동물 모델의 초기 진단 및 골관절염 조절에 의한 치료 효능평가 연구
Animal Model for Early Diagnosis and Evaluating Therapeutic Efficacy in Management of Osteoarthritis
- 주제(키워드) Osteoarthritis; animal model; destabilization of medial meniscus (DMM); subchondral bone change; cartilage degeneration; hyaluronic acid (HA); Micro-CT , Type II collagen , Immuno-liposomes
- 발행기관 아주대학교
- 지도교수 민병현
- 발행년도 2014
- 학위수여년월 2014. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000016234
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Osteoarthritis (OA) is a multifactorial disease with many risk factors and it results from a complex interplay between mechanical, cellular and biochemical forces. Animal models of OA are an important surrogate that provides a means to study OA pathophysiology, and develop therapeutic agents and biomarkers for diagnosing and prognosing OA. CHAPTER I: Osteoarthritis (OA) is a multifactorial disease affecting both the cartilage and the subchondral bone. However, animal models of OA cannot represent both of the changes and show variations depending on induction methods and animal species/strains. This study first investigated subchondral bone changes and its correlation with cartilage degeneration in two different OA models in C57Bl/6 mice. Experimental OA was produced by type II collagenase-induced osteoarthritis (CIOA) and destabilization of the medial meniscus (DMM). The subchondral plate and trabecular bone of the tibia were analyzed by micro-computed tomography (µCT) and cartilage degeneration was analyzed histologically after safranin-O staining at 2, 4, 6 and 8 weeks. In DMM model, cartilage degeneration was induced reproducibly and progressively along with time. Progressive increase in subchondral bone volume but not in bone thickness was also observed in both subchondral bone plate (SBP) and subchondral trabecular bone (STB) in the medial tibial area. The changes in subchondral bone volume was correlated well with the histological cartilage degeneration (R2=0.7870). In contrast to CIOA model, cartilage degeneration was not relatively unpredictable with being increased only until 4 weeks and decreased thereafter. No significant changes in subchondral bone were observed in all area at all time points. These results suggest that DMM model of mice OA is more reliable and useful than CIOA model by representing better both of cartilage degeneration and subchondral bone change with high correlation coefficient. CHAPTER II: We established an OA model of C57BL/6 mice that is more reproducible and amenable to therapeutic intervention by controlling their movement. OA was induced in 9 week-old C57BL/6 mice by destabilizing the medial meniscus (DMM). The mice were then raised either in the standard cage for free movement or in a confine cage customized to restrict mice movement. Mice in the confine cage were subjected to no exercise or an exercise of 400, 800 and 1200 m/day, respectively. OA lesions of mice in confine cage were much severer in the exercise group and showed much less variation. The validity of our novel OA model with the movement control system was proven by successfully discriminating the therapeutic effect of hyaluronic acid (HA) in the histological scores, while the OA model using standard cage showed just statistically insignificant difference. In conclusion, the mice OA model could be more reproducible and reliable by adopting the confine cage and enforced periodic exercise of mice. CHAPTER III: The extracellular matrix (ECM) of cartilage is dense, avascular and aneurogenic tissues, these early lesions are not painful and often go undetected until significant damage has occurred. At present, there are no appropriate treatments to cure cartilage degradation so early detection is critical. One of the cause of limitation for developing a preventive therapy for OA is the lack of good tools for efficiently diagnosing the disease and monitoring its progression during the early stages. We have developed MabCII (monoclonal antibody collagen type II) nanosomes encapsulated the XenoFluor dye for diagnosis of early OA. These MabCII-nanosomes increased the binding efficacy to tissue according to the severity of cartilage damage. Here, we show that MabCII-nanosomes can monitor sensitively identify minimal cartilage damage in the DMM (destabilization of the medial meniscus) model of OA. In conclusion, MabCII-nanosomes techniques could potentially be used non-invasive method for diagnosis the early onset of osteoarthritis disease. In conclusion, our results suggest that developed osteoarthritis model and diagnostic tools could be a useful tool for various therapeutic applications instead of human on regenerative medicine field.
