Dry Cartilage Research Articles

Decreased Sulfate Content and Zeta Potential Distinguish Glycosaminoglycans of the Extracellular Matrix of Osteoarthritis Cartilage.

We aimed to determine the characteristics that distinguish glycosaminoglycans (GAGs) from osteoarthritis (OA) and normal cartilage and from men and women. Cartilage samples from 30 patients subjected to total joint arthroplasty secondary to OA or fracture (control) were evaluated, and the GAG content (μg/mg dry cartilage) after proteolysis was determined by densitometry, using agarose-gel electrophoresis. Relative percentages of carbon (C), nitrogen (N), and sulfur (S) in GAGs were determined by elemental microanalysis, as well as the zeta potential. Seventeen samples (56.6%) were from patients >70 years old, with 20 (66.6%) from women, and most [20 (66.6%)] were from the hip. The GAG content was similar regardless of patients being >/≤ 70 years old with 96.5 ± 63.5 and 78.5 ± 38.5 μg/mg (P = 0.1917), respectively. GAG content was higher in women as compared to men, with 89.5 ± 34.3 and 51.8 ± 13.3 μg/mg, respectively (P = 0.0022), as well as in OA than fracture samples, with 98.4 ± 63.5 and 63.6 ± 19.6 μg/mg, respectively (P = 0.0355). The GAG extracted from the cartilage of patients >70 years old had increase in N, and there were no gender differences regarding GAG elemental analysis. GAG from OA had a highly significant (P = 0.0005) decrease in S% (1.79% ± 0.25%), as compared to fracture samples (2.3% ± 0.19%), with an associated and significant (P = 0.0001) reduction of the zeta potential in the OA group. This is the first report of a reduced S content in GAG from OA patients, which is associated with a reduced zeta potential.

Increase in molar mass distinguishes chondroitin sulfate from osteoarthritis and normal extracellular cartilage matrix

ABSTRACT Purpose To determine alterations of chondroitin sulfate (CS) that reflect cartilage damage in an experimental osteoarthritis (OA) model as well as in human OA samples. Materials and methods Rats were subjected to anterior cruciate ligament transection (ACLT; OA) or a sham procedure and sacrificed 14, 28, or 70 days after ACLT for histopathology and analysis of extracted CS. Cartilage samples from 14 patients undergoing hip or shoulder arthroplasty secondary to OA or fracture (control) were subjected to the same protocol. The CS content (µg/mg dry cartilage) after proteolysis was determined by densitometry, using agarose-gel electrophoresis. Molar mass (MM) and peak MM of CS were determined using high-performance size-exclusion chromatography (HPSEC). Results OA and sham joints at 70 d had 24 [22–24] and 3 [1–6] median histopathology scores, respectively (p < 0.001). Relative CS content (77.7 ± 8.3 µg/mg) was significantly increased in OA samples 70 d after ACLT, as compared to sham (53.5 ± 10.0 µg/mg). Peak MM of CS was higher in OA than in sham samples (P < 0.05). Similarly, CS content and peak MM were higher in cartilage from human OA patients, as compared to fracture samples, reproducing experimental data. Conclusion Cartilage matrix from experimental and human OA samples has increased in the relative CS content. Increase in the peak MM distinguishes CS of the extracellular matrix of OA from normal cartilage.

Wave velocities in articular cartilage measured by micro-Brillouin scattering technique

Micro-Brillouin scattering was used to measure gigahertz ultrasonic wave velocities in the articular cartilage of a bovine femur. Velocities propagating parallel to the surface of the subchondral bone were 3.36-3.83 × 103 m/s in a dry cartilage sample. Anisotropy measurements were also performed in a 10-μm-diameter local area of the cartilage matrix. A weak velocity anisotropy reflected characteristics of the layers. The velocity also depended on the water content. In the middle layer, the velocity in the dry sample was 3.58 × 103 m/s, whereas that for a fully wet sample was 2.04 × 103 m/s.

