Ultrastructural localization of calcium in normal and pathologic cartilage

Part of the Electron Microscopy in Biology and Medicine book series (EMBM, volume 7)


This chapter intends to provide the reader with a review of some of the basic techniques concerned with the visualization of calcium at the ultrastructural level in normal mineralizing cartilage and in cases of cartilage pathologies. In general, calcium can be found in various forms in mineralizing tissues: ionic, covalently bound to a carrier (such as enzymes, calcium-binding proteins, glycoproteins, or glycolipids), or in a solid phase such as a crystal [1,2]. In recent years several methodologies have been developed in trying to demonstrate, both qualitatively and/or quantitively, the in-situ distribution of calcium in in-vivo models.


Growth Plate Matrix Vesicle Mandibular Condyle Epiphyseal Cartilage Hypertrophic Zone 
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  1. 1.
    Boskey AL: Current concepts ot the physiology and biochemistry of calcification. Clin Orthop Relat Res 157: 225–257, 1981.PubMedGoogle Scholar
  2. 2.
    Wuthier RE: A review of the primary mechanism of endochondral calcification with special emphasis on the role of cells, mitochondria, and matrix vesicles. Clin Orthop Relat Res 169: 219–257, 1982.PubMedGoogle Scholar
  3. 3.
    Cameron DA: The fine structure of bone and calcified cartilage. Clin Orthop 26: 199–228, 1963.PubMedGoogle Scholar
  4. 4.
    Scott BL, Pease DS: Electron microscopy of the epiphyseal apparatus. Anat Rec 126: 465–495, 1956.PubMedCrossRefGoogle Scholar
  5. 5.
    Robinson RA, Cameron DA: Electron microscopy of cartilage and bone matrix at the distal epiphyseal line of the femur in the newborn infant. J Biophys Biochem Cvtol 2: 253–260, 1956.CrossRefGoogle Scholar
  6. 6.
    Takuma S: Electron microscopy of the developing cartilagenous epiphysis. Arch Oral Biol 2: 111–119, 1960.PubMedCrossRefGoogle Scholar
  7. 7.
    Bonucci E: Fine structure of early cartilage calcification. J Ultrastruct Res 20: 33–50, 1967.PubMedCrossRefGoogle Scholar
  8. 8.
    Anderson HC: Vesicles associated with calcification in the matrix of epiphyseal cartilage. J Cell Biol 41: 59–72, 1969.PubMedCrossRefGoogle Scholar
  9. 9.
    Anderson HC, Matsuzawa T, Sajdera SW, Ali SY: Membranous particles in calcifying cartilage matrix. Trans NY Acad Sci 32: 619–628, 1970.Google Scholar
  10. 10.
    Bonucci E: The locus of initial calcification in cartilage and bone. Clin Orthop 78: 108–139, 1971.PubMedCrossRefGoogle Scholar
  11. 11.
    Thyberg J: Electron microscopic studies on the initial phases of calcification in guinea pig epiphyseal cartilage. J Ultrastruct Res 46: 206–218, 1974.PubMedCrossRefGoogle Scholar
  12. 12.
    Crissman RS, Low RN: A study of fine structural changes in the cartilage-to-bone transition within the developing chick vertebrae. Am J Anat 140: 451–470, 1974.PubMedCrossRefGoogle Scholar
  13. 13.
    Simon DR, Berman I, Howell DS: Relationship of extracellular matrix vesicles to calcification in normal and healing rachitic epiphyseal cartilage. Anat Rec 176: 167–180, 1973.PubMedCrossRefGoogle Scholar
  14. 14.
    Atkin I, Pita JC, Ornoy A, Agundez A, Castiglione G, Howell DS: Effects of vitamin D metabolites on healing of low-phosphate, vitamin D-deficient induced rickets in rats. Bone 6: 113–123, 1985.PubMedCrossRefGoogle Scholar
  15. 15.
    Silbermann M, Lewinson D: An electron microscopic study of the premineralizing zone of the condylar cartilage of the mouse mandible. J Anat 125: 55–70, 1978.PubMedGoogle Scholar
  16. 16.
