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

Uranium Bioreduction and Biomineralization

Following the development of nuclear science and technology, uranium contamination has been an ever increasing concern worldwide because of its potential for migration from the waste repositories and long-term contaminated environments. Physical and chemical techniques for uranium pollution are expensive and challenging. An alternative to these technologies is microbially mediated uranium bioremediation in contaminated water and soil environments due to its reduced cost and environmental friendliness. To date, four basic mechanisms of uranium bioremediation-uranium bioreduction, biosorption, biomineralization, and bioaccumulation-have been established, of which uranium bioreduction and biomineralization have been studied extensively. The objective of this review is to provide an understanding of recent developments in these two fields in relation to relevant microorganisms, mechanisms, influential factors, and obstacles

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

Tumor-associated macrophages promote prostate cancer migration through activation of the CCL22-CCR4 axis

Previous studies have found that tumor-associated macrophages (TAMs) promote cancer progression. We previously reported that TAMs promote prostate cancer metastasis via activation of the CCL2-CCR2 axis. The CCR4 (receptor of CCL17 and CCL22) expression level in breast cancer was reported to be associated with lung metastasis. The aim of this study was to elucidate the role of CCR2 and CCR4 in prostate cancer progression. CCR2 and CCR4 were expressed in human prostate cancer cell lines and prostate cancer tissues. In vitro co-culture of prostate cancer cells and macrophages resulted in increased CCL2 and CCR2 levels in prostate cancer cells. The addition of CCL2 induced CCL22 and CCR4 production in prostate cancer cells. The migration and invasion of prostate cancer cells via enhanced phosphorylation of Akt were promoted by CCL17 and CCL22. CCR4 may be a potential candidate for molecular-targeted therapy

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

MicroRNA miR-146a-5p inhibits the inflammatory response and injury of airway epithelial cells via targeting TNF receptor-associated factor 6

Bronchial asthma is a common respiratory disease, which is characterized by airway inflammation, remodeling and hyperresponsiveness. MicroRNAs (miRNAs), as reported, are implicated in the pathogenesis of many diseases, but how miRNAs-146a-5p (miR-146a-5p) works in asthma remains inconclusive. In this work, we proved that miR-146a-5p expression was inhibited in asthma patients’ plasma and platelet activating factor (PAF)-induced human small airway epithelial cells (HSAECs). MiR-146a-5p up-regulation ameliorated the inflammatory reaction and cell barrier damage of HSAECs induced by PAF, and inhibited the apoptosis; besides, miR-146a-5p down-regulation functioned oppositely. In addition, miR-146a-5p could target TNF receptor-associated factor 6 (TRAF6) and negatively regulate its expression. TRAF6 overexpression could counterract the impact of miR-146a-5p up-regulation on PAF-induced inflammation, cell barrier damage and apoptosis of HSAECs. Collectively, miR-146a-5p may protect airway epithelial cells and inhibit the pathogenesis of asthma via targeting TRAF6

Isolation and Degradation Characteristics of PBAT Film Degrading Bacteria

In recent years, PBAT (polybutylene adipate-co-terephthalate) mulch has become one of the most commonly used biodegradable mulching films. In this paper, five potential strains of PBAT film degrading bacteria were screened from the soil sample using PBAT film as the sole carbon source. A highly efficient PBAT degrading strain JZ1 was isolated by comparing the degradation performance of PBAT mulching film identified as Peribacillus frigoritolerans S2313 by 16S rDNA sequence analysis. The capacity of the strain to degrade PBAT film was optimized by adjusting the cultivation conditions such as nitrogen source, pH, and inoculum volume. After 8 weeks of cultivation, the actual degradation rate of the strain to PBAT mulch film reached 12.45%. SEM (scanning electron microscopy) coupled with EDX (energy dispersive spectroscopy) analysis showed that microbial degradation is an oxidation process and is mainly due to the amorphous regions of the PBAT film. The biodegradation of PBAT film by Peribacillus frigoritolerans may provide a promising method for regulating the degradation progress of PBAT film in the farmlands

Cadmium Caused Different Toxicity to Photosystem I and Photosystem II of Freshwater Unicellular Algae <i>Chlorella</i> <i>pyrenoidosa</i> (Chlorophyta)

Heavy metals such as Cd pose environmental problems and threats to a variety of organisms. The effects of cadmium (Cd) on the growth and activities of photosystem I (PSI) and photosystem II (PSII) of Chlorella pyrenoidosa were studied. The growth rate of cells treated with 25 and 100 µM of Cd for longer than 48 h were significantly lower than the control, accompanying with the inhibition of photosynthesis. The result of quantum yields and electron transport rates (ETRs) in PSI and PSII showed that Cd had a more serious inhibition on PSII than on PSI. Cd decreased the efficiency of PSII to use the energy under high light with increasing Cd concentration. In contrast, the quantum yield of PSI did not show a significant difference among different Cd treatments. The activation of cyclic electron flow (CEF) and the inhibition of linear electron flow (LEF) due to Cd treatment were observed. The photochemical quantum yield of PSI and the tolerance of ETR of PSI to Cd treatments were due to the activation of CEF around PSI. The activation of CEF also played an important role in induction of non-photochemical quenching (NPQ). The binding features of Cd ions and photosystem particles showed that Cd was easier to combine with PSII than PSI, which may explain the different toxicity of Cd on PSII and PSI