M.W. Lark

Biosynthetic response and mechanical properties of articular cartilage after injurious compression

Traumatic joint injury is known to produce osteoarthritic degeneration of articular cartilage. To study the effects of injurious compression on the degradation and repair of cartilage in vitro, we developed a model that allows strain and strain rate‐controlled loading of cartilage explants. The influence of strain rate on both cartilage matrix biosynthesis and mechanical properties was assessed after single injurious compressions. Loading with a strain rate of 0.01 s−1 to a final strain of 50% resulted in no measured effect on the cells or on the extracellular matrix, although peak stresses reached levels of about 12 MPa. However, compression with strain rates of 0.1 and 1 s−1 caused peak stresses of approximately 18 and 24 MPa, respectively, and resulted in significant decreases in both proteoglycan and total protein biosynthesis. The mechanical properties of the explants (compressive and shear stiffness) were also reduced with increasing strain rate. Additionally, cell viability decreased with increasing strain rate, and the remaining viable cells lost their ability to exhibit an increase in biosynthesis in response to low‐amplitude dynamic mechanical stimulation. This latter decrease in reparative response was most dramatic in the tissue compressed at the highest strain rates. We conclude that strain rate (like peak stress or strain) is an important parameter in defining mechanical injury, and that cartilage injuriously compressed at high strain rates can lose its characteristic anabolic response to low‐amplitude cyclic mechanical loading.

Phenylbutyrates as potent, orally bioavailable vitronectin receptor (integrin αvβ3) antagonists

In our continuing efforts to identify small molecule vitronectin receptor antagonists, we have discovered a series of phenylbutyrate derivatives, exemplified by 16, which have good potency and excellent oral bioavailability (approximately 100% in rats). This new series is derived conceptually from opening of the seven-membered ring of SB-265123.

Novel bone antiresorptive approaches

Inhibition of bone resorption is a mechanism that has been clinically validated as a means to control bone loss in diseases such as postmenopausal osteoporosis. The development of marketable drugs in this area has resulted in significant clinical benefits; however, improvements can still be made. Several novel antiresorptive mechanisms are currently under consideration in the pharmaceutical industry, which will hopefully result in the development of improved bone antiresorptive therapies.

A53 MECHANICAL INJURY POTENTIATES THE COMBINED EFFECTS OF TNF-α AND IL-6/sIL-6R ON PROTEOGLYCAN CATABOLISM IN BOVINE CARTILAGE