    Appleton J: The ultrastructure of the articular tissue of the mandibular condyle in the rat. Arch Oral Biol 20: 823–826, 1975.PubMedCrossRefGoogle Scholar
  17. 17.
    Meickle MC: The mineralization of condylar cartilage in the rat mandible: An electron microscopic enzyme histochemical study. Arch Oral Biol 21: 33–43, 1976.CrossRefGoogle Scholar
  18. 18.
    Dearden LC: Enhanced mineralization of the tibial epiphyseal plate in the rat following propylthiouracil treatment: A histochemical, light and electron microscopic study. Anat Rec 178: 671–690, 1974.PubMedCrossRefGoogle Scholar
  19. 19.
    Bonucci E: Matrix vesicle formation in cartilage of scorbutic guinea pigs: Electron microscope study of serial sections. Metab Bone Dis Relat Res 1: 205–212, 1978.CrossRefGoogle Scholar
  20. 20.
    Silbermann M, Lewinson D, Toister Z: Early cartilage response to systemic glucocorticoid administration: An ultrastructural study. Metab Bone Dis Relat Res 2: 267–279, 1980.CrossRefGoogle Scholar
  21. 21.
    Bonucci E, Dearden LC: Matrix vesicles in aging cartilage. Fed Proc 35: 163–168, 1976.PubMedGoogle Scholar
  22. 22.
    Martin JH, Matthews JL: Mitochondrial granules in chon-drocytes. Calcif Tissue Res 3: 184–193, 1969.PubMedCrossRefGoogle Scholar
  23. 23.
    Matthews JL, Martin JH, Lynn JA, Collins EJ: Calcium incorporation in the developing cartilaginous epiphysis. Calcif Tissue Res 1: 330–336, 1968.PubMedCrossRefGoogle Scholar
  24. 24.
    Matthews JL, Martin JH, Collins EJ: Metabolism of radioactive calcium by cartilage. Clin Orthop 58: 213–223, 1968.PubMedGoogle Scholar
  25. 25.
    Brighton CT, Hunt RM: Mitochondrial calcium: Its role in calcification. Clin Orthop Relat Res 100: 406–416, 1973.CrossRefGoogle Scholar
  26. 26.
    Brighton CT, Hunt RM: Histochemical localization of calcium in growth plate mitochondria and matrix vesicles. Fed Proc 35: 143–147, 1976.PubMedGoogle Scholar
  27. 27.
    Lewinson D, Silbermann M: Chondrocyte involvement in condylar cartilage calcification utilizing potassium pyroantimonate-osmium tetroxide. Metab Bone Dis Relat Res 1: 243–250, 1978.CrossRefGoogle Scholar
  28. 28.
    Appleton J, Morris DC: The use of potassium pyroantimonate-osmium method as a means of identifying and localizing calcium at the ultrastructural level in the cells of calcifying systems. J Histochem Cytochem 27: 676–680, 1979.PubMedCrossRefGoogle Scholar
  29. 29.
    Morris DC, Appleton J: Ultrastructural localization of calcium in the mandibular condylar growth cartilage of the rat. Calcif Tissue Int 30: 27–34, 1980.PubMedCrossRefGoogle Scholar
  30. 30.
    Lewinson D, Silberman M: 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: 143–150, 1982.PubMedCrossRefGoogle Scholar
  31. 31.
    Burger EH, de Bruijn WC: Mitochondrial calcium of intact and mechanically damaged bone and cartilage cells studied with K-pyroantimonate. Histochemistry 62: 325–336, 1979.PubMedCrossRefGoogle Scholar
  32. 32.
    Barnard T: Mitochondrial matrix granules, dense particles, and the sequestration of calcium by mitochondria. Scanning Electron Microsc II: 419–433, 1981.Google Scholar
  33. 33.
    Lewinson D, Silbermann M: In vitro precocious accumulation of calcium and matrix vesicles formation in young cartilage cells: Specific effects of corticosteroids. Calcif Tissue Int 36: 702–710, 1984.PubMedCrossRefGoogle Scholar
  34. 34.
