活性氧及过渡金属对关节软骨蛋白聚糖降解的影响

在温育的小牛关节软骨切片介质中加入黄嘌呤氧化酶/次黄嘌呤活性氧产生系统,可使关节软骨蛋白聚糖的降解显著增高。这种作用不能被超氧化物歧化酶或铁络合剂二乙三胺戊乙酸抑制,但可被过氧化氢酶抑制。在温育的介质中加入H_2O_2,也可使关节软骨蛋白聚糖降解增高,Cu~(2+)或Co~(2+)对此有显著促进作用,其它过渡金属则作用不明显。应用Sepharose 6B柱层析,分析软骨蛋白聚糖降解产物,多数实验组只在kD=0处有一个大分子的洗脱峰,但Cu~(2+)+H_2O_2组则在kD=0及0.57处出现两个洗脱峰(分别称为峰Ⅰ及峰Ⅱ)。醋酸纤维素薄膜双向电泳证实峰Ⅱ为硫酸软骨素,提示Cu~(2+)与H_2O_2复合物可导致蛋白聚糖分子的断裂。本研究结果可能有助于阐明某些变性型骨关节疾病的发病机理,例如因缺硒所致的大骨节病等。     

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.     

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.     

The Pathological Role of the ERK Pathway in Human Adult Articular Cartilage

This article has no abstract.     

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...     

Study on the Microstructure of Human Articular Cartilage/Bone Interface

For improving the theory of gradient microstructure of cartilage/bone interface, human distal femurs were studied. Scanning Electron Microscope (SEM), histological sections and MicroCT were used to observe, measure and model the microstructure of cartilage/bone interface. The results showed that the cartilage/bone interface is in a hierarchical structure which is composed of four different tissue layers. The interlocking of hyaline cartilage and calcified cartilage and that of calcified cartilage and subchondral bone are in the manner of “protrusion-pore” with average diameter of 17.0 μm and 34.1 μm respectively. In addition, the cancellous bone under the cartilage is also formed by four layer hierarchical structure, and the adjacent layers are connected by bone trabecula in the shape of H, I and Y, forming a complex interwoven network structure. Finally, the simplified structure model of the cartilage/bone interface was proposed according to the natural articular cartilage/bone interface. The simplified model is a 4-layer gradient biomimetic structure, which corresponds to four different tissues of natural cartilage/bone interface. The results of this work would be beneficial to the design of bionic scaffold for the tissue engineering of articular cartilage/bone.     

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.     

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.     

Collagen Metabolism of Human Osteoarthritic Articular Cartilage as Modulated by Bovine Collagen Hydrolysates

Destruction of articular cartilage is a characteristic feature of osteoarthritis (OA). Collagen hydrolysates are mixtures of collagen peptides and have gained huge public attention as nutriceuticals used for prophylaxis of OA. Here, we evaluated for the first time whether different bovine collagen hydrolysate preparations indeed modulate the metabolism of collagen and proteoglycans from human OA cartilage explants and determined the chemical composition of oligopeptides representing collagen fragments. Using biophysical techniques, like MALDI-TOF-MS, AFM, and NMR, the molecular weight distribution and aggregation behavior of collagen hydrolysates from bovine origin (CH-Alpha®, Peptan™ B 5000, Peptan™ B 2000) were determined. To investigate the metabolism of human femoral OA cartilage, explants were obtained during knee replacement surgery. Collagen synthesis of explants as modulated by 0–10 mg/ml collagen hydrolysates was determined using a novel dual radiolabeling procedure. Proteoglycans, NO, PGE₂, MMP-1, -3, -13, TIMP-1, collagen type II, and cell viability were determined in explant cultures. Groups of data were analyzed using ANOVA and the Friedman test (n = 5–12). The significance was set to p≤0.05. We found that collagen hydrolysates obtained from different sources varied with respect to the width of molecular weight distribution, average molecular weight, and aggregation behavior. None of the collagen hydrolysates tested stimulated the biosynthesis of collagen. Peptan™ B 5000 elevated NO and PGE₂ levels significantly but had no effect on collagen or proteoglycan loss. All collagen hydrolysates tested proved not to be cytotoxic. Together, our data demonstrate for the first time that various collagen hydrolysates differ with respect to their chemical composition of collagen fragments as well as by their pharmacological efficacy on human chondrocytes. Our study underscores the importance that each collagen hydrolysate preparation should first demonstrate its pharmacological potential both in vitro and in vivo before being used for both regenerative medicine and prophylaxis of OA.     

