Abstract
A biotinylated complex of aggrecan G1-domain and link protein was used to characterize the distribution of hyaluronan in paraffin-embedded sections of adult human and canine intervertebral disc and cartilage endplate. Limited chondroitinase ABC and trypsin digestions of the sections before staining was utilized to expose hyaluronan potentially masked by aggrecan. Hyaluronan concentration and hyaluronan to uronic acid ratio in different parts of the discs were measured as a background for the histological analysis.
Hyaluronan staining was strong in the nucleus pulposus and inner parts of annulus fibrosus of both species, corroborated by biochemical assays of the same compartments. Particularly in human samples, hyaluronan in the interterritorial matrix of nucleus pulposus and annulus fibrosus was readily accessible to the probe without enzyme treatments. In contrast, the cell-associated hyaluronan signal was enhanced after trypsin or limited chondroitinase ABC-treatment of the sections, suggesting that pericellular hyaluronan was more masked by aggrecan than in the distant matrix. A puzzling feature of canine cartilage endplate cells was their intensive cell-associated hyaluronan signal, part of which appeared intracellular. Hyaluronan was abundant between the collagenous lamellae in annulus fibrosus, perhaps important in the plasticity of this tissue.
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Antoniou J, Goudsouzian NM, Heathfield TF, Winterbottom N, Steffen T, Poole AR, Aebi M, Alini M (1996) The human lumbar endplate. Evidence of changes in biosynthesis and denaturation of the extracellular matrix with growth, maturation, aging, and degeneration. Spine 21: 1153-1161.
Arokoski J, Kiviranta I, Laakkonen J, Tiihonen A, Sopanen E, Ikonen J, Haapanen K, Nevalainen T, Tammi M, Helminen HJ (1991) A ten-track treadmill for running training of dogs. Lab Animal Sci 41: 246-250.
Asari A, Miyauchi S, Miyazaki K, Hamai A, Horie K, Takahashi T, Sekiguchi T, Machida A, Kohno K, Uchiyama Y (1992) Intra-and extracellular localization of hyaluronic acid and proteoglycan constituents (chondroitin sulfate, keratan sulfate, and protein core) in articular cartilage of rabbit tibia. J Histochem Cytochem 40: 1693-1703.
Bayliss MT, Venn M, Maroudas A, Ali SY (1983) Structure of proteoglycans from different layers of human articular cartilage. Biochem J 209: 387-400.
Bernick S, Cailliet R (1982) Vertebral end-plate changes with aging of human vertebrae. Spine 7: 97-102.
Bishop PB, Pearce RH (1993) The proteoglycans of the cartilaginous endplate of the human intervertebral disc change after maturity. J Orthop Res 11: 324-331.
Blumenkrantz N, Asboe-Hansen G (1973) New method for quantitative determination of uronic acids. Anal Biochem 54: 484-489.
Boos N, Nerlich AG, Wiest I, von der Mark K, Aebi M (1997) Immunolocalization of type X collagen in human lumbar intervertebral discs during ageing and degeneration. Histochem Cell Biol 108: 471-480.
Chelberg MK, Banks GM, Geiger DF, Oegema TR, Jr (1995) Identification of heterogeneous cell populations in normal human intervertebral disc. J Anat 186: 43-53.
Dowthwaite GP, Edwards JCW, Pitsillides AA (1998) An essential role for the interaction between hyaluronan and hyaluronan binding proteins during joint development. J Histochem Cytochem 46: 641-651.
Engström-Laurent A, Hällgren R (1987) Circulating hyaluronic acid varies with physical activity in health and rheumatoid arthritis. Relationship to synovitis mass and morning stiffness. Arthritis Rheum 30: 1333-1338.
Fenderson BA, Stamenkovic I, Aruffo A (1993) Localization of hyaluronan in mouse embryos during implantation, gastrulation and organogenesis. Differentiation 54: 85-98.
Franzén A, Inerot S, Hejderup S-O, Heinegård D (1981) Variations in the composition of bovine hip articular cartilage with distance from the articular surface. Biochem J 195: 535-543.
Fraser JRE, Laurent TC (1996) Hyaluronan. In Comper WD, ed. Extracellular Matrix Vol. 2. 1st edn. Amsterdam: Harwood Academic Publishers GmbH. pp. 141-199.
Hardingham TE, Adams P (1976) A method for the determination of hyaluronate in the presence of other glycosaminoglycans and its application to human intervertebral disc. Biochem J 159: 143-147.
Heinegård D, Hascall VC (1974) Aggregation of cartilage proteoglycans: Characteristics of the proteins isolated from trypsin digests of aggregates. J Biol Chem 249: 4250-4256.
Heinegård D, Björnsson S, Mörgelin M, Sommarin Y (1998) Hyaluronan-binding proteins. In Laurent TC, ed. The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives. 1st edn. London: Portland Press. pp. 113-122.
Hua Q, Knudson CB, Knudson W (1993) Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis. J Cell Sci 106: 365-375.
Inkinen RI, Lammi MJ, Lehmonen S, Puustjärvi K, Kääpä E, Tammi MI (1998) Relative increase of biglycan and decorin and altered chondroitin sulfate epitopes in the degenerating human intervertebral disc. J Rheum 25: 506-514.
Jackson P (1994) High-resolution polyacrylamide gel electrophoresis of fluorophore-labeled reducing saccharides. Meth Enzym 230: 250-265.
