Elementary School Teachers' Awareness of Forest Welfare Services and Promotion of Strategies for School-based Health Promotion Programs Using the Forest

Purpose: This study was conducted to identify the perceptions toward school forest programs related to forest welfare services in elementary schools and suggest strategies to activate new programs. Methods: A mixed method research was performed. Four teachers and one forest therapist participated in a focus group interview162 teachers answered a survey. Results: The teachers were aware of the effects of the forest program, but there were some barriers, including the question of whether there was an accessible forest, school forest management problems, the risk of teachers work overload, and the lack of program diversification for elementary students. Solutions included the expansion of school forests and forest facilities available to students, development of a variety of programs, provision of appropriate information on available facilities, and cooperation with educational institutions for institutionalization and increased effectiveness of school-based forest utilization programs. In addition, a scientific basis for data accumulation is needed. Conclusion: The Ministry of Forestry is cooperating with the Ministry of Education and local education offices to activate a forest-use health promotion program for elementary school students. Additionally, to utilize the forests in regular education courses, teachers should strive to spread positive awareness of forests.본 논문은 2016년 산림청의 지원을 받아 수행되었음

Differentiation of Tonsil derived Mesenchymal Stem Cells in Bioactive Thermogelling Systems

온도가 증가함에 따라 졸-젤 전이를 보이는 온도 민감성 고분자는 조직 공학과 재생 의약 시스템으로 응용될 수 있다. 이러한 온도 민감성 고분자는 간단한 스캐폴드 제조법으로 수술을 하지 않아도 되는 임플란트 과정과 살아있는 조직 상태를 모사할 수 있는 장점을 가지고 있다. 이 논문에서는 효과적인 조직 공학 응용을 위한 무기물이나 관능기가 도입된 바이오액티브 온도민감성 고분자를 살펴보았다. 첫번째 연구에서는, 주사가능한 칼슘 인산염 메조 결정체 (4 - 8 m)와 폴리펩타이드 온도 민감성 고분자의 무기/유기 복합 시스템이 편도 줄기세포 (TMSC)의 골화 분화를 촉진한다고 밝혔다. 나노 입자 (10 - 100 nm)나 순수 하이드로젤 시스템과 비교했을 때, 무기/유기 복합 시스템은 골 분화지표인 알칼리성 인산가수분해효소 (ALP), 뼈형성단백질2 (BMP2), 오스테오칼신 (OCN)이 mRNA와 단백질 레벨에서 높게 발현 되었다. 분화된 골세포의 ALP 활동도 역시 대조군과 비교하여 높은 수치를 보였다. 메조 복합체 시스템은 무기물에 의한 세포/단백질의 부착을 위한 단단한 표면뿐만 아니라, 하이드로젤에 의한 부드러운 세포 지지 매트릭스를 제공한다. 두번째 연구에서는, 락토비온산 (LB)로 컨주게이션된 온도 민감성 고분자를 이용하여 TMSC의 간 분화를 유도하였다. MatrigelTM과 2차원배양 시스템과 비교하였을 때, 간세포핵인자4 (HNF4 ), 알부민 (ALB), 글루코스-6-인산분해효소 (G6P)와 아시알로당단백질수용체1 (ASGR1)의 간 분화지표가 LB 컨주게이션 된 온도민감성 고분자 시스템에서 더 높은 것을 관찰하였다. ALB 합성, 요소 생성, 인도시아닌그린 흡수의 주요 간 기능 검사 역시 대조군 보다 갈락토실화 온도민감성 고분자에서 분화된 간세포가 더 효과적임을 관찰하였다. 이 연구에서는 무기/온도민감성 복합 시스템과 갈락토실화 온도민감성 고분자 시스템이 TMSC의 골과 간으로의 분화를 효과적으로 촉진함을 확인하였다. 