Identification of important interactions between subchondral bone ...

More documents

Recommendations

Info

Methods Sources of proteases, inhibitors and cytokines CHAPTER 7: PAPER IV Purified human matrix metalloproteinase-1 (MMP-1) and recombinant human MMP-7 were purchased from Enzo life science, DK. Recombinant human Cathepsin-L and purified human Cathepsin-S and Cathepsin-B were from BioVision, US. Recombinant human aggrecanases; ADAMTS-4 and ADAMTS-5, were from Chemicon international, DK. Purified human MMP-9 and recombinant human MMP-3, MMP-13 and Cathepsin-K, were purchased from Biocol, DE. The matrix metalloprotease inhibitor (R)-N4-Hydroxy-N1-[(S)-2-(1H-indol-3-yl)-1- methylcarbamoyl-ethyl]-2-isobutyl-succinamide (GM6001) and the cysteine protease inhibitor trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane, L-trans-3-Carboxyoxiran-2-carbonyl-L- leucylagmatine, N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E64) were both purchased from Sigma, DK. The cytokines, oncostatin M (OSM) was from Sigma, DK, and the Tumor Necrosis Factor-α (TNF-α) was from R&D systems, UK. Buffers To evaluate endogenous activity of metalloproteinases (MMPs and ADAMTS’), cartilage explants were incubated in an MMP buffer [50 mM Tris-HCL, 200 mM NaCl, 5 mM CaCl 2, 1 µM ZnCl 2, 0.05% NaN 3, 0.05% Brij-35, pH 7]. Activation of latent MMPs was performed by adding 1 mM freshly made p-Aminophenylmercurie (APMA). To evaluate endogenous activity of cysteine proteinases, cartilage explants were incubated in a cysteine buffer [100 mM Na-Acetate, 5 mM EDTA, 0.05% NaN 3, pH 5]. Activation of latent cysteine proteinases was performed by adding 20 mM cysteine just before use. Both buffers were prepared with inspiration from Tabassi et al. 13 . Ex vivo models Human articular cartilage experiments The human OA cartilage was obtained from female patients between the ages of 65-77 years undergoing total knee arthroplasty. The patients were informed by both oral and written communication at the hospital and gave their written consent before entering study, which was approved by the Capital Region of Denmark, DK-3400, approval number HD-2007-0084. The cartilage was metabolically inactivated (MI) by freeze-thaw cycles and cut into homologous explants. Half of the MI explants were incubated in MMP buffer for 3, 6, and 9 h in the presence or absence of the inhibitors, GM6001 [10 µM] and E64 [40uM], and the proteases [100 ng/ml]; MMP-1, -3, -7, -9, -13 and ADAMTS-4, -5. The other half were incubated in cysteine buffer for 3, 6, and 9 h in the presence or absence of the inhibitors, GM6001 [10 µM] and E64 [40 µM], and the proteases [100 ng/ml]; Cathepsin-K, -B, -L, -S. At the end of each incubation time (3, 6, 9h), conditioned buffer (about 300 µl) was extracted and stored frozen at -20°C until assayed for specific biomarkers. 92
Bovine articular cartilage experiments CHAPTER 7: PAPER IV Bovine knees (Hereford) were bought at the local slaughter house (Slangerup, DK) just after slaughtering. Articular cartilage explants were isolated under semi-sterile conditions and washed in PBS. One third of the cartilage explants were MI (t=0) and stored in -80°C until use. The rest of the cartilage explants were pre-cultured with pro-inflammatory cytokines, OSM [10 ng/ml] and TNF-α [20 ng/ml] (O+T), for 4 or 11 days to increase aggrecanase or MMP activity, respectively 3,4 . The cartilage explants pre-cultured in cytokines for 4 or 11 days were after each termination washed in PBS and MI, and stored in -80°C until use. The frozen explants without cytokine pre-culture (t=0) and the pre-cultured catabolic induced explants (t=4 and t=11), were thawed and incubated in MMP or cysteine buffer for 3, 6, and 9 h in the presence or absence of the inhibitors, GM6001 [10 µM] and E64 [40 µM]. At the end of each incubation time (3h, 6h, 9h), conditioned buffer (about 300 µl) was extracted and stored frozen at -20°C until assayed for specific biomarkers. Biomarkers of cartilage degradation The type II collagen telopeptide fragment, CTX-II (urine cartilaps, IDS Ltd, DK), were measured accordingly to the manufacturer’s instructions 7 . AGNx-II measures MMP-mediated aggrecan degradation accordingly to Sumer et al. 8 . The type II collagen inter helical neo-epitope fragment (…PKGARG 707 ), NBC2, was measured accordingly to Bay-Jensen et al. 2011 (unpublished). Statistic analysis Results in fig. 1 are shown as mean + standard error of mean (SEM). Statistic analysis in fig. 1 was performed by ANOVA; nonparametric test, don’t assuming Gaussian distribution, n=4, Kruskal-Wallis test with Dunn’s multiple comparison post test. Statistic significance is indicated by the number of asterisks, * p < 0.05, ** p < 0.01, and *** p < 0.001. The Tables show mean value of n=4 ± standard deviation (SD). Results Detection of key proteases to degrade human OA cartilage ex vivo; evaluated by collagen type II degradation markers Human OA cartilage explants incubated in buffer optimal for MMPs, increased the levels of CTX-II when incubated with MMP-7 (p
Page 1 and 2:
F A C U L T Y O F S C I E N C E U N
Page 3 and 4:
Supervisors University of Copenhage
Page 5 and 6:
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 and 58: 268 Cartilage 2(3) Figure 1. Charac
Page 59 and 60: 270 Cartilage 2(3) Figure 4. The ca
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]
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: Introduction CHAPTER 7: PAPER IV Ca
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
Page 115: Biochemical Markers of Joint Tissue
show all

Identification of important interactions between subchondral bone ...

Create successful ePaper yourself

Delete template?

Save as template?