Quantitative Polarized Light Microscopy Research Articles

Incomplete restoration of immobilization induced softening of young beagle knee articular cartilage after 50-week remobilization.

The aim of this study was to characterize the biomechanical and structural changes in canine knee cartilage after an initial 11-week immobilization and subsequent remobilization period of 50 weeks. Cartilage from the immobilized and remobilized knee was compared with the tissue from age-matched control animals. Compressive stiffness, in the form of instant shear modulus (ISM) and equilibrium shear modulus (ESM) of articular cartilage, was investigated using an in situ indentation creep technique. The local variations in cartilage of glycosaminoglycan (GAG) concentration were measured with a microspectrophotometer after safranin O staining of histological sections. Using a computer-based quantitative polarized light microscopy method, collagen-related optical retardation, gamma, of cartilage zones were performed to investigate the collagen network of cartilage. Macroscopically, cartilage surfaces of the knee joint remained intact both after immobilization and remobilization periods. Immobilization caused significant softening of the lateral femoral and tibial cartilages, as expressed by ESM (up to 30%, p < 0.05). Remobilization restored the biomechanical properties of cartilage in the lateral condyle of tibia, but in the lateral condyle of femur ESM remained 15% below the control level (p = 0.05). The instant shear modulus was not changed either after immobilization or remobilization. The GAG content of the cartilage was slightly decreased after immobilization, especially in the superficial zone of cartilage, but the change was not statistically significant. After remobilization the intensity of safranin O content rose to control level. Neither immobilization nor remobilization had any effect on the gamma value of collagen fibril network either in the superficial or the deep zone at any of the test points. The changes of ESM were positively correlated with the alterations in GAG content of the superficial and deep zones after immobilization and remobilization. This confirms the key role of protoglycans in the regulation of the equilibrium stiffness of articular cartilage. As a conclusion, immobilization of the joint of a young individual may cause long-term, if not permanent, alterations of cartilage biomechanical properties. This may predispose joint to degenerative changes later in life.

Biomechanical and structural characteristics of canine femoral and tibial cartilage.

To analyze the interrelationships between the structure, composition, and mechanical properties of articular cartilage, canine knee (n = 10) femoral and tibial cartilages were used as experimental tissues. The biomechanical properties, instant shear modulus (IM), and equilibrium shear modulus (EM) of articular cartilage were investigated using an in situ indentation creep technique. The local variations in the concentration of glycosaminoglycans (GAGs) in the cartilage were measured with a microspectrophotometer after safranin-O staining of histological sections. Using a computer-based quantitative polarized light microscopy method, area-specific measurements of the optical path difference were performed to quantitate collagen-related optical retardation (gamma) of cartilage zones. The IM and EM were 131.3 and 51.2% higher (p < 0.001) in the femoral cartilage than in the tibial cartilage, respectively. The mean thickness of the superficial zone and the relative proportion of the superficial zone from the total uncalcified cartilage was 107.1 and 155.3% higher (p < 0.001) at the femoral test points than in the tibial ones, respectively. The mean thickness of the tibial uncalcified cartilage was 21.1% higher (p < 0.001) than the thickness of the femoral cartilage. The GAG concentration of the tibial cartilage was higher (14.8%, p < 0.001) than that of the femoral cartilage, especially in the superficial zone (50.0%, p < 0.05), whereas the gamma of the collagen network in the superficial zone of the femoral cartilage was 64.7% higher (p < 0.001) than in the tibial cartilage. The percent relative thickness and retardation gamma of the superficial zone correlated positively with the indentation stiffness of the canine knee articular cartilage. These observations indicate that cartilage is structurally inhomogenous and layered tissue and the local organization of collagen and GAG concentration of the articular cartilage regulate the biomechanical properties of the tissue. The structure and composition of the superficial articular cartilage significantly affects the indentation response of the canine knee articular cartilage.

