Degradation of hyaluronic acid by photosensitized riboflavin in vitro. Modulation of the effect by transition metals, radical quenchers, and metal chelators.

The effect of photoexcited riboflavin (RF) on the viscosity of hyaluronic acid (HA) solutions has been investigated. UV irradiation of RF causes under aerobic conditions fragmentation of HA and a decrease in the viscosity of its solutions. A decrease of HA viscosity occurs in PO(4)-buffered solutions and is accelerated by high pH, Fe2+ (but much less so by Fe3+), certain metal chelators, and horseradish peroxidase (HRP); it is partially inhibited by catalase and less so by superoxide dismutase (SOD). The reactivity of the system was completely blocked by Tris, ethanol, aspirin, d-manitol, dimethylthiourea (DMTU), dimethylsulfoxide (DMSO), and sodium azide. These results indicate that the most likely chemical species involved in the reaction is the hydroxyl radical. Singlet oxygen ((1)O(2)) generation is suggested by the ability of NaN3 and DMSO to completely inhibit the reactivity of the system. These two agents, however, may also interact with OH. radical, as well and suppress the reactivity of the system. H(2)O(2) and O(2).- seem also to be produced in significant amounts, because catalase and SOD partially block the reactivity of the system. The effect of HRP may be due to hydrogen subtraction from HA and H(2)O(2) reduction to water. Photoexcitation of RF may potentially occur in vitro and in vivo in the organs and tissues that are permeable to light, such as the eye or skin, and damage HA and other cell-matrix components causing inflammation and accelerating aging.

Metabolic and biochemical abnormalities of articular cartilage induced by implantation of a sterile sheet of polyethylene in the rabbit patellofemoral joint.

A polyethylene sheet was implanted in the patellofemoral joint of the right knee of the rabbit and the biochemical and metabolic changes in the articular cartilage from femoral trochleas (in contact with the implant) and femoral condyles (free of direct contact) were compared with those in their sham-operated counterparts 7, 15, and 30 days after joint implantation. The results showed that there was an increase in the water content; the extraction yields of uronic acid--, 35SO4-, and [3H]glycine-containing compounds; and the incorporation of [3H]thymidine, [3H]glycine, and 35SO4. Concomitantly, the contents of uronic acid--, hexosamine-, neutral sugars-, and hydroxyproline-containing substances decreased in the femoral trochlear cartilage and, to a much lesser extent, in the femoral condylar cartilage from implanted joints. The increased capacity of viable chondrocytes to incorporate metabolic radiolabeled precursors into newly synthesized macromolecules may represent a reparative cell response to the tissue injury induced by the implant. This is therefore a useful model for studying the response of chondrocytes to mechanical injury and tissue tolerance to intraarticularly implanted prosthetic materials.

Tophus-derived monosodium urate monohydrate crystals are biologically much more active than synthetic counterpart.

Monosodium urate monohydrate (MSUM) crystals derived from a tophus surgically removed from patients suffering from gout and MSUM prepared from a supersaturated solution of sodium urate were studied and compared with respect to their ability to: (1) stimulate chemiluminescence (CL) production by human polymorphonuclear (PMN) cells, (2) induce hemolysis of the human red blood cells and (3) induce inflammation when injected in the rat paw and knee joint. Human MSUM crystals were considerably more active in stimulating CL production by PMN cells and in inducing synovial inflammation. Both serum and papain pretreatment of human MSUM crystals caused inhibition of their enhancing effect on CL production by PMN cells. Papain pretreatment only reduced their phlogogenic activity. Uncoated and, to a much lesser extent, serum-coated human MSUM crystals induced secretion by mononuclear cells (MNC) of the factor(s) that considerably enhanced CL production by PMN cells. Both tophus-derived and synthetic crystals appeared to be weak hemolytic agents. Serum pretreatment of synthetic MSUM crystals reduced their hemolytic activity. These results suggest that surface coating, destroyed by papain treatment, was probably responsible for cell activation induced by human MSUM crystals.

Effect of cytochrome c concentration on the ultrastructural appearance of bovine nasal cartilage proteoglycans.

Bovine nasal cartilage proteoglycan monomers were studied by Kleinschmidt and Zahn's molecular spreading technique as modified by Rosenberg et al. By decreasing the cytochrome c concentration in the epiphase to 2 micrograms per 100 microliters we were able on nitrocellulose-coated grids routinely to obtain highly contrasted and well spread proteoglycan monomers with a characteristic brush-like appearance and, sometimes, a clearly distinguishable hyaluronic acid binding region. Previously, a hyaluronic acid binding region has only been observed routinely in spread proteoglycan aggregates, and a brush-like structure of proteoglycan monomers on carbon-coated grids, but with considerably less precision due to the poor contrast of the molecules. Molecular spreading was further improved by decreasing the cytochrome c concentration in the epiphase to less than 2 micrograms per 100 microliters, but contrast was reduced making visualization of molecular details difficult.