    Carson FL, Davis WL, Matthews JL, Martin JH: Calcium localization in normal, rachitic, and D3-treated chicken epiphyseal chondrocytes utilizing potassium pyroantimonate-osmium tetroxide. Anat Rec 190: 23–40, 1978.PubMedCrossRefGoogle Scholar
  35. 35.
    Brighton CT, Hunt RM: Electron microscopic pyroantimonate studies of matrix vesicles and mitochondria in the rachitic growth plate. Metab Bone Dis Relat Res 1: 199–204, 1978.CrossRefGoogle Scholar
  36. 36.
    Brighton CT, Hunt RM: The role of mitochondria in growth plate calcification as demonstrated in a rachitic model. J Bone Joint Surg 60A: 630–639, 1978.PubMedGoogle Scholar
  37. 37.
    Dougherty WJ: Ca-enriched amorphous mineral deposits associated with the plasma membranes of chondrocytesand matrix vesicles of rat epiphyseal cartilage. Calcif Tissue Int 35: 486–495, 1983.PubMedCrossRefGoogle Scholar
  38. 38.
    Brighton CT, Hunt RM: Histochemical localization of calcium in the fracture callus with potassium pyroantimonate. J Bone Joint Surg 68A: 703–715, 1986.PubMedGoogle Scholar
  39. 39.
    Kashiwa HK, Thiersch NJ: Evaluation of potassium pyroantimonate/sucrose/glutaraldehyde concentration and incubation time as essential variates for localizing calcium bound to organic compounds in epiphyseal chondrocytes. J Histochem Cytochem 32: 1055–1065, 1984.PubMedCrossRefGoogle Scholar
  40. 40.
    Morris DC, Appleton J: The effects of lanthanum on the ultrastructure of hypertrophic chondrocytes and the localization of lanthanum precipitates in condylar cartilages of rats fed on normal and rachitogenic diets. J Histochem Cytochem 32: 239–247, 1984.PubMedCrossRefGoogle Scholar
  41. 41.
    Weiss GB: Cellular pharmacology of lanthanum. Annu Rev Pharmacol 14: 343–354, 1974.CrossRefGoogle Scholar
  42. 42.
    de Bernard B: Glycoproteins in the local mechanism of calcification. Clin Orthop Relat Res 162: 233–244, 1982.PubMedGoogle Scholar
  43. 43.
    Lewinson D, Silbermann M: Quantitative and distributional changes in the activity of alkaline phosphatase during the maturation of cartilage. J Histochem Cytochem 30: 261–269, 1982.PubMedCrossRefGoogle Scholar
  44. 44.
    Boothroyd K: The problem of demineralization in thin sections of fully calcified bone. J Cell Biol 20: 165–173, 1964.PubMedCrossRefGoogle Scholar
  45. 45.
    Anderson CE, Parker J: Electron microscopy of the epiphyseal cartilage plate. Clin Orthop Relat Res 58: 225–241, 1968.PubMedCrossRefGoogle Scholar
  46. 46.
    Hòhling HJ, Steffens H, Ashton BA, Nicholson WAP: Zeitschrift fur Molekularbiologie der Hartgewebesbildung. Verh Dtsch Ges Pathol 58: 54–71, 1974.PubMedGoogle Scholar
  47. 47.
    Hòhling HJ, Steffens H, Stamm G, Mays U: Transmission microscopy of freeze-dried, unstained epiphyseal cartilage of the guinea pig. Cell Tissue Res 167: 243–263, 1976.PubMedCrossRefGoogle Scholar
  48. 48.
    Hòhling HJ, Barckhaus RH, Krifting ER, Quint P, Althoff J: Quantitative electron microscopy of the early stages of cartilage mineralization. Metab Bone Dis Relat Res 1: 109–114, 1978.CrossRefGoogle Scholar
  49. 49.
    Ali SY, Wisby A: Mitochondrial granules of chondrocytes in cryosections of growth cartilage. Am J Anat 144: 243–247, 1975.PubMedCrossRefGoogle Scholar
  50. 50.