Lammi P, Inkinen RI, von der Mark K, Puustjärvi K, Arokoski J, Hyttinen MM, Lammi MJ (1998) Localization of type X collagen in the intervertebral disc of mature beagle dogs. Matrix Biol 17: 449-453.
Laurent TC, Fraser JRE (1992) Hyaluronan. FASEB J 6: 2397-2404.
Noonan KJ, Stevens JW, Tammi R, Tammi M, Hernandez JA, Midura RJ (1996) Spatial distribution of CD44 and hyaluronan in the proximal tibia of the growing rat. J Orthop Res 14: 573-581.
Parkkinen JJ,Häkkinen TP, Savolainen S, Wang C, Tammi R, Ågren UM, Lammi MJ, Arokoski J, Helminen HJ, Tammi MI (1996) Distribution of hyaluronan in articular cartilage as probed by a biotinylated binding region of aggrecan. Histochem Cell Biol 105: 187-194.
Pitsillides AA, Archer CW, Prehm P, Bayliss MT, Edvards JCW (1995) Alterations in hyaluronan synthesis during developing joint cavitation. J Histochem Cytochem 43: 263-273.
Puustjärvi K, Lammi M, Kiviranta I, Helminen HJ, Tammi M (1993a) Flat bed scanner in the quantitative assay of 35SO4-incorporation by X-ray film autoradiography of intervertebral disc sections. Histochem 99: 67-73.
Puustjärvi K, Lammi MJ, Helminen HJ, Inkinen R, Tammi M (1993b) Proteoglycans in the intervertebral disc of young dogs following long term running exercise. Connect Tissue Res 30: 225-240.
Rauch U, Karthikeyan L, Maurel P, Margolis RU, Margolis RK (1992) Cloning and primary structure of neurocan, a developmentally regulated, aggregating chondroitin sulfate proteoglycan of brain. J Biol Chem 267: 19536-19547.
Ripellino JA, Klinger MM, Margolis RU, Margolis RK (1985) The hyaluronic acid binding region as a specific probe for the localization of hyaluronic acid in tissue sections. Application on chick embryo and rat brain. J Histochem Cytochem 33: 1060-1066.
Roberts S, Urban JPG, Evans H, Eisenstein SM (1996) Transport properties of the human cartilage endplate in relation to its composition and calcification. Spine 21: 415-420.
Ropponen K,Tammi M, Parkkinen J, Eskelinen M, Tammi R, Lipponen P, Ågren U, Alhava E, Kosma V-M(1998) Tumor cell-associated hyaluronan as an unfavorable prognostic factor in colonrectal cancer. Cancer Res 58: 342-347.
Scott JE, Bosworth TR, Cribb AM, Taylor JR (1994) The chemical morphology of age-related changes in human intervertebral disc glycosaminoglycans from cervical, thoracic and lumbar nucleus pulposus and annulus fibrosus. J Anat 184: 73-82.
Shinomura T, Nishida Y, Ito K, Kimata K (1993) cDNA cloning of PG-M, a large chondroitin sulfate proteoglycan expressed during chondrogenesis in chick limb buds. Alternative spiced multiforms of PG-M and their relationships to versican. J Biol Chem 268: 14461-14469.
Suzuki S, Saito H, Yamagata T, Anno K, Seno N, Kawai Y, Furuhashi T (1968) Formation of three types of disulfated disaccharides from chondroitin sulfates by chondroitinase digestion. J Biol Chem 243: 1543-1550.
Säämänen A-M, Tammi M, Kiviranta I, Jurvelin J, Helminen HJ (1989) Levels of chondroitin-6-sulfate and nonaggregating proteoglycans at articular cartilage contact sites in the knees of young dogs subjected to moderate running exercise. Arthritis Rheum 32: 1282-1292.
Tammi R, Ripellino JA, Margolis RU, Tammi M (1988) Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe. J Invest Derm 90: 412-414.
Tammi R, Ågren U, Tuhkanen A-L, Tammi M (1994) Hyaluronan metabolism in skin. Progr Histochem Cytochem 29: 1-81.
Tammi R, Maccallum D, Hascall VC, Pienimäki J-P, Hyttinen M, Tammi M (1998) Hyaluronan bound to CD44 on keratinocytes is displaced by hyaluronan decasaccharides and not hexasaccharides. J Biol Chem 273: 28878-28888.
Thompson JP, Pearce RH, Schechter MT, Adams ME, Tsang IKY, Bishop PB (1990) Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc. Spine 15: 411-415.
Toole BP (1991) Proteoglycans and hyaluronan in morphogenesis and differentiation. In Hay ED, ed. Cell Biology of the Extracellular Matrix. 2nd edn. New York: Plenum Publishing Corp. pp. 305-341.
Wang C, Tammi M, Guo H, Tammi R (1996) Hyaluronan distribution in the normal epithelium of esophagus, stomach, and colon and their cancers. Am J Path 148: 1861-1869.
Yamada H, Watanabe K, Shimonaka M, Wamaguchi Y (1994) Molecular cloning of brevican, a novel brain proteoglycan of aggrecan/versican family. J Biol Chem 269: 10119-10126.
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Inkinen, R., Lammi, M., Ågren, U. et al. Hyaluronan Distribution in the Human and Canine Intervertebral Disc and Cartilage Endplate. Histochem J 31, 579–588 (1999). https://doi.org/10.1023/A:1003898923823
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DOI: https://doi.org/10.1023/A:1003898923823