more목차
TABLE OF CONTENTS………………………………………………………………………. i
LIST OF FIGURES…………………………………………………………………………….iv
LIST OF TABLES……………………………………………………………………………...vi
ABBREVIATIONS……………………………………………………………………………vii
ABSTRACT…………………………………………………………………………………...viii
BACKGROUND
1. Osteoarthritis………………………………………………………………………………..01
2. Imaging Biomarkers of Osteoarthritis.................................................................................03
3. Animal models of osteoarthritis……………………………………………………………04
3.1 Surgical models…………………………………………………………………….……07
3.2 Chemical models………………………………………………………………………...07
3.3 Spontaneous models……………………………………………………………………..08
4. Aim and title of this study………………………………………………………………......10
CHAPTER I
Comparison between subchondral bone change and cartilage degeneration in collagenase- and DMM- induced osteoarthritis (OA) models in mice
1. Introduction…………………………………………………………………………………12
2. Materials and Methods……………………………………………………………………14
2.1 Animals...............................................................................................................................14
2.2 Collagenase-induced OA model.........................................................................................14
2.3 Destabilization of the medial meniscus (DMM) model of OA..........................................14
2.4 Histological analysis...........................................................................................................16
2.5 Micro-computed tomography (CT)....................................................................................16
2.6 Statistical analysis...............................................................................................................18
3. Results.....................................................................................................................................19
3.1 Cartilage degradation in collagenase- and DMM-induced OA mice.................................19
3.2 Changes of subchondral bone in the animal OA models....................................................21
3.3 Correlation between the cartilage damage and subchondral bone change.........................23
3.4 Longitudinal analysis of bone change................................................................................25
4. Discussion................................................................................................................................27
CHAPTER II
Development of a reliable and reproducible murine osteoarthritis model
1. Introduction…………………………………………………………………………………31
2. Materials and Methods……………………………………………………………………34
2.1 Animal care........................................................................................................................34
2.2 Surgical construction of OA...............................................................................................36
2.3 Exercise model of OA mice................................................................................................36
2.4 Measurement of movement................................................................................................38
2.5 Histology............................................................................................................................38
2.6 Hyaluronic acid (HA) injection..........................................................................................39
2.7 Statistical Analysis..............................................................................................................39
2. Results......................................................................................................................................40
3.1 Confine cage significantly reduces mouse movement.......................................................40
3.2 Fewer but more severe OA lesions in mice in the confine cage.........................................42
3.3 Combination of movement restriction and regular exercise produces stable and linear induction of OA lesions in DMM mice...............................................................................44
3.4 Novel OA model with the movement control better represents the therapeutic effect of HA......................................................................................................................................46
4. Discussion................................................................................................................................51
CHAPTER III
Targeted Nanosomes for Detection of Early Stage of Osteoarthritis in DMM Mouse Model of a reliable and reproducible murine osteoarthritis model
1. Introduction…………………………………..………………………………………..……56
2. Materials and Methods..........................................................................................................58
2.1 Animals care and surgery ...................................................................................................58
2.2 Liposome synthesis and antibody coupling........................................................................58
2.3 In vivo test of immunoliposome ........................................................................................61
2.4 Histological analysis……. .................................................................................................62
2.5 Statistics ............................................................................................................................62
3. Results......................................................................................................................................64
3.1 Characterization of nanosomes...........................................................................................64
3.2 In vitro binding efficacy of nanosomes..............................................................................64
3.3 In vivo binding efficacy of nanosomes..............................................................................67
3.4 In vivo targeting of MabCII-nanosomes............................................................................67
3.5 Detection of fluorescence in knee joint after systemic injection…………………………70
4. Discussion................................................................................................................................73
CONCLUSION...........................................................................................................................76
REFERENCE.............................................................................................................................78
국문요약......................................................................................................................................86
감사의 글…………………………………………………………………………………….88
