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Type III collagen in normal human articular cartilage has been detected biochemically and its location in a diffuse area around the chondrocytes demonstrated by immunofluorescence. It can be found pericellularly throughout the depth of the cartilage and is evident in specimens ranging in age from 17 to 81 years.
IL1-stimulated pig articular cartilage fragments were cultured in the and absence of various metalloproteinase inhibitors. Tissue inhibitor of metalloproteinases (TIMP) was unable to stop the release of proteoglycan from the cartilage. Incubation of cartilage with a potent synthetic metalloproteinase inhibitor inhibited the release of proteoglycan in a dose-dependent fashion. The results suggest that low-M(r) metalloproteinase inhibitors may have therapeutic potential in limiting connective tissue breakdown in conditions such as rheumatoid arthritis.
The role of the fluid within articular cartilage as affected by the load-sharing mechanism and its potential, beneficial effects were assessed with the u-p finite element method. The mechanical behavior of cartilage as it covers the surface of a diarthrodial joint was evaluated when the partitioning of an applied stress to the solid and fluid phases of the tissue was varied. Comparisons were made in the response of the cartilage when 0%, 25%, 50%, or 75% of the applied stress was supported by the fluid at the surface. Substantial changes in the behavior of the tissue were observed for each load case. As the fluid sustained a larger portion of the applied stress, several parameters were affected; the fluid pressure within the cartilage layer remained at a higher value, the stress and strain generated in the solid matrix decreased while the compression of the cartilage layer decreased. These findings indicate that an increased load-partitioning to the fluid phase in cartilage may perform the function of shielding the solid matrix from excessive stresses. This could also potentially alter the mechanical environment around the chondrocytes, influencing metabolic activity and homeostasis.
Normal male articular cartilage (34 specimens, age range 1--30 years) has been examined in vitro for response to somatomedin (SM) activity. Basal 3H-thymidine and 35S-sulfate incorporation both decreased with increasing age of the cartilage donor. However, enhancement of isotope incorporation which was attained on addition of 10% normal plasma (containing IU SM/ml) was greatest in cartilage from adolescents in the age range 12--17 years. The mean enhancement of 3H-thymidine incorporation (expressed as % basal) was as follows: age 1--10 years = 184 +/- 28 (SE), N = 9; 12--17 years = 436 +/- 101 (11); 18--30 years = 231 +/- 49 (8); and for 35S-sulfate incorporation was 1--10 years = 389 +/- 100 (8); 12--17 years = 824 +/- 273 (11); and 18--30 years = 572 +/- 56 (8). The increased response of cartilage in the 12--17 year group suggests that a greater sensitivity to the somatomedins may contribute to the increased skeletal growth during adolesence.
Proteoglycans in cultures of adult bovine articular cartilage labeled with [35S]sulfate after 5 days in culture and maintained in medium containing 20% fetal calf X serum had longer half-lives (average 11 days) compared with those of the same tissue maintained in medium alone (average 6 days). The half-lives of proteoglycans in cultures of calf cartilage labeled after 5 days in culture and maintained in medium with serum were considerably longer (average 21 days) compared to adult cartilage. If 0.5 mM cycloheximide was added to the medium of cultures of adult cartilage, or the tissue was maintained at 4 degrees C after labeling, the half-lives of the proteoglycans were greater, 24 and greater than 300 days, respectively. Analyses of the radiolabeled proteoglycans remaining in the matrix of the tissue immediately after labeling the tissue and at various times in culture revealed two main populations of proteoglycans; a large species eluting with Kav of 0.21-0.24 on Sepharose CL-2B, of high bouyant density and able to form aggregates with hyaluronate, and a small species eluting with a Kav of 0.63-0.70 on Sepharose CL-2B, of low buoyant density, containing only chondroitin sulfate chains, and unable to form aggregates with hyaluronate. The larger proteoglycan had shorter half-lives than the smaller proteoglycan; in cartilage maintained with serum, the half-lives were 9.8 and 14.5 days, respectively. Labeling cartilage with both [3H]leucine and [35S]sulfate showed the small proteoglycan to be a separate synthetic product. The size distribution of 35S-labeled proteoglycans lost into the medium was shown to be polydisperse on Sepharose CL-2B, the majority eluting with a Kav of 0.27 to 0.35, of high buoyant density, and unable to aggregate with hyaluronate. The size distribution of glycosaminoglycans from 35S-labeled proteoglycans appearing in the medium did not differ from that associated with labeled proteoglycans remaining in the matrix.
