Identification of important interactions between subchondral bone ...

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1480 S.H. Madsen et al. / Steroids 76 (2011) 1474–1482 Fig. 5. The effect of dexamethasone in pre-osteoclasts and mature osteoclasts. Osteoclasts were cultured in on either bone (black bars) or plastic (gray bars) in the presence or absence of 100 nM dexamethasone (DEX). Pre-osteoclasts were cultured for 21 days (A–C) and mature osteoclasts for 14 days (D–F). Cell viability was measured with alamar blue after 14 or 21 days (A and D). The number of osteoclasts was measured with TRAP activity (B and E) and bone resorption was measured by calcium release from bone (C and F). DEX [100 nM] treatment decreased cell viability, osteoclast number and bone resorption in both pre-osteoclasts and mature osteoclasts. However, the effects of DEX [100 nM] on pre-osteoclasts were higher than the effects on mature osteoclasts. Each treatment n P 5. we used the bovine articular cartilage explants model that is presently the preferred model for studying cartilage turnover ex vivo [30,33]. The strong indications that OSM + TNF-a reduced the ability to induce cartilage degradation in the presence of DEX or PRED, evaluated by biomarkers and histology, suggest a chondroprotective effect by GCs independent of bone cells. Furthermore, histology showed that GC treatment had a positive effect on proteoglycans under anabolic conditions, which is in line with a study by Van der Kraan et al. that showed that PRED stimulates proteoglycan synthesis in patellar cartilage, validated by [ 35 S] incorporation [43]. However, the catabolic stimulated cartilage explants in the presence or absence of DEX, were both close to be depleted for proteoglycans, measured by the intensity of color (sGAG), which was not as convincing as the 70% reduction of MMP-mediated aggrecan degradation shown with the biomarker, 342-G2. However, depletion of color may results from an early loss of aggrecanase-mediated highly-sGAG-saturated aggrecan degradation, leaving low-sGAG-saturated aggrecan to be degraded by MMPs at day 19 [44], as we did not see any MMP-mediated degradation at days 5 and 12. This suggests that the anti-catabolic effects of GC on cartilage may be limited to the degradation mediated by MMPs. Interestingly, GC treatment in resting conditions resulted in a small loss of proteoglycans from the articular cartilage explants that we were unable to detect using the MMP-mediated aggrecan degradation marker, 342-G2. Thus, the two opposite effects of GCs treatment (protecting in activated cartilage and degrading in resting cartilage) indicate that the activation stage of the chondrocytes is important in relation to the GC response. If the GC-mediated protective effect results from an alteration of the chondrocyte phenotype (preventing the induction/release of proteases) or that the 74 GCs inhibit the cytokine pathway, still remains unclear. Yet, our data indicate that a reduction of MMP activity is involved, as the MMP-dependent biomarkers, 342-G2 and CIIM, are reduced. This is in line with a recent study by Garvican et al. suggesting that GCs reduce MMP-mediated cartilage degradation [9], although other enzymatic pathways, such as the u-PA/plasminogen/plasmin-pathway, also have been indicated [45]. Thus, in the search for new disease modifying osteoarthritic drugs (DMOADs), we suggest that the inhibition of protease pathways mediated by GCs could be of interest. We did not see any protective effects of GCs on the non-stimulated bovine cartilage explants, as opposed to the results with cartilage from the femoral head explants. The bovine cartilage explants represent ‘‘healthy’’ cartilage, whereas the femoral heads consist of cartilage in development, which contains more proliferating, pre-hypertrophic and hypertrophic chondrocytes (which are observed in OA cartilage) than the bovine cartilage explants [29]. This could explain why we have to induce a catabolic milieu with cytokines in the bovine cartilage explants, before we see a protective effect of GCs, and suggest that GCs mainly affect chondrocytes which are activated, whereas resting chondrocytes appear to respond less. We do acknowledge that we deal with cross-species comparisons, which may cause the different effects observed in this study. However, the anabolic and catabolic controls have the same effects on the different species, suggesting the effects of GC, or lack of same, are due to action from the GC and not a species-dependent effect. The present study clearly shows that GCs have demonstrably large effects on cells of bone. While it for long has been known that GCs induce apoptosis of osteoblasts and osteocytes in vivo, there is
