Abstract
To establish an optimal method for analysis of the collagen structures from unstained tissue sections, a computerized image analysis system using a charge coupled device camera coupled to a polarizing light microscope was used. Retardation values of birefringence, which are proportional to the content and fibril orientation of collagen in the extracellular matrix of articular cartilage, were determined from sections prepared in different ways. In the superficial zone of articular cartilage, the highest retardation values were recorded from sections cut parallel to the so-called split lines indicating the anisotropic arrangement of collagen. Complete digestion of glycosaminoglycans reduced the retardation value by approximately 6.0%, suggesting a minor, but not insignificant, contribution of glycosaminoglycans to the birefringence of the matrix. The use of a mounting medium with a refractive index close to that of the collagen (e.g. DPX) increased the specificity of the method, since the optical anisotropy of collagen derives predominantly from the intrinsic (structural) birefringence. In conclusion, analysis of unstained sections after careful removal of paraffin and glycosaminoglycans from the tissues provides a sensitive and rapid quantitative assessment of oriented collagen structures in articular cartilage
Similar content being viewed by others
References
Arokoski, J., Hyttinen, M., LapvetelÄinen, T., TakÁcs, P., KosztÁczky, B., MÓdis, L., Kovanen, V. & Helminen, H. (1996) Decreased birefringence of the superficial zone and collagen network in the canine knee (stifle) articular cartilage after long distance running training detected by quantitative polarized light microscopy. Ann. Rheum. Dis. 55, 253–64.
Bennett, H. (1950) Methods Applicable to the Study of both Fresh and Fixed Materials with Polarized Light. New York: Paul B. Hoeber.
Benninghoff, A. (1925) Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion. Erste Mitteilung: Die modellierenden und formerhaltenden Faktoren des Knorpelreliefs. Zeitschrift für die gesamte Anatomie 76, 43–63.
Bullough, P. & Goodfellow, J. (1968) The significance of the fine structure of articular cartilage. J. Bone Joint Surg. 50B, 852–7.
Clarke, I. (1974) Articular cartilage: a review and scanning electron microscope study. II. The territorial fibrillar architecture. J. Anat. 118, 261–80.
Constantine, V.S. & Mowry, R.W. (1968) Selective staining of human dermal collagen. II. The use of picrosirius red F3BA with polarization microscopy. J. Invest. Dermatol. 50, 419–23.
Eyre, D., Dickson, I. & Van Ness, K. (1988) Collagen cross-linking in human bone and articular cartilage. Age-related changes in the content of mature hydroxypyridinium residues. Biochem. J. 252, 495–500.
Eyre, D., Wu, J. & Woods, P. (1992) Cartilage Specific Collagens — Structural Studies. New York: Raven Press.
Frey-Wyssling, A. (1953) Submicroscopic Morphology of Protoplasm, 2nd edn. Amsterdam: Elsevier.
Hamperl, H. (1961) Remnants of paraffin ('paraffin-inclusions') in histological sections. Virchows Arch. Path. Anat. 334, 79–80.
Inoue, S. (1981) Video image processing greatly enhance contrast, quality and speed in polarization-based microscopy. J. Cell. Biol. 89, 346–56.
Jeffery, A.K., Blunn, G.W., Archer, C.W. & Bentley, G. (1991) Three-dimensional collagen architecture in bovine articular cartilage. J. Bone Joint Surg. 73B, 795–801.
Junqueira, L., Bignolas, G. & Brentani, R. (1979) Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem. J. 11, 447–55.
Kimura, A., Kawaguchi, T., Ono, T., Sakuma, A., Yokoya, Y., Kochi, H. & Nakamura, K. (1988) Cell surface heparan sulphate and adhesive property of sublines of rat ascites hepatoma AH7974. J. Cell. Sci. 90, 683–9.
Kiviranta, I., Jurvelin, J., Tammi, M., SÄÄmÄnen, A.-M. & Helminen, H. (1985) Microspectrophotometric quantitation of glycosaminoglycans in articular cartilage sections stained with Safranin O. Histochemistry 82, 249–55.
Kuettner, K., Aydelotte, M. & Thonar, E. (1991) Articular cartilage matrix and structure: a minireview. J. Rheumol. Suppl. 18, 46–8.
Li, S.-W., Helminen, H., Faessler, R., LapvetelÄinen, T., KirÁly, K., Pelttari, A., Arokoski, J., Arita, M., Khillan, J. & Prockop, D. (1995) Transgenic mice with targeted inactivation of the Col2a1 gene for collagen II develop a skeleton with membranous and periosteal bone but no endochondral bone. Genes Devel. 9, 2821–30.