Biomechanical and structural characteristics of canine femoral and tibial cartilage

To analyze the interrelationships between the structure, composition, and mechanical properties of articular cartilage, canine knee (n = 10) femoral and tibial cartilages were used as experimental tissues. The biomechanical properties, instant shear modulus (IM), and equilibrium shear modulus (EM) of articular cartilage were investigated using an in situ indentation creep technique. The local variations in the concentration of glycosaminoglycans (GAGs) in the cartilage were measured with a microspectrophotometer after safranin-O staining of histological sections. Using a computer-based quantitative polarized light microscopy method, area-specific measurements of the optical path difference were performed to quantitate collagen-related optical retardation (Γ) of cartilage zones. The IM and EM were 131.3 and 51.2% higher (p < 0.001) in the femoral cartilage than in the tibial cartilage, respectively. The mean thickness of the superficial zone and the relative proportion of the superficial zone from the total uncalcified cartilage was 107.1 and 155.3% higher (p < 0.001) at the femoral test points than in the tibial ones, respectively. The mean thickness of the tibial uncalcified cartilage was 21.1% higher (p < 0.001) than the thickness of the femoral cartilage. The GAG concentration of the tibial cartilage was higher (14.8%, p < 0.001) than that of the femoral cartilage, especially in the superficial zone (50.0%, p < 0.05), whereas the Γ of the collagen network in the superficial zone of the femoral cartilage was 64.7% higher (p < 0.001) than in the tibial cartilage. The percent relative thickness and retardation Γ of the superficial zone correlated positively with the indentation stiffness of the canine knee articular cartilage. These observations indicate that cartilage is structurally inhomogenous and layered tissue and the local organization of collagen and GAG concentration of the articular cartilage regulate the biomechanical properties of the tissue. The structure and composition of the superficial articular cartilage significantly affects the indentation response of the canine knee articular cartilage. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 48: 99–107, 1999

Decreased birefringence of the superficial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training, detected by quantitative polarised light microscopy.

To investigate the effects of a one year programme of running training (up to 40 km/day for 15 weeks) on the spatial orientation pattern of collagen and glycosaminoglycans in articular cartilage in different parts of the knee (stifle) and shoulder joints of young beagle dogs. Area specific measurements of the optical path difference (= retardation, gamma) and the cartilage zone thickness were performed using conventional procedures and a new computer based quantitative polarised light microscopy method. Transmission electron microscopy was used to determine the zonal volume density of collagen fibrils. The concentrations of collagen and hydroxypyridinium crosslinks were investigated biochemically. Running training decreased gamma by 24-34% (p < 0.05) in the superficial zone of the lateral femoral condyle articular cartilage and at the centre of the tibial condyles. Gamma of glycosaminoglycans decreased by 26% (p < 0.05) in the superficial zone of the lateral condyle of the femur, but at the same site the volume density of collagen fibrils was unchanged. Neither the collagen concentration nor the concentration of hydroxypyridinium crosslinks was altered as a result of running. In both control and runner dogs, the thickness and gamma values of the superficial zone were greater in the humerus and the femur than in the tibia. Endurance type running exercise in beagles caused a reduction in the superficial zone birefringence of the articular cartilage, which indicates either a disorganisation or a reorientation of the superficial zone collagen network. Articular cartilage showed marked variability of collagen network organisation over the different knee (stifle) joint articular surfaces.

Major components of bone in subcapital and trochanteric fractures. A comparative study

Quantitative polarised light microscopy was applied to sections of unfixed, undecalcified bone taken at operation from patients with two types of proximal femoral fracture, subcapital and trochanteric. Specimens were also taken from the equivalent sites in otherwise normal subjects at autopsy, and from various other sites of traumatic fractures; these two latter groups acted as controls. Analysis of the 57 specimens disclosed changes in the nature of the bone at the site of subcapital fractures, namely the presence of relatively large crystals of hydroxyapatite and a change in the molecular orientation, but not total content, of the acidic proteoglycans of the bone matrix. Our results have confirmed and extended the findings of others on subcapital fractures, and have also shown very similar changes in the trochanteric fractures. It thus appears that the bony changes in the two types of proximal femoral fracture are not as different as has been suggested.

Altered orientation of glycosaminoglycans and cellular changes in the tibial cartilage in the first two weeks of experimental canine osteoarthritis

Changes in the cellularity and in the nature of the matrix were studied in the cartilages of the tibial plateau in experimentally induced arthritis in the dog, 7 and 14 days after section of the anterior cruciate ligament. Samples from the different regions of these cartilages were chilled and sectioned in a cryostat, with a variable microtome chuck to allow precise orientation of the specimens. The samples were examined by normal light microscopy, by microscopic interferometry, and by quantitative polarized light microscopy. The orientation of the glycosaminoglycans was assessed by the new "induced birefringence" method. The results indicated that only the region of the medial tibial cartilage that was unprotected by the meniscus was affected, showing increased water content, loss of superficial cells, and a decrease in orientation of the glycosaminoglycans. Whereas the birefringence of the collagen was unaffected, the superficial area that lacked oriented glycosaminoglycans was markedly increased; this may be a useful indicator of early osteoarthritic changes.

