효과적인 골재생을 위한 hydroxyapatite-collagen 스케폴드를 이용한 BMP-2 와 alendronate 의 순차적 이중 약물 전달

학위논문 (박사) -- 서울대학교 대학원 : 의과대학 의학과, 2021. 2. 장학.The clinical use of bioactive molecules in bone regeneration has been known to have side effects, which result from uncontrolled and supraphysiological doses. In this study, we demonstrated the synergistic effect of two bioactive molecules, bone morphogenic protein-2 (BMP-2) and alendronate (ALN), by releasing them in a sequential manner. Collagen-hydroxyapatite composite scaffolds functionalized using BMP-2 are loaded with biodegradable microspheres where ALN is encapsulated. The results indicate an initial release of BMP-2 for a few days, followed by the sequential release of ALN after two weeks. The composite scaffolds significantly increase osteogenic activity owing to the synergistic effect of BMP-2 and ALN. Enhanced bone regeneration was identified at eight weeks post-implantation in the rat 8-mm critical-sized defect. Our findings suggest that the sequential delivery of BMP-2 and ALN from the scaffolds results in a synergistic effect on bone regeneration, which is unprecedented. Therefore, such a system exhibits potential for the application of cell-free tissue engineering.골형성을 위해 사용하는 생리활성 분자는 생리학적으로 사용되는 용량을 초과하여 사용할 경우 임상적인 부작용이 있는 것으로 알려졌다. 본 연구에서는 생체 활성 분자인 BMP-2 (Bone morphogenic protein-2) 와 alendronate (ALN)를 순차적으로 방출하여 in vitro 및 in vivo에서의 시너지 효과를 보여줍니다. 개발된 약물전달체는 BMP-2를 탑재한 Collagen-hydroxyapatite scaffold에 ALN 탑재된 생분해성 마이크로 스피어를 삽이하여 제작되었습니다. 개발된 약물전달체의 약동학적 특성을 분석해보니, BMP-2는 0~7일 사이 방출이 완료되었고 ALN은 14~21일 사이 방출이 완료되어 순차방출이 되는 것을 확인 할 수 있었습니다. 또한 단일 약물의 사용보다 BMP-2와 ALN의 순차방출시 골 재생 효과가 뛰어났음을 8 mm의 임계 골결손 rat 모델을 8주간 관찰한 결과 확인 할 수 있었습니다. 본 연구는 BMP-2 와 ALN의 순차적 전달이 효과적인 골 재생에 뛰어난 효과를 가져옴을 시사합니다. 이러한 시간차 약물방출 시스템은 세포를 사용하지 않는 조직 공학 재료로써의 응용 가능성을 보여줍니다.ABSTRACT ⅰ CONTENTS ⅲ LIST OF FIGURES AND TABLES. ⅳ INTRODUCTION. 1 MATERIALS AND METHODS Materials 5 CHAS fabrication. 5 Drug release kinetics of BMP-2 in CHAS. 6 Drug release kinetics of ALN in CHAS 7 Preparation of PLGA microspheres 8 Cell cultures & Cell viability test 8 Alkaline phosphatase (ALP) activity assay. 9 Animal model and surgical procedures. 9 Micro-computed tomography (μ-CT) Analysis 11 Sample preparation and histological analysis 12 Statistical analysis. 12 RESULTS Development of a sequential dual delivery system for BMP-2 and ALN in CHAS. 13 In vitro release of BMP-2 and ALN from CHAS 14 In vitro osteogenesis study. 14 μ-CT analysis 16 Histological analysis. 17 DISCUSSION 18 CONCLUSION 21 REFERENCE. 23 ABSTRACT IN KOREAN. 42 LIST OF FIGURES Figur1. Schematic illustration of sequential dual-drug delivery using BMP-2 and ALN. 29 Figur2. Morphological analysis of CHAS. 31 Figur3. Drug release test. 32 Figur4. Cell viability evaluation. 33 Figur5. μ-CT radiographic evaluation.34 Figur6. Quantitative analysis of new bone volume 35 Figur7. Histological evaluation of bone regeneration in rat calvarial defects at 4 and 8 weeks postoperatively. 36 Supplementary Figure S1. SEM image and pore size distribution of PLGA microspheres 38 Supplementary Figure S2. SEM image and pore size distribution of hydroxyapatite nanoparticles (nHAps) 38 Supplementary Figure S3. Surgical procedure for creating an 8 mm cranial defect in rat and implantation of the scaffold at the calvarial defect site. 40 Supplementary Figure S1. Micro-CT imaging procedures to detectbone regeneration 41 LIST OF TABLES Table1. Sample abbreviations used in this study 30Docto