이러한 바이오액티브 온도민감성 하이드로젤 시스템은 줄기세포 분화에 적합한 인공 ECM 설계의 중요한 단서를 제공할 뿐만 아니라, 주사용조직공학 응용으로써의 가능성을 입증하였다.;A thermogel system that undergoes a sol-to-gel transition as the temperature increses has been suggested as tissue engineering or regenerative medicine system. Due to its simple fabrication and non-surgical implanting procedure and mimic the 3D living organism conditions. In this thesis, bioactive thermogelling system including inorganic/thermogel, and functional group modified thermogel for more effective tissue engineering are investigated. First work, injectable inorganic/organic composite systems consisting of well-defined meso crystals (4 - 8 μm) of calcium phosphate and polypeptide thermogel significantly enhance the osteogenic differentiation of the tonsil derived mesenchymal stem cells (TMSCs). Compared to composite systems incorporating nano particles (10 -100 nm) or pure hydrogel systems, osteogenic biomarkers including alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP2), and osteocalcin (OCN) highly expressed at both the mRNA level and the protein level in the meso crystal composite systems. ALP activity of differentiated osteocytes, is also significantly higher in the meso composite systems compared to the nano composite systems or the pure hydrogel systems. The meso composite systems provides not only hard surfaces for binding the cells/proteins by the inorganic meso crystals but also a soft matrix for holding the cells by the hydrogel. Second work, galactosylated thermogel systems can induce the hepatogenic differentiation of the TMSCs. Compared to 3D MatrigelTM and 2D culture system, hepatogenic biomarkers involving hepatocyte nuclear factor 4 (HNF4 ), albumin (ALB), glucose 6-phosphatase (G6P), and asialoglycoprotein receptor 1 (ASGR1) highly expressed at both the mRNA level and the protein level in galactosylated thermogel. Hepatic functions of differentiatied hepatocytes including ALB synthesis, urea production, indocyanin green uptake, are also significantly higher in the galactosylated thermogel compared to control groups. Through the current research, the osteogenic and hepatogenic differentiation of TMSCs in vitro were investigated by bioactive thermogelling systems. It can be promised tissue engineering tools to create a suitable environment which induces the desired tissue formation. The thesis suggested that bioactive thermogelling systems not noly provide important clues in designing an artificial extracellular matrix for stem cell differentiation, but also proved the potential as an injectable tissue engineering applications.I. Background 1 A. Themogelling Systems 1 B. Tissue Engineering Systems 32 C. Tonsil derived Mesenchymal Stem Cells 36 D. Thermogelling System for Tissue Engineering Systems 38 II. Rationale for Study 43 III. Differentiation of Tonsil derived Mesenchymal Stem Cells in Bioactive Thermogelling Systems 45 A. Nano versus Meso Composite for Osteogenic Differentiation of Tonsil-derived Mesenchymal Stem Cells Materials and method 45 B. Galactosylated Thermogel as a Three Dimensional Culture Scaffold for Hepatogenic Differentiation of Tonsil Mesenchymal Stem Cells 79 IV. Conclusions and Future Research 113 Bibliography 116 Abstract (in Korean) 137박03

