scholarly journals Monoclonal antibodies to different protein-related epitopes of human articular cartilage proteoglycans

1986 ◽  
Vol 234 (1) ◽  
pp. 31-41 ◽  
Author(s):  
T T Glant ◽  
K Mikecz ◽  
A R Poole
Keyword(s):  
Monoclonal Antibodies ◽  
Articular Cartilage ◽  
Alkali Treatment ◽  
Buoyant Density ◽  
Limb Bud ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Keratan Sulphate ◽  
Human Adult ◽  
Cartilage Proteoglycans

Monoclonal antibodies produced against chondroitinase-treated human adult cartilage proteoglycans were selected for their ability to recognize epitopes on native proteoglycans. Binding analyses revealed that four of these monoclonal antibodies (BCD-4, BCD-7, EFG-4 and KPC-190) each recognized a different epitope on the same proteoglycan molecule which represents a subpopulation of a high buoyant density (D1) fraction of human articular cartilage proteoglycans (10, 30, 50 and 60% in fetal-newborn, 1.5 years old, 15 years old and 52-56 years old cartilages, respectively). Analysis of epitope specificities revealed that BCD-7 and EFG-4 monoclonal antibodies recognized epitopes on proteoglycan monomer which are associated with the protein structure in that they are sensitive to cleavage by Pronase, papain and alkali treatment and do not include keratan sulphate, chondroitin sulphate or oligosaccharides. The BCD-4 and KPC-190 epitopes also proved to be sensitive to Pronase or papain digestion or to alkali treatment, but keratanase or endo-beta-galactosidase also reduced the immunoreactivity of these epitopes. These observations indicate that the BCD-4 and KPC-190 epitopes represent peptides substituted with keratan sulphate or keratan sulphate-like structures. The BCD-4 epitope is, however, absent from a keratan sulphate-rich fragment of human adult proteoglycan, while the other three epitopes were detected in this fragment. None of these four epitopes were detected in the link proteins of human cartilage, in the hyaluronic acid-binding region of human newborn cartilage proteoglycan, in Swarm rat chondrosarcoma proteoglycan, in chicken limb bud proteoglycan monomer and in the small dermatan sulphate-proteoglycan of bovine costal cartilage. EFG-4 and KPC-190 epitopes were not detected in human fetal cartilage proteoglycans, although fetal molecules contained trace amounts of epitopes reactive with BCD-4 and BCD-7 antibodies.

scholarly journals Age-related changes in the composition and structure of human articular-cartilage proteoglycans

1978 ◽  
Vol 176 (3) ◽  
pp. 683-693 ◽  
Author(s):  
M T Bayliss ◽  
S Y Ali
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Age Groups ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Keratan Sulphate ◽  
Age Related ◽  
Composition And Structure ◽  
Normal Human ◽  
Cartilage Proteoglycans

1. Analysis of the purified proteoglycans extracted from normal human articular cartilage with 4M-guanidinium chloride showed that there was an age-related increase in their content of protein and keratan sulphate. 2. The hydrodynamic size of the dissociated proteoglycans also decreased with advancing age, but there was little change in the proportion that could aggregate. 3. Results suggested that some extracts of aged-human cartilage had an increased content of hyaluronic acid compared with specimens from younger patients. 4. Dissociated proteoglycans, from cartilage of all age groups, bind to hyaluronic acid and form aggregates in direct proportion to the hyaluronic acid concentration. 5. Electrophoretic heterogeneity of the dissociated proteoglycans was demonstrated on polyacrylamide/agarose gels. The number of proteoglycan species observed was also dependent on the age of the patient.

scholarly journals Age-related changes in protein-related epitopes of human articular-cartilage proteoglycans

1986 ◽  
Vol 236 (1) ◽  
pp. 71-75 ◽  
Author(s):  
T T Glant ◽  
K Mikecz ◽  
P J Roughley ◽  
E Buzás ◽  
A R Poole
Keyword(s):  
Core Protein ◽  
Buoyant Density ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Keratan Sulphate ◽  
Cartilage Proteoglycan ◽  
Age Related ◽  
Age Dependent ◽  
Old Adult ◽  
Cartilage Proteoglycans