still some controversy regarding the mechanism of action. Some data indicate a role for osteoclasts and others data indicate direct effects on osteoblasts [17,46]. Controversially, a recent study showed that the endogenous GC-induced signaling in osteoblasts is needed for proper bone formation in vivo in mice [47]. In vitro data regarding bone formation are also confusing, as data showing that GCs support stimulation of RANKL production, increase nodule formation, as well as inhibiting bone formation [18,20,21,48]. Our data shows that GC-mediated effects on osteoblasts in cell cultures are dependent on the ‘‘activation state’’ of the osteoblasts, with a toxic effect on non-activated cells, and a pro-osteoblastic effect on BMP-2-induced cells. This is in line with the results from the femoral heads, which decreased bone formation with PRED treatment, but increased bone formation in combination with IGF-I. The conflicting in vitro and in vivo data could indicate that we need to look more closely into GCs-mediated effects in the different functional subgroups of osteoblasts, and more importantly, how to modulate these with respect to preventing GC-induced bone damage [48,49]. Several studies have indicated that GCs stimulate osteoclastogenesis in vitro [50,51]; however, most of these studies were conducted in the presence of stromal/osteoblastic cells, which are known to produce RANKL in response to GCs [14,18]. We found that in pure osteoclast precursor cell cultures, GCs inhibited osteoclastogenesis by reducing the viability of the cells, which is in line with the decreased viability and osteoclast number found in PREDstimulated femoral heads. These data correlate to data showing decreased osteoclastogenesis in vivo in mice, and strongly indicate that the primary effect of GCs on osteoclastogenesis is inhibitory [15,16]. However, an interesting observation in this regard is that the effect of GCs on the mature osteoclasts, on cell viability, osteoclast number and resorption, was smaller than the effect on the differentiating osteoclasts. This could indicate that osteoclasts and their precursors have different levels of sensitivity to GC treatment. As all osteoclast cultures are a mix of mature multinucleated osteoclasts and their precursor cells, and the ratio of mature to pre-osteoclastic cells is dependent on several different aspects, such as density, donor, serum, etc. [31] we speculate that the reason for the predominantly toxic effect could be that we have more osteoclast precursors present in the cultures than Soe and Delaisse, who described a prolongation of the resorption cycle, when treating mature osteoclasts derived from CD14 + cells with PRED [52]. A limitation to the study is that the effects observed with DEX and PRED have not been tested for steroids specificity with a non-GC control (e.g. estrogen or cholesterol). However, Kim et al. [17] showed that GR°C / mice were spared the impact of DEX on osteoclasts and their precursors, indicating a true GR-mediated action of DEX. This steroid specificity is furthermore supported by Battista et al. [3], which showed that the effect from DEX was dependent on the percentage of the GR in the chondrocytes. Nonetheless, future studies should include a non-GC control to ensure that the effects observed are indeed specific to the GC class of steroids. In conclusions, we found that GCs did not appear to affect resting chondrocyte lifespan and function. Furthermore, GCs showed a protective effect on catabolic-induced cartilage and showed a tendency to increase cartilage formation under anabolic conditions. These results are encouraging for the use of intra-articular GC injections in OA knees. However, considering the well-established consequences on bone it is important to remember the tight functional relationship between bone and cartilage under both physiologically and pathologically conditions [27]. Thus, further studies into the functional relationship between these compartments are needed, especially with the focus on the possibility to rescue bone damage by the addition of anabolic therapy, such as parathyroid hormone [48]. S.H. Madsen et al. / Steroids 76 (2011) 1474–1482 1481 75 Acknowledgment This work was partly funded by a grant from the Danish Research Foundation, The Ministry of Science Technology and Innovation, Denmark (08-036109) with a stipend for the corresponding author. References [1] Engvall IL, Svensson B, Tengstrand B, Brismar K, Hafstrom I. Impact of low-dose prednisolone on bone synthesis and resorption in early rheumatoid arthritis: experiences from a two-year randomized study. Arthritis Res Ther 2008;10(6):R128. [2] Hepper CT, Halvorson JJ, Duncan ST, Gregory AJ, Dunn WR, Spindler KP. The efficacy and duration of intra-articular corticosteroid injection for knee osteoarthritis: a systematic review of level I studies. J Am Acad Orthop Surg 2009;17(10):638–46. [3] DiBattista JA, Martel-Pelletier J, Antakly T, Tardif G, Cloutier JM, Pelletier JP. Reduced expression of glucocorticoid receptor levels in human