Microstructural Modeling of Collagen Network Mechanics and Interactions with the Proteoglycan Gel in Articular Cartilage

Cartilage matrix mechanical function is largely determined by interactions between the collagen fibrillar network and the proteoglycan gel. Although the molecular physics of these matrix constituents have been characterized and modern imaging methods are capable of localized measurement of molecular densities and orientation distributions, theoretical tools for using this information for prediction of cartilage mechanical behavior are lacking. We introduce a means to model collagen network contributions to cartilage mechanics based upon accessible microstructural information (fibril density and orientation distributions) and which self-consistently follows changes in microstructural geometry with matrix deformations. The interplay between the molecular physics of the collagen network and the proteoglycan gel is scaled up to determine matrix material properties, with features such as collagen fibril pre-stress in free-swelling cartilage emerging naturally and without introduction of ad hoc parameters. Methods are developed for theoretical treatment of the collagen network as a continuum-like distribution of fibrils, such that mechanical analysis of the network may be simplified by consideration of the spherical harmonic components of functions of the fibril orientation, strain, and stress distributions. Expressions for the collagen network contributions to matrix stress and stiffness tensors are derived, illustrating that only spherical harmonic components of orders 0 and 2 contribute to the stress, while orders 0, 2, and 4 contribute to the stiffness. Depth- and compression-dependent equilibrium mechanical properties of cartilage matrix are modeled, and advantages of the approach are illustrated by exploration of orientation and strain distributions of collagen fibrils in compressed cartilage. Results highlight collagen-proteoglycan interactions, especially for very small physiological strains where experimental data are relatively sparse. These methods for determining matrix mechanical properties from measurable quantities at the microscale (composition, structure, and molecular physics) may be useful for investigating cartilage structure-function relationships relevant to load-bearing, injury, and repair.     

Autophagy Modulates Articular Cartilage Vesicle Formation in Primary Articular Chondrocytes

Chondrocyte-derived extracellular organelles known as articular cartilage vesicles (ACVs) participate in non-classical protein secretion, intercellular communication, and pathologic calcification. Factors affecting ACV formation and release remain poorly characterized; although in some cell types, the generation of extracellular vesicles is associated with up-regulation of autophagy. We sought to determine the role of autophagy in ACV production by primary articular chondrocytes. Using an innovative dynamic model with a light scatter nanoparticle counting apparatus, we determined the effects of autophagy modulators on ACV number and content in conditioned medium from normal adult porcine and human osteoarthritic chondrocytes. Healthy articular chondrocytes release ACVs into conditioned medium and show significant levels of ongoing autophagy. Rapamycin, which promotes autophagy, increased ACV numbers in a dose- and time-dependent manner associated with increased levels of autophagy markers and autophagosome formation. These effects were suppressed by pharmacologic autophagy inhibitors and short interfering RNA for ATG5. Caspase-3 inhibition and a Rho/ROCK inhibitor prevented rapamycin-induced increases in ACV number. Osteoarthritic chondrocytes, which are deficient in autophagy, did not increase ACV number in response to rapamycin. SMER28, which induces autophagy via an mTOR-independent mechanism, also increased ACV number. ACVs induced under all conditions had similar ecto-enzyme specific activities and types of RNA, and all ACVs contained LC3, an autophagosome-resident protein. These findings identify autophagy as a critical participant in ACV formation, and augment our understanding of ACVs in cartilage disease and repair.     

Interactions between Subunits in Heterodimeric Ncd Molecules

The nonprocessive minus-end-directed kinesin-14 Ncd is involved in the organization of the microtubule (MT) network during mitosis. Only one of the two motor domains is involved in the interaction with the MT. The other head is tethered to the bound one. Here we prepared, purified, and characterized mutated Ncd molecules carrying point mutations in one of the heads, thus producing heterodimeric motors. The mutations tested included substitutions in Switch I and II: R552A, E585A, and E585D; the decoupling mutant N600K; and a deletion in the motor domain in one of the subunits resulting in a single-headed molecule (NcN). These proteins were isolated by two sequential affinity chromatography steps, followed by measurements of their affinities to MT, enzymatic properties, and the velocity of the microtubule gliding test in vitro. A striking observation is a low affinity of the single-headed NcN for MT both without nucleotides and in the presence of 5′-adenylyl-β,γ-imidodiphosphate, implying that the tethered head has a profound effect on the structure of the Ncd-MT complex. Mutated homodimers had no MT-activated ATPase and no motility, whereas NcN had motility comparable with that of the wild type Ncd. Although the heterodimers had one fully active and one inactive head, the ATPase and motility of Ncd heterodimers varied dramatically, clearly demonstrating that interactions between motor domains exist in Ncd. We also show that the bulk property of dimeric proteins that interact with the filament with only one of its heads depends also on the distribution of the filament-interacting subunits.     

Comparison of Gene Expression Patterns in Articular Cartilage and Xiphoid Cartilage

Biochemical Genetics - Cartilage is a resilient and smooth connective tissue that is found throughout the body. Among the three major types of cartilage, namely hyaline cartilage, elastic...     

