Summary
The ferrocyanide-reduced osmium (FRO) fixation method was applied to neonatal mouse mandibular condylar cartilage for its processing for electron microscopy. The results were compared to those obtained by the conventional glutaraldehyde—osmium tetroxide fixation method. Three different stages in the life cycle of condylar cartilage cells were examined. FRO enabled the visualization of delicate fibrillar mesh in the matrix of all three zones of the cartilage, resulting in a dense appearance of the intercellular matrix. The classical stellate shape of matric granules seen in cartilage fixed with glutaraldehyde—osmium tetroxide was not observed in FRO-processed tissues. Chondrocytes that were FRO-processed almost entirely filled their lacunar space. In their pericellular area, fibrillar material and electron-dense aggregates could be demonstrated by the FRO method. As a conclusion of this study, it is recommended to supplement a conventional protocol with the FRO fixation method for routine and research purposes.
Similar content being viewed by others
References
AKISAKA, T. & SHIGENAGA, Y. (1983) Ultrastructure of growing epiphyseal cartilage processed by rapid freezing and freeze-substitution.J. Electron Microsc. (Tokyo) 32, 305–20.
ANDERSON, H. C. & SAJDERA, S. W. (1971) The fine structure of bovine nasal cartilage.J. Cell Biol. 49, 650–63.
ARSENAULT, L. A., OTTENSMEYER, P. F. & HEATH, B. I. (1988) An electron microscopic and spectroscopic study of murine epiphyseal cartilage: analysis of fine structure and matrix vesicles preserved by slam-freezing and freeze-substitution.J. Ultrastruct. Mol. Struct. Res. 98, 32–47.
BRIGHTON, C. T. & HUNT, R. M. (1976) Histochemical localization of calcium in growth plate mitochondria and matrix vesicles.Fed. Proc. 35, 143–7.
CARSON, F. L., DAVID, W. L., MATTHEWS, J. L. & MARTIN, J. H. (1978) Calcium localization in normal, rachitic, and D3-treated chicken epiphyseal chondrocytes utilizing potassium pyroantimonate—osmium tetroxide.Anat. Rec. 190, 23–40.
COLTOFF-SCHILLER, B. & GOLDFISCHER, S. (1981) Glycosaminoglycans in the rat aorta: ultrastructural localization with toluidine blue and osmium-ferrocyanide procedures.Amer. J. Pathol. 105, 232–40.
DAVIS, W. L., JONES, R. G., KNIGHT, J. P. & HAGLER, H. K. (1982) Cartilage calcification: an ultrastructural, histochemical, and analytical X-ray microprobe study of the zone of calcification in the normal avian epiphyseal growth plate.J. Histochem. Cytochem. 30, 221–34.
DEBERNARD, B. (1982) Glycoproteins in the local mechanism of calcification.Clin. Orthop. 162, 233–44.
DE BRUIJN, W. C. & DEN BREEJEN, P. (1976) Glycogen, its procedure which selectively contrasts glycogen. InFourth European Regional Conference on Electron Microscopy (edited by BOCCIARELLI, D. S.), Vol. II, pp. 5–11. Rome: Tipografia Polyglotta Vaticana.
DE BRUIJN, W. C. & DEN BREEKJEN, P. (1976) Glycogen, its chemistry and morphological appearance in the electron microscope. III. Identification of the tissue ligands involved in the glycogen contrast-staining reaction with the osmium (VI)—iron (II) complex.Histochem. J. 8, 121–42.
DE BRUIJN, W. C. & VAN BUITENEN, J. M. H. (1980) X-ray microanalysis of aldehyde-fixed glycogen contraststained by OsVI·FeII and OsVI·RuIV complexes.J. Histochem. Cytochem. 28, 1242–50.
DVORAK, A. M., HAMMOND, M. E., DVORAK, H. F. & KARNOVSKY, M. H. (1972) Loss of cell surface-associated material from peritoneal exudate cells associated with lymphocyte-mediated inhibition of macrophage migration from capillary tubes.Lab. Invest. 27, 561–74.
