Adsorption and Distribution of Fluorescent Solutes near the Articular Surface of Mechanically Injured Cartilage

The development of cartilage-specific imaging agents supports the improvement of tissue assessment by minimally invasive means. Techniques for highlighting cartilage surface damage in clinical images could provide for sensitive indications of posttraumatic injury and early stage osteoarthritis. Previous studies in our laboratory have demonstrated that fluorescent solutes interact with cartilage surfaces strongly enough to affect measurement of their partition coefficients within the tissue bulk. In this study, these findings were extended by examining solute adsorption and distribution near the articular surface of mechanically injured cartilage. Using viable cartilage explants injured by an established protocol, solute distributions near the articular surface of three commonly used fluorophores (fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and carboxytetramethylrhodamine (TAMRA)) were observed after absorption and subsequent desorption to assess solute-specific matrix interactions and reversibility. Both absorption and desorption processes demonstrated a trend of significantly less solute adsorption at surfaces of fissures compared to adjacent intact surfaces of damaged explants or surfaces of uninjured explants. After adsorption, normalized mean surface intensities of fissured surfaces of injured explants were 6%, 40%, and 32% for FITC, TRITC, and TAMRA, respectively, compared to uninjured surfaces. Similar values were found for sliced explants and after a desorption process. After desorption, a trend of increased solute adsorption at the site of intact damaged surfaces was noted (316% and 238% for injured and sliced explants exposed to FITC). Surface adsorption of solute was strongest for FITC and weakest for TAMRA; no solutes negatively affected cell viability. Results support the development of imaging agents that highlight distinct differences between fissured and intact cartilage surfaces.     

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.     

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.     

可磷酸化短肽偶联壳聚糖介导IGF-1和IL-1RA双基因联合治疗兔关节软骨损伤

非病毒基因转移载体--壳聚糖被广泛用于基因转染,然而相对较低的转染效率限制了其在基因治疗中的应用.本课题组曾经报告可磷酸化短肽修饰壳聚糖(hosphorylatable short peptide coupled chitosan,pSP-CS)可增加体外培养细胞的DNA转染效率.本研究中采用pSP-CS作为基因载体介导人白细胞介素-1受体拮抗剂基因(interleukin-1 receptor antagonist gene, IL-1RA)和人胰岛素样生长因子-1基因(insulin-like growth factor 1 gene, IGF-1) 局部转染, 联合治疗兔关节软骨损伤.将pSP-CS与单基因表达质粒pBudCE4.1-IGF-1、pBudCE4.1-IL-1RA和共表达质粒pBudCE4.1-IGF-1+IL-1RA制成pSP-CS/pDNA复合物,制备股骨外侧髁全层软骨损伤模型,pSP-CS/pDNA 复合物关节腔内注射4周. ELISA分析发现,转基因组关节腔灌洗液中含有大量外源蛋白IGF-1和IL-1RA. 定量PCR检测mRNA显示, 各转基因组明显下调基质金属蛋白酶-3(matrix metallo-proteinase-3, Mmp-3)基因表达; 上调基质金属蛋白酶抑制剂-1(matrix metallo-proteinase inhibitor-1, Timp-1)和二型胶原(Collagen II) 基因表达(P < 0.05);双基因转染组作用明显优于单基因转染组(P< 0.05). HE及Collagen II免疫组化染色显示, 各转基因组软骨损伤处出现不同程度的软骨性修复,以双基因转染组作用最优. 本研究表明,pSP-CS可以携带外源基因进入软骨组织并局部大量表达,IGF-1与IL-1RA协同作用明显促进损伤软骨修复,为今后临床多基因治疗软骨损伤提供了实验基础.     

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.     

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.     

Structure-Function Relations and Rigidity Percolation in the Shear Properties of Articular Cartilage

Among mammalian soft tissues, articular cartilage is particularly interesting because it can endure a lifetime of daily mechanical loading despite having minimal regenerative capacity. This remarkable resilience may be due to the depth-dependent mechanical properties, which have been shown to localize strain and energy dissipation. This paradigm proposes that these properties arise from the depth-dependent collagen fiber orientation. Nevertheless, this structure-function relationship has not yet been quantified. Here, we use confocal elastography, quantitative polarized light microscopy, and Fourier-transform infrared imaging to make same-sample measurements of the depth-dependent shear modulus, collagen fiber organization, and extracellular matrix concentration in neonatal bovine articular cartilage. We find weak correlations between the shear modulus |G^∗| and both the collagen fiber orientation and polarization. We find a much stronger correlation between |G^∗| and the concentration of collagen fibers. Interestingly, very small changes in collagen volume fraction v_c lead to orders-of-magnitude changes in the modulus with |G^∗| scaling as (v_c – v₀)^ξ. Such dependencies are observed in the rheology of other biopolymer networks whose structure exhibits rigidity percolation phase transitions. Along these lines, we propose that the collagen network in articular cartilage is near a percolation threshold that gives rise to these large mechanical variations and localization of strain at the tissue’s surface.     

