IL-8 more than by MCP-1 or SDF-1α. IL-8 and MIP-3α significantly enhanced the chemotaxis of BMSCs compared with MCP-1, SDF-1α, or PBS. Human BMSC recruitment to transplanted scaffolds containing either IL-8 or MIP-3α significantly increased in vivo compared to scaffolds containing PBS. Furthermore, IL-8- and MIP-3α-containing scaffolds enhanced tissue regeneration of an osteochondral defect site in beagle knee articular cartilage. Therefore, this study suggests that IL-8 and MIP-3α are the candidates that induce the regeneration of damaged articular cartilage.ope

FAK mediates signal crosstalk between type II collagen and TGF-beta 1 cascades in chondrocytic cells

The purpose of this study was to evaluate the mechanism of crosstalk between the type II collagen and TGF-beta1 signaling pathways in chondrocytic cells. Articular chondrocytes, isolated from porcine knee cartilage, and the SW1353 cell line were cultured on either type II collagen-coated or -uncoated plates in the presence or absence of TGF-beta1. Expression of pSMAD 2, pSMAD 3, pFAK(Y397) and pFAK(Y925) in articular chondrocytes and the SW1353 cell line was analyzed by immunoblotting. Cell proliferation rates and glycosaminoglycan (GAG) content was determined after treatment with type II collagen or/and TGF-beta1. For inhibition study, human FAK-specific RNA small interference (siFAK) in SW1353 cell line was performed. In this study, expression of pSMAD 2, pSMAD 3, pFAK(Y397) and pFAK(Y925) were synergistically increased by co-treatment with type II collagen and TGF-beta1 in articular chondrocytes. The proliferation of porcine articular chondrocytes and GAG secretion in SW1353 cells were synergistically increased by co-stimulation with type II collagen and TGF-beta1. Synergistically increased expression and nuclear translocation of pSMAD 2 and pSMAD 3 and GAG secretion of SW1353 cells were significantly inhibited by siFAK transfection. Therefore, we suggest that FAK-SMAD 2/3 mediates signal crosstalk between type II collagen and TGF-beta1 and regulates GAG secretion in chondrocytic cellsope

공공정책과 기업가정신의 관계성 규명에 대한 연구

학위논문(석사) - 한국과학기술원 : 산업공학과, 2008.2, [ iv, 44 p. ]SME and entrepreneurship policy makers who are confronted with economic crises take into account which policy is the first to be implemented as well as those that come. Hence, three questions are of concern here: 1. What policies are effective for entrepreneurship activity and how are they effective? 2. What effects of those policies differ corresponding to the different intensity of entrepreneurship activity? 3. Why was Asian entrepreneurship activity low from 1998 to 2004? This study aims to propose which variables of public policies have an effect on promoting start-up entrepreneurship. Therefore, the relationship between public policies and firm birth rate across 27 countries is examined using ordinary least squared regression analysis. This paper conducts this analysis using OECD annual data from 1998 to 2004. 75 independent variables were selected from following OECD databases: Finance, ; National Accounts, ; Labor, ; Demography and Population, ; Science, Technology and Patents, ; and Public Sector, Taxation and Market Regulation. Four equations are examined. Three equations capture the relationship between firm birth rate and each field concerning public policy, and one equation considers all fields. In order to obtain an overall picture with meaningful factors regressions were computed, and each time adding one variable was added each time to the baseline model. As a result, a complete multivariate model was formulated. Subsequently a `developed country` dummy was included in the model to observe the group effect of developed and developing countries. It was that ‘Short-term interest rate’, ‘Public expenditure on startup incentives’ and ‘Institutional investor assets’ have strong positive effects on the variable of firm birth rate. Similarly, differences are shown between countries with high and low firm birth rate. In Eq. (2), which analyzes the relationship between firm birth rate and labor policies, ‘Public expenditure on labor ma...한국과학기술원 : 산업공학과

Evaluation of Efficacy and Safety of Stem Cells as a Therapeutics, Especially Focusing on Bone Marrow Derived Stromal Cells

Bone marrow derived-stromal cells (BMSCs) are pluriplotent progenitors for a variety of cell types, including osteoblast, chondrocyte, adipocyte, and so on. Stem cell research has enormous potential in the future clinical treatment of a wide range of diseases. Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. However, we don’t have exact evaluation methods and safeguards for clinical application. In this study, we investigated tracking BMSCs differentiation, localization, toxicity, and migration in vivo. The fluorescent vector used in our studies did not affect BMSCs viability or their ability to undergo osteogenic and adipogenic differentiation in vitro. During differentiation, EGFP-BMSCs by Oil Red O and Alizarin Red S were stained. EGFP-BMSCs were transplanted into the femoral region in autologous rabbit. After one month, these cells were detectable by confocal microscopy and RTPCR. Transplanted EGFP-BMSCs were not detected another organs (spleen, kidney, liver, and muscle) in immunohistochemistry and RT-PCR. In organ-function test and cell-toxicity examination, there is no difference between the before and after EGFP-BMSCs transplantation. we observed that transplanted EGFP-BMSCs were not affected cell-toxicity and migration. This results offer some evaluation methods and safeguards for clinical application using BMSCs. In further study, it will be needed to test reproductive and developmental toxicity after transplantation and observe migration of EGFP-BMSCs after transplantation.ope