Changes in crystal size and orientation of acidic glycosaminoglycans at the fracture site in fractured necks of femur.

The aim of this study was to try to elucidate the increased susceptibility of the neck of femur to fracture. Quantitative polarised light microscopy has been applied to fresh, undecalcified sections of samples of bone taken from the site of fracture, in specimens taken at operation from patients with fractures of the femoral neck or osteoarthritic femoral heads or from the equivalent site from otherwise normal subjects at necropsy. In all 21 specimens of fractured necks of femur, but in none of the other specimens, relatively large crystals (up to 2.5 X 0.5 micrometres) were found close to the site of fracture; the properties of these crystals were compatible with their being apatite. Measurement of the natural birefringence of the collagen showed no difference in the orientation of the collagen in all three types of specimen. However, the orientation of acidic glycosaminoglycans, measured by the birefringence of alcian blue bound to these moieties, was 45 per cent lower in the specimens from fractured necks of femur than in the other specimens, even though the total content of acidic glycosaminoglycans was unchanged. Although the decreased orientation was most marked close to the site of fracture, it was still apparent 15 millimetres from that site. These changes were unlikely to be simply the sequelae of fracture since they were not found in traumatic fractures of other bones. Thus it is conceivable that changes in the orientation of the ground substance allow formation of relatively large crystals of apatite and that such crystals, in the microcrystalline mass of apatite, are the cause of the increased fragility of such bones.

Quantitative polarized-light microscopy of sound surface enamel.

Quantitative polarized-light microscopy of sound surface enamel.

Birefringence of Protein Solutions and Biological Systems: II. Studies on TMV, Tropocollagen, and Paramyosin

The intrinsic birefringences of TMV, tropocollagen, and paramyosin were calculated from flow birefringence measurements using the theory of Peterlin and Stuart. The values are -0.029, -0.029, and -0.030, respectively. The intrinsic birefringences of TMV and tropocollagen were measured as a function of the refractive index of the solvent in glycerol-water mixtures. In both cases the values were not constant and became less negative as the refractive index increased. Theoretical calculations showed that the large solvent effect could not be caused by a hydration shell of index different from that of the bulk solvent. It is concluded that either (a) the intrinsic birefringence calculated from the Peterlin-Stuart theory is incorrect or (b) the intrinsic birefringence depends markedly on the solvent. These results are of importance to the problem of quantitative polarized light microscopy since the separation of form and intrinsic birefringence contributions is based on the assumption that intrinsic birefringence is independent of solvent.

Circular structures in the Arctic Islands

OBJECTIVE: To investigate the effects of a one year programme of running training (up to 40 km/day for 15 weeks) on the spatial orientation pattern of collagen and glycosaminoglycans in articular cartilage in different parts of the knee (stifle) and shoulder joints of young beagle dogs. METHODS: Area specific measurements of the optical path difference (= retardation, gamma) and the cartilage zone thickness were performed using conventional procedures and a new computer based quantitative polarised light microscopy method. Transmission electron microscopy was used to determine the zonal volume density of collagen fibrils. The concentrations of collagen and hydroxypyridinium crosslinks were investigated biochemically. RESULTS: Running training decreased gamma by 24-34% (p &lt; 0.05) in the superficial zone of the lateral femoral condyle articular cartilage and at the centre of the tibial condyles. Gamma of glycosaminoglycans decreased by 26% (p &lt; 0.05) in the superficial zone of the lateral condyle of the femur, but at the same site the volume density of collagen fibrils was unchanged. Neither the collagen concentration nor the concentration of hydroxypyridinium crosslinks was altered as a result of running. In both control and runner dogs, the thickness and gamma values of the superficial zone were greater in the humerus and the femur than in the tibia. CONCLUSION: Endurance type running exercise in beagles caused a reduction in the superficial zone birefringence of the articular cartilage, which indicates either a disorganisation or a reorientation of the superficial zone collagen network. Articular cartilage showed marked variability of collagen network organisation over the different knee (stifle) joint articular surfaces.