重组人中期因子midkine对大鼠膝关节软骨部分损伤的修复作用

目的:通过外源注射不同剂量的重组人中期因子midkine(rhMK),研究其对大鼠膝关节软骨部分损伤的修复作用。方法:雄性SD大鼠双侧膝关节建立软骨部分损伤的动物模型,术后24小时分别向关节腔内注射生理盐水或rhMK (20μg/kg、60μg/kg、180μg/kg)。于术后8周将大鼠全部处死,取材进行组织学观察,从而确定最佳注射剂量;在药代动力学研究中,按最佳注射剂量向正常大鼠膝关节腔内注射rhMK,分别于注射后1小时、1天、3天、6天、9天、12天和15天处死大鼠,检测膝关节软骨组织中rhMK的含量。结果:不同剂量的重组蛋白对膝关节软骨部分损伤均有不同程度的修复作用,其中180μg/kg的剂量效果最佳;以180μg/kg的剂量向正常大鼠膝关节腔内注射rhMK后,经过Kinetica5.0药代动力学软件拟合后,计算得rhMK在软骨组织中的消除相半衰期为8.69天。结论:rhMK对大鼠膝关节软骨部分损伤有明显的修复作用,最佳注射剂量为180μg/kg,最佳注射时间间隔为8天。     

Characterization of Human DSPG3, a Small Dermatan Sulfate Proteoglycan

PG-Lb is a small dermatan sulfate proteoglycan that has been previously characterized in chicken. In the developing limb, chick PG-Lb appears to be exclusively expressed in the zone of flattened chondrocytes. We have cloned and sequenced the human homolog to chick PG-Lb from two human chondrocyte cDNA libraries and a human chondrocyte RNA sample. The human homolog has been named DSPG3, as it is the third member of the small dermatan sulfate proteoglycan family to be identified and characterized along with biglycan (PG-I) and decorin (PG-II). DSPG3 maps to chromosome 12q21 and is composed of 1515 nucleotides of cDNA that code for a 322-amino-acid protein. The protein contains three potential glycosaminoglycan attachment sites, two N-glycosylation sites, a poly- glutamic acid stretch, and six cysteines. By Northern analysis, we have demonstrated that DSPG3 is expressed in cartilage, as well as ligament and placental tissues.     

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…     

Interactions between Nearest-neighboring Glycosaminoglycan Molecules of Articular Cartilage

The electrostatic interaction effects including the interaction potential, force and torque between the neighboring chondroitin sulfate glycosaminoglycan (CS-GAG) molecular chains in the bottle brush conformation of proteoglycan aggrecan are obtained as the functions of the minimum separation distance and the mutual angle between the molecular chains based on an asymptotic solution of the Poisson-Boltzmann equation that the CS-GAGs satisfy under the normal physiological conditions of articular cartilage. The present study indicates that the electrostatic interactions are not only associated intimately with the separation distance and the mutual angle, which are shown as purely exponential in separation distance and decrease with increasing mutual angle, but also dependent sensitively on the saline concentration in the electrolyte solution within the tissue. Further analysis shows that in the range of the separation distance between two neighboring CS-GAG molecular chains in the bottle brush conformation of the aggrecan in vivo (2~4 nm), if the saline concentration in the electrolyte solution is not less than a value of concentration (~0.1 M), the interactions between the molecular chains will monotonically increase with decreasing the saline concentration, however, if the saline concentration is less than the value of concentration, the relationship between the interactions and the saline concentration will not be simply monotonic. Some present results are in agreement with the existed relevant conclusions.