Association of a lower molecular weight protein to the mu-opioid receptor demonstrated by (125)I-beta-endorphin cross-linking studies

Cross-linking experiments using the (125)I-beta-endorphin revealed the presence of several receptor-related species in cell lines expressing endogenous opioid receptors, including a small molecular mass protein (approximately 22 kDa). Previous reports have suggested that this 22-kDa (125)I-beta-endorphin cross-linked protein could be the degradative product from a higher molecular mass species, i.e., a fragment of the receptor. To determine if this protein is indeed a degraded receptor fragment, (125)I-beta-endorphin was cross-linked to the (His)(6) epitope-tagged mu-opioid receptor (His-mu) stably expressed in the murine neuroblastoma Neuro(2A) cells. Similar to earlier reports with cell lines expressing endogenous receptors, two major bands of 72- and 25-kDa proteins were specifically cross-linked. Initial cross-linking experiments indicated the absolute requirement of the high-affinity (125)I-beta-endorphin binding to the mu-opioid receptor prior to the appearance of the low molecular weight species, suggesting that the 22-kDa protein could be a degraded fragment of the receptor. However, variations in the ratios of these protein bands being cross-linked by several homo- or heterobifunctional cross-linking agents were observed. Although neither the carboxyl terminus mu-opioid receptor-specific antibodies nor the antibodies against the epitope at the amino terminus of the receptor could recognize the 22-kDa protein, this (125)I-beta-endorphin cross-linked species could be coimmunoprecipitated with the receptor antibodies or could be isolated with a nickel resin affinity chromatography. The direct physical association of the 22-kDa protein with the receptor was demonstrated also by the observation that the 22-kDa protein could not bind to the nickel resin alone, but that its binding to the nickel resin was restored in the presence of the His-mu. Taken together, these results suggest that the 22-kDa protein cross-linked by (125)I-beta-endorphin is not a degradative product, but a protein located within the proximity of the mu-opioid receptor, and that it is tightly associated with the receptor.     

The insulin-like growth factors (IGFs) I and II bind to articular cartilage via the IGF-binding proteins

Bovine articular cartilage discs (3 mm diameter x 400 micrometer thick) were equilibrated in buffer containing (125)I-insulin-like growth factor (IGF)-I (4 degrees C) +/- unlabeled IGF-I or IGF-II. Competition for binding to cartilage discs by each unlabeled IGF was concentration-dependent, with ED(50) values for inhibition of (125)I-IGF-I binding of 11 and 10 nM for IGF-I and -II, respectively, and saturation by 50 nM. By contrast, an analog of IGF-I with very low affinity for the insulin-like growth factor-binding proteins (IGF-BPs), des-(1-3)-IGF-I, was not competitive with (125)I-IGF-I for cartilage binding even at 100-400 nM. Binding of the (125)I-labeled IGF-II isoform to cartilage was competed for by unlabeled IGF-I or -II, with ED(50)s of 160 and 8 nM, respectively. This probably reflected the differential affinities of the endogenous IGF-BPs (IGF-BP-6 and -2) for IGF-II/IGF-I. Transport of (125)I-IGF-I was also measured in an apparatus that allows diffusion only across the discs (400 micrometer), by addition to one side and continuous monitoring of efflux on the other side. The time lag for transport of (125)I-IGF was 266 min, an order of magnitude longer than the theoretical prediction for free diffusion in the matrix. (125)I-IGF-I transport then reached a steady state rate (% efflux of total added (125)I-IGF/unit time), which was subsequently accelerated approximately 2-fold by addition of an excess of unlabeled IGF-I. Taken together, these results indicate that IGF binding to cartilage, mostly through the IGF-BPs, regulates the transport of IGFs in articular cartilage, probably contributing to the control of their paracrine activities.     