Concentration profiles of collagen and proteoglycan in articular cartilage by Fourier transform infrared imaging and principal component regression

Fourier-transform infrared imaging (FT-IRI) technique with the principal component regression (PCR) method was used to quantitatively determine the 2D images and the depth-dependent concentration profiles of two principal macromolecular components (collagen and proteoglycan) in articular cartilage. Ten 6μm thick sections of canine humeral cartilage were imaged at a pixel size of 6.25μm in FT-IRI. The infrared spectra extracted from FT-IRI experiments were imported into a PCR program to calculate the quantitative distributions of both collagen and proteoglycan in dry cartilage, which were subsequently converted into the wet-weight based concentration profiles. The proteoglycan profiles by FT-IRI and PCR significantly correlated in linear regression with the proteoglycan profiles by the non-destructive μMRI (the goodness-of-fit 0.96 and the Pearson coefficient 0.98). Based on these concentration relationships, the concentration images of collagen and proteoglycan in both healthy and lesioned articular cartilage were successfully constructed two dimensionally. The simultaneous construction of both collagen and proteoglycan concentration images demonstrates that this combined imaging and chemometrics approach could be used as a sensitive tool to accurately resolve and visualize the concentration distributions of macromolecules in biological tissues.

Chondrocyte viability and metabolic activity after treatment of bovine articular cartilage with bipolar radiofrequency: An in vitro study

Purpose: Some controversy exists regarding the effects of radiofrequency (RF) probes on articular cartilage. To further elucidate these effects, we examined the chondrocyte viability and metabolic activity after treatment of fresh bovine articular cartilage with bipolar RF probes. Type of Study: In vitro assessment. Methods: Three fresh bovine knees served as a baseline control for chondrocyte viability, yielding 6 samples (1 from each medial femoral condyle and 1 from each lateral femoral condyle). After the baseline expected chondrocyte viability was determined, 3 additional bovine knees served as the experimental specimens for the study. Under sterile conditions, 2 different bipolar RF probes were used to treat the articular surface in a light contact mode, moving at a linear rate of 3 to 4 mm/s to provide tissue debridement. Full-thickness articular cartilage was then harvested from each of the treatment areas. Six samples per probe were then assessed for chondrocyte viability using fluorescent double-staining followed by confocal microscopy; 6 samples per probe were assessed for metabolic activity using an 35SO 4 incorporation assay; and 12 additional untreated samples were obtained to serve as controls for viability (n = 6) and metabolic activity (n = 6). Results: The depth of chondrocyte death (mean ± standard deviation) was 109.4 ± 22.1 μm after treatment with the ACD-50 probe, and was 172.3 ± 34.3 μm after treatment with the 2.5-mm/90° probe. The 35SO 4 uptake (mean ± standard deviation) was 2584 ± 1388 cpm/mg dry cartilage for the ACD-50 probe and 1995 ± 852 cpm/mg of dry cartilage for the 2.5-mm/90° probe. The 35SO 4 uptake for the control was 2647 ± 1380 cpm/mg dry cartilage. Conclusions: The 2 probes tested created a well-controlled debridement with smooth edges and a defined margin of chondrocyte death that extended approximately 100 to 200 μm deep to the treatment area. There does not appear to be a significant effect on the metabolic activity of the chondrocytes adjacent to the treatment zone, but with the small sample size we lacked sufficient statistical power to definitively determine these effects. Clinical Relevance: The 2 bipolar radiofrequency probes tested created a well-controlled debridement in normal articular cartilage with smooth edges and a defined margin of chondrocyte death that extended approximately 100 to 200 μm into the treatment area.

The measurement of negative charge content in cartilage using a colloid titration technique

A colloid titration technique has been used to determine the sulfate and carboxylate content of various glycosaminoglycans and has been validated by comparing the results with data obtained using well-established techniques. The method has been applied to the measurement of the negative charge content of cartilage slices at various depths from the articular surface and to the determination of sulfate and carboxylate contents in bovine nasal septa. Titrations of nasal septa were performed on milled cartilage, on cartilage digested with papain and on proteoglycans purified by cesium chloride gradient centrifugation of guanidinium chloride extracts. The sulfate content was similar for all three preparations (0.5 μeq per milligram dry cartilage). However, the carboxylate content determined on milled cartilage was 40% higher than that obtained for cartilage digested with papain or for purified proteoglycans; this implies the possible contribution of carboxyl groups from structural glycoproteins present in the extracellular matrix. The carboxylate content determined on purified proteoglycans was in excellent agreement with values calculated from chemical analyses.

