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Biomedical subjects

K E Kuettner

Publications and source records attributed to K E Kuettner.

At least 19 recordsLinked to original sources
PubMed1992

Biochemistry of articular cartilage in health and disease.

Articular cartilage covers the ends of long bones in synovial joints, providing smooth articulation and cushioning of the underlying bone during joint movement. The tissue can be viewed as a viscoelastic, composite material composed of collagen type II (and smaller amounts of other collagens) entrapping compressed (underhydrated) proteoglycan aggregates which generate a high osmotic/swelling pressure. This abundant extracellular matrix (ECM) is synthesized and turned over by relatively few cells, the chondrocytes. These cells produce a compartmentalized ECM, the components of which are heterogeneous and vary with anatomical location. They also undergo changes with age and altered functional requirements. Articular cartilage contains no separating basement membranes, nerves, lymphatics, or blood vessels. Access to nutrients and elimination of waste products occur via diffusion through the extracellular matrix. The turnover of collagen is much slower than that of proteoglycans. Products of the metabolic turnover of the matrix macromolecules are released continuously into the synovial cavity and ultimately reach the blood circulation where they can be measured as "markers" of metabolic changes.

Animals↗
PubMed1992

Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads.

Human and bovine adult articular chondrocytes cultured in alginate beads or agarose gel retain their spherical shape and typical chondrocytic appearance for at least 5 weeks. Aggrecan is always the major population of proteoglycans (PGs) synthesized; its size varies depending upon the age of the cartilage from which the cells are derived but it is not influenced by the culture system used. Studies of human chondrocytes cultured in alginate showed that the majority of the newly-synthesized aggrecan molecules are rapidly incorporated into aggregates which can be extracted from the gel in their native form. About one-third of the aggregates formed during a 4-hour period of incubation in the presence of [35S]-sulfate are packed at high concentration in the cell-associated matrix; the remainder is present in areas of the gel further removed from the cells. Aggrecan appears to be turned over more slowly by human than by bovine cells in both culture systems; in both species, the rate of turnover is slower in alginate than in agarose. Decorin is also synthesized in small amounts but it is rapidly lost from the agarose or alginate gel.

Adolescent↗
PubMed1992

Influence of interleukin-1 on the morphology and proteoglycan metabolism of cultured bovine articular chondrocytes.

Bovine articular chondrocytes cultured in agarose gel in the presence of serum elaborated a highly organized extracellular matrix rich in proteoglycans and collagens. The cultures were evaluated quantitatively by radiosulfate labeling of proteoglycans, and by densitometry following staining with alcian blue. In addition, immunohistochemical methods were used to demonstrate the presence of several components of cartilage proteoglycan molecules. Treatment with Interleukin-1 (Il-1) or retinol resulted in diminished synthesis and enhanced catabolism of matrix proteoglycans, but the chondrocytes were more sensitive to human recombinant Il-1 alpha than to Il-1 beta. Treatment with Il-1 alpha or retinol resulted in a profound disorganization of the residual matrix around the majority of the chondrocytes, while Il-1 beta caused much less severe changes. Some variation in cellular response to Il-1 alpha may result from the heterogeneity previously reported among articular chondrocytes.

Animals↗
PubMed1992

Isolation and characterization of an abundant elastase inhibitor from NaCl extracts of bovine nasal septa and articular cartilage.

Extracts of cartilage have been reported to inhibit many serine proteinases and metalloenzymes. Such inhibition may be important in protecting cartilage against degradation by chondrocytic proteinases such as collagenase, stromelysin and by leukocytic proteases, such as elastase. We report here isolation and partial characterization of a 17-kD elastase inhibitor from 0.5 M NaCl extracts of both nasal septum cartilage and articular cartilage, which inhibits elastase and represents 0.08% of the weight of nasal cartilage and 0.002% of the weight of articular cartilage. The protein was highly specific for elastase and did not inhibit cartilage metalloproteinases, suggesting that it may be mainly directed toward protecting cartilage against leukocytic proteases. The inhibitor had a blocked amino-terminus, was high in serine and glycine and lacked carbohydrate. The ease with which the inhibitor was extracted from cartilage suggests that it may function in vivo as a highly abundant elastase inhibitor which is secreted into synovial fluid from cartilage. The inhibitor was shown to be synthesized by bovine articular cartilage in explant culture and nearly all of the metabolically labeled material was secreted into the culture media. The inhibitor cross-reacted with polyclonal antibodies to bovine neck ligament alpha-elastin and antibodies to the inhibitor reacted with bovine neck ligament elastin. The properties of this inhibitor are different than those of any other reported cartilage derived inhibitor.

Amino Acids↗
PubMed1991

Novel method of purification of human leukocytic elastase using adsorption on a high-performance liquid chromatography gel permeation column.