    Ali SY, Craig-Gray J, Wisby A, Philips M: Preparation of thin cryosections for electron probe analysis of calcifying cartilage. J Microsc 111: 65–76, 1977.PubMedCrossRefGoogle Scholar
  51. 51.
    Ali SY, Wisby A, Craig-Gray J: Electron probe analysis of cryosections of epiphyseal cartilage. Metab Bone Dis Relat Res 1: 97–103, 1978.CrossRefGoogle Scholar
  52. 52.
    Appleton J, Lyon K, Swindin KJ, Chesters J: Ultrastructure and energy-dispersive x-ray microanalysis of cartilage after rapid freezing, low-temperature freeze-drying, and embedding in Spurr’s resin. J Histochem Cytochem 33: 1073–1079, 1985.PubMedCrossRefGoogle Scholar
  53. 53.
    Appleton J: x-ray microanalysis of growth cartilage after rapid freezing, low-temperature freeze-drying, and embedding in resin. Scanning Microsc 1: 1135–1144, 1987.Google Scholar
  54. 54.
    Hargest TE, Gay CV, Schraer H, Wasserman AJ: Vertical distribution of elements in cells and matrix of epiphyseal growth plate cartilage determined by quantitative electron probe analysis. J Histochem Cytochem 33: 275–286, 1985.PubMedCrossRefGoogle Scholar
  55. 55.
    Wuthier RE: Electrolytes of isolated epiphyseal chondrocytes, matrix vesicles, and extracellular fluid. Calcif Tissue Res 23: 125–133, 1977.PubMedCrossRefGoogle Scholar
  56. 56.
    Barnard T: Mitochondrial matrix granules, dense particles, and the sequestration of calcium by mitochondria. Scanning Electron Microsc II: 419–434, 1981.Google Scholar
  57. 57.
    Lehninger AL: Mitochondria and calcium transport. Biochem. J 119: 129–138, 1970.PubMedGoogle Scholar
  58. 58.
    Landis WJ: Application of electron probe x-ray analysis to calcification studies of bone and cartilage. Scanning Electron Microsc II: 555–570, 1979.Google Scholar
  59. 59.
    Landis WJ, Glimcher MJ: Electron optical and analytical observations of rat growth plate cartilage prepared by ultracryomicrotomy: The failure to detect a mineral phase in matrix vesicles and the identification of heterodispersed particles and the initial solid phase of calcium phosphate deposited in the extracellular matrix. J Ultrastruct Res 78: 227–268, 1982.PubMedCrossRefGoogle Scholar
  60. 60.
    Ozawa H, Yamamoto T: An application of energy-dispersive x-ray microanalysis for the study of biological calcification. J Histochem Cytochem 31: 210–213, 1983.PubMedCrossRefGoogle Scholar
  61. 61.
    Boyde A, Shapiro IM: Energy-dispersive x-ray elemental analysis of isolated epiphyseal growth plate chondrocyte fragments. Histochemistry 69: 85–94, 1980.PubMedCrossRefGoogle Scholar
  62. 62.
    Shapiro IM, Boyde A: Microdissection-elemental analysis of the mineralizing growth cartilage of the normal and rachitic chick. Metab Bone Dis Relat Res 5: 317–326, 1984.PubMedCrossRefGoogle Scholar
  63. 63.
    Boyde A, Jones SJ: Scanning electron microscopic studies of the formation of mineralized tissues. In: Developmental Aspects of Oral Biology. HC Slavkin, LA Bavetta (eds). New York: Academic Press, p 243–274, 1972.Google Scholar
  64. 64.
    Ornoy A, Langer Y: Scanning electron microscopy studies on the origin and structure of matrix vesicles in epiphyseal cartilage from young rats, Isr J Med Sci 14: 745–752, 1978.PubMedGoogle Scholar
  65. 65.
    Boyde A, Sela J: Scanning electron microscope study of separated calcospherites from the matrices of different mineralizing systems. Calcif Tissue Res 26: 47–49, 1978.PubMedCrossRefGoogle Scholar
  66. 66.