EFFECT OF INCREASED WEIGHT STRESS ON JOINT INTEGRITY, SYNOVIAL FLUID COMPOSITION AND CATHEPSIN B-LIKE ACTIVITY OF ARTICULAR CARTILAGE IN GROWING SWINE

Twenty crossbred gilts (20.5 kg) were individually penned in 1.2 × 1.5-m pens and fed ad libitum a 20% crude protein diet. An initial load of 15% of bodyweight was placed in shoulder bags on 50% of the animals and this ratio was maintained by weekly increases in load weight. Eight gilts from each treatment group were observed for 5 h on each of days 6, 11 and 16 to estimate the amount of time spent standing or walking. After 27 d the pigs were slaughtered. Elbow joints were examined grossly and histologically for osteochondrotic lesions. Lesions associated with a reduced proteoglycan staining occurred at the articular surface of the medial humeral condyle of 50% of the loaded animals but were not observed in the controls. Average daily gain, percent of time when pigs were standing, and elbow joint synovial fluid hyaluronate concentrations were 0.82 vs. 0.82 kg (P &gt; 0.05), 12.8 vs. 17.0% (P &gt; 0.05), and 2.42 vs. 2.58 mg mL−1 (P &gt; 0.05) for loaded vs. control groups, respectively. Cathepsin B-like activity of humeral condylar cartilage was 2.25 and 2.56 μmoles of benzyloxycarbonyl-arginine-arginine-4 methoxy-2-naphthylamide hydrolyzed h−1 g dry cartilage−1 (P &gt; 0.05) for the loaded and control groups, respectively. The results indicate that mild mechanical stress causes focal degeneration of humeral condylar cartilage which could predispose these animals to more severe osteoarthrosis and osteochondrosis at maturity. Key words: Swine, cartilage, cathepsin B, osteochondrosis, stress

MORPHOLOGICAL AND BIOCHEMICAL ANALYSIS OF BIOPSY SPECIMENS IN DISORDERS OF SKELETAL DEVELOPMENT

A programme for combined morphological and biochemical analysis of cartilage-bone biopsy material is described. The structural analysis includes light and electron microscopy, and the biochemistry is focused on polysaccharides. A procedure for isolation and characterization of proteoglycans and glycosaminoglycans from dry cartilage in amounts down to 1 mg is described. The procedure is exemplified by analyses of material from patients with osteogenesis imperfecta, mucopolysaccharidosis IV-Morquio and metaphyseal chondrodysplasia type McKusick. In osteogenesis imperfecta characteristic morphological alterations were demonstrated. Apart from increased chondroitin sulphate molecular weights, the biochemical analyses showed values within the assumed normal range. In Morquio's disease extensive intracellular deposits in single membrane vacuoles were observed in both chondrocytes and osteocytes. There were indications that these deposits contained keratan sulphate, which also occurred in increased amounts in the total tissue preparation. Furthermore, the tendency to formation of proteoglycan aggregates was decreased. In the tissue from the patients with the McKusick syndrome insignificant ultrastructural changes, mainly affecting the mitochondria, were found. The biochemical results were within the assumed normal range. The significance of these findings and the possibilities of further methodological development are discussed.

Growth inhibition induced by chronic dexamethasone treatment of foals

Summary Pony and horse foals were given daily intramuscular injections of dexamethasone, beginning at 6 months of age. Ponies were injected with 0 or 0.5 mg/100 kg bodyweight for 3, 8, or 11 months, or with 5.0 mg/100 kg for 11 months. Horses were treated with 0 or 5.0 mg/100 kg for 20 weeks. Rates of gain were inhibited (P . 10). Urinary nitrogen loss increased within 3 months (P Samples of left femur growth cartilage taken from ponies killed after 3 and 8 months were analyzed. Deoxyribonucleic acid content ( u g/ mg dry cartilage) was doubled by treatment (P Growth inhibition appeared to result from the production of abnormal growth cartilage, deficient in collagen and glycosaminoglycans. A metabolic shift from energy production to intra-chondrocyte energy storage, as reflected by decreased lactate dehydrogenase activities, appeared responsible for the decreased production of cartilage components.