Biodistribution study of a monoclonal antibody specific to link proteins from human cartilage

We report the use of monoclonal antibody 6F3 prepared against link proteins from human articular cartilage to elucidate the distribution of these glycoproteins within connective and other tissues. By immunohistochemical analysis, we showed that only the Fab fragment could reach the antigenic site in human articular sections. Cross-reactivity of the antibody 6F3 with link proteins purified from rat articular cartilage allowed us to carry out a biodistribution analysis in vivo in the rat. The time course of whole blood and plasma showed maximal activity 6h after the 20 μg i.v. injection of [¹²⁵I]Fab-6F3. Urinary excretion seems to be a high route of elimination. Moreover, we noticed no radioactive uptake across the blood-brain barrier. A significant fixation of labeled antibody Fab-6F3 was observed in noncartilaginous connective tissues such as aorta, skin and lung. As expected, specific and increased radioactivity was observed in all cartilage tissues, this increase was significantly higher 6 h after the [¹²⁵I]Fab-6F3 injection than in the other connective tissues.     

Del1: a new protein in the superficial layer of articular cartilage

Articular cartilage contains four distinct zones, extending from the surface to the subchondral bone. Freshly isolated chondrocytes from the superficial zone of articular cartilage retain a collagenase-P-resistant cell-associated matrix. In the studies described here, the protein Del1 was identified as a component of the cell-associated matrix of superficial zone chondrocytes from adult bovine articular cartilage. Very little Del1 was associated with freshly isolated deep zone chondrocytes. Western blot analysis of articular cartilage cell and tissue extracts using polyclonal antibodies specific for Del1 showed Del1 was present in an insoluble cell-associated fraction. Extracts of the superficial zone of articular cartilage were found to be enriched in Del1 compared to the deeper layers of the tissue. Immunohistochemical staining of full-thickness articular cartilage with anti-Del1 antibodies also showed an enrichment of Del1 in the superficial zone. These observations are the first to describe the protein Del1 in a nonendothelial, nonfetal tissue.     

Transport and binding of insulin-like growth factor I through articular cartilage

This study focused on the role of insulin-like growth factor (IGF) binding proteins (IGFBPs) in cartilage on the transport and binding of IGF-I within the tissue. We have developed experimental and theoretical modeling techniques to quantify and contrast the roles of diffusion, binding, fluid convection, and electrical migration on the transport of IGF-I within cartilage tissue. Bovine articular cartilage disks were equilibrated in buffer containing 125I-IGF-I and graded levels of unlabeled IGF-I. Equilibrium binding, as measured by the uptake ratio of 125I-IGF-I in the tissue (free plus bound) to the concentration of labeled species in the buffer, was found to be consistent with a first-order reversible binding model involving one dominant family of binding sites within the matrix. Western ligand blots revealed a major IGF binding doublet around 23 kDa, which has been previously shown to coincide with IGFBP-6. Diffusive transport of 125I-IGF-I through cartilage was measured and found to be consistent with a diffusion-limited reaction theoretical model incorporating first-order reversible binding. Addition of excess amounts of unlabeled IGF-I during steady state transport of 125I-IGF-I resulted in release of bound 125I-IGF-I from the tissue, as predicted by the diffusion-reaction model. In contrast, addition of the low-affinity Des(1-3)IGF-I analog did not result in release of bound 125I-IGF-I. Application of electric current was used to augment transport of IGF-I through cartilage via electroosmosis and electrophoresis. Taken together, our results suggest that a single dominant substrate family, the high-affinity IGFBPs, is responsible for much of the observed binding of IGF-I within cartilage. The data suggest that intratissue fluid flow, such as that induced by mechanical loading of cartilage in vivo may be expected to enhance IGF transport by an order of magnitude and that this increment may help to counterbalance the restrictions encountered by the immobilization of IGFs by the binding proteins.     