Structure-Function Relations and Rigidity Percolation in the Shear Properties of Articular Cartilage

Among mammalian soft tissues, articular cartilage is particularly interesting because it can endure a lifetime of daily mechanical loading despite having minimal regenerative capacity. This remarkable resilience may be due to the depth-dependent mechanical properties, which have been shown to localize strain and energy dissipation. This paradigm proposes that these properties arise from the depth-dependent collagen fiber orientation. Nevertheless, this structure-function relationship has not yet been quantified. Here, we use confocal elastography, quantitative polarized light microscopy, and Fourier-transform infrared imaging to make same-sample measurements of the depth-dependent shear modulus, collagen fiber organization, and extracellular matrix concentration in neonatal bovine articular cartilage. We find weak correlations between the shear modulus |G^∗| and both the collagen fiber orientation and polarization. We find a much stronger correlation between |G^∗| and the concentration of collagen fibers. Interestingly, very small changes in collagen volume fraction v_c lead to orders-of-magnitude changes in the modulus with |G^∗| scaling as (v_c – v₀)^ξ. Such dependencies are observed in the rheology of other biopolymer networks whose structure exhibits rigidity percolation phase transitions. Along these lines, we propose that the collagen network in articular cartilage is near a percolation threshold that gives rise to these large mechanical variations and localization of strain at the tissue’s surface.     

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.     

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.     

Distribution of Small Integrin-binding Ligand, N-linked Glycoproteins (SIBLING) in the Articular Cartilage of the Rat Femoral Head

The small integrin-binding ligand, N-linked glycoprotein (SIBLING) family is closely related to osteogenesis. Until recently, little was known about their existence in articular cartilage. In this study, we systematically evaluated the presence and distribution of four SIBLING family members in rat femoral head cartilage: dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), osteopontin (OPN), and dentin sialophosphoprotein (DSPP). First, non-collagenous proteins were extracted and then separated by ion-exchange chromatography. Next, the protein extracts eluted by chromatography were analyzed by Stains-all staining and Western immunoblotting. IHC was used to assess the distribution of these four SIBLING family members in the femoral head cartilage. Both approaches showed that all the four SIBLING family members are expressed in the femoral head cartilage. IHC showed that SIBLING members are distributed in various locations throughout the articular cartilage. The NH₂-terminal fragments of DMP1, BSP, and OPN are present in the cells and in the extracellular matrix, whereas the COOH-terminal fragment of DMP1 and the NH₂-terminal fragment of DSPP are primarily intracellularly localized in the chondrocytes. The presence of the SIBLING family members in the rat femoral head cartilage suggests that they may play important roles in chondrogenesis. (J Histochem Cytochem 58:1033–1043, 2010)     

Characterization and Localization of Citrullinated Proteoglycan Aggrecan in Human Articular Cartilage

Background

Rheumatoid arthritis (RA) is an autoimmune disease of the synovial joints. The autoimmune character of RA is underscored by prominent production of autoantibodies such as those against IgG (rheumatoid factor), and a broad array of joint tissue-specific and other endogenous citrullinated proteins. Anti-citrullinated protein antibodies (ACPA) can be detected in the sera and synovial fluids of RA patients and ACPA seropositivity is one of the diagnostic criteria of RA. Studies have demonstrated that RA T cells respond to citrullinated peptides (epitopes) of proteoglycan (PG) aggrecan, which is one of the most abundant macromolecules of articular cartilage. However, it is not known if the PG molecule is citrullinated in vivo in human cartilage, and if so, whether citrulline-containing neoepitopes of PG (CitPG) can contribute to autoimmunity in RA.

Methods

CitPG was detected in human cartilage extracts using ACPA+ RA sera in dot blot and Western blot. Citrullination status of in vitro citrullinated recombinant G1 domain of human PG (rhG1) was confirmed by antibody-based and chemical methods, and potential sites of citrullination in rhG1 were explored by molecular modeling. CitPG-specific serum autoantibodies were quantified by enzyme-linked immunosorbent assays, and CitPG was localized in osteoarthritic (OA) and RA cartilage using immunohistochemistry.

Findings

Sera from ACPA+ RA patients reacted with PG purified from normal human cartilage specimens. PG fragments (mainly those containing the G1 domain) from OA or RA cartilage extracts were recognized by ACPA+ sera but not by serum from ACPA- individuals. ACPA+ sera also reacted with in vitro citrullinated rhG1 and G3 domain-containing fragment(s) of PG. Molecular modeling suggested multiple sites of potential citrullination within the G1 domain. The immunohistochemical localization of CitPG was different in OA and RA cartilage.

Conclusions

CitPG is a new member of citrullinated proteins identified in human joints. CitPG could be found in both normal and diseased cartilage specimens. Antibodies against CitPG may trigger or augment arthritis by forming immune complexes with this autoantigen in the joints of ACPA+ RA patients.     

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.     

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

This article has no abstract.