Abstract
A technique to correlate the ultrastructural distribution of mineral with its organic material in identical sections of mineralized turkey leg tendon (MTLT) and human bone was developed. Osmium or ethanol fixed tissues were processed for transmission electron microscopy (TEM). The mineralized tissues were photographed at high, intermediate, and low magnifications, making note of section features such as fibril geometry, colloidal gold distribution, or section artifacts for subsequent specimen realignment after demineralization. The specimen holder was removed from the microscope, the tissue section demineralized in situ with a drop of 1 N HCl, then stained with 2% aqueous vanadyl sulfate. The specimen holder was reinserted into the microscope, realigned with the aid of the section features previously noted, and rephotographed at identical magnification used for the mineralized sections. A one to one correspondence was apparent between the mineral and its demineralized crystal “ghost” in both MTLT and bone. The fine structural periodic banding seen in unmineralized collagen was not observed in areas that were fully mineralized before demineralization, indicating that the axial arrangement of the collagen molecules is altered significantly during mineralization. Regions that had contained extrafibrillar crystallites stained more intensely than the intrafibrillar regions, indicating that the noncollagenous material surrounded the collagen fibrils. The methodology described here may have utility in determining the spatial distribution of the noncollagenous proteins in bone.
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Arsenault AL (1989) A comparative electron microscopic study of apatite crystals in collage fibrils of rat bone, dentin and calcified turkey leg tendons. Bone Miner 6:165–177
Landis WJ, Song MJ, Leith A, McEwen L, McEwem BF (1993) Mineral and organic matrix interaction in normally calcifying tendon visualized in three dimensions by high-voltage electron microscopic tomography and graphic image reconstruction. J Struct Biol 110:39–54
Traub W, Arad T, Weiner S (1989) Three-dimensional ordered distribution of crystals in turkey tendon collagen fibers. Proc Natl Acad Sci USA 86:9822–9826
Arsenault AL (1988) Crystal-collagen relationships in calcified turkey leg tendons visualized by selected-area dark field electron microscopy. Calcif Tissue Int 43:202–212
Arsenault AL, Frankland BW, Ottensmeyer FP (1991) Vectorial sequence of mineralization in the turkey leg tendon determined by electron microscopic imaging. Calcif Tissue Int 48:46–55
Lee DD, Glimcher MJ (1991) Three-dimensional spatial relationship between the collagen fibrils and the inorganic calcium phosphate crystals of pickerel (Americanus americanus) and Herring (Clupea harengus) bone. J Mol Biol 217:487–504
Lees S, Prostak KS, Ingle VK, Kjoller K (1994) The loci of mineral in turkey leg tendon as seen by atomic force microscope and electron microscopy. Calcif Tissue Int 55:180–189
Ichijo T, Yamashita Y, Terashima T (1993) Observation on structural features and characteristics of biological apatite crystals. 4. Observation on ultrastructure of human bone crystals. Bull Tokyo Med Dent Univ 40:93–112.
McKee MD, Nanci A, Landis WJ, Gerstenfeld LC, Gothoh Y, Glimcher MJ (1989) Ultrastructural immunolocalization of a major phosphoprotein in embryonic chick bone. Connect Tissue Res 21:21–29
McKee MD, Farach-Carson MC, Butler WT, Hauschka PV, Nanci A (1993) Ultrastructural immunulocalization of noncollagenous (osteopontin and osteocalcin) and plasma (albumin and alpha 2HS-glycoprotein) proteins in rat bone. J Bone Miner Res 8:485–496
Sawada T, Nanci A (1995) Spatial distribution of enamel proteins and fibronectin at early stages of rat incisor tooth formation. Arch Oral Biol 40:1029–1038
Hultenby K, Reinholt FP, Norgard M, Oldberg A, Wendel M, Heinegard D (1994) Distribution and synthesis of bone sialoprotein in metaphyseal bone of young rats show a distinctly different pattern from that of osteopontin. Eur J Cell Biol 63:230–239
Garant PR (1978) Microanatomy of the oral mineralized tissues. In: Shaw JH, Sweeney EA, Cappucino CC, Meller SM (eds) Textbook of oral biology. Saunders Company. Philadelphia, p 181
Riminucci M, Silvestrini G, Bonucci E, Fisher LW, P Gehron Robey, Bianco P (1995) The anatomy of bone sialoprotein immunoreactive sites in bone as revealed by combined ultrastructural histochemistry and immunohistochemistry. Calcif Tisue Int 57:277–284
Landis WJ, Arsenault AL (1989) Vesicle- and collagen-mediated calcification in the turkey leg tendon. Connect Tissue Res 22:35–42
Hulmes DJS, Holmes DF, Cummings C (1985) Crystalline regions in collagen fibrils. J Mol Biol 184:473–477
Katsura N (1991) Nanospace theory for biomineralization. In: Suga S, Nakahara H (eds) Mechanisms and phylogeny of mineralization in biological systems. Springer-Verlag, Tokyo, p 193
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Prostak, K.S., Lees, S. Visualization of crystal-matrix structure. In situ demineralization of mineralized turkey leg tendon and bone. Calcif Tissue Int 59, 474–479 (1996). https://doi.org/10.1007/BF00369213
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DOI: https://doi.org/10.1007/BF00369213