MR水激励序列显示膝关节软骨的临床应用研究

目的：探讨三维快速梯度回波水激励膝关节软骨成像序列（3D-FFE-WATS）相对于三维快速梯度回波预饱和反转恢复法脂肪抑制序列（3D-FFE-SPIR）在显示膝关节软骨方面的优势,选择显示膝关节软骨的最佳序列。方法：应用3D-FFE-WATS及3D-FFE-SPIR序列组合对20名志愿者及30例疑诊关节软骨损伤的单膝关节进行检查,获得膝关节各软骨的3D图像,并利用3D最大密度投影法（MIP）进行横断面和冠状面3D重建。分析上述2种序列对软骨病变的显示及检出能力,计算其对关节软骨的信噪比（SNR）和对比噪声比（CNR）,并进行统计学分析。结果：两序列在显示膝关节软骨SNR方面,无统计学意义（P〉0.05）;两种序列在显示软骨与关节液的CNR、软骨与骨皮质的CNR、软骨与骨髓的CNR、软骨与肌肉的CNR差异方面t值分别为（-30.619;2.348;-2.408;2.216）,有统计学意义（P〈0.05）。结论：3D-FFE-WATS序列可作为膝关节软骨成像的首选序列。     

Polyclonal anti-idiotypic opioid receptor antibodies generated by the monoclonal beta-endorphin antibody 3-E7

Anti-idiotypic antibodies were raised in rabbits against the monoclonal beta-endorphin antibody 3-E7. These antibodies inhibit beta-endorphin binding to the 3-E7 antibody, binding of 3H-diprenorphine to solubilized opioid receptors and the binding of 125I-beta-endorphin to rat brain membranes. Exposure of NG-108CC15 hybrid cells to anti-idiotypic antibodies produces an opioid-like inhibition of PGE1-stimulated cAMP accumulation. These data suggest that the antibodies raised by the anti-idiotypic route both bind to and activate opioid receptors.     

The low permeability of healthy meniscus and labrum limit articular cartilage consolidation and maintain fluid load support in the knee and hip

The knee meniscus and hip labrum appear to be important for joint health, but the mechanisms by which these structures perform their functions are not fully understood. The fluid phase of articular cartilage provides compressive stiffness and aids in maintaining a low friction articulation. Healthy fibrocartilage, the tissue of meniscus and labrum, has a lower fluid permeability than articular cartilage. In this study we hypothesized that an important function of the knee meniscus and the hip labrum is to augment fluid retention in the articular cartilage of a mechanically loaded joint. Axisymmetric hyperporoelastic finite element models were analyzed for an idealized knee and an idealized hip. The results indicate that the meniscus maintained fluid pressure and inhibited fluid exudation in knee articular cartilage. Similar, but smaller, effects were seen with the labrum in the hip. Increasing the fibrocartilage permeability relative to that of articular cartilage gave a consolidation rate and loss of fluid load support comparable to that predicted by meniscectomy or labrectomy. The reduced articular cartilage fluid pressure that was calculated for the joint periphery is consistent with patterns of endochondral ossification and osteophyte formation in knee and hip osteoarthritis. High articular central strains and loss of fluid load support after meniscectomy could lead to fibrillation. An intact low-permeability fibrocartilage is important for limiting fluid exudation from articular cartilage in the hip and knee. This may be an important aspect of the role of fibrocartilage in protecting these joints from osteoarthritis.     

Arthritis induced immunologically with cationic amidated bovine serum albumin in the Guinea pig

Arthritis was induced by injecting cationic amidated bovine serum albumin (aBSA) (pI approximately 9.2) into the knee joint of immunized guinea pigs and the mechanisms of articular cartilage destruction were studied morphologically and biochemically. Marked synovitis associated with polymorphonuclear leukocyte (PML) infiltration occurred within 1 day of the challenge. Articular cartilage infiltrated by PMLs was almost completely destroyed after 2 weeks. During the initial destructive process, proteoglycans were depleted from the cartilage and later collagen fibers disappeared. Granulation tissue growing in the inflamed synovium and bone marrow replaced the destroyed cartilage and joint cavity and formed fibrous scar tissue (fibrous ankylosis) by 8 weeks. Subsequently, the knee joints developed cartilagenous ankylosis by 12 weeks and finally bony ankylosis at 28 weeks. Autoradiography using 125I-aBSA and immunofluorescence studies for immunoglobulin (IgG) and complement (C3) demonstrated that the antigen is trapped in all zones of the articular cartilage and serves as a trigger for immune complex formation. Significantly increased neutral proteinase activities against substrates of proteoglycan subunits, [3H]carboxymethylated transferrin and L-pyroglutamyl-L-prolyl-L-valine-paranitroanilide were detected in homogenates of the synovium and cartilage from arthritic knee joints 1 and 2 weeks after induction. Inhibitor studies and pH curves suggested that the proteinase is leukocyte elastase. Measurable amounts of gelatinolytic activity, detected by activation with 4-aminophenylmercuric acetate and inhibited with EDTA, were also present in the same samples, but there was no detectable collagenase activity. The data on SDS-gelatin substrate gel showed that the proteinase is gelatinase derived from PMLs. These results suggest that in aBSA-induced arthritis, elastase and gelatinase from PMLs invading articular cartilage may play important roles in cartilage destruction.     