osteoarthritic chondrocytes. Role in the suppression of metalloprotease synthesis. J Clin Endocrinol Metab 1993;76(5):1128–34. [4] Chrysis D, Ritzen EM, Savendahl L. Growth retardation induced by dexamethasone is associated with increased apoptosis of the growth plate chondrocytes. J Endocrinol 2003;176(3):331–7. [5] Chrysis D, Zaman F, Chagin AS, Takigawa M, Savendahl L. Dexamethasone induces apoptosis in proliferative chondrocytes through activation of caspases and suppression of the Akt–phosphatidylinositol 3 0 -kinase signaling pathway. Endocrinology 2005;146(3):1391–7. [6] Sadowski T, Steinmeyer J. Effects of non-steroidal antiinflammatory drugs and dexamethasone on the activity and expression of matrix metalloproteinase-1, matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 by bovine articular chondrocytes. Osteoarthritis Cartilage 2001;9(5):407–15. [7] Nakazawa F, Matsuno H, Yudoh K, Watanabe Y, Katayama R, Kimura T. Corticosteroid treatment induces chondrocyte apoptosis in an experimental arthritis model and in chondrocyte cultures. Clin Exp Rheumatol 2002;20(6):773–81. [8] Richardson DW, Dodge GR. Dose-dependent effects of corticosteroids on the expression of matrix-related genes in normal and cytokine-treated articular chondrocytes. Inflamm Res 2003;52(1):39–49. [9] Garvican ER, Vaughan-Thomas A, Redmond C, Gabriel N, Clegg PD. MMPmediated collagen breakdown induced by activated protein C in equine cartilage is reduced by corticosteroids. J Orthop Res 2010;28(3):370–8. [10] Eastell R, Reid DM, Compston J, Cooper C, Fogelman I, Francis RM, et al. A UK Consensus Group on management of glucocorticoid-induced osteoporosis: an update. J Intern Med 1998;244(4):271–92. [11] Hooyman JR, Melton III LJ, Nelson AM, O’Fallon WM, Riggs BL. Fractures after rheumatoid arthritis. A population-based study. Arthritis Rheum 1984;27(12):1353–61. [12] Caplan L, Saag KG. Glucocorticoids and the risk of osteoporosis. Expert Opin Drug Saf 2009;8(1):33–47. [13] Canalis E, Mazziotti G, Giustina A, Bilezikian JP. Glucocorticoid-induced osteoporosis: pathophysiology and therapy. Osteoporos Int 2007;18(10):1319–28. [14] Hofbauer LC, Zeitz U, Schoppet M, Skalicky M, Schuler C, Stolina M, et al. Prevention of glucocorticoid-induced bone loss in mice by inhibition of RANKL. Arthritis Rheum 2009;60(5):1427–37. [15] Jia D, O’Brien CA, Stewart SA, Manolagas SC, Weinstein RS. Glucocorticoids act directly on osteoclasts to increase their life span and reduce bone density. Endocrinology 2006;147(12):5592–9. [16] Weinstein RS, Chen JR, Powers CC, Stewart SA, Landes RD, Bellido T, et al. Promotion of osteoclast survival and antagonism of bisphosphonate-induced osteoclast apoptosis by glucocorticoids. J Clin Invest 2002;109(8):1041–8. [17] Kim HJ, Zhao H, Kitaura H, Bhattacharyya S, Brewer JA, Muglia LJ, et al. Glucocorticoids suppress bone formation via the osteoclast. J Clin Invest 2006;116(8):2152–60. [18] Hofbauer LC, Gori F, Riggs BL, Lacey DL, Dunstan CR, Spelsberg TC, et al. Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells: potential paracrine mechanisms of glucocorticoid-induced osteoporosis. Endocrinology 1999;140(10):4382–9. [19] Sivagurunathan S, Muir MM, Brennan TC, Seale JP, Mason RS. Influence of glucocorticoids on human osteoclast generation and activity. J Bone Miner Res 2005;20(3):390–8. [20] Oshina H, Sotome S, Yoshii T, Torigoe I, Sugata Y, Maehara H, et al. Effects of continuous dexamethasone treatment on differentiation capabilities of bone marrow-derived mesenchymal cells. Bone 2007;41(4):575–83. [21] Jager M, Fischer J, Dohrn W, Li X, Ayers DC, Czibere A, et al. Dexamethasone modulates BMP-2 effects on mesenchymal stem cells in vitro. J Orthop Res 2008;26(11):1440–8. [22] Espina B, Liang M, Russell RG, Hulley PA. Regulation of bim in glucocorticoidmediated osteoblast apoptosis. J Cell Physiol 2008;215(2):488–96.
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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 ........
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Abstract Abstract Osteoarthritis (O
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Sammendrag på dansk Sammendrag på
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CHAPTER 1 Objectives CHAPTER 1: Obj
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CHAPTER 2 Introduction CHAPTER 2: I
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2.2 Biology of the joint CHAPTER 2:
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CHAPTER 2: Introduction Also osteob
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CHAPTER 2: Introduction follows nor
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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: use a range of assays ranging from
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
Page 115: Biochemical Markers of Joint Tissue
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