EGGLI, P. S., HERMANN, W., HUNZIKER, E. B. & SCHENK, R. K. (1985) Matrix compartments in the growth plate of the proximal tibia of rats.Anat. Rec. 211, 246–57.
EINSTEIN, R., SORGENTE, N. & KUETTNER, K. E. (1971) Organization of extracellular matrix in epiphyseal growth plate.Amer. J. Pathol. 65, 515–18.
ENGFELDT, B., HULTENLEY, K. & MULLER, M. (1986) Ultrastructure of hyaline cartilage.Acta Pathol. Microbiol. Immunol. Scand. [A] 94, 313–23.
FARNUM, C. E. & WILSMAN, N. J. (1983) Pericellular matrix of growth plate chondrocytes: a study using postfixation with osmium-ferrocyanide.J. Histochem. Cytochem. 31, 765–75.
FARNUM, C. E. & WILSMAN, N. J. (1987) Morphologic stages of the terminal hypertrophic chondrocyte of growth plate cartilage.Anat. Rec. 219, 221–32.
HASCALL, G. K. (1980) Cartilage proteoglycans: comparison of sectioned and spread whole molecules.J. Ultrastruct. Res. 70, 369–75.
HOWLETT, C. R. (1979) The fine structure of the proximal growth plate of the avian tibia.J. Anat. 128, 377–99.
HUZIKER, E. B., HERRMANN, W. & SCHENK, R. K. (1982) Improved cartilage fixation by ruthenium hexamine trichloride (RHT): a prerequisite for morphometry in growth cartilage.J. Ultrastruct. Res. 81, 1–12.
HUNZIKER, E. B., HERRMANN, W. & SCHENK, R. K. (1983) Ruthenium hexamine trichloride (RHT)-mediated interaction between plasmalemmal components and pericellular matrix proteoglycans is responsible for the preservation of chondrocyte plasma membranesin situ during cartilage fixation.J. Histochem. Cytochem. 31, 717–27.
HUNZIKER, E. B. & SCHENK, K. R. (1984) Cartilage ultrastructure after high-pressure freezing, freeze-substitution, and low-temperature embedding. II. Intercellular matrix ultrastructure: preservation of proteoglycans in their natural state.J. Cell Biol. 98, 277–82.
KARNOVSKY, M. J. (1971) Use of ferrocyanide-reduced osmium tetroxide in electron microscopy (abstract).J. Cell Biol. 54, 284.
KASHIWA, H. K., LUCHTEL, D. L. & PARK, H. Z. (1975) Chondroitin sulfate and electron-lucent bodies in the pericellular rim about unshrunken hypertrophied chondrocytes of chick long bones.Anat. Rec. 183, 359–72.
KHAN, T. A. & OVERTON, J. (1970) Lanthanum staining of developing chick cartilage and reaggregating cartilage cells.J. Cell Biol. 44, 433–8.
LAROS, G. S. & COOPER, R. R. (1972) Electron microscopic visualization of proteinpolysaccharides.Clin. Orthop. Relat. Res. 84, 179–92.
LEWINSON, D. & SILBERMANN, M. (1978) Chondrocyte involvement in condylar cartilage calcification utilizing potassium pyroantimonate—osmium tetroxide.Metab. Bone Dis. Relat. Res. 1, 243–50.
LEWINSON, D. & SILBERMANN, M. (1982) Landmarks in chondrocyte differentiation and maturation as envisaged by changes in the distribution of calcium complexes: an ultrastructural—histochemical study.Metab. Bone Dis. Relat. Res. 4, 143–50.
MATUKAS, V. J., PANNER, B. J. & ORBISON, J. L. (1967) Studies on ultrastructural identification and distribution of proteinpolysaccharide in cartilage matrix.J. Cell Biol. 32, 367–77.
MAUPIN-SZAMIER, P. & POLLARD, T. D. (1978) Actin filament destruction by osmium tetroxide.J. Cell Biol. 77, 837–52.
OI, T. & UTSUMI, N. (1980) Ultrastructure of hypertrophic chondrocytes of rat mandibular condyles using lanthanum-containing fixatives.Arch. Oral Biol. 25, 77–81.
