Identification of important interactions between subchondral bone ...

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Madsen et al. 271 Table 1. Biochemical Markers Measured from the Conditioned Medium at Days 4, 7, and 10 in the Different Age Groups Age of Culture Stimulated Mice, wk Day with HP, µg/mL sGAG, µg/mL TRAP CTX-I, ng/mL PIINP, ng/mL CTX-II, pg/mL W/O 5.22 ± 0.81 113.12 ± 3.46 0.47 ± 0.06 2.97 ± 0.82 93.61 ± 4.65 48.69 ± 3.73 4 O + T 6.21 ± 1.22 143.84 ± 8.45** 0.81 ± 0.12* 3.23 ± 0.27 54.92 ± 9.58** 74.96 ± 19.19 IGF 5.16 ± 0.59 92.61 ± 10.11* 0.63 ± 0.06 3.79 ± 0.84 100 ± 0.00 26.10 ± 3.87** W/O 2.59 ± 0.46 49.51 ± 8.51 0.27 ± 0.02 4.61 ± 0.54 68.78 ± 5.55 6.30 ± 1.40 3 7 O + T 2.41 ± 0.35 87.18 ± 5.04** 0.33 ± 0.01* 11.72 ± 3.54 40.83 ± 3.02** 17.44 ± 3.83* IGF 4.38 ± 0.46* 58.96 ± 4.34 0.26 ± 0.01 4.49 ± 0.39 74.38 ± 1.76 15.28 ± 1.86** W/O 3.56 ± 0.53 70.74 ± 4.85 0.22 ± 0.001 2.55 ± 0.56 46.01 ± 3.05 0.48 ± 0.38 10 O + T 3.09 ± 0.29 83.18 ± 9.70** 0.25 ± 0.01** 3.62 ± 0.47 28.95 ± 2.11** 7.90 ± 1.93** IGF 4.53 ± 0.55 89.71 ± 3.88* 0.21 ± 0.01 2.26 ± 0.17 69.24 ± 1.38*** 8.51 ± 1.50** W/O 1.34 ± 0.16 40.52 ± 5.05 0.29 ± 0.03 3.21 ± 0.39 54.55 ± 6.48 10.70 ± 0.86 4 O + T 1.69 ± 0.11 50.53 ± 0.68 0.36 ± 0.03 5.29 ± 0.76* 64.01 ± 4.38 29.66 ± 4.14** IGF 1.27 ± 0.09 24.16 ± 3.10 0.21 ± 0.01* 4.26 ± 0.16* 82.18 ± 6.23* 8.38 ± 2.30 W/O 1.48 ± 0.28 24.22 ± 4.24 0.24 ± 0.01 3.11 ± 0.65 74.48 ± 2.48 16.26 ± 1.68 6 7 O + T 1.57 ± 0.13 20.86 ± 1.01 0.46 ± 0.05** 13.77 ± 4.18* 59.43 ± 3.58** 139.18 ± 27.05** IGF 1.77 ± 0.06 24.86 ± 2.79 0.18 ± 0.002*** 4.21 ± 0.47 64.86 ± 5.31 14.81 ± 2.50 W/O 1.53 ± 1.02 6.42 ± 1.32 0.19 ± 0.01 2.08 ± 0.14 56.22 ± 1.54 12.94 ± 1.70 10 O + T 2.37 ± 0.38 10.81 ± 1.00 0.45 ± 0.06** 6.02 ± 1.07** 48.12 ± 4.02 160.84 ± 45.55* IGF 2.02 ± 0.45 10.35 ± 0.51 0.19 ± 0.02 4.58 ± 0.29*** 44.61 ± 5.05 14.47 ± 2.19 W/O 2.26 ± 0.40 25.56 ± 1.35 0.33 ± 0.03 10.82 ± 0.84 65.45 ± 3.70 68.75 ± 11.89 4 O + T 1.61 ± 0.38 28.26 ± 2.72 0.36 ± 0.04 14.46 ± 2.72 54.89 ± 3.22 59.61 ± 9.14 IGF 2.05 ± 0.37 22.08 ± 2.46 0.24 ± 0.004** 4.80 ± 0.29*** 78.57 ± 2.69* 36.53 ± 8.24 W/O 1.49 ± 0.35 9.70 ± 1.93 0.36 ± 0.04 14.86 ± 2.50 53.16 ± 4.19 32.18 ± 3.13 9 7 O + T 3.01 ± 0.88 15.95 ± 1.56* 0.48 ± 0.06 18.71 ± 1.98 46.87 ± 2.82 309.83 ± 34.52*** IGF 1.49 ± 0.27 11.39 ± 2.18 0.23 ± 0.01** 14.87 ± 2.68 66.78 ± 2.62* 18.63 ± 5.09 12 a W/O 2.34 ± 0.41 14.12 ± 2.34 0.30 ± 0.02 9.09 ± 1.07 29.98 ± 3.36 30.86 ± 6.86 10 O + T 6.76 ± 0.13*** 20.10 ± 0.91* 0.55 ± 0.07** 24.13 ± 3.33** 33.73 ± 2.81 361.52 ± 2.09*** IGF 3.05 ± 0.81 13.32 ± 1.35 0.23 ± 0.03 6.25 ± 0.49* 33.09 ± 1.78 37.77 ± 13.16 W/O 4.79 ± 0.53 19.74 ± 1.31 0.19 ± 0.008 27.84 ± 8.69 20.31 ± 3.61 62.88 ± 28.45 10 O + T 13.89 ± 1.59*** 24.00 ± 1.39* 0.30 ± 0.037* 341.39 ± 78.25*** 0.80 ± 0.80* 1714.80 ± 379.31** IGF 7.15 ± 0.43** 23.67 ± 1.11 0.17 ± 0.011 42.60 ± 16.09 67.78 ± 0.73*** 54.07 ± 22.04 Note: Values are significantly different from W/O. a The biochemical markers