Effect of prednisolone on the glycosaminoglycan components of the regenerating articular cartilage.

Investigations were performed on the effect of prednisolone (0.5 mg/kg) on the regenerating femoral articular cartilage of the knee joint in dogs that had been subjected to semiarthroplasty. After 70 days of prednisolone treatment the dogs were killed and the regenerating articular cartilage was removed, minced, and dried with acetone. The acetone-dried material was used for the determination of galactosamine, glucosamine, uronic acid, sulphate, sialic acid and hydroxyproline. Prednisolone treatment elicited a quantitative increase in galactosamine (30.2%), uronic acid (76.2%), and sulphate (9.1%), while no difference was observed in sialic acid content between the treated and untreated groups. From the molar ratio of the measured components it appears that prednisolone produced an increase in chondroitin sulphate and hyaluronic acid, and a decrease in the keratosulphate content of cartilage. By comparing the values measured in the regenerating articular cartilage of control and prednisolone-treated dogs with the values obtained in the mature articular cartilage, we may conclude that prednisolone--at least as regards the glycosaminoglycans of the ground substance--exerts an accelerating effect on cartilage regeneration.     

Comparison of MRI-based estimates of articular cartilage contact area in the tibiofemoral joint

Knee osteoarthritis (OA) detrimentally impacts the lives of millions of older Americans through pain and decreased functional ability. Unfortunately, the pathomechanics and associated deviations from joint homeostasis that OA patients experience are not well understood. Alterations in mechanical stress in the knee joint may play an essential role in OA; however, existing literature in this area is limited. The purpose of this study was to evaluate the ability of an existing magnetic resonance imaging (MRI)-based modeling method to estimate articular cartilage contact area in vivo. Imaging data of both knees were collected on a single subject with no history of knee pathology at three knee flexion angles. Intra-observer reliability and sensitivity studies were also performed to determine the role of operator-influenced elements of the data processing on the results. The method's articular cartilage contact area estimates were compared with existing contact area estimates in the literature. The method demonstrated an intra-observer reliability of 0.95 when assessed using Pearson's correlation coefficient and was found to be most sensitive to changes in the cartilage tracings on the peripheries of the compartment. The articular cartilage contact area estimates at full extension were similar to those reported in the literature. The relationships between tibiofemoral articular cartilage contact area and knee flexion were also qualitatively and quantitatively similar to those previously reported. The MRI-based knee modeling method was found to have high intra-observer reliability, sensitivity to peripheral articular cartilage tracings, and agreeability with previous investigations when using data from a single healthy adult. Future studies will implement this modeling method to investigate the role that mechanical stress may play in progression of knee OA through estimation of articular cartilage contact area.     

Ultrastructure of the mineralizing metacarpophalangeal joint of progressive ankylosis (ank/ank) mice

The metacarpophalangeal joint of the progressive ankylosis mouse was examined at 4, 6 and 8 weeks of age by using electron microscopy and electron probe microanalysis. These studies describe the progression of the disorder, and they reveal three overlapping phases: pannus proliferation, chondrophyte formation and mineralization, and articular cartilage mineralization. The chondrophyte mineralized in a manner fairly similar to endochondrial bone, whereas articular cartilage mineralized by advancement of a calcification front across the cartilage. Synovial crystal phagocytosis was also observed. The progressive ankylosis mouse should be an important model for studying both crystal deposition and abnormal articular cartilage calcification.     