Meachim, G., Denham, D., Emery, I. & Wilkinson, P. (1974) Collagen alignments and artificial splits at the surface of human articular cartilage. J. Anat. 118, 101–18.
Minns, R. & Steven, F. (1977) The collagen fibril organization in human articular cartilage. J. Anat. 123, 437–57.
MÓdis, L. (1991) Organization of the Extracellular Matrix: A Polarisation Microscopic Approach. Boca Raton: CRC Press.
MÓdis, L., ÁdÁny, R. & Lakatos, I. (1982) Polarisation-soptische Analyse der menschlichen embryonalen Knorpelmatrix. Acta Histochem. Suppl. 26, 305–12.
Nedzel, G. A. (1951) Intranuclear birefringent inclusions, an artifact occurring in paraffin sections. J. Microsc. Sci. 92, 343–6.
Newton, R.H., Haffegec, J.P. & Ho, M.W. (1995) Polarized light microscopy of weakly birefringent biological specimens. J. Microsc. 180, 127–30.
Oldenburg, R. & Mei, G. (1995) New polarized light microscope with precision universal compensator. J. Microsc. 180, 140–7.
Pickering, J.G. & Boughner, D.R. (1991) Quantitative assessment of the age of fibrotic lesions using polarized light microscopy and digital image analysis. Am. J. Pathol. 138, 1225–31.
Prockop, D.J. & Kivirikko, K.J. (1995) Collagens: molecular biology, diseases, and potentials for therapy. Annu. Rev. Biochem. 64, 403–34.
Puchtler, H., Waldrop, F. & Valentine, L. (1973) Polarization microscopic studies of connective tissue stained with picro-sirius red FBA. Beitr. Path. Bd. 150, 174–87.
Puchtler, H., Meloan, S. & Waldrop, F. (1988) Are picro-dye reactions for collagen quantitative? Chemical and histochemical considerations. Histochemistry 88, 243–56.
Reeves, W., Kanwar, Y. & Farquhar, M. (1980) Assembly of the glomerular filtration surface: differentiation of anionic sites in glomerular capillaries of newborn rat kidney. J. Cell. Biol. 85, 735–53.
RomhÁnyi, G. (1963) Über die submikroskopische struckturelle Grundlage der metachromatischen Reaktion. Acta Histochem. 15, 201–33.
Schaap, C.J. & Forer, A. (1984) Video digitizer analysis of birefringence along the length of single chromosomal spindle fibres. I. Description of the system and general results. J. Cell. Sci. 65, 21–40.
Speer, D. & Dahners, L. (1979) The collagenous architecture of articular cartilage. Clin. Orthop. Rel. Res. 139, 267–75.
Vidal, B. C. (1964) The part played by the mucopoly-saccharides in the form birefringence of collagen. Protoplasma 59, 472–9.
Vidal, B. C. (1980) The part played by proteoglycans and structural glycoproteins in the molecular orientation of collagen bundles. Cell. Mol. Biol. 26, 415–21.
Vidal, B.C. & Vilarta, R. (1988) Articular cartilage: collagen II-proteoglycan interactions. Availability of reactive groups. Variation in birefringence and differences as compared to collagen I. Acta Histochem. 83, 189–205.
Vlachos, J.D. (1968) Birefringence and paraffinophilia of cell nuclei. Stain Technol. 43, 89–95.
Whittaker, P., Boughner, D.R., Perkins, D.G. & Canham, P.B. (1987) Quantitative structural analysis of collagen in chordae tendineae and its relation to floppy mitral values and proteoglycan infiltration. Br. Heart. J. 57, 264–9.
Whittaker, P., Kloner, R.A., Boughner, D.R. & Pickering, J.G. (1994) Quantitative assessment of myocardial collagen with picrosirius red staining and circularly polarized light. Basic Res. Cardiol. 89, 397–410.
Yamada, K., Fujita, Y. & Shimizu, S. (1982) The effect of digestion with keratanase (Pseudomonas sp.) on certain histochemical reactions for glycosaminoglycans in cartilaginous and corneal tissues. Histochem. J. 14, 897–910.
Yamada, K. & Hoshino, M. (1973) Digestion with chondroitinases of acid mucopolysaccharides in rabbit cartilages as revealed by electron microscopy. Histochem. J. 5, 195–7.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kiraly, K., Hyttinen, M.M., Lapvetelainen, T. et al. Specimen preparation and quantification of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy. J Mol Hist 29, 317–327 (1997). https://doi.org/10.1023/A:1020802631968
Issue Date:
DOI: https://doi.org/10.1023/A:1020802631968