    Lester KS, Ash MM Jr: Scanning electron microscopy of mineralized cartilage in rat mandibular condyle. J Ultrastruct Res 72: 141–150, 1980.PubMedCrossRefGoogle Scholar
  67. 67.
    Shapiro IM, Boyde A: Mineralization of normal and rachitic chick growth cartilage: Vascular canals, cartilage calcification, and osteogenesis. Scanning Microsc 1: 599–606, 1987.PubMedGoogle Scholar
  68. 68.
    Hall TA, Hohling HJ, Bonucci E: Electron probe x-ray analysis of osmiophilic globules as possible sites of early mineralization in cartilage. Nature 231: 535–536, 1971.CrossRefGoogle Scholar
  69. 69.
    Hall TA, Anderson HC, Appleton J: The use of thin specimens for x-ray microanalysis in biology. J Microsc 99: 177–182, 1973.CrossRefGoogle Scholar
  70. 70.
    Ali SY; Analysis of matrix vesicles and their role in the calcification of epiphyseal cartilage. Fed Proc 35: 135–142, 1976.PubMedGoogle Scholar
  71. 71.
    Ali SY, Wisby A, Evans L, Craig-Gray J: The sequence of calcium and phosphorus accumulation by matrix vesicles. Calcif Tissue Res [Suppl]: 490–493, 1977.Google Scholar
  72. 72.
    Davis WL, Jones RG, Knight JP, Hagler HK: Cartilage calcification: An ultrastructural, histochemical. and analytic x-ray microprobe study of the zone of calcification in the normal avian epiphyseal growth plate. J Histochem Cytochem 30: 221–234, 1982.PubMedCrossRefGoogle Scholar
  73. 73.
    Simmons DJ, Arsenis C, Whitson W, Kahn SE, Boskey AL, Gollob N: Mineralization of rat epiphyseal cartilage: A circadian rhythm. Miner Electrolyte Metab 9: 28–37, 1983.PubMedGoogle Scholar
  74. 74.
    Wroblewski J: Elemental changes associated with chon¬drocyte differentiation in rat rib growth plate. Histochemistry 87: 145–149, 1987.PubMedCrossRefGoogle Scholar
  75. 75.
    Althoff J, Quint P, Kreftig ER, Höhling HJ: Morphological studies on the epiphyseal growth plate combined with biochemical and x-ray microprobe analyses. Histochemistry 74: 541–552, 1982.PubMedCrossRefGoogle Scholar
  76. 76.
    Jeanguillaume C: Electron energy-loss spectroscopy and biology. Scanning Microsc 1: 437–450, 1987.PubMedGoogle Scholar
  77. 77.
    Arsenault AL, Ottensmeyer FP: Quantitative spatial distributions of calcium, phosphorus, and sulfur in calcifying epiphysis by high-resolution electron spectroscopic imaging. Proc Natl Acad Sei USA 80: 1322–1326, 1983.CrossRefGoogle Scholar
  78. 78.
    Arsenault AL, Ottensmeyer FP: Visualization of early intramembranous ossification by electron microscopic and spectroscopic imaging. J Cell Biol 98: 911–921, 1984.PubMedCrossRefGoogle Scholar
  79. 79.
    Livne E, Oliver C, Leapman RD, Rosenberg LC, Poole AR, Silbermann M: Age-related changes in the role of matrix vesicles in the mandibular condylar cartilage. J Anat 150: 61–74, 1987.PubMedGoogle Scholar
  80. 80.
    Silbermann M, Reddi AH, Hand AR, Leapman RD, von der Mark K, Franzen A: Further characterization of the extracellular matrix in the mandibular condyle of neonatal mice. J Anat 151: 169–168, 1987.PubMedGoogle Scholar
  81. 81.
    McCarthy DJ, Kohn NN, Faires SS: The significance of calcium phosphate crystals in the synovial fluid of arthritis patients: The “pseudogout” syndrome. I. Clinical aspects. Ann Intern Med 56: 711–737, 1962.Google Scholar
  82. 82.
