Modeling the Insulin-Like Growth Factor System in Articular Cartilage

IGF signaling is involved in cell proliferation, differentiation and apoptosis in a wide range of tissues, both normal and diseased, and so IGF-IR has been the focus of intense interest as a promising drug target. In this computational study on cartilage, we focus on two questions: (i) what are the key factors influencing IGF-IR complex formation, and (ii) how might cells regulate IGF-IR complex formation? We develop a reaction-diffusion computational model of the IGF system involving twenty three parameters. A series of parametric and sensitivity studies are used to identify the key factors influencing IGF signaling. From the model we predict the free IGF and IGF-IR complex concentrations throughout the tissue. We estimate the degradation half-lives of free IGF-I and IGFBPs in normal cartilage to be 20 and 100 mins respectively, and conclude that regulation of the IGF half-life, either directly or indirectly via extracellular matrix IGF-BP protease concentrations, are two critical factors governing the IGF-IR complex formation in the cartilage. Further we find that cellular regulation of IGF-II production, the IGF-IIR concentration and its clearance rate, all significantly influence IGF signaling. It is likely that negative feedback processes via regulation of these factors tune IGF signaling within a tissue, which may help explain the recent failures of single target drug therapies aimed at modifying IGF signaling.National Health and Medical Research Council (Australia) (APP1051455

Charge based intra-cartilage delivery of single dose dexamethasone using Avidin nano-carriers suppresses cytokine-induced catabolism long term

Objective: Avidin exhibits ideal characteristics for targeted intra-cartilage drug delivery: its small size and optimal positive charge enable rapid penetration through full-thickness cartilage and electrostatic binding interactions that give long half-lives in vivo. Here we conjugated Avidin with dexamethasone (DEX) and tested the hypothesis that single-dose Avidin-delivered DEX can ameliorate catabolic effects in cytokine-challenged cartilage relevant to post-traumatic OA. Methods: Avidin was covalently conjugated with DEX using fast (ester) and slow, pH-sensitive release (hydrazone) linkers. DEX release kinetics from these conjugates was characterized using 3H-DEX-Avidin (scintillation counting). Cartilage explants treated with IL-1α were cultured with or without Avidin-DEX conjugates and compared to soluble DEX. Sulfated-glycosaminoglycan (sGAG) loss and biosynthesis rates were measured using DMMB assay and 35S-incorporation, respectively. Chondrocyte viability was measured using fluorescence staining. Results: Ester linker released DEX from Avidin significantly faster than hydrazone under physiological buffer conditions. Single dose Avidin-DEX suppressed cytokine-induced sGAG loss over 3-weeks, rescued IL-1α-induced cell death, and restored sGAG synthesis levels without causing cytotoxicity. The two Avidin-DEX conjugates in 1:1 combination (fast:slow) had the most prominent bioactivity compared to single dose soluble-DEX, which had a shorter-lived effect and thus needed continuous replenishment throughout the culture period to ameliorate catabolic effects. Conclusion: Intra-cartilage drug delivery remains inadequate as drugs rapidly clear from the joint, requiring multiple injections or sustained release of high doses in synovial fluid. A single dose of Avidin-conjugated drug enables rapid uptake and sustained delivery inside cartilage at low intratissue doses, and potentially can minimize unwanted drug exposure to other joint tissues.Deshpande Center for Technological InnovationNational Science Foundation (U.S.). Materials Research Science and Engineering Centers (Program) (Award DMR-1419807

Poroelasticity is the dominant energy dissipation mechanism in cartilage at the nano-scale

Recent studies of micro- and nano-scale mechanics of cartilage and chondrocyte pericellular matrix have begun to relate matrix molecular structure to its mechanical response. AFM-based indentation has revealed rate-dependent stiffness at the micro-scale. While multi-scale elastic behavior has been studied, and poro-viscoelastic properties have been extensively documented at the tissue-level, time-dependent behavior and energy dissipation mechanisms of cartilage matrix at the nano-scale are not well understood. Here, we used AFM-based dynamic compression in conjunction with poroelastic finite element modeling to study the frequency-dependent behavior of cartilage using nano-scale oscillatory displacement amplitudes. We introduce the characteristic frequency f[subscript peak] at which the maximum energy dissipation occurs as an important parameter to characterize matrix time-dependent behavior. Use of micron-sized AFM probe tips with nano-scale oscillatory displacements over a 3-decade frequency range enabled clear identification of this characteristic frequency f[subscript peak]. The length-scale dependence of poroelastic behavior combined with judicious choice of probe tip geometry revealed flow-dependent and flow-independent behavior during matrix displacement amplitudes on the order of macromolecular dimensions and intermolecular pore-sizes.National Science Foundation (U.S.) (Grant CMMI-0758651)National Institutes of Health (U.S.) (National Institute of Arthritis and Musculoskeletal and Skin Diseases (U.S.) Grant AR33236

