A new microspectrophotometric method was developed for quantitation of glycosaminoglycans with Safranin O dye in articular cartilage matrix. From histological sections molar extinction coefficient of Safranin O was determined and used to measure the dye content of the sections. The amount of glycosaminoglycans was determined with depth of bovine articular cartilage by both gas chromatography and thin layer chromatography to calculate the fixed negative charge content. Comparison between the results revealed that binding of Safranin O to glycosaminoglycan polyanions was stoichiometric and showed minimal nonspecific staining. The method provides an accurate technique for quantitation and localization of fixed negative charge content of glycosaminoglycans in the articular cartilage matrix. Specific enzyme digestions enable detection of separate glycosaminoglycans.
The effects of long distance running training (up to 40km/day) on the biomechanical properties of young canine articular cartilage were investigated using in situ indentation creep technique. The stiffness of articular cartilage was determined in the form of instantaneous and equilibrium shear moduli. The rate of cartilage deformation was also computed. Microspectrophotometric and polarized light microscopic analyses were made for determination of local glycosaminoglycan content and collagen organization, respectively. During a period of one year, the runner dogs (n = 10) underwent a gradually increased treadmill exercise up to 40 km/day, while the littermate control dogs (n = 10) lived normally in their cages. The equilibrium shear modulus of articular cartilage decreased significantly by 12 to 14% (p < 0.05) in the lateral, but not in the medial, condyles of femur and tibia. In the lateral condyles of the runner dogs, the deformation rate of cartilage increased by 16% (p < 0.05). Consequently, the articular cartilage response to running training was site-dependent. The simultaneous changes of the equilibrium shear modulus or the retardation time spectrum and the glycosaminoglycan content confirm the key role of proteoglycans in modulating the cartilage equilibrium stiffness and creep rate. The changes in the instant shear modulus appeared to be predominantly related to the alterations in the organization of the superficial collagen network. Although the running training did not create cartilage damage, we assume that the softening of the cartilage may with time jeopardize the ability of articular cartilage to maintain its normal structural and functional properties.
The right knees of 4-month-old NZW rabbits were splinted in extension for 1 to 8 weeks. Biochemical changes of the knee articular cartilage were noted after decreased (splinted leg) and increased loading (created by the shift of body weight onto the left, contralateral limb). Increased loading accelerated changes associated with maturation of articular cartilage, which include accumulation of hyaluronic acid (HA) and keratan sulfate-rich proteoglycans (KS, PG) that are tightly bound to the tissue. After 8-weeks of splinting the content of extractable PGs in the tibial medial condyle decreased. The lost material was apparently replaced by PGs with a higher degree of sulfation of the chondroitin sulfate (Ch-S) chains. Reduced loading disturbed normal maturation as evidenced by inhibition of the accumulation of KS-rich, non-extractable PGs. Collagen content increased in all samples of different joint sites and groups during the 8-week experiment. The content of extractable PGs decreased slightly, while the content of non-extractable, especially KS-rich PGs increased. The greatest changes occurred in the tibial medial condyle, where the KS content was highest.
Casting of the right knee (stifle) joints of young beagle dogs for 11 weeks caused up to 48% reduction in the glycosaminoglycan (GAG) concentration of the uncalcified articular cartilage, as assessed by a new microspectrophotometric method. The GAGS were depleted mainly in the superficial zone of the cartilage. Although the thickness of the uncalcified cartilage was not decreased, the calcified cartilage under the tidemark was thinned by 6 2 5 % at the femoral condyles. The increased weight-bearing in the limb opposite the one in the splint caused uncalcified cartilage thickness to be augmented by 19% and GAG concentration by 25-35 % in the intermediate, deep, and calcified zones of the summits of the femoral condyles; the changes were smaller in other, less loaded parts of the joint. It is concluded that in young dogs, increased weight-bearing augments local proteoglycan content of the articular cartilage matrix, while unloading reduces it.
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