Monoclonal antibodies to alpha-endorphin effective in immunohistochemistry and immunoblotting

Four mouse monoclonal antibodies (E11, A8, F8, H5) to alpha-endorphin have been produced. The antibodies bind 12.5, 20.6, 9.6, 6.6% of 125I-beta-endorphin and 35.5, 15.1, 12.8, 12.2% of 125I-gamma-endorphin; the binding of 125I-alpha-endorphin being taken for 100%. The binding of antibodies E11, A8, F8 and H5 to 125I-alpha-endorphin was 50% inhibited by unlabeled ligand in concentrations 5, 50, 30 and 35 nM respectively. Using tissue sections of rat pituitary it was shown that antibody E11 can be used for the localization of endorphin producing cells by immunofluorescence. The antibodies F8 and H5 effectively detected endorphin precursor proopiomelanocortin by immunoblotting.     

Mechanical and physicochemical regulation of the action of insulin-like growth factor-I on articular cartilage

The development and maintenance of healthy joints is a complex process involving many physical and biological stimuli. This study investigates the interaction between insulin-like growth factor-I (IGF-I) and static mechanical compression in the regulation of articular cartilage metabolism. Bovine cartilage explants were treated with concentrations of IGF-I from 0 to 300 ng/ml in the presence or absence of 0-50% static compression, and the transient and steady-state incorporation of [(3)H]proline and [(35)S]sulfate into matrix components were measured. In parallel studies, cartilage explants were treated with 0-300 ng/ml IGF-I at media pH ranging from 6.4 to 7.2 and the steady-state incorporation of [(3)H]proline and [(35)S]sulfate was measured. The effect of 50% static compression on IGF-I transport was determined by measuring the uptake of (125)I-labeled IGF-I into cartilage explants. Static compression decreased both [(3)H]proline and [(35)S]sulfate incorporation in a dose-dependent manner in the presence or absence of IGF-I. IGF-I increased [(3)H]proline and [(35)S]sulfate incorporation in a dose-dependent manner in the presence or absence of compression, but the anabolic effect of the growth factor was lessened when the tissue was compressed by 50%. The response of cartilage explants to IGF-I was similarly lessened in unstrained tissue cultured in media at pH 6.4, a condition which results in a similar intratissue pH to that when cartilage is compressed by 50%. The characteristic time constant (tau) for IGF-I stimulation of cartilage explants was approximately 24 h, while tau for inhibition of biosynthesis by static compression was approximately 2 h. Samples which were both compressed and treated with IGF-I demonstrated an initial decrease in biosynthetic activity at 2 h, followed by an increase at 24 h. Static compression did not alter tau for (125)I-labeled IGF-I transport into cartilage but decreased the concentration of (125)I-labeled IGF-I in the tissue at equilibrium.     

High-resolution measurements of the multilayer ultra-structure of articular cartilage and their translational potential

Current musculoskeletal imaging techniques usually target the macro-morphology of articular cartilage or use histological analysis. These techniques are able to reveal advanced osteoarthritic changes in articular cartilage but fail to give detailed information to distinguish early osteoarthritis from healthy cartilage, and this necessitates high-resolution imaging techniques measuring cells and the extracellular matrix within the multilayer structure of articular cartilage. This review provides a comprehensive exploration of the cellular components and extracellular matrix of articular cartilage as well as high-resolution imaging techniques, including magnetic resonance image, electron microscopy, confocal laser scanning microscopy, second harmonic generation microscopy, and laser scanning confocal arthroscopy, in the measurement of multilayer ultra-structures of articular cartilage. This review also provides an overview for micro-structural analysis of the main components of normal or osteoarthritic cartilage and discusses the potential and challenges associated with developing non-invasive high-resolution imaging techniques for both research and clinical diagnosis of early to late osteoarthritis.     