The Effect of Aging on the Synthesis of Hexosamine-Containing Substances from Rat Costal Cartilage

The amount of glycosaminoglycan (GAG) in dry costal cartilage tissue of rats decreased with aging, while the GAG content in mg DNA (unit cartilage cell) remained the same with aging. These results can be explained by the finding that the total number of cartilage cells decreased with aging. Electrophoretic analysis showed that chondroitin 4-sulfate was the major GAG in rat costal cartilage of various ages. Rat costal cartilage of different ages was incubated with radioactive precursors, and newly synthesized GAG was prepared and the radioactivity analyzed to determine the biosynthetic activity. As to changes in the radioactivity uptake with aging per mg dry cartilage tissue, aging influenced [35S]sulfate incorporation into GAG more significantly than [3H]glucosamine incorporation into GAG. There was a significant decrease in the specific radioactivity of [35S]sulfate per mg DNA (unit cartilage cell), whereas the specific radioactivity of [3H]glucosamine per mg DNA did not change significantly with aging. Both the total sulfotransferase activity and the specific activity per mg DNA decreased significantly with aging. Analysis of disaccharide units formed after chondroitinase ABC digestion of labeled GAG isolated from young and old cartilage showed that the percentage of incorporation of [3H]glucosamine into deltaDi-OS increased significantly with aging. These results suggested that the appearance of nonsulfated positions in the structure of the chondroitin sulfate chain increased with aging. On the basis of gel chromatography on Bio-Gel A-1.5 m no significant difference in the approximate molecular size of chondroitin sulfate was observed between the young and old GAG samples. The present study indicated that the sulfation of chondroitin sulfate chains from rat costal cartilage decreased with the process of aging.

Growth of Snell dwarfmice with hGH, insulin and peptide fractions containing SM-activity

In hypopituitary Snellmice, we studied the effects of hGH, insulin and peptide fractions containing SM-activity(SM-P) and insulin-like activity as measured by radioreceptorassay (RRA), but no L-thyroxine,prolactin,testosterone,insulin and GH,on body length,weight,cartilagemetabolism and serum glucose(SG). Cartilage activity (CA) was estimated by 21 hrs incubation in medium with 35S-sulfate. Treatment for 4 weeks with saline, hGH and SM-P induced an increase(as % of initial value ± SEM) in length of resp.104 ± 1.0, 113.8 ± 1.3 and 109.8 ± 1.3%, and in weight of 114.6 ± 3.3 135.4 ± 5.3, 132.8 ± 1.5%. Insulin, at a dose equal to the insulin-like activity of SM-P in RRA units had no effect on length and weight. After three days of treatment CA is resp.1672 ± 223; 5088 ± 444; 4225 ± 163 dpm/mg dry cartilage; Insulin induced a decrease of SG. SM-P had an equal effect at the same dose level in RRA-units, hGH had no effect.