Monoclonal antibodies were prepared that recognize different age-related epitopes on proteoglycan subunits of high buoyant density isolated from human epiphysial and articular cartilages. Antibody EFG-4 (IgG1) recognizes a proteinase-sensitive segment associated with the core protein. Antibody BCD-4 (IgG1) reacts with keratan sulphate bound to core protein. Both epitopes are minimally expressed in foetal cartilage and increase with age after birth to become maximally expressed in adult cartilage by about 30 years of age. In contrast, monoclonal antibody alpha HFPG-846 (IgM) recognizes a core-protein-related epitope that is maximally expressed in young foetal cartilage, declines up to birth and thereafter and is almost absent after about 30 years of age. Antibody alpha HFPG-846 was used to isolate by immuno-affinity chromatography two subpopulations of proteoglycan subunits from a 16-year-old-human cartilage proteoglycan subunit preparation. Only the antibody-unbound population showed a significant reaction with antibodies EGF-4 and BCD-4. The amino acid and carbohydrate compositions of these proteoglycan fractions were different, and one (antibody-bound) resembled those of foetal and the other (antibody-unbound) resembled those of adult proteoglycans isolated from 24-27-week-old-foetal and 52-56-year-old-adult cartilage respectively. These observations demonstrate that human cartilages contain at least two chemically and immunochemically distinct populations of proteoglycans, the proportions and content of which are age-dependent. It is likely that these populations represent the products of different genes, though their heterogeneity may be compounded by the result of different post-translation modifications.

scholarly journals Immunochemical analysis of cartilage proteoglycans. Cross-reactivity of molecules isolated from different species

1981 ◽  
Vol 199 (1) ◽  
pp. 81-87 ◽  
Author(s):  
J Wieslander ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Limb Bud ◽  
Binding Region ◽  
Human Cartilage ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycans ◽  
Rabbit Articular Cartilage ◽  
Hyaluronic Acid Binding

Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.

scholarly journals The identification and characterization of two populations of aggregating proteoglycans of high buoyant density isolated from post-natal human articular cartilages of different ages

1987 ◽  
Vol 248 (3) ◽  
pp. 735-740 ◽  
Author(s):  
C Webber ◽  
T T Glant ◽  
P J Roughley ◽  
A R Poole
Keyword(s):  
Hyaluronic Acid ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Buoyant Density ◽  
Human Articular Cartilage ◽  
Keratan Sulphate ◽  
Different Ages ◽  
Cartilage Proteoglycans ◽  
Two Populations

After chromatography on Sepharose CL-2B under associative conditions, high-buoyant-density human articular-cartilage proteoglycans were analysed biochemically and by radioimmunoassay with monoclonal antibodies to a core-protein-related epitope and to keratan sulphate. An examination of proteoglycans from individuals of different ages revealed the presence at 1 year of mainly a single polydisperse population containing chondroitin sulphate (uronic acid) and keratan sulphate. From 4 years onwards a smaller keratan sulphate-rich and chondroitin sulphate-deficient population appears in increasing amounts until 15 years. At the same time the larger population shows a progressive decrease in size from 1 year onward. By 23 years and after the proportion of keratan sulphate in the larger chondroitin sulphate-rich proteoglycan increases. Both adult proteoglycan populations are shown immunologically to aggregate with hyaluronic acid, with the smaller showing a greater degree of interaction. The larger population is richer in serine and glycine, and the smaller population contains more glutamic acid/glutamine, alanine, phenylalanine, lysine and arginine; its protein content is also higher. Whether the larger post-natal population represents a different gene product from the single polydisperse population found in the human fetus, which has a different amino acid composition, remains to be established. The smaller population, which represents approximately one-third the mass of the larger population in the adult, may represent a degradation product of the larger population, in which the hyaluronic acid-binding region and keratan sulphate-rich region are conserved.

scholarly journals Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage

1985 ◽  
Vol 231 (1) ◽  
pp. 129-138 ◽  
Author(s):  
P J Roughley ◽  
R J White ◽  
A R Poole
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Binding Protein ◽  
Buoyant Density ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Acid Binding Protein ◽  
Adult Human ◽  
Proteoglycan Aggregates ◽  
Hyaluronic Acid Binding

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.

scholarly journals Biosynthesis of proteoglycan in vitro by cartilage from human osteochondrophytic spurs

1982 ◽  
Vol 206 (2) ◽  
pp. 329-341 ◽  
Author(s):  
Charles J. Malemud ◽  
Victor M. Goldberg ◽  
Roland W. Moskowitz ◽  
Lee L. Getzy ◽  
Robert S. Papay ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Deae Cellulose ◽  
Culture Conditions ◽  
Human Articular Cartilage ◽  
Guanidinium Chloride ◽  
Tissue Slices ◽  
Keratan Sulphate ◽  
Defined Culture

