Identification of important interactions between subchondral bone ...

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Madsen et al. 269 Figure 2. Microscopic changes after catabolic and anabolic stimulation. Sections from femoral heads from 3-, 6-, 9-, and 12-week-old mice were stained with safranin O (proteoglycans) and fast green (collagens) after culture for 10 days in the absence (W/O) or presence of IGF-I (anabolic) and OSM + TNF-α (catabolic) stimulation. The black bars represent 250 µM (A-D, F-I, K-N) and 20 µM (E, J, O). The black squares in the 12-week-old femoral heads (D, I, N) represent the magnification of the cartilage surface (E, J, O), which is divided in rows after treatment. from the cartilage surface. In 12-week-old femoral heads, the proteoglycans seem to be almost depleted from the cartilage compartment in the nonstimulated (Fig. 2D) and the OSM + TNF-α–stimulated (Fig. 2I) explants. Anabolic stimulation with IGF-I protects against the proteoglycan loss seen in W/O (Fig. 2A-D and K-N). Additionally, in 12-week-old mice, anabolic stimulation shows proteoglycan staining around the chondrocytes (Fig. 2O) compared to the W/O (Fig. 2E). Next, we measured the size of the growth plate to investigate whether morphological changes could be induced (Fig. 3A). The size of the growth plate in IGF- I–stimulated 12-week-old femoral heads is increased by approximately 48% (P < 0.001) compared to W/O, whereas OSM + TNF-α stimulation decreases the growth plate size by approximately 34% (P < 0.001) compared to W/O (Fig. 3B). This indicates that we are able to induce alteration to the femoral heads ex vivo with catabolic (degenerative) or anabolic (generative) factors. Catabolic Stimulation Increases Collagen and Proteoglycan Turnover Levels of hydroxyproline, an indicator of total collagen turnover, in the conditioned medium indicate that stimulation by OSM + TNF-α increases the total release of collagens by approximately 49% (P < 0.05) to approximately 190% (P < 0.001) in femoral heads aged 6, 9, and 12 weeks, but not in femoral heads from 3-week-old mice (Fig. 4A). The longitudinal effects from the different stimulations are shown in Table 1. OSM + TNF-α increases the release of collagens 56 Figure 3. Quantification of growth plate zone in femoral heads. Sections from femoral heads from 12-week-old mice were stained with safranin O (proteoglycans) and fast green (collagens) after culture for 10 days in the absence (W/O) or presence of IGF-I (anabolic) and OSM + TNF-α (catabolic) stimulation. The growth plate size was measured in one femoral head per treatment from 4 different experiments using the annotation system (see Methods). The growth plate thickness (µm) is calculated by the average of the black lines in the growth plate (A). The catabolic stimulation decreased the growth plate size, whereas the anabolic stimulation increased the size of the growth plate (B). ***P < 0.001. over time in 6- and 9-week-old mice. In femoral heads from 12-week-old mice, the anabolic stimulation also increases total collagen release by approximately 49% (P < 0.01) compared to W/O (Fig. 4A). OSM + TNF-α stimulation increases the total release of sulfated glycosaminoglycans (sGAG) to the conditioned media by approximately 22% (P < 0.05) to approximately 40% (P < 0.01), except from the experiment with 6-week-old mice (Fig. 4B); however, similar trends were observed.
270 Cartilage 2(3) Figure 4. The catabolic control induces a release of collagens and proteoglycans. The experiments were assessed by the collagen degradation marker, hydroxyproline (A), and the proteoglycan turnover marker, sGAG (B). Each graph represents the accumulated measurements from days 4, 7, and 10 except from 12-week-old mice (only day 10). Cytokine stimulation increases the collagen release in experiments with 6-, 9-, and 12-week-old mice (A). Cytokine stimulation also increases proteoglycan release in experiments with 3-, 9-, and 12-week-old mice (B). Asterisks indicate statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001. 57
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F A C U L T Y O F S C I E N C E U N
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Supervisors University of Copenhage
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Preface Preface The work presented
Page 7 and 8: Contents Contents Abstract ........
Page 9 and 10: Abstract Abstract Osteoarthritis (O
Page 11 and 12: Sammendrag på dansk Sammendrag på
Page 13 and 14: CHAPTER 1 Objectives CHAPTER 1: Obj
Page 15 and 16: CHAPTER 2 Introduction CHAPTER 2: I
Page 17 and 18: 2.2 Biology of the joint CHAPTER 2:
Page 19 and 20: CHAPTER 2: Introduction Also osteob
Page 21 and 22: CHAPTER 2: Introduction follows nor
Page 23 and 24: CHAPTER 2: Introduction When the jo
Page 25 and 26: CHAPTER 2: Introduction Cartilage i
Page 27 and 28: 2.3 The pathology of Osteoarthritis
Page 29 and 30: CHAPTER 2: Introduction number of a
Page 31 and 32: CHAPTER 2: Introduction Cysteine pr
Page 33 and 34: CHAPTER 2: Introduction include car
Page 35 and 36: 2.4 The effect of Glucocorticoids C
Page 37 and 38: 2.5 References for CHAPTER 2 1 WebM
Page 39 and 40: 51 Lorenz H. and Richter W. Osteoar
Page 41 and 42: 97 Nakamura H., Tanaka M., Masuko-H
Page 43 and 44: 139 Garnero P., Ayral X., Rousseau
Page 45 and 46: CHAPTER 3 Overview of OA models CHA
Page 47 and 48: CHAPTER 3: Overview of OA models of
Page 49 and 50: 3.3 Ex vivo controls CHAPTER 3: Ove
Page 51 and 52: 19 Brandt K.D. Animal models of ost
Page 53 and 54: CHAPTER 4 CHAPTER 4: PAPER I PAPER
Page 55 and 56: 266 Cartilage 2(3) therapy for oste
Page 57: 268 Cartilage 2(3) Figure 1. Charac
Page 61 and 62: 272 Cartilage 2(3) Figure 5. Cytoki
Page 63 and 64: 274 Cartilage 2(3) Figure 6. The an
Page 65 and 66: 276 Cartilage 2(3) supports the inc
Page 67 and 68: 278 Cartilage 2(3) release and rece
Page 69 and 70: CHAPTER 5 CHAPTER 5: PAPER II PAPER
Page 71 and 72: leading to fragility fractures [12]
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Page 75 and 76: use a range of assays ranging from
Page 77 and 78: still some controversy regarding th
Page 79 and 80: CHAPTER 6 CHAPTER 6: PAPER III PAPE
Page 81 and 82: Biomarkers Downloaded from informah
Page 91 and 92: CHAPTER 7 CHAPTER 7: PAPER IV PAPER
Page 93 and 94: Introduction CHAPTER 7: PAPER IV Ca
Page 95 and 96: Bovine articular cartilage experime
Page 97 and 98: CHAPTER 7: PAPER IV Detection of ke
Page 99 and 100: CHAPTER 7: PAPER IV The MMP inhibit
Page 101 and 102: CHAPTER 7: PAPER IV The pre-stimula
Page 103 and 104: CHAPTER 8 CHAPTER 8: General discus
Page 105 and 106: Future perspectives CHAPTER 8: Futu
Page 107 and 108: Appendix I Co-author declarations f
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Appendix I 107
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Appendix I 109
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Appendix I 111
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Biochemical Markers of Joint Tissue
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