A polypeptide proteinase inhibitor from human articular cartilage has been purified to homogeneity by stepwise Sephadex G-75, heparin-Sepharose and octyl-Sepharose affinity chromatography. The inhibitor is strongly cationic (pI greater than or equal to 10.5) and consists of two non-identical polypeptides associated by means of electrostatic and/or hydrophobic interactions. Amino acid analysis of the aggregate confirmed that the polypeptide was rich in basic, and hydrophobic amino acids and contained only one disulphide bridge. Sedimentation equilibrium studies showed that the aggregate had MW congruent to 7000 which could be dissociated into two polypeptides each of MW congruent to 3500. While the subunits were primarily serine proteinase inhibitors the aggregate form could also inhibit bacterial collagenase and pepsin but not thermolysin nor the cysteine proteinases, ficin or bromelain. Binding of 125I-labelled human cartilage inhibitor to heparin, keratan sulphate and proteoglycan subunit was demonstrated using gel exclusion chromatography but no interaction was detected with chondroitin 6-sulphate or hyaluronic acid. Binding of cartilage inhibitor subunits to link proteins was also shown by polyacrylamide electrophoresis. These data suggest that the human cartilage inhibitor may be localised at specific sites on the proteoglycan complex where it would be ideally placed to attenuate degradation by matrix proteinases or constitute part of an enzyme-inhibitor complex.
The proteoglycan subunits isolated from human articular cartilage of various ages (ranging from the newborn to the mature adult) were analyzed for their content of histidinoalanine. It was noted that while the young juveniles and adolescents contained about 1 nmole of histidinoalanine per mg protein, the skeletally mature adults contained about twice this amount. Unlike many age-related changes in proteoglycan structure that occur prior to the end of growth, this variation would appear to be more related to maturation of the adult.
In explant cultures of articular cartilage from cattle of different ages radiolabeled leucine was shown to be incorporated into link proteins 1, 2 and 3. The newly synthesized link proteins were incorporated into and lost from the cartilage extracellular matrix with time. The levels of radiolabeled link proteins 1 and 2 remaining in the matrix declined over the culture period, but there was an initial increase in the amount of radiolabeled link protein 3, before its level declined. The turnover time of the radiolabeled link proteins 1 and 2 were similar, indicating that neither link protein was preferentially processed to generate link protein 3, nor lost from the extracellular matrix. The majority of the radiolabeled link protein lost from the cartilage matrix could not be recovered from the culture medium, suggesting that turnover of the radiolabeled aggrecan complexes involves the newly synthesized link protein being internalized by the chondrocytes. Inclusion of cytotoxic proteinase inhibitors to the culture medium resulted in a marked decrease in the rate of loss of link protein from the cartilage, suggesting that the catabolism of link protein is cell-mediated and dependent on metabolically active cells.
Proteoglycans were extracted from normal human articular cartilage of various ages with 4M-guanidinium chloride and were purified and characterized by using preformed linear CsCl density gradients. With advancing age, there was a decrease in high-density proteoglycans of low protein/uronic acid weight ratio and an increase in the proportion of lower-density proteoglycans, richer in keratan sulphate and protein. Proteoglycans of each age were also shown to disaggregate in 4M-guanidinium chloride and at low pH and to reaggregate in the presence of hyaluronic acid and/or low-density fractions. Osteoarthrotic-cartilage extracts had an increased content of higher-density proteoglycans compared with normal cartilage of the same age, and results also suggested that these were not mechanical or enzymic degradation products, but were possibly proteoglycans of an immature nature.