Proteoglycan biosynthesis by human osteochondrophytic spurs (osteophytes) obtained from osteoarthritic femoral heads at the time of surgical joint replacement was studied under defined culture conditions in vitro. Osteophytes were primarily present in two anatomic locations, marginal and epi-articular. Minced tissue slices were incubated in the presence of [35S]sulphate or [14C]glucosamine. Osteophytes incorporated both labelled precursors into proteoglycan, which was subsequently characterized by CsCl-isopycnic-density-gradient ultracentrifugation and chromatography on Sepharose CL-2B. The material extracted with 0.5m-guanidinium chloride showed 78.1% of [35S]sulphate in the A1 fraction after centrifugation. Only 23.0% of the [35S]sulphate in this A1 fraction was eluted in the void volume of Sepharose CL-2B under associative conditions. About 60–80% of the [35S]sulphate in the tissue 4m-guanidinium chloride extract was associated with monomeric proteoglycan (fraction D1). The average partition coefficient (Kav.) of the proteoglycan monomer on Sepharose CL-2B was 0.28–0.33. Approx. 12.4% of this monomer formed stable aggregates with high-molecular-weight hyaluronic acid in vitro. Sepharose CL-2B chromatography of fractions with lower buoyant densities (fractions D2–D4) demonstrated elution profiles on Sepharose CL-2B substantially different than that of fraction D1, indicative of the polydisperse nature of the newly synthesized proteoglycan. Analysis of the composition and chain size of the glycosaminoglycans showed the following: (1) preferential elution of both [35S]sulphate and [14C]glucosamine in the 0.5m-LiCl fraction on DEAE-cellulose; (2) the predominant sulphated glycosaminoglycan was chondroitin 6-sulphate (60–70%), with 9–11% keratan sulphate in the monomer proteoglycan; (3) Kav. values of 0.38 on Sephadex G-200 and 0.48 on Sepharose CL-6B were obtained with papain-digested and NaBH4-treated D1 monomer respectively. A comparison of the synthetic with endogenous glycosaminoglycans indicated similar types. These studies indicated that human osteophytes synthesized in vitro sulphated proteoglycans with some characteristics similar to those of mature human articular cartilage, notably in the size of their proteoglycan monomer and predominance of chondroitin 6-sulphate. They differed from articular cartilage primarily in the lack of substantial quantities of keratan sulphate and aggregation properties associated with monomer interaction with hyaluronic acid.

Effects of Radiofrequency Energy on Human Articular Cartilage

2005 ◽  
Vol 33 (7) ◽  
pp. 1035-1039 ◽  
Author(s):  
Sean Caffey ◽  
Edward McPherson ◽  
Brian Moore ◽  
Thomas Hedman ◽  
C. Thomas Vangsness
Keyword(s):  
Cell Death ◽  
Articular Cartilage ◽  
Dead Cell ◽  
Radiofrequency Energy ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Cellular Death ◽  
Significant Difference ◽  
Grade Ii ◽  
Grade Iii

Background Previous radiofrequency work has not rigidly controlled energy application to the articular cartilage, giving uncertain results published to date. Hypothesis At minimal settings, radiofrequency probes cause cell death in measurable areas when applied to human articular cartilage. Study Design Controlled laboratory study. Methods Simulating operating room conditions, 5 commercially available radiofrequency probes were attached to a customized jig to standardize a minimal contact pressure of each probe tip to 2.0 g. Keeping all variables the same, probes were placed on specific points of fresh grade II human cartilage with treatment times of 1 and 3 seconds at the manufacturer's recommended settings. Grade III cartilage was also tested with a treatment time of 3 seconds, and grade II cartilage was studied with the probe held 1 mm off the cartilage surface. Cartilage was blindly analyzed by confocal microscopy using a live/dead cell viability assay to determine the extent of cell death. Results Radiofrequency probes produced significant cellular death in the form of a half-circle into the cartilage to variable depths. For treatment times of 1 and 3 seconds, cell death measurements ranged from 404 to 539 μm and 1034 to 1283 μm, respectively. One probe failed to show any effect, with minimal evidence of cell death or cartilage smoothing. When probes were kept a 1.0-mm distance above the cartilage, no cell death or cartilage smoothing was noted. Radiofrequency treatment of grade III cartilage penetrated to the subchondral bone. There was no statistically significant difference between the damage caused by monopolar and bipolar probes when tested under these rigidly controlled conditions. Conclusion These results showed significant cellular death at these minimal conditions to the underlying chondrocytes with radiofrequency probes. Surgeons using this technology need to be aware of the power and dangerous potential these probes can have on articular cartilage.