Regeneration of full-thickness skin defects by differentiated adipose-derived stem cells into fibroblast-like cells by fibroblast-conditioned medium

Background Fibroblasts are ubiquitous cells in the human body and are absolutely necessary for wound healing such as for injured skin. This role of fibroblasts was the reason why we aimed to differentiate human adipose-derived stem cells (hADSCs) into fibroblasts and to test their wound healing potency. Recent reports on hADSC-derived conditioned medium have indicated stimulation of collagen synthesis as well as migration of dermal fibroblasts in wound sites with these cells. Similarly, human fibroblast-derived conditioned medium (F-CM) was reported to contain a variety of factors known to be important for growth of skin. However, it remains unknown whether and how F-CM can stimulate hADSCs to secrete type I collagen. Methods In this study, we obtained F-CM from the culture of human skin fibroblast HS27 cells in DMEM media. For an in-vivo wound healing assay using cell transplantation, balb/c nude mice with full-thickness skin wound were used. Results Our data showed that levels of type I pro-collagen secreted by hADSCs cultured in F-CM increased significantly compared with hADSCs kept in normal medium for 72 h. In addition, from a Sircol collagen assay, the amount of collagen in F-CM-treated hADSC conditioned media (72 h) was markedly higher than both the normal medium-treated hADSC conditioned media (72 h) and the F-CM (24 h). We aimed to confirm that hADSCs in F-CM would differentiate into fibroblast cells in order to stimulate wound healing in a skin defect model. To investigate whether F-CM induced hADSCs into fibroblast-like cells, we performed FACS analysis and verified that both F-CM-treated hADSCs and HS27 cells contained similar expression patterns for CD13, CD54, and CD105, whereas normal medium-treated hADSCs were significantly different. mRNA level  analysis for Nanog, Oct4A, and Sox2 as undifferentiation markers and vimentin, HSP47, and desmin as matured fibroblast markers supported the characterization that hADSCs in F-CM were highly differentiated into fibroblast-like cells. To discover the mechanism of type I pro-collagen expression in hADSCs in F-CM, we observed that phospho-smad 2/3 levels were increased in the TGF-β/Smad signaling pathway. For in-vivo analysis, we injected various cell types into balb/c nude mouse skin carrying a 10-mm punch wound, and observed a significantly positive wound healing effect in this full-thickness excision model with F-CM-treated hADSCs rather than with untreated hADSCs or the PBS injected group. Conclusions We differentiated F-CM-treated hADSCs into fibroblast-like cells and demonstrated their efficiency in wound healing in a skin wound model

Evaluation of fatty acids in groomed fingerprint by gas chromatographic analysis using various extraction solvents and treatment methods

Extremely small amounts of fatty acids detected in latent fingerprints are important for studying fingerprint visualization and age determination through changes in composition over time. However, methods for efficiently extracting or recovering fatty acids from fingerprints have not been extensively studied. If accurate and stable quantitative estimations are established, age estimates will be possible through a better understanding of the fatty acid composition. The extraction solvent and treatment method are essential factors for achieving a reliable analysis of fatty acids. There have been few previous studies that efficiently compared fatty acids. In this study, fatty acids from sebaceous fingerprint residues were quantified with various extraction solvents and treatment methods and were evaluated with gas chromatography flame ionization detection (GC-FID). All data were analyzed using a statistical method.Center for Research and Development of Police science and Technology and Korean National Police Agency (PA-H000001) The Korea government (Ministry of Education) (NRF-2017R1D1A1B03030163) The Korea government (Ministry of Science, ICT and Future Planning) (MSIP) (No. NRF-2018M3C1B7020722) The Ministry of Health & Welfare, Republic of Korea (Grant Number:) (HI14C1277) Ministry of Science, ICT & Future Planning (NRF-2017M3A9E9072939) Seoul National University Hospital Research Fund (Grant 26-2015-0030