    Reginato AJ, Schumacher HR, Martinez VA: The articular cartilage in familial chondrocalcinosis: Light and electron microscopic study. Arthritis Rheum 17: 977–992. 1974.PubMedCrossRefGoogle Scholar
  83. 83.
    Boivin G, Lagier R: An ultrastructural study of articular chondrocalcinosis in cases of knee osteoarthritis. Virchows Arch [A] 400: 13–29, 1983.CrossRefGoogle Scholar
  84. 84.
    Bjelle AO: Morphological study of articular cartilage in pyrophosphate arthropathy. Ann Rheum Dis 31: 449–456, 1972.PubMedCrossRefGoogle Scholar
  85. 85.
    Schumacher HR: Ultrastructural findings in chondrocalcinosis and pseudogout. Arthritis Rheum 19: 413–425, 1976.PubMedCrossRefGoogle Scholar
  86. 86.
    Pritzker KPH: Calcium-pyrophosphate crystal arthropathy: A biomineralization disorder. Hum Pathol 17: 543–545, 1986.PubMedCrossRefGoogle Scholar
  87. 87.
    Howell DS, Martell-Pelletier J, Pelletier J-P, Morales S, Munitz O: NTP pyrophosphohydrolase in human chondrocalcinotic and osteoarthritic cartilage. II. Further studies on histologic and subcellular distribution. Arthritis Rheum 27: 193–199, 1984.PubMedCrossRefGoogle Scholar
  88. 88.
    Tenenbaum J, Muniz O, Schumacher HR, Good AE, Howell DS: Comparison of phosphohydrolase activities from articular cartilage in calcium pyrophosphate deposition disease and primary osteoarthritis. Arthritis Rheum 24: 492–500, 1981.PubMedCrossRefGoogle Scholar
  89. 89.
    Renlund RC, Pritzker KP, Cheng PT, Kessler MJ: Rhesus monkey (Macaca mulatta) as a model for calcium pyrophosphate dihydrate crystal deposition disease. J Med Primatol 15, 11–16, 1986.PubMedGoogle Scholar
  90. 90.
    Ali SY, Griffiths S: Matrix vesicles and apatite deposition in osteoarthritis. In: Matrix Vesicles: Proceedings of the 3rd International Conference on Matrix Vesicles. E Ascenzi, E Bonucci, B de Bernard (eds), Milan: Wichtig Editore, p 241–247, 1981.Google Scholar
  91. 91.
    Ali SY, Griffiths S: Formation of calcium phosphate crystals in normal and osteoarthritic cartilage. Ann Rheum Dis [Suppl]: 45–48, 1983.Google Scholar
  92. 92.
    Ali SY: Apatite-type crystal deposition in arthritic cartilage. Scanning Electron Microsc 4: 1555–1566, 1985.Google Scholar
  93. 93.
    Diepe PA: Crystal-induced arthropathies and osteoarthritis. In: Recent Advances in Rheumatology. WW Buchanan, W Carson-Dick, (eds), London: Churchill-Livingstone, p 1–18, 1981.Google Scholar
  94. 94.
    Schumacher HR, Somlyo AP, Rose LT, Maurer K: Arthritis associated with apatite crystals. Ann Intern Med 27: 411–416, 1977.Google Scholar
  95. 95.
    Ohvia T, Ishikawa K: Hydroxyapatite deposition in articular cartilage by intra-articular injections of methylprednisolone. J Bone Joint Surg 68A: 509–520, 1986.Google Scholar
  96. 96.
    Silbermann M, Mordohovich D, Toister Z, Azaria N: Mechanisms involved in mandibular condylopathy secondary to intra-articular injections of glucocorticoids. J Oral Surg 36: 112–117, 1978.PubMedGoogle Scholar
  97. 97.
    Ornov A, Sekeles E, Smith P, Simkin A, Kohn G: Achondrogenesis type I in three sibling fetuses. Am J Pathol 82: 71–84, 1976.Google Scholar

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© Kluwer Academic Publishers 1990

Authors and Affiliations

  1. 1.Faculty of MedicineTechnion-Israel Institute of TechnologyHaifaIsrael

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