효소분해 가능한 온도 민감성 고분자 PAL-PLX-PAL 의 연구

Aqueous solutions of thermogelling polymers undergo sol-to-gel transition as the temperature increases. Therefore, the drug or cells are mixed as a sol state , followed by injection at a target site of subcutaneous layer to make an implanted depot system. The conventional syringe injection without the need of surgical procedure makes the thermogelling polymers solution as a minimally invasive delivery system. Homo- and co-polymers of lactic acid, glycolic acid, and caprolactone, chitosan, polyphosphazene, poloxamer derivatives, polycarbonate, polycyanoacrylate, β-lactoglobulin, elastin-like polypeptide (ELP) based polymer, and silk-elastin-like polymer have been reported as biodegradable thermogelling polymer aqueous solutions.1-9 However, most of thermogelling polymers suffer from reconstitution of the formulation due to the slow dissolution kinetics of the polymer in water. As the temperature increases, the aqueous solubility of the thermogelling polymer decreases. Therefore, the traditional method to prepare the aqueous solution of the themrogelling polymer is to dissolve the polymer at cold water, requiring a long time for reconstitution of the drug formulation as a solution. On the other hand, thermogelling polymers with enzymatic degradability can detour the solubility problem by storing the polymer as an enzyme-free buffer solution. The polymer begins to be degraded by the enzymes only after in vivo application, suggesting the storage stability and ready-to-use formulation of the thermogelling polymer as an aqueous solution, rendering a significant improvement of the convenience for medical application. Recently, we reported a poly(ethylene glycol)-poly(alanine-co-phenylalanine) (PEG-PAF) that underwent degradation by cathepsin B, cathepsin C, and elastase.10,11 The polymer showed stability in phosphate buffered saline (in vitro) without any significant mass loss (< 10 %), whereas 90 % of mass loss in the subcutaneous layer of rat (in vivo). As an extension of the enzymatic degradable sequence for a thermogelling system, here, we are reporting poly(alanine-co-leucine)-poloxamer-poly(alanine-co-leucine) (PAL-PLX-PAL). The polypeptide is expected not only to develop self-assembly with specific secondary structures but also to show unique enzymatic degradability.12-14 Physicochemical properties of the polymer aqueous solution was investigated by dynamic mechanical analysis, circular dichroism, dynamic light scattering, transmission electron microscopy, FTIR, and 13C-NMR. The degradation of the gel was studied in buffer solutions containing enzymes (in vitro) as well as in the subcutaneous layer of rats (in vivo). Tissue compatibility of the in-situ formed gel depot was investigated. In addition, the drug release profiles of a model protein drug, bovine serum albumin (BSA), were investigated by a preset gel-injection strategy to reduce the initial burst.;이 연구는 효소적으로 분해 가능한 온도 민감성 고분자에 대한 것으로, 폴리(알라닌-co-류신)이 양끝에 씌워져 있는 폴리(프로필렌 글리콜)-폴리(에틸렌 글리콜)-폴리(프로필렌 글리콜)) (PAL-PLX-PAL)에 대해 보고하고자 한다. 이 고분자 수용액은 온도가 증가함에 따라 온도겔화를 나타낸다. 실험결과, 수용액의 농도가 3.0 wt.% ~ 10.0 wt.% 일 때 20~40oC의 온도 범위에서 졸-겔 전이를 보인다. 이러한 양친매성 고분자는 소수성인 PAL (폴리(알라닌-co-류신)) 펩타이드 블록이 중심부를 형성하고 친수성인 PLX(폴리(프로필렌 글리콜)-폴리(에틸렌 글리콜)-폴리(프로필렌 글리콜))가 껍질부분을 형성함으로써 수용액상에서 미셸을 형성한다. 폴리(폴리(알라닌-co-류신))의 α-나선 이차구조는 20~50oC에서 안정한 반면, PLX는 졸-겔 전이를 하는 동안 분자 운동이 현저하게 감소됨을 보인다. 이 고분자는 MMP (매트릭스 메탈로프로테인아제), Elastase (엘라스타아제)같은 단백질 가수분해 효소에 분해되는 반면, Cathepsin B (카텝신 B), Cathepsin C (카텝신 C)와 Chymotrypsin (키모트립신)효소와 PBS 버퍼에 대해서는 비교적 안정하였다. In-situ에서 형성된 겔을 쥐에 피하주사 하였을 때, 겔 지속성, 조직적합성을 45일 동안 알아보았다. 이 연구는 PAL-PLX-PAL가 주사 가능한 생체 물질로서 매우 유망하다는 것을 보여준다.CHAPTER 1 INTRODUCTION 1 1.1 Hydrogel 2 1.2 Stimuli-responsive Polymer 4 1.3 Thermosensitive Polymers 6 1.3.1 Principle of Thermosensitivity 6 1.3.2 Thermosensitive Polymers Based on Amphiphilic Balance 8 1.4 Biodegradable Polymers 13 1.4.1 Determination of Biodegradable Polymers 13 1.4.2 Biodegradable Poloxamer 16 1.4.3 Enzymatically Degradable Peptide-based Polymers 21 1.5 Drug Delivery Systems 23 1.5.1 Controlled drug delivery system 23 1.5.2 Thermosensitive Polymers for Drug Delivery System 26 1.6 Rationale for Study 28 1.7 References 30 CHAPTER 2 Enzymatically Degradable Thermogelling PAL-PLX-PAL 33 2.1 Introduction 34 2.2 Experimental Section 37 2.2.1 Materials 37 2.2.2 Synthesis 38 2.2.3 Instruments and Measurements 39 2.3 Results and Discussion 46 2.3.1 Synthesis and Characterization 46 2.3.2 Sol-to-Gel Transition 51 2.3.3 Micellization Study 54 2.3.4 Micelle Size Study 58 2.3.5 Secondary Structure Study 60 2.3.6 13C-NMR Study 64 2.3.7 Control of Phase Diagram and Secondary Structure 66 2.3.8 Enzymatic Degradation Study 68 2.3.9 in-vivo Biocompatibility Study 70 2.3.10 in-vitro Drug Release Study 75 2.4 Conclusions 77 2.5 References 79 국문 요약 8