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.National Institute of Arthritis and Musculoskeletal and Skin Diseases (U.S.) (Grant AR60331

Size- and speed-dependent mechanical behavior in living mammalian cytoplasm

Active transport in the cytoplasm plays critical roles in living cell physiology. However, the mechanical resistance that intracellular compartments experience, which is governed by the cytoplasmic material property, remains elusive, especially its dependence on size and speed. Here we use optical tweezers to drag a bead in the cytoplasm and directly probe the mechanical resistance with varying size a and speed V. We introduce a method, combining the direct measurement and a simple scaling analysis, to reveal different origins of the size- and speed-dependent resistance in living mammalian cytoplasm. We show that the cytoplasm exhibits size-independent viscoelasticity as long as the effective strain rate V/a is maintained in a relatively low range (0.1 s −1 < V/a < 2 s −1 ) and exhibits size-dependent poroelasticity at a high effective strain rate regime (5 s −1 < V/a < 80 s −1 ). Moreover, the cytoplasmic modulus is found to be positively correlated with only V/a in the viscoelastic regime but also increases with the bead size at a constant V/a in the poroelastic regime. Based on our measurements, we obtain a full-scale state diagram of the living mammalian cytoplasm, which shows that the cytoplasm changes from a viscous fluid to an elastic solid, as well as from compressible material to incompressible material, with increases in the values of two dimensionless parameters, respectively. This state diagram is useful to understand the underlying mechanical nature of the cytoplasm in a variety of cellular processes over a broad range of speed and size scales. Keywords: cell mechanics; poroelasticity; viscoelasticity; cytoplasmic state diagra

Transport and equilibrium uptake of a peptide inhibitor of PACE4 into articular cartilage is dominated by electrostatic interactions

The availability of therapeutic molecules to targets within cartilage depends on transport through the avascular matrix. We studied equilibrium partitioning and non-equilibrium transport into cartilage of Pf-pep, a 760 Da positively charged peptide inhibitor of the proprotein convertase PACE4. Competitive binding measurements revealed negligible binding of Pf-pep to sites within cartilage. Uptake of Pf-pep depended on glycosaminoglycan charge density, and was consistent with predictions of Donnan equilibrium given the known charge of Pf-pep. In separate transport experiments, the diffusivity of Pf-pep in cartilage was measured to be ~1 × 10[superscript −6] cm[superscript 2]/s, close to other similarly-sized non-binding solutes. These results suggest that small positively charged therapeutics will have a higher concentration within cartilage than in the surrounding synovial fluid, a desired property for local delivery; however, such therapeutics may rapidly diffuse out of cartilage unless there is additional specific binding to intra-tissue substrates that can maintain enhanced intra-tissue concentration for local delivery.National Institutes of Health (U.S.) (Grant AR45779)National Institutes of Health (U.S.) (Grant AR33236)Pfizer Inc

Proliferative remodeling of the spatial organization of human superficial chondrocytes distant from focal early osteoarthritis