Histochemical and cellular changes induced in the rabbit knee joint by an intraarticular implantation of a sheet of polyethylene.

Implantation of a sterile rigid sheet of polyethylene into the right patellofemoral rabbit joint caused changes that resemble those found in human osteoarthritis. Initial synovial proliferation resulted in the isolation of the implant. Marginal osteophytes developed from soft cellular tissue which later underwent chondrification and finally became ossified. Articular cartilage under the area in contact with the implant showed early cell necrosis and diffuse loss of metachromasia. Focal superficial fibrillation followed and later developed into deep erosions and bone exposure. The first clefts appeared tangentially to the surface within an acellular electron-dense material. In spite of numerous mitotic divisions of viable chondrocytes, mainly in clusters or clones, cell density progressively decreased in every layer of articular cartilage beneath the implant. The present study suggests that necrosis of chondrocytes, and the later failure of viable cells to respond adequately to the altered environment, can lead to cartilage fibrillation and then to more severe lesions.

Anti-inflammatory drugs, prostanoid and proteoglycan production by cultured bovine articular chondrocytes.

The effect of various anti-inflammatory drugs on the production of prostaglandins E2 and F2 alpha, 6 keto PGF1 alpha and thromboxane B2 by bovine articular chondrocytes was measured by radioimmunoassay. While indomethacin and meclofenamic acid caused a dose-dependent inhibition of all prostanoids measured, the effects of hydrocortisone and colchicine varied with respect to different prostanoids. Hydrocortisone (10(-7)M - 10(-13)M) both in the presence and absence of added arachidonic acid, resulted in an inhibition of prostaglandins E2 and F2 alpha, and to a lesser extent, 6 keto PGF 1 alpha, but TxB2 production was only slightly inhibited by the drug in the absence of arachidonic acid and markedly increased in its presence. Colchicine (10(-7)M-10(-3)M) had the opposite effect, causing an inhibition of TxB2 and stimulating PGE2 and 6 keto PGF1 alpha production. These findings suggest that certain anti-inflammatory drugs may, in addition to their action on phospholipase A2 and cyclo-oxygenases, exert potent effects at the level of the different synthetases. In order to see whether these alterations in relative prostanoid levels affected proteoglycan metabolism, the effect of anti-inflammatory drugs on proteoglycan synthesis by cultured chondrocytes was tested using 35SO4 labeling methodology. The results showed that at the concentrations tested (10(-5)M to 10(-7)M), indomethacin, dexamethasone, hydrocortisone and colchicine inhibited 35SO4 incorporation into newly synthesized proteoglycan molecules both in the presence (10(-6)M) and absence of exogenous arachidonic acid. In the same concentration range chloroquine had no effect. These results do not support the hypothesis of direct prostanoid involvement in the modulation of proteoglycan synthesis in articular cartilage.

Age-related changes in the synthesis of matrix macromolecules by bovine articular cartilage.

Calf and mature cow articular cartilage was labeled in vitro with [35S]SO4 and [3H]glycine and kinetics of incorporation of both isotopes by cartilage fragments was determined by scintillation spectroscopy. The cartilage fragments were then extracted in sequence with 4M GuHCl (Guanidium chloride) and pepsin. The pepsin digest was adjusted to 1.3 M NaCl and pepsin-solubilized collagen salted out. The 4M GuHCl extract, collagen and pepsin-resistent residue were then freeze-dried. The 4M GuHCl extract was further fractionated by DEAE (Diethylaminoethyl) 52 ion exchange chromatography to obtain protein and PG (Proteoglycan) fractions. The protein fraction was also characterised by SDS-PAGE and PG fraction by Sepharose C1-2B chromatography under associative conditions in the presence and absence of an exogenous HA (Hyaluronic acid). The GAG (Glycosaminoglycan) side chains of the PG samples were analysed by Sephadex G-200 column chromatography and their composition determined by paper chromatography after chondroitinase ABC digestion. Linear incorporation of both isotopes was observed from 1 to 18 hours of incubation and roughly equal amounts of [35S]SO4 counts were found on per cell bases in both cartilages although less [3H]glycine was incorporated by cow chondrocytes. It was also found that calf chondrocytes synthesize much greater proportion of the collagen whereas the cow cells synthesize PGs of smaller hydrodynamic sizes, bearing shorter GAG side chains that are enriched in KS (Keratan sulfate) and Ch-6S (Chondroitin-6 sulfate isomer). A failure of cow 35S-PGs monomers to interact with an exogenous HA in the presence of other extracted components was also demonstrated. The relevance of these findings for the mechanism of cartilage damage in aging and osteoarthritis is discussed.