scholarly journals Collagen Growth Pattern in Human Articular Cartilage of the Knee

Cartilage ◽  
2020 ◽  
pp. 194760352097101
Author(s):  
Adam E.M. Jørgensen ◽  
Peter Schjerling ◽  
Michael R. Krogsgaard ◽  
Michael M. Petersen ◽  
Jesper Olsen ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Growth Pattern ◽  
Age Determination ◽  
Joint Surface ◽  
Lateral Condyle ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Medial Condyle ◽  
Cartilage Formation ◽  
Tibia Plateau

Objective During skeletal growth, the articular cartilage expands to maintain its cover of bones in joints, however, it is unclear when and how cartilage grows. We aim to determine the expanding growth pattern and timing across the tibia plateau in human knees. Design Six human tibia plateaus (2 healthy, 2 with osteoarthritis, and 2 with posttraumatic osteoarthritis) were used for full-depth cartilage sampling systematically across the joint surface at 12 medial and 4 lateral sites. Methodologically, we took advantage of the performed nuclear bomb tests in the years 1955 to 1963, which increased the atmospheric 14C that was incorporated into human tissues. Cartilage was treated enzymatically to extract collagen, analyzed for 14C content, and year at formation was determined from historical atmospheric 14C concentrations. Results By age-determination, each tibia condyle had central points of formation surrounded by later-formed cartilage toward the periphery. Furthermore, the tibia plateaus contained collagen with 14C levels corresponding to mean donor age of 11.7 years (±3.8 SD). Finally, the medial condyle had lower 14C levels corresponding to formation 1 year later than the lateral condyle ( P = 0.009). Conclusions Human cartilage on the tibia plateau contains collagen that has experienced little if any turnover since school-age. The cartilage formation develops from 2 condyle centers and radially outward with the medial condyle finishing slightly later than the lateral condyle. This suggests a childhood programmed cartilage formation with a very limited adulthood collagen turnover.

scholarly journals Separation and characterization of two populations of aggregating proteoglycans from cartilage

1985 ◽  
Vol 225 (1) ◽  
pp. 95-106 ◽  
Author(s):  
D Heinegård ◽  
J Wieslander ◽  
J Sheehan ◽  
M Paulsson ◽  
Y Sommarin
Keyword(s):  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Rich Region ◽  
Keratan Sulphate ◽  
Core Proteins ◽  
Lower Mobility ◽  
Cartilage Proteoglycans

Intermediary gel immunoelectrophoresis was used to show that purified aggregating cartilage proteoglycans from 2-year-old steers contain two distinct populations of molecules and that only one of these is immunologically related to non-aggregating cartilage proteoglycans. The two types of aggregating proteoglycans were purified by density-gradient centrifugation in 3.5M-CsCl/4M-guanidinium chloride and separated by zonal rate centrifugation in sucrose gradients. The higher-buoyant-density faster-sedimenting proteoglycan represented 43% of the proteoglycans in the extract. It had a weight-average Mr of 3.5 × 10(6), did not contain a well-defined keratan sulphate-rich region, had a quantitatively dominant chondroitin sulphate-rich region and contained 5.9% protein and 23% hexosamine. The lower-buoyant-density, more slowly sedimenting, proteoglycan represented 15% of the proteoglycans in the extract. It had a weight-average Mr of 1.3 × 10(6), contained both the keratan sulphate-rich and the chondroitin sulphate-rich regions and contained 7.3% protein and 23% hexosamine. Each of the proteoglycan preparations showed only one band on agarose/polyacrylamide-gel electrophoresis. The larger proteoglycan had a lower mobility than the smaller. The distribution of chondroitin sulphate chains along the chondroitin sulphate-rich region was similar for the two types of proteoglycans. The somewhat larger chondroitin sulphate chains of the larger proteoglycan could not alone account for the larger size of the proteoglycan. Peptide patterns after trypsin digestion of the proteoglycans showed great similarities, although the presence of a few peptides not shared by both populations indicates that the core proteins are partially different.

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