released from 12-week-old mice at day 10 represent total release from the entire culture period. *P < 0.05. **P < 0.01. ***P < 0.001. OSM + TNF-α Increases the Osteoclast Number and Bone Resorption Measurements of the conditioned media show that stimulation with OSM + TNF-α increases the tartrate-resistant acid phosphatase (TRAP) activity for all the age groups in a range from approximately 41% (P < 0.05) to approximately 78% (P < 0.01) compared with their respective W/O, indicating increased osteoclast numbers (Fig. 5A). OSM + TNF-α increases the osteoclast number over time in 6- and 9-week-old mice. However, the osteoclast number decreases over time in 3-week-old mice (Table 1). Conversely to OSM + TNF-α stimulation, IGF-I stimulation shows approximately 17% (P < 0.05) to approximately 29% (P < 0.01) decrease in the osteoclast number 58 in 6- and 9-week-old mice (Fig. 5A), which results from constant low values measured at all 3 days (Table 1). The collagen type I resorption measurements reflect the respective osteoclast number during catabolic stimulation. The resorption increases approximately 65% (P < 0.05) to approximately 1200% (P < 0.001) in the presence of OSM + TNF-α stimulation compared to W/O (Fig. 5B). However, in 6-week-old mice, the collagen type I resorption measurements do not reflect the TRAP activity when stimulated with IGF-I. In 3- and 6-week-old mice, the resorption of collagen type I peaks at day 7, whereas it peaks at or after day 10 in 9-week-old mice (Table 1). These measurements show that it is possible to measure osteoclast number and activity via biochemical markers in our ex vivo model.
272 Cartilage 2(3) Figure 5. Cytokines increase the osteoclast number and resorption of bone. The conditioned media from the 4 experiments with 4 individual age groups were assessed by the osteoclast number measured by TRAP activity (A) and the collagen type I resorption marker, CTX-I (B). Each graph represents the accumulated measurements from days 4, 7, and 10 except from 12-week-old mice (only day 10). Catabolic stimulation increases the osteoclast number in all 4 experiments, whereas the anabolic stimulation decreases the osteoclast number in the experiments with 6- and 9-week-old mice (A). Cytokines (O + T) also increase the total collagen type I resorption in the experiments with 6-, 9-, and 12-week-old mice (B). *P < 0.05. **P < 0.01. ***P < 0.001. 59
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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
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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 and 58: 268 Cartilage 2(3) Figure 1. Charac
Page 59: 270 Cartilage 2(3) Figure 4. The ca
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]
Page 73 and 74: after 1 week (Fig. 2F). The additio
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
Page 109 and 110: 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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