Neutrophil elastases are serine proteinases released during acute and chronic inflammatory states. We have developed a novel isolation method for neutrophil elastase, involving conventional gel chromatography followed by adsorption of protein at low ionic strength on a high-performance liquid chromatography gel permeation column. The bound elastase is then eluted by application of higher ionic strength. This adsorption step at low ionic strength, a step to be avoided in most purification methods, was used to advantage here to allow isolation of homogeneous material. This purification procedure should be useful for quick, simple bulk preparation of the enzyme.

Chromatography, Gel↗
PubMed1991

Circulating keratan sulfate: a marker of cartilage proteoglycan catabolism in osteoarthritis.

The serum level of a highly sulfated epitope present on long keratan sulfate chains provides a direct measure of the rate of catabolism of cartilage proteoglycans. Levels of the keratan sulfate epitope are elevated in patients with generalized osteoarthritis (OA), indicating these individuals have elevated rates of cartilage proteoglycan catabolism. In the Pond-Nuki model of canine OA, the serum level of the keratan sulfate epitope rises rapidly after the transection of the anterior cruciate ligament, long before OA lesions can be detected, and remains high for at least 13 weeks.

Animals↗
PubMed1991

Articular cartilage matrix and structure: a minireview.

The ability of articular cartilage to undergo reversible deformation is dependent upon the organization of specific macromolecules within the extracellular matrix. This abundant matrix is elaborated by a small number of chondrocytes which maintain homeostasis via a synchronized balance between anabolism and catabolism. Type II collagen together with smaller amounts of other collagens form the fibrous network of the tissue in which are "entrapped" the aggregating proteoglycans in an underhydrated form. Small amounts of nonaggregating dermatan sulfate containing proteoglycans and matrix proteins are also present in the matrix but their function is less well understood.

Cartilage, Articular↗
PubMed1990

Biochemical characterization of long-term culture of the Swarm rat chondrosarcoma chondrocytes in agarose.

Difficulty in maintaining phenotypic stability of the Swarm rat chondrosarcoma in long-term monolayer cultures has prompted investigation of alternative conditions that would enable extended maintenance of these cells, permitting use of the tumor as a model system for the long-term study of proteoglycan metabolism. Morphological analysis of the growth of the chondrosarcoma chondrocytes in agarose has shown stability of the culture over a 20 day period with respect to the ability of the cells to proliferate and synthesize an Alcian blue-positive extracellular matrix. The present study confirms these findings through analysis of the growth characteristics of the culture and the pattern of proteoglycan and collagen synthesis. The chondrocytes actively synthesize a proteoglycan-rich matrix at a rate dependent on the initial plating density and concentration of serum in the culture medium. These factors similarly affect the proliferative capabilities of the culture as demonstrated by the growth curves obtained at different culture conditions. During 20 days in culture, the cells synthesize an aggregating chondroitin sulfate proteoglycan and collagen type II, typical of cartilage and this chondrosarcoma. In addition, small molecular weight proteoglycans were found to be present at concentrations of up to 10% of the total proteoglycan population. Degradative rates are slow, the proteoglycan half-life is about 30 days, but can be enhanced with retinol, reducing the half-life to 2 days.

Animals↗
PubMed1988

Serum keratan sulfate levels in osteoarthritis patients.

Serum levels of keratan sulfate (KS), measured by an enzyme-linked immunosorbent-inhibition assay, were found to be significantly higher in 31 patients with hypertrophic osteoarthritis (OA) than those in 41 adults without joint disease. Seventy-seven percent of patients with OA, but only 12% of control subjects, had serum levels which were more than 1 SD above the mean of the control group. Following replacement of a single osteoarthritic hip joint, serum KS levels decreased, at first, in all patients. Subsequently, the concentration of serum KS progressively increased; 6 months following surgery, KS levels were similar or close to the preoperative levels in virtually all patients. The results suggest that patients with hypertrophic OA may have a generalized imbalance of cartilage proteoglycan metabolism. Measurements of serum KS are likely to prove most useful in studying this particular subset of patients with generalized OA.

Aged↗
PubMed1988

Age related changes in the concentration of serum keratan sulphate in children.

Serum was obtained from 155 children at the time of admission to hospital for elective surgery. The concentration of serum keratan sulphate was determined by an ELISA which uses an antibody specific for keratan sulphate, a molecule found predominantly in cartilage. Concentrations of keratan sulphate rise progressively during the first four years of life (0-2: mean = 357 micrograms/l; 2-4: mean = 422 micrograms/l) and then remain high until 12 years of age (mean = approx. 500 micrograms/l). At this time, concentrations drop markedly (13-year olds: mean = 377 micrograms/l; 14-year olds: mean = 318 micrograms/l). After age 15, concentrations continue to fall toward the concentrations found in normal adults. Serum concentrations did not show significant differences with respect to disease category, sex or race but were found to vary, sometimes markedly, from child to child at any one age. The results suggest human cartilage undergoes significant changes in metabolic activities during maturation. Measurements of keratan sulphate concentration in serum may prove useful in studying the biochemical and physiological bases of these changes and in monitoring growth or endochondral ossification during maturation.