Considerations on Joint and Articular Cartilage Mechanics

When studying joint degeneration leading to osteoarthritis (OA), it seems imperative that local joint tissue loading is known during normal everyday movement and that the adaptive/degenerative effects of this loading are quantified systematically. Philosophically, we believe the best way to approach this problem is by studying joint degeneration and osteoarthritis in long-term experimental models and by representing diarthrodial joints and the associated tissues with accurate, geometric and structural, theoretical models. Here, we present selected examples of our work representing this approach. Experimentally, we demonstrate that the local loading of joints changes continuously in experimental models of OA, not only because of the changing external and internal loading, but also because of the continuous alterations in joint contact geometry and tissue mechanical properties. Furthermore, we show that single bouts of joint loading affect gene expression, and that gene expression, as well as subsequent joint degeneration is site-specific. In fact, opposing articular surfaces that are exposed to the same loading may degenerate at completely different rates. Finally, we propose a series of theoretical models of articular cartilage and contact mechanics, demonstrating that many of the anisotropic and inhomogeneous properties can be explained by structural elements and their orientation and volumetric concentration across the tissue.     

Testing Drugs on Human Osteoarthritic Articular Cartilage

New drugs are generally developed against animal models of the human disease. Before they are subjected to clinical trials it might be helpful to be able to test whether they are as effective against the disease in human tissue as they were in animals. It is proposed that this can be achieved by the use of organ maintenance culture of the human diseased tissue, the relevant biochemical parameters being measured by quantitative cytochemistry. In the present studies differences between the effect of indomethacin and of the ‘chondroprotective’ drug diclofenac sodium, on human osteoarthritic cartilage, have been measured.     

Focal Articular Defects Modify Cartilage Contact Mechanics

This article has no abstract.     

En Bloc Staining of Articular Cartilage and Bone

Blocks of canine and porcine articular cartilage were stained en bloc with Weigert's iron hematoxylin or Harris' hematoxylin with or without eosin Y counterstaining and cleared in methyl salicylate. The morphology and three-dimensional relationships of chondrocytes were best demonstrated with Weigert's iron hematoxylin. The morphology of the cartilage and chondrocytes was superior to that in sections of routine hematoxylin and eosin stained, paraffin processed samples. The three-dimensional localization of intracellular lipids in individual and clones of chondrocytes was observed when cartilage samples were stained with oil red O and mounted directly in a water-based medium. Blocks of decalcified bone were stained en bloc with Weigert's iron hematoxylin and cleared with methyl salicylate. The three-dimensional orientation of osteocytes around osteonal canals, in circumferential lamellae, and in interstitial lamellae was demonstrated. The morphology of “cutting cones” in cortical bone also was observed.     

The Frictional Coefficient of Bovine Knee Articular Cartilage

1 Introduction Cartilage has excellent biomechanical and tri- bological properties with low friction and minimum wear in diarthrodial joints throughout the lifetime of most people, and the lifetime of articular cartilage can be 40 years or longer. This has inspired material and bionic scientists to study the mechanism of such excellent tri- bological characteristics in order to develop artificial joints. Various mechanisms have been proposed to ex- plain the remarkable low friction behavior of…     

Elastoviscous Transitions of Articular Cartilage Reveal a Mechanism of Synergy between Lubricin and Hyaluronic Acid

When lubricated by synovial fluid, articular cartilage provides some of the lowest friction coefficients found in nature. While it is known that macromolecular constituents of synovial fluid provide it with its lubricating ability, it is not fully understood how two of the main molecules, lubricin and hyaluronic acid, lubricate and interact with one another. Here, we develop a novel framework for cartilage lubrication based on the elastoviscous transition to show that lubricin and hyaluronic acid lubricate by distinct mechanisms. Such analysis revealed nonspecific interactions between these molecules in which lubricin acts to concentrate hyaluronic acid near the tissue surface and promotes a transition to a low friction regime consistent with the theory of viscous boundary lubrication. Understanding the mechanics of synovial fluid not only provides insight into the progression of diseases such as arthritis, but also may be applicable to the development of new biomimetic lubricants.     

Storage of Porcine Articular Cartilage at High Subzero Temperatures

Objective: Transplantation of osteochondral allograft tissue can treat large joint defects but is limited by tissue availability, surgical timing, and infectious disease transmission. Fresh allografts perform the best but requirements for infectious disease testing delay the procedure with subsequent decrease in cell viability and function. Hypothermic storage at lower temperatures can extend tissue banking time without loss of cell viability and, therefore, increase the supply of allograft tissue. This study investigated the effects of different cryoprotectant solutions on intact AC at various subzero temperatures. Design: 10 mm porcine osteochondral dowels were immersed for 30 minutes in various combinations of solutions [(XVIVO, propylene glycol (51% w/w), sucrose (46% w/w)] cooled to various subzero temperatures (−10, −15, and −20 °C), and held for 30 min. After warming, 70 μm slices were stained with membrane integrity dyes, viewed under fluorescence microscopy and cell recovery calculated relative to fresh controls. Results: Results demonstrated excellent cell recovery (>75%) at −10°C provided ice did not form. Excellent cell recovery (>70%) occurred at −15°C in solutions containing 51% propylene glycol but formation of extra-matrix ice in other solutions resulted in significant cell loss. All groups had <6% cell recovery at −20°C and propylene glycol did not provide a protective effect even though extra-matrix ice did not form Conclusions: These results suggest that extra-matrix ice plays an important role in cell damage during cryopreservation. Excellent cell recovery can be obtained after storage at subzero temperatures if ice does not form. Hypothermic preservation at high subzero temperatures may extend AC storage time in tissue banks compared to current techniques.     