Adult human articular cartilage has been maintained in organ culture for three days. The uptake of 35SO4 has been studied over a 48-hour period. The incorporation of 35SO4 was significantly increased in osteoarthrotic cartilage compared to normal cartilage from the same joints. The uronic acid content of the osteoarthrotic cartilage was significantly reduced which reflects the proteoglycan loss in the diseased tissue. The DNA content, which is an indirect measurement of the cell population, was not significantly different in the diseased and normal cartilage.
In both young and old human articular cartilage explants, TNF alpha induced a concentration-dependent, reversible suppression of the proteoglycan (PG) synthesis. Young cartilage was more sensitive to TNF alpha than old cartilage: 50% suppression of PG synthesis was reached at a TNF alpha concentration of 5 U/ml for young and 30 U/ml for old cartilage, whereas at 10(3) U/ml the PG synthesis of young cartilage was blocked and that of old cartilage suppressed by 80%. These inhibition levels of PG synthesis resulted in 25% PG depletion of the explants after 8 days of culture. The release of cartilage PG was not enhanced. TNF alpha induced no detectable amounts of IL-1 (less than 0.01 U) in young or old cartilage but did induce IL-6 production. The induced amounts of IL-6 were higher in young than in old cartilage but no dose-dependency was evident. Antibodies to neither IL-1 nor IL-6 had any influence on the TNF alpha-induced suppression of PG synthesis. The combination of TNF alpha and IL-1 led to an additive inhibition of PG synthesis which had no relationship to induced IL-6. TNF alpha was about 100-fold less active than IL-1.
The role of aluminum accumulation in articular tissues of patients affected by dialysis-associated arthropathy (DAA) is questioned. The aim of this work is to identify the nature of these aluminum accumulations by the use of secondary ion mass spectrometry (SIMS). Al/Si ratios of about 1, measured by SIMS, strongly suggest for the first time the presence of aluminum silicates and possibly aluminum hydroxides in amyloid synovial tissue and articular cartilage of 1 patient with DAA and aluminum intoxication. This is thermodynamically consistent with the total dissolved Al and Si contents and pH measured in the synovial fluids. These results are similar to the abnormal Al distribution recently found by SIMS in the forebrain of chronic renal dialysis patients and to the amorphous aluminum silicates identified in the core of senile plaques in Alzheimer's disease.
The physical and morphological properties of articular cartilage have been used as a model for the preparation of hydrogel based synthetic analogues of this complex high water content natural hydrogel. The relatively poor strength and stiffness of simple homogeneous hydrogels have been enhanced by semi-interpenetrating polymer network (semi-IPN) technology to a level which enables the mechanical properties of natural cartilage to be approached. Maintenance of chondrocytic phenotypes at the implant interface in vitro has been found to require careful control of pore size and distribution in the hydrogel matrix. The study of synthetic techniques for the fabrication of macroporous semi-IPNs has enabled hydrogel semi-IPNs with appropriate pore sizes and mechanical properties to be produced. A range of in vitro testing techniques have been developed to enable the physico-chemical properties of these materials to be optimised prior to animal studies.
Proteoglycans were extracted from bovine articular cartilage with guanidine-HCl and fractionated in cesium chloride density gradients by equilibrium ultracentrifugation. The acidic glycosaminoglycan (AGAG) components were then determined enzymatically with chondroitinase-ABC and streptomyces hyaluronidase. Under associative and dissociative conditions, the distribution of the AGAG components was as follows: the ratio of 4-sulfated disaccharide units to total AGAG increased with decreasing density gradients whereas that of 6-sulfated disaccharide units to total AGAG increased with increasing density gradients. The ratio of disulfated disaccharide units to total AGAG increased somewhat with decreasing density gradients whereas that of non-sulfated disaccharide units tended to decrease. Although the cartilage proteoglycan macromolecules were heterogeneous, a certain regularity was observed with respect to the distribution of sulfate and the degree of sulfation in the chondroitin sulfate chains of the proteoglycans.