Adolescent↗
PubMed1988

Isolation and partial characterization of neutrophil elastase inhibitors from bovine vitreous and aorta.

Bovine vitreous body and aorta contain extractable leukocyte elastase inhibitors, which were purified by gel filtration and affinity chromatography on agarose-pancreatic elastase. The purified inhibitor preparation from aorta was resolved by polyacrylamide gel electrophoresis into a main band migrating slightly faster than commercial Trasylol and a more weakly stained band migrating close to chymotrypsinogen. The purified inhibitor preparation from both sources inhibited, in a competitive fashion, purified human leukocyte elastase and was ineffective against bovine trypsin and leukocyte cathepsin G or collagenase. These inhibitors from vitreous body and aorta were distinguishable by several criteria from serum inhibitors.

Animals↗
PubMed1988

Quantification of hen egg white lysozyme in cartilage by an enzyme-linked immunosorbent assay.

We have developed an enzyme-linked immunosorbent assay (ELISA) with an inhibition step to quantify hen egg white lysozyme (HEWL) on a weight basis. The assay is relatively insensitive to changes in ionic strength or pH. Quantification is not affected by the presence or large amounts of mammalian lysozymes or inhibitors of enzymatic activity such as soluble chitin oligomers. We used this ELISA to show that the HEWL content of chick cartilages increases progressively between days 14 and 18 of embryonic life. The maturation-related increase does not appear to be the result of increased synthesis by the chondrocytes for the latter did not synthesize detectable amounts of lysozyme in vitro. Evidence was obtained to suggest that most, if not all, the lysozyme in cartilage is derived from the surrounding fluids which come in contact with the matrix.

Animals↗
PubMed1988

Differences between sub-populations of cultured bovine articular chondrocytes. I. Morphology and cartilage matrix production.

Bovine articular chondrocytes cultured in agarose gel comprise a heterogeneous population when judged by morphological and histochemical criteria. The purpose of the present experiments was to compare, under the same conditions of culture, sub-populations of chondrocytes derived from different depths of articular cartilage. Sub-populations of chondrocytes were cultured separately following their isolation from slices of articular cartilage cut from successive depths of the tissue. Chondrocytes derived from superficial and deep zones differed significantly in morphology, rate of proliferation, and activity in secreting a proteoglycan-rich extracellular matrix. The differences are sufficient to account for the heterogeneity observed in cultures of the entire cell population, and the correlate well with known variations with depth in morphology and histochemistry of intact articular cartilage. These results demonstrate that articular chondrocytes continue in culture to express metabolic differences which reflect their original anatomical location; such differences may have important functional significance.

Animals↗
PubMed1988

Differences between sub-populations of cultured bovine articular chondrocytes. II. Proteoglycan metabolism.

Sub-populations of bovine articular chondrocytes derived from different depths of the cartilage showed differences in accumulation of proteoglycan-rich extracellular matrix in culture. To extend these morphological studies, the synthesis and catabolism of 35S-labeled proteoglycans have been examined in similar cultures. Chondrocytes from deep zones synthesized significantly more proteoglycans than cells from the superficial zone. While all populations of chondrocytes synthesized predominantly aggregating proteoglycans, a higher proportion of isotope was present in non-aggregating proteoglycans in cultures of superficial chondrocytes, by comparison with those of deep cells. Proteoglycans were degraded more rapidly by superficial cells than by chondrocytes from deeper layers. These results correlate both with previous histochemical studies of similar cultures, and with known depth-related variations in biochemical composition of intact articular cartilage.

Animals↗
PubMed1987

The effect of zwitterionic detergents on the extraction and functional properties of cartilage proteoglycans.

A range of structurally related zwitterionic detergents, Zwittergents 3-06, 3-08, 3-10, and 3-12, and a derivative of cholic acid (Chaps) were examined for their ability to enhance the extraction of newly synthesized, intracellular proteoglycans and for their effect on the functional properties of cartilage proteoglycan. Although none of the detergents could extract greater than 4% of the intracellular proteoglycans when used alone, Zwittergents 3-10, 3-12, and Chaps proved equally as effective when used in combination with 4 M guanidine HCl extracting greater than 90% of newly synthesized proteoglycans. Rate zonal centrifugation of aggregates containing either 3H-link protein or 3H-monomer, which had been incubated with 2% (w/v) detergent indicated that none of the test detergents caused a disassembly of intact aggregates. However, both Zwittergents 3-10 and 3-12 prevented the reaggregation of components dissociated with 4 M guanidine HCl. Similar to the finding with aggregate, none of the detergents caused a disassembly of monomer-link protein complexes prepared from purified 3H-link protein and proteoglycan monomer, while Zwittergents 3-10 and 3-12 prevented their assembly from free link protein and monomer. However, monomer-link protein complexes once formed were able to associate with hyaluronic acid to form link-stable ternary complexes in the presence of all detergents tested including Zwittergents 3-10 and 3-12.

Animals↗