Abstract
The effect of cationic dyes on the ultrastructure of hypertrophic cartilage was compared with results obtained with modern cryotechniques in studies on rat epiphyseal growth plate. Addition of alcian blue, acridine orange, cupromeronic blue, ruthenium hexamine trichloride, ruthenium red, or safranin O to conventional glutaraldehyde/ osmium tetroxide fixatives to a large extent resulted in prevention of chondrocyte shrinkage except for alcian blue which showed poor tissue penetration. The fine structure of the matrix in pericellular and territorial compartments appeared very coarse with areas of high contrast in tissue exposed to fixatives containing cationic dyes. This indicates structural collapse and precipitation of electron-dense material, a pattern clearly differing from that observed in specimens prepared by the cryotechniques. The dyes giving a pattern most similar to that seen after high pressure freezing, freeze substitution, and low temperature embedding were acridine orange and safranin O. It is concluded that studies of matrix ultrastructure down to the molecular level necessitate the application of cryotechniques.
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References
Gordon MK, Olsen BR (1990) The contribution of collagenous proteins to tissue-specific matrix assemblies. Curr Opin Cell Biol 2:833–838
Vaughan L, Mendler M, Huber S, Bruckner P, Winterhalter KH, Irwin MI, Mayne R (1988) D-periodic distribution of collagen type IX along cartilage fibrils. J Cell Biol 106:991–997
Schmid TM, Linsenmayer TF (1985) Immunohistochemical localization of short chain cartilage collagen (type X) in avian tissues. J Cell Biol 100:598–605
Heinegård D, Oldberg Å (1989) Structure and biology of cartilage and bone matrix noncollagenous macromolecules. FASEB J 3:2042–2051
Hedbom E, Heinegård D (1989) Interactions of a 59 kDa connective tissue matrix protein with collagen I and collagen II. J Biol Chem 264:6898–6905
Engfeldt B, Hjertquist S-O (1968) Studies on the epiphyseal growth zone. I. The preservation of acid glycosaminglycans in tissues in some histochemical procedures for electron microscopy. Virchows Arch Abt B Zellpath 1:222–229
Hunziker EB, Herrmann W, Schenk RK (1982) Improved cartilage fixation by ruthenium hexammine trichloride (RHT). A prerequisite for morphometry in growth cartilage. J Ultrastruct Res 81:1–12
Hunziker EB, Herrman W (1987) In situ localization of cartilage extracellular matrix components by immunoelectron microscopy after cryotechnical tissue processing. J Histochem Cytochem 35:647–655
Szirmai JA, Doyle J (1961) Quantitative histochemical and chemical studies on the composition of cartilage. J Histochem Cytochem 9:611–618
Luft JH (1971) Ruthenium red and violet. 1. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anat Rec 171:347–368
Shepard N, Mitchell N (1976) Localization of proteoglycan by light and electron microscopy using safranin O. A study of epiphyseal cartilage. J Ultrastruct Res 54:451–460
Chen K, Wight TN (1984) Proteoglycans in arterial smooth muscle cell cultures: an ultrastructural histochemical analysis. J Histochem Cytochem 32:347–357
Scott JE (1986) Proteoglycan—collagen interactions. Ciba Foundation Symp 124:104–116
Steinbrecht RA, Zierhold K (1987) Cryotechniques in biological electron microscopy. Springer-Verlag, Heidelberg, New York, London, Paris, Tokyo
Moor H, Bellin G, Sandri C, Akert K (1980) The influence of high pressure freezing on mammalian nerve tissue. Cell Tissue Res 209:201–216
Hunziker EB, Herrmann W, Schenk RK, Müller M, Moor H (1984) Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. I. Chondrocyte ultrastructure. Implications for the theories of mineralization and vascular invasion. J Cell Biol 98:267–276
Hunziker EB, Schenk RK (1984) Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. II. Intercellular matrix ultrastructure—preservation of proteoglycans in their native state. J Cell Biol 98:277–282
Engfeldt B, Hultenby K, Müller M (1986) Ultrastructure of hyaline cartilage. I. A comparative study of cartilage from different species and locations using cryofixation, freeze substitution and low-temperature embedding techniques. Acta Pathol Microbiol Immunol Scand (Sect A) 94:313–323
Engfeldt B, Reinholt FP (1992) Structure and calcification of epiphyseal growth cartilage. In: Bonucci E (ed) Calcification in biological systems. CRC Press, Boca Raton, Ann Arbor, London, Tokyo, p 217
Hunziker EB, Ludi A, Herrmann W (1992) Preservation of cartilage matrix proteoglycans using cationic dyes chemically related to ruthenium hexaammine trichloride. J Histochem Cytochem 40:909–917
Shepard N (1992) Role of proteoglycans in calcification. In: Bonucci E (ed) Calcification in biological systems. CRC Press, Boca Raton, Ann Arbor, London, Tokyo, p 41
Humbel B, Müller M (1984) Freeze substitution and lowtemperature embedding. Proc 8th Eur Congr Elec Microsc 3: 1789–1798
Scott JE, Hughes EW (1982) Differential staining of polyanions according to critical electrolyte concentration principles in mixed solvents. J Microsc 129:209–219
Eggli PS, Herrmann W, Hunziker EH, Schenk RK (1985) Matrix compartments in the growth plate of the proximal tibia of rats. Anat Rec 211:246–257
Paulsson M, Mörgelin M, Wiedeman H, Beardmore-Gray M, Dunham D, Hardingham T, Heinegård D, Timpl R, Engel J (1987) Extended and globular protein domains in cartilage proteoglycans. Biochem J 245:763–772
Michel M, Hillmann T, Müller M (1991) Cryosectioning of plant material frozen at high pressure. J Microsc 163:3–18
Takagi M (1990) Ultrastructural cytochemistry of cartilage proteoglycans and their relation to the calcification process. In: Bonucci E, Motta PM (eds) Ultrastructure of skeletal tissues. Bone and cartilage in health and disease. Kluwer Academic Publishers, Boston, Dordrecht, London, p 111
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Engfeldt, B., Reinholt, F.P., Hultenby, K. et al. Ultrastructure of hypertrophic cartilage: Histochemical procedures compared with high pressure freezing and freeze substitution. Calcif Tissue Int 55, 274–280 (1994). https://doi.org/10.1007/BF00310406
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DOI: https://doi.org/10.1007/BF00310406