POLLARD, T. D. (1976) The role of actin in the temperaturedependent gelation and contraction of extracts ofAcanthamoeba.J. Cell Biol. 68, 579–601.
RIEMERSA, J. C., ALSBACH, E. J. J. & DE BRUIJN, W. C. (1984) Chemical aspects of glycogen contrast-staining by potassium osmate.Histochem. J. 16, 123–36.
SCHOFIELD, B. H., WILLIAMS, B. R. & DOTY, S. B. (1975) Alcian Blue staining for electron microscopy: application of the critical electrolyte concentration principle.Histochem. J. 7, 139–49.
SHEPARD, N. & MITCHELL, N. (1976a) The localization of proteoglycan by light and electron microscopy using Safranin O.J. Ultrastruct. Res. 54, 451–60.
SHEPARD, N. & MITCHELL, N. (1976b) Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O: a study of epiphyseal cartilage.J. Histochem. Cytochem. 4, 621–9.
SHEPARD, N. & MITCHELL, N. (1977) The use of ruthenium red andp-phenylenediamine to stain cartilage simultaneously for light and electron microscopy.J. Histochem. Cytochem. 25, 1163–8.
SHEPARD, N. & MITCHELL, N. (1981) Acridine orange stabilization of glycosaminoglycans in beginning endochondral ossification.Histochemistry 70, 107–14.
SHEPARD, N. & MITCHELL, N. (1985) Ultrastructural modifications of proteoglycans coincident with mineralization in local regions of rat growth plate.J. Bone Joint Surg. 67, 455–64.
SILBERMANN, M. & LEWINSON, D. (1978) An electron microscopic study of the premineralizing zone of the condylar cartilage of the mouse mandible.J. Anat. 125, 55–70.
SILBERMANN, M. REDDI, A. H. & HAND, A. R. (1987) Further characterization of the extracellular matrix in the mandibular condyle in neonatal mice.J. Anat. 151, 169–88.
STAGNI, N., FURLAN, G., VITTUR, F., ZANETTI, M. & DeBERNARD, B. (1979) Enzymatic properties of Ca++-binding glycoprotein isolated from preosseous cartilage.Calcif. Tissue Int. 29, 27–32.
TAKAGI, M., PARMLEY, R. T., DENYS, F. R. & KAGEYAMA, M. (1983a) Ultrastructural visualization of complex carbohydrates in epiphyseal cartilage with the tannic acid-salt methods.J. Histochem. Cytochem. 31, 783–90.
TAKAGI, M., PARMLEY, R. T. & DENYS, F. R. (1983b) Ultrastructural cytochemistry and immunocytochemistry of proteoglycans associated with epiphyseal cartilage calcification.J. Histochem. Cytochem. 31, 1089–100.
THYBERG, J. (1977) Electron microscopy of cartilage proteoglycans.Histochem. J. 9, 259–66.
THYBERG, J., LOHMANDER, S. & FRIBERG, U. (1973) Electron microscopic demonstration of proteoglycans in guinea pig epiphyseal cartilage.J. Ultrastruct. Res. 45, 407–27.
TRILLO, A. (1977) Ultrastructure of proteoglycans in human renal arteries from end-stage renal disease.Atherosclerosis 28, 161–9.
WEISS, R. E. & REDDI, A. H. (1981) Appearance of fibronectin during the differentiation of cartilage, bone, and bone marrow.J. Cell Biol. 88, 630–7.
WHITE, D. L., MAZURKIEVICZ, J. E. & BARNETT, R. J. (1979) A chemical mechanism for tissue staining by osmium tetroxide-ferrocyanide mixtures.J. Histochem. Cytochem. 27, 1084–91.
WILSMAN, N. J., FARNUM, C. E., HILLEY, H. D. & CARLSON, C. S. (1981) Ultrastructural evidence of a functional heterogeneity among physeal chondrocytes in growing swine.Amer. J. Vet. Res. 42, 1547–53.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lewinson, D. Application of the ferrocyanide-reduced osmium method for mineralizing cartilage: Further evidence for the enhancement of intracellular glycogen and visualization of matrix components. Histochem J 21, 259–270 (1989). https://doi.org/10.1007/BF01757178
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01757178