Effect of articular cartilage resection on the growth and formation of the femoral and acetabular epiphyses]

Different parts of the articular cartilage were resected in 46 rabbits at the age of 2.5 months. The resected narrow stripe of the articular cartilage completely restored by the 60--90th day and the growth of the condyles was not disturbed. Resection of considerable areas of the articular cartilage on the condyles and on the femoral head was accompanied by a certain disturbance of the osseous tissue growth in these areas with resulted impression of the condyles, deformation of the head and further formation of coxa vara. The removal of 1/3 of the articular cartilage of the cotyloid cavity resulted in a certain increase of its diamter, uneven development at the site of resection; the femoral head of this joint increased, its spherical shape was altered. The restored cartilage did not restore its original structure characteristic for a growing bone. The newly formed articular cartilage lost its ability to participate in endochondral bone formation during the growth of the animal.     

Opiate receptor mediated internalization of 125I-beta-endorphin in human polymorphonuclear leucocytes

The early events in the interaction of (125I)-Tyr27-beta-endorphin with human polymorphonuclear leucocytes were investigated. Using ultrastructural autoradiography we found that the labeled peptide specifically bound to the plasma membrane and was internalized within two minutes of incubation at 37 degrees C. Both processes could be inhibited by unlabeled beta-endorphin or by the opiate antagonist diprenorphine. This finding was confirmed by radioreceptorassays. With longer incubation times the specific association of the labeled beta-endorphin with the cells decreased. About 10% of the tracer was degraded within 10 min of incubation as shown by gel chromatography. The morphological changes induced by 125I-beta-endorphin in the granulocytes were investigated under the microscope. The labeled peptide had the same biological effect as unlabeled beta-endorphin.     

Inhibition of endogenous TGF-beta during experimental osteoarthritis prevents osteophyte formation and impairs cartilage repair

Osteoarthritis has as main characteristics the degradation of articular cartilage and the formation of new bone at the joint edges, so-called osteophytes. In this study enhanced expression of TGF-beta1 and -beta3 was detected in developing osteophytes and articular cartilage during murine experimental osteoarthritis. To determine the role of endogenous TGF-beta on osteophyte formation and articular cartilage, TGF-beta activity was blocked via a scavenging soluble TGF-beta-RII. Our results clearly show that inhibition of endogenous TGF-beta nearly completely prevented osteophyte formation. In contrast, treatment with recombinant soluble TGF-beta-RII markedly enhanced articular cartilage proteoglycan loss and reduced the thickness of articular cartilage. In conclusion, we show for the first time that endogenous TGF-beta is a crucial factor in the process of osteophyte formation and has an important function in protection against cartilage loss.     

Pathways of load-induced cartilage damage causing cartilage degeneration in the knee after meniscectomy

Results of both clinical and animal studies show that meniscectomy often leads to osteoarthritic degenerative changes in articular cartilage. It is generally assumed that this process of cartilage degeneration is due to changes in mechanical loading after meniscectomy. It is, however, not known why and where this cartilage degeneration starts. Load induced cartilage damage is characterized as either type (1)--damage without disruption of the underlying bone or calcified cartilage layer--or type (2), subchondral fracture with or without damage to the overlying cartilage. We asked the question whether cartilage degeneration after meniscectomy is likely to be initiated by type (1) and/or type (2) cartilage damage. To investigate that we applied an axisymmetric biphasic finite element analysis model of the knee joint. In this model the articular cartilage layers of the tibial and the femoral condyles, the meniscus and the bone underlying the articular cartilage of the tibia plateau were included. The model was validated with data from clinical studies, in which the effects of meniscectomy on contact areas and pressures were measured. It was found that both the maximal values and the distributions of the shear stress in the articular cartilage changed after meniscectomy, and that these changes could lead to both type (1) and type (2) cartilage damage. Hence it likely that the cartilage degeneration seen after meniscectomy is initiated by both type (1) and type (2) cartilage damage.