The turnover of proteoglycans in bovine articular cartilage was determined in explant cultures, maintained at 32 degrees C or 37 degrees C. Both the rate of proteoglycan synthesis and the release of newly synthesized proteoglycans were decreased in cultures incubated at 32 degrees C compared to 37 degrees C. At both temperatures the newly synthesized proteoglycans were similar in hydrodynamic size and chain length of the glycosaminoglycans. However, the ratio of 6-sulfated disaccharides over 4-sulfated disaccharides of the newly synthesized glycosaminoglycans, differed less from the endogenous ratio at 32 degrees C than at 37 degrees C. At both temperatures, the incorporated 35S-sulfate is released from explants in two pools. Twenty-three percent of the 35S-radiolabel was released into the culture medium during an initial short phase (t1/2 = 1.1 day at 32 degrees C, 1.3 day at 37 degrees C), 77% had a much longer half-life. The lowered temperature markedly decreased the release of 35S-sulfate with a slow turnover (t1/2 = 60 days at 32 degrees C, 38 days at 37 degrees C).
Scanning and transmission microscopy of the articular cartilage was performed in femoral condyles of persons at the age of 30-50 years. It was demonstrated that hyaline cartilage is covered with a protective fibrillar layer consisting of tightly pressed collagenous fibrillae with an underlying layer of fibroblastic cells. In the intracellular substance of the hyaline cartilage fibrillar structures form a complex reticular web with vertical arrangement of the main collagenous fasiculi. In the superficial layer of the hyaline cartilage the collagenous fibrillae and their fasciculi form arcade-like structures. Lacunar chondrocytes have a rough villose surface, cellular secrete is discharged as round granules through cytoplasmic membrane. Ultrastructural changes in chondrocytes are observed simultaneously with their degenerative-dystrophic changes.
PURPOSE: To evaluate T1-weighted magnetic resonance (MR) imaging after diffusion of gadopentetate dimeglumine for visualization of articular cartilage lesions. MATERIALS AND METHODS: MR imaging was performed in eight human cadaveric patella specimens immediately and 4 hours after placement into a vessel filled with gadopentetate dimeglumine solution (2.5 mmol/L). T1-weighted spin-echo and inversion-recovery turbo spin-echo MR sequences with nulled cartilage signal (inversion time of 300 msec) were used. In a total of 128 articular cartilage areas, MR imaging findings were compared with macroscopic and histopathologic findings. Pathologic evaluation was performed by one musculoskeletal pathologist. With knowledge of pathologic observations, MR images were analyzed by one musculoskeletal radiologist with regard to intrinsic signal intensity characteristics and surface abnormalities of articular cartilage. RESULTS: Histopathologic findings demonstrated 67 areas of normal articular cartilage and 66 cartilage lesions (grade 1, n = 19; grade 2, n = 15; grade 3, n = 26; grade 4, n = 6). All grade 3 and 4 lesions could be identified on MR images obtained immediately after submersion and after 4 hours. Ninety-four percent of grade 1 and 2 lesions were identified as areas of predominantly decreased contrast enhancement on delayed MR images obtained with both sequences. MR images obtained immediately after submersion demonstrated abnormal signal intensity in only 9% and 12% of grade 1 and 2 lesions, respectively. CONCLUSION: T1-weighted MR images obtained in vitro after gadopentetate dimeglumine diffusion allow demonstration of articular cartilage surface lesions and early stages of cartilage degradation.
Normal and stored articular cartilage from the medial tibial plateaus of mature canine knee joints were evaluated histologically and biomechanically. The medial plateaus from the right knee (control) were assessed fresh, while the left (stored) were preserved in culture media at 4 degrees C for 3, 7, 14, or 28 days and then evaluated. Biomechanically, confined compression tests were performed on all specimens to determine the aggregate modulus and apparent permeability of the articular cartilage. Histologically, Safranin O- and hematoxylin and eosin (H&E)-stained sections were evaluated. All stored cartilage specimens had an aggregate modulus on average lower than normal, but the differences were not significant (p greater than 0.10). The apparent permeability was on average higher than but also not significantly different from normal (p greater than 0.10). Time in storage (up to 28 days) did not have a significant effect on the biomechanical properties of stored cartilage normalized by control values (p greater than 0.50). Safranin O and H&E histological evaluation also showed no overall changes in cell appearance or staining of the stored cartilage when compared with control for the time periods studied.