Objective Human superficial chondrocytes show distinct spatial organizations, and they commonly aggregate near osteoarthritic (OA) fissures. The aim of this study was to determine whether remodeling or destruction of the spatial chondrocyte organization might occur at a distance from focal (early) lesions in patients with OA. Methods Samples of intact cartilage (condyles, patellofemoral groove, and proximal tibia) lying distant from focal lesions of OA in grade 2 joints were compared with location-matched nondegenerative (grade 0–1) cartilage samples. Chondrocyte nuclei were stained with propidium iodide, examined by fluorescence microscopy, and the findings were recorded in a top-down view. Chondrocyte arrangements were tested for randomness or significant grouping via point pattern analyses (Clark and Evans Aggregation Index) and were correlated with the OA grade and the surface cell densities. Results In grade 2 cartilage samples, superficial chondrocytes were situated in horizontal patterns, such as strings, clusters, pairs, and singles, comparable to the patterns in nondegenerative cartilage. In intact cartilage samples from grade 2 joints, the spatial organization included a novel pattern, consisting of chondrocytes that were aligned in 2 parallel lines, building double strings. These double strings correlated significantly with an increased number of chondrocytes per group and an increased corresponding superficial zone cell density. They were observed in all grade 2 condyles and some grade 2 tibiae, but never in grade 0–1 cartilage. Conclusion This study is the first to identify a distinct spatial reorganization of human superficial chondrocytes in response to distant early OA lesions, suggesting that proliferation had occurred distant from focal early OA lesions. This spatial reorganization may serve to recruit metabolically active units as an attempt to repair focal damage.National Institutes of Health (U.S.) (grant P5O-AR39239)National Institutes of Health (U.S.) (grant R01-AR33236)Deutsche Forschungsgemeinschaft (DFG) (grant RO 2511/1-1)Deutsche Forschungsgemeinschaft (DFG) (grant RO 2511/2-1

Effects of Dexamethasone on Mesenchymal Stromal Cell Chondrogenesis and Aggrecanase Activity: Comparison of Agarose and Self-Assembling Peptide Scaffolds

Objective: Dexamethasone (Dex) is a synthetic glucocorticoid that has pro-anabolic and anticatabolic effects in cartilage tissue engineering systems, though the mechanisms by which these effects are mediated are not well understood. We tested the hypothesis that the addition of Dex to chondrogenic medium would affect matrix production and aggrecanase activity of human and bovine bone marrow stromal cells (BMSCs) cultured in self-assembling peptide and agarose hydrogels. Design: We cultured young bovine and adult human BMSCs in (RADA)[subscript 4] self-assembling peptide and agarose hydrogels in medium containing TGF-β1±Dex and analyzed extracellular matrix composition, aggrecan cleavage products, and the effects of the glucocorticoid receptor antagonist RU-486 on proteoglycan content, synthesis, and catabolic processing. Results: Dex improved proteoglycan synthesis and retention in agarose hydrogels seeded with young bovine cells but decreased proteoglycan accumulation in peptide scaffolds. These effects were mediated by the glucocorticoid receptor. Adult human BMSCs showed minimal matrix accumulation in agarose, but accumulated ~50% as much proteoglycan and collagen as young bovine BMSCs in peptide hydrogels. Dex reduced aggrecanase activity in (RADA)[subscript 4] and agarose hydrogels, as measured by anti-NITEGE Western blotting, for both bovine and human BMSC-seeded gels. Conclusions: The effects of Dex on matrix production are dependent on cell source and hydrogel identity. This is the first report of Dex reducing aggrecanase activity in a tissue engineering culture system.National Science Foundation (U.S.). Graduate Research FellowshipNational Institutes of Health (U.S.) (Grant EB003805

Molecular Adhesion between Cartilage Extracellular Matrix Macromolecules

In this study, we investigated the molecular adhesion between the major constituents of cartilage extracellular matrix, namely, the highly negatively charged proteoglycan aggrecan and the type II/IX/XI fibrillar collagen network, in simulated physiological conditions. Colloidal force spectroscopy was applied to measure the maximum adhesion force and total adhesion energy between aggrecan end-attached spherical tips (end radius R ≈ 2.5 μm) and trypsin-treated cartilage disks with undamaged collagen networks. Studies were carried out in various aqueous solutions to reveal the physical factors that govern aggrecan–collagen adhesion. Increasing both ionic strength and [Ca2+] significantly increased adhesion, highlighting the importance of electrostatic repulsion and Ca2+-mediated ion bridging effects. In addition, we probed how partial enzymatic degradation of the collagen network, which simulates osteoarthritic conditions, affects the aggrecan–collagen interactions. Interestingly, we found a significant increase in aggrecan–collagen adhesion even when there were no detectable changes at the macro- or microscales. It is hypothesized that the aggrecan–collagen adhesion, together with aggrecan–aggrecan self-adhesion, works synergistically to determine the local molecular deformability and energy dissipation of the cartilage matrix, in turn, affecting its macroscopic tissue properties.National Science Foundation (U.S.) (Grant CMMI-0758651)National Institutes of Health (U.S.) (Grant AR60331)United States. Dept. of Defense (National Defense Science and Engineering Graduate Fellowship (Grant N00244-09-1-0064))Shriners of North AmericaDrexel University (Faculty Start-up Grant