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.     

玻璃化法与低温冷冻法保存兔关节软骨异体移植的实验对比研究

目的:观察玻璃化法保存同种异体软骨移植修复兔关节软骨缺损的效果,并与低温冷冻法作比较.方法:18只健康成年日本大白兔,其中6只用于异体骨软骨柱的制备,另外12只随机分成2组,每组各6只.软骨移植物标本取材后,采用低温冷冻法和玻璃化法保存2w.同种异体骨软骨移植物相应复温后移植于兔膝关节软骨缺损模型的相应软骨缺损区.移植术后12w处死动物,移植区软骨组织后固定、切片、脱钙及石蜡包埋.采用苏木精-伊红染色及蕃红-O染色后进行组织学观察及Mankin评分.结果:玻璃化组软骨移植区颜色与周围正常软骨组织基本一致,软骨完整,无坍陷及裂纹;镜下观察可见其组织深层透明软骨样组织,浅层软骨区可见纤维样软骨组织,细胞成熟,大量分泌软骨基质.低温冷冻组软骨移植区表面欠光整,存在裂隙及轻度坍陷;镜下观察可见少量纤维样软骨组织,大部分为纤维结缔组织,细胞排列相对紊乱,软骨基质分泌量少.两组的组织学Mankin评分分别为9.6± 2.4和3.0± 1.0,低温冷冻组显著高于玻璃化组(t=2.014,P=0.000).结论:采用玻璃化法保存兔同种异体软骨移植物是一种可行的方法.相比低温冷冻法,玻璃化法保存的同种异体软骨其移植术后,软骨修复区的组织更接近正常软骨.     

A Phase-Field Model for Articular Cartilage Regeneration in Degradable Scaffolds

Degradable scaffolds represent a promising solution for tissue engineering of damaged or degenerated articular cartilage which due to its avascular nature, is characterized by a low self-repair capacity. To estimate the articular cartilage regeneration process employing degradable scaffolds, we propose a mathematical model as the extension of Olson and Haider’s work (Int. J. Pure Appl. Math. 53:333–353, 2009). The simulated tissue engineering procedure consists in (i) the explant of a cylindrical sample, (ii) the removal of the inner core region, and (iii) the filling of the inner region with hydrogels, degradable scaffolds enriched with nutrients, such as oxygen and glucose. The phase-field model simulates the cartilage regeneration process at the scaffold-cartilage interface. It embeds reaction-diffusion equations, which are used to model the nutrient and regenerated extracellular matrix. The equations are solved using an unconditionally stable hybrid numerical scheme. Cartilage repair processes with full-thickness defects, which are controlled by properties of hydrogel materials and cartilage explant culture based on biological interest are observed. The implemented mathematical model shows the capability to simulate cartilage repairing processes, which can be virtually controlled evaluating hydrogel and cartilage material properties including nutrient supply and defected magnitude. In particular, the adopted methodology is able to explain the regeneration time of cartilage within hydrogel environments. With the numerical scheme, the numerical simulations are demonstrated for the potential improvement of hydrogel structures.     

Of Mice,Men and Elephants: The Relation between Articular Cartilage Thickness and Body Mass

Mammalian articular cartilage serves diverse functions, including shock absorption, force transmission and enabling low-friction joint motion. These challenging requirements are met by the tissue’s thickness combined with its highly specific extracellular matrix, consisting of a glycosaminoglycan-interspersed collagen fiber network that provides a unique combination of resilience and high compressive and shear resistance. It is unknown how this critical tissue deals with the challenges posed by increases in body mass. For this study, osteochondral cores were harvested post-mortem from the central sites of both medial and lateral femoral condyles of 58 different mammalian species ranging from 25 g (mouse) to 4000 kg (African elephant). Joint size and cartilage thickness were measured and biochemical composition (glycosaminoclycan, collagen and DNA content) and collagen cross-links densities were analyzed. Here, we show that cartilage thickness at the femoral condyle in the mammalian species investigated varies between 90 µm and 3000 µm and bears a negative allometric relationship to body mass, unlike the isometric scaling of the skeleton. Cellular density (as determined by DNA content) decreases with increasing body mass, but gross biochemical composition is remarkably constant. This however need not affect life-long performance of the tissue in heavier mammals, due to relatively constant static compressive stresses, the zonal organization of the tissue and additional compensation by joint congruence, posture and activity pattern of larger mammals. These findings provide insight in the scaling of articular cartilage thickness with body weight, as well as in cartilage biochemical composition and cellularity across mammalian species. They underscore the need for the use of appropriate in vivo models in translational research aiming at human applications.