+Sui, Y; **Song, X-Y; +**Lee, J H; *DiMicco, M; **Blake, S M; *Hung, H; **James, I; ** Lark, M W; +§¶*Grodzinsky, A J +Biological, §Mechanical, ¶Electrical Engineering, *Center for Biomedical Engineering, MIT, Cambridge, MA; **Centocor R&D, Inc, Radnor, PA ysui@mit.edu INTRODUCTION: Acute traumatic joint injury increases the risk of developing osteoarthritis (OA) [1]. The mechanisms by which injury causes chronic cartilage degradation in vivo are not fully understood, but elevated levels of injury-induced pro-inflammatory cytokines [2], including TNF-α and IL-6 [3], may play pivotal roles in the pathogenesis of OA. TNF-α causes a synergistic loss of PG from mechanically-injured cartilage in vitro [4], but the pathways regulating this synergy are unclear. The objectives of this study were to (1) examine the combined effect of TNF-α and IL-6/sIL6R on proteoglycan degradation in mechanically-injured cartilage, and (2) to determine the role of endogenous IL-6 in the cartilage catabolism induced by both TNF-α and mechanical injury. METHODS: Cartilage disks (3 mm diam., 1 mm thick) were harvested from the middle zone of the femoropatellar grooves of 1-2-week old calves, and equilibrated for 2 days in normal medium (DMEM + 1% ITS) prior to treatment. Cytokine and Mechanical Injury Treatments: Location-matched disks were either injuriously compressed (50% strain, 100%/second strain rate), cultured in medium with rhTNF-α (25 ng/ml), treated with rhIL-6 (50 ng/ml) plus soluble IL-6 receptor (sIL-6R, 250ng/ml), or treated with combinations of these three conditions (Fig.1). Culture was terminated after 6 days of treatment. In a separate experiment, half the cartilage disks (Fig. 3) were pre-equilibrated for 6 days with an IL-6 blocking Fab fragment (50 ug/ml, Centocor, J&J) prior to treatment; the remaining disks were incubated in normal medium during this period. Afterward, disks were either injuriously compressed, incubated with rhTNF-α (25 ng/ml), or treated with combined injury + TNF-α. Disks that were pre-treated with the IL-6 blocking Fab fragment continued to receive Fab fragments until the termination of the experiment. Aggrecan Western Blotting, GAG Content and Histology: Culture medium from each condition was collected on day 2, 4 and 6 after the initial injury and/or cytokine treatments. Concentrated medium was used to perform Western blot analysis using a monoclonal Ab specific to the G1NITEGE fragment of aggrecan (kindly provided by C. Flannery, Wyeth). DMMB dye was used to quantify sGAG released into the medium. Selected cartilage samples were fixed in gluteraldehyde with RHT, paraffin-embedded, sectioned, and stained with Toluidine Blue. Additional disks were radiolabeled during days 4-6 with 5 μCi/ml SO4 to assess proteoglycan synthesis. RESULTS: 3-way ANOVA analyses followed by post-hoc Tukey’s pairwise comparisons showed that TNF-α formed interactions with both IL-6/sIL-6R (p<0.001) and injurious compression (p<0.001) causing increased sGAG release (Fig. 1(i)) and decreased proteoglycan synthesis (data not shown). While IL-6/sIL-6R significantly augmented TNF-αinduced proteoglycan degradation (Fig.1(i)B,G), the largest amount of GAG loss was caused by the combination of injury+TNF-α + IL-6/sIL6R (Fig.1(i)D). Histology showed that GAG loss was not uniform across the disk cross-section, but instead was initiated at the disk periphery and progressed towards the disk center with time (Fig. 2). The most rapid, severe progression of GAG loss was observed in disks treated with the combination of injury + TNF-α + IL-6/sIL-6R (Fig. 2e,f). Analysis of conditioned medium for aggrecan fragments by Western blotting demonstrated that the most dramatic release of aggrecanase-generated cleavage products occurred in response to treatment with TNF-α + IL6/sIL-6R, both with and without injurious compression (Fig. 1(ii)B,D). The IL-6 blocking Fab fragment was effective in neutralizing exogenous rhIL-6 in the medium, and was not toxic to cells (data not shown). In separate studies, TNF-α + mechanical injury caused greater GAG loss than either treatment alone (Fig.3G,H,I). Importantly, the IL-6 blocking Fab fragment significantly reduced the combined catabolic effects of TNF-α + mechanical injury on GAG loss, with no exogenous IL-6 present (Fig.3D,I). DISCUSSION: We found that the combined treatment with TNF-α and IL-6/sIL-6R induced significantly more GAG loss than either cytokine alone did, consistent with previous studies of TNF-α/IL-6 treatment [3], and suggesting this catabolic response was associated with aggrecanase (but not MMP) activity. Additionally, we now report that the catabolic effect of TNF-α, and the combined effect of TNF-α + IL-6/sIL-6R are both highly potentiated by mechanical injury. The degradative effects of injury + TNF-α appear to be due, in part, to the action of endogenous IL-6, as sGAG loss was partly abrogated by the IL-6 blocking Fab fragment. This result is also consistent with the increased loss of sGAG upon addition of exogenous IL-6 to the combination of TNF-α and mechanical injury. Histology observations (Fig. 2) suggest that the kinetics of cartilage degradation is not merely a consequence of the activities of proteolytic enzymes, but it also depends strongly on the transport of cytokines, proteases, anti-IL-6 Fab and other cartilage biomolecules, which may be altered by overload injury. In conclusion, our study suggests that pro-inflammatory cytokines, whose productions are elevated by traumatic joint injury, can interact to potentiate cartilage catabolism. The mechanobiological (cell-mediated) responses to overload [5], as well as altered transport of cytokines and proteases in the damaged matrix, may both be affected by joint injury, making the damaged cartilage tissue more susceptible to further degradation by biochemical mediators. REFERENCES:[1] Gelber+, Ann Intern Med 133:3211, ‘00; [2] Irie+, Knee 10:93, ‘03; [3] Flannery+, Matrix Biol, 19:549, ‘00. [4] Patwari+, Arth Rheum, 48:1292, ‘03 [5] Lee+, Arth Rheum, 52:2386, ‘05. Acknowledgements: Supported by Centocor and NIH Grant AR45779