The application of heat-deproteinization to the morphological study of cortical bone: A contribution to the knowledge of the osteonal structure.
Observation of heat-deproteinized cortical bone specimens in incident light enabled the high definition documentation of the osteonal pattern of diaphyseal Haversian bone. This prompted a study to compare these images with those revealed by polarized light microscopy, carried out either on decalcified or thin, undecalcified, resin-embedded sections. Different bone processing methods can reveal structural aspects of the intercellular matrix, depending on the light diffraction mode: birefringency in decalcified sections can be ascribed to the collagen fibrils orientation alone; in undecalcified sections, to both the ordered layout of collagen and the inorganic phase; in the heat-deproteinized samples, exclusively to the hydroxyapatite crystals aggregation mode. The elemental chemical analysis documented low content of carbon and hydrogen, no detectable levels of nitrogen and significantly higher content of calcium and phosphorus in heat-deproteinized samples, as compared with dehydrated controls. In both samples, the X-ray diffraction (XRD) pattern did not show any significant difference in pattern of hydroxyapatite, with no peaks of any possible decomposition phases. Scanning electron microscopic (SEM) morphology of heat-deproteinized samples could be documented with the fracturing technique facilitated by the bone brittleness. The structure of crystal aggregates, oriented in parallel and with marks of time periods, was documented. Comparative study of deproteinized and undecalcified samples showed that the matrix inorganic phase did not undergo a coarse grain thermal conversion until it reached 500°C, maintaining the original crystals structure and orientation. Incident light stereomicroscopy, combined with SEM analysis of deproteinized bone fractured surfaces, is a new enforceable technique which can be used in morphometric studies to improve the understanding of the osteonal dynamics. Microsc. Res. Tech. 79:691-699, 2016. © 2016 Wiley Periodicals, Inc.
Department of Medical and Surgical Specialities, Radiological Sciences and Public Health, University of Brescia, Brescia, Italy.
Ugo E
UE
Pazzaglia
Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy.
Terenzio
T
Congiu
Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy.
Petra
P
Basso
Department of Mechanical and Industrial Engineering, Chemistry for Technologies Lab, University of Brescia, Brescia, Italy.
Ivano
I
Alessandri
Department of Chemistry, University of Pavia, Pavia, Italy.
Lucia
L
Cucca
Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy.
Mario
M
Raspanti
D000328:Adult; D003094:Collagen / Q000737:chemistry / Q000302:isolation & purification; D000071538:Cortical Bone / Q000648:ultrastructure*; D006253:Haversian System / Q000648:ultrastructure*; D006358:Hot Temperature; D006801:Humans; D006882:Hydroxyapatites / Q000737:chemistry; D008297:Male; D008855:Microscopy, Electron, Scanning
D006882:Hydroxyapatites; D003094:Collagen
collagen fibrils; cortical bone; heat-deproteinization; hydroxyapatite; osteon