Proteoglycans from Bovine Proximal Humeral Articular Cartilage

Abstract To provide methods applicable to the study of human articular cartilage, we have studied the extraction, fractionation, and physical characterization of proteoglycans from bovine articular cartilage. Two dissociative solvents, MgCl2 and GuHCl, differ sharply in the amount of the proteoglycan extracted from articular cartilage and the macromolecular species present in the initially extracted crude proteoglycan. PG(MgCl2), the crude proteoglycan extracted with 3 m MgCl2, is obtained in a yield of 0.16 g per g of dry cartilage, and accounts for 50% of the uronate and 23% of the hexose of the whole cartilage. PG(GuHCl), the crude proteoglycan extracted with 4 m GuHCl, is obtained in a yield of 0.26 g per g of dry cartilage, and accounts for 81% of the uronate and 61% of the hexose of the whole cartilage. On analytical ultracentrifugation in associative solvents, PG(MgCl2) shows three components: a 4 S protein, a 16 S proteoglycan species, and a 60 to 70 S proteoglycan aggregate. Equilibrium density gradient centrifugation of PG(MgCl2) under associative conditions in 4 m CsCl results in a sharp separation of the 4 S protein into the top sixth of the gradient, and of the 16 S species and 70 S aggregate into the bottom sixth of the gradient. On analytical ultracentrifugation in associative solvents, PG(GuHCl) shows a 4 S protein, a 16 S proteoglycan species, and a 56 S proteoglycan aggregate. The proteoglycan aggregate in either PG(MgCl2) or PG(GuHCl) is completely and reversibly dissociable in either 3 m MgCl2 or 3 m GuHCl. However, while dissociation in 3 m MgCl2 yields the 16 S species, dissociation in 3 m GuHCl yields a 10.5 S proteoglycan subunit. The isolated 16 S proteoglycan species has an intrinsic viscosity of 184 ml per g, a partial specific volume of 0.594 ml per g, and a molecular weight of 1.6 x 106. PG(GuHCl) contains a high molecular weight complex, not present in PG(MgCl2). The complex differs from the 70 S proteoglycan aggregate in size, as indicated by its sedimentation properties; in composition, as indicated by its lower proteoglycan and higher protein and collagen contents; and, in the noncovalent bonds involved in its formation, as indicated by its dissociation in 3 m GuHCl but not in 3 m MgCl2. Analytical ultracentrifugation of the dissociated complex in 3 m GuHCl shows that a 2.7 S protein is a major component of the complex. None of the 2.7 S protein is detectable in preparations of PG(MgCl2) or of the 70 S aggregate. Two states of macromolecular organization of proteoglycans appear to exist in articular cartilage. The first, and lower level of organization is that of the 70 S proteoglycan aggregate. The second and higher level of organization is that of the complex formed by the noncovalent association of proteoglycans and collagen in the presence of the 2.7 S protein.

Incorporation and disappearance of sulfate in different regions of mouse and rat costal cartilage in vivo and in vitro.

AbstractA rapid and simple method for liquid scintillation counting of 33S‐sulfate in biological tissues is described. Sulfate incorporation per mg dry weight into selected lateral, medial and intermediate regions of ribs was studied using costal cartilage from rats and mice. In vitro, cartilage pieces embracing the osteochondral junction had 2–4 times larger incorporation rate than the remaining homogeneous parts of the ribs, both if entire rib cages or stripped and diced cartilage were incubated. After in vivo injection to rats and mice the incorporation rates of sulfate into the region of the osteochondral junction was 2.6 times that found in “resting” cartilage in rats and 4.4 times in mice. The 35S‐sulfate uptake in the ribs per mg dry cartilage diminished in the caudal direction. Growth of the chest cage was mainly a longitudinal growth of the bony segments. Marked differences in the disappearance rates of previously incorporated sulfate were found in different regions along the rib. Rapid sulfate disappearance was found in the bony segment which, however, had incorporated only one seventh of the amount per mg taken up by “resting” cartilage. The “resting” cartilage segments exhibited a steady slow disappearance rate for sulfate and the osteochondral junction region consisting of several differently active tissues showed a rapid sulfate disappearance in the beginning followed by a slow clearance after 2 weeks similar to that of “resting” cartilage. The necessity of careful selection of costal cartilage samples with respect to regional differences is emphasized.

The Separation of New Forms of the Proteinpolysaccharides of Bovine Nasal Cartilage

Abstract The principal proteinpolysaccharide, called PP-L, of bovine nasal cartilage has been fractionated centrifugally to give four products. The method requires low concentration of PP-L, high concentration of salt, and makes use of a remarkable difference between the effects of salts with monovalent and divalent cations on the sedimentation behavior of these proteinpolysaccharides. A fifth product, PP-L2, has previously been isolated from cartilage residue. Of these five, three are almost identical analytically, but differ in the stage of the whole procedure at which their separation becomes possible. The protein contents of the five lie in the range 9 to 36%, but the amino acid profiles of all five are very similar. Of the five forms, the one called PP-L3 behaves in the analytical ultracentrifuge as a single component; the others do not. The two forms with high protein contents sediment most easily and have the highest limiting viscosity numbers. These five products account for 40% of the original dry cartilage weight and 85% of its uronic acid content.