Han-Hwa Hung

Mechanical injury potentiates proteoglycan catabolism induced by interleukin‐6 with soluble interleukin‐6 receptor and tumor necrosis factor α in immature bovine and adult human articular cartilage

OBJECTIVE Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL-6 plays a role in proteoglycan loss caused by a combination of injury and TNFalpha. METHODS Injured or uninjured cartilage disks were incubated with or without TNFalpha and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6-blocking antibody Fab fragment and subjected to injury and TNFalpha treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme-linked immunosorbent assay to determine chondrocyte production of IL-6. RESULTS In bovine cartilage samples, injury combined with TNFalpha and IL-6/sIL-6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL-6/sIL-6R, injury plus TNFalpha exposure up-regulated chondrocyte production of IL-6, but incubation with the IL-6-blocking Fab significantly reduced proteoglycan degradation. CONCLUSION Our findings indicate that mechanical injury potentiates the catabolic effects of TNFalpha and IL-6/sIL-6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFalpha appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6-blocking Fab.

Mechanical motion promotes expression of Prg4 in articular cartilage via multiple CREB-dependent, fluid flow shear stress-induced signaling pathways.

Lubricin is a secreted proteoglycan encoded by the Prg4 locus that is abundantly expressed by superficial zone articular chondrocytes and has been noted to both be sensitive to mechanical loading and protect against the development of osteoarthritis. In this study, we document that running induces maximal expression of Prg4 in the superficial zone of knee joint articular cartilage in a COX-2-dependent fashion, which correlates with augmented levels of phospho-S133 CREB and increased nuclear localization of CREB-regulated transcriptional coactivators (CRTCs) in this tissue. Furthermore, we found that fluid flow shear stress (FFSS) increases secretion of extracellular PGE2, PTHrP, and ATP (by epiphyseal chondrocytes), which together engage both PKA- and Ca(++)-regulated signaling pathways that work in combination to promote CREB-dependent induction of Prg4, specifically in superficial zone articular chondrocytes. Because running and FFSS both boost Prg4 expression in a COX-2-dependent fashion, our results suggest that mechanical motion may induce Prg4 expression in the superficial zone of articular cartilage by engaging the same signaling pathways activated in vitro by FFSS that promote CREB-dependent gene expression in this tissue.

High-bandwidth AFM-based rheology is a sensitive indicator of early cartilage aggrecan degradation relevant to mouse models of osteoarthritis

Murine models of osteoarthritis (OA) and post-traumatic OA have been widely used to study the development and progression of these diseases using genetically engineered mouse strains along with surgical or biochemical interventions. However, due to the small size and thickness of murine cartilage, the relationship between mechanical properties, molecular structure and cartilage composition has not been well studied. We adapted a recently developed AFM-based nano-rheology system to probe the dynamic nanomechanical properties of murine cartilage over a wide frequency range of 1 Hz to 10 kHz, and studied the role of glycosaminoglycan (GAG) on the dynamic modulus and poroelastic properties of murine femoral cartilage. We showed that poroelastic properties, highlighting fluid-solid interactions, are more sensitive indicators of loss of mechanical function compared to equilibrium properties in which fluid flow is negligible. These fluid-flow-dependent properties include the hydraulic permeability (an indicator of the resistance of matrix to fluid flow) and the high frequency modulus, obtained at high rates of loading relevant to jumping and impact injury in vivo. Utilizing a fibril-reinforced finite element model, we estimated the poroelastic properties of mouse cartilage over a wide range of loading rates for the first time, and show that the hydraulic permeability increased by a factor ~16 from knormal=7.80×10(-16)±1.3×10(-16) m(4)/N s to kGAG-depleted=1.26×10(-14)±6.73×10(-15) m(4)/N s after GAG depletion. The high-frequency modulus, which is related to fluid pressurization and the fibrillar network, decreased significantly after GAG depletion. In contrast, the equilibrium modulus, which is fluid-flow independent, did not show a statistically significant alteration following GAG depletion.

Effects of the Combination of Microfracture and Self-Assembling Peptide Filling on the Repair of a Clinically Relevant Trochlear Defect in an Equine Model

BACKGROUND The goal of this study was to test the ability of an injectable self-assembling peptide (KLD) hydrogel, with or without microfracture, to augment articular cartilage defect repair in an equine cartilage defect model involving strenuous exercise. METHODS Defects 15 mm in diameter were created on the medial trochlear ridge and debrided down to the subchondral bone. Four treatment groups (n = 8 each) were tested: no treatment (empty defect), only defect filling with KLD, only microfracture, and microfracture followed by filling with KLD. Horses were given strenuous exercise throughout the one-year study. Evaluations included lameness, arthroscopy, radiography, and gross, histologic, immunohistochemical, biochemical, and biomechanical analyses. RESULTS Overall, KLD-only treatment of defects provided improvement in clinical symptoms and improved filling compared with no treatment, and KLD-only treatment protected against radiographic changes compared with microfracture treatment. Defect treatment with only microfracture also resulted in improved clinical symptoms compared with no treatment, and microfracture treatment resulted in repair tissue containing greater amounts of aggrecan and type-II collagen compared with KLD-only treatment. Microfracture treatment also protected against synovial fibrosis compared with no treatment and KLD-only treatment. Treatment with the self-assembling KLD peptide in combination with microfracture resulted in no additional improvements over microfracture-only treatment. In general, the nature of the predominant tissue in the defects was a mix of noncartilaginous and fibrocartilage tissue, with no significant differences among the treatments. CONCLUSIONS Treatment of defects with only KLD or with only microfracture resulted in an improvement in clinical symptoms compared with no treatment; the improvement likely resulted from different causes depending on the treatment. Whereas microfracture improved the quality of repair tissue, KLD improved the amount of filling and protected against radiographic changes. CLINICAL RELEVANCE Treatment of defects with only microfracture and with KLD only resulted in clinical improvements compared with untreated defects, despite differing with respect to the structural improvements that they induced.

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

Cartilage tissue engineering using a new self-assembling peptide gel scaffold

Emerging therapies for repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defect site to initiate de novo tissue regeneration. Biocompatible scaffolds assist in providing a template for cell distribution and extracellular matrix accumulation in a three-dimensional defect geometry. A variety of scaffolds have been investigated for cartilage repair in tissue culture and/or in animals. However, no scaffold-based cartilage construct is yet available for clinical application. In this study, we have explored the use of a novel self-assembling peptide hydrogel as a three-dimensional tissue engineering scaffold for cartilage repair [1].