A. Larry Arsenault

Crystals in calcified epiphyseal cartilage and cortical bone of the rat

SummaryThe size of hydroxyapatite (HAP) crystals in calcified cartilage and cortical bone of the rat has been studied and compared with that of synthetic poorly crystalline hydroxyapatite (PCHA). Crystal size was determined by X-ray diffraction and selected-area dark field imaging, and their elemental compositions were determined by emission spectroscopy. The crystal size of cartilage, bone, and PCHA were found to be between 120 and 170 Å in length by 50 Å in width as determined by both X-ray diffraction and dark field imaging. Cartilage had a lower ash weight than bone but both have the same Ca/P ratio of 1.6. These findings, though in agreement with the X-ray diffraction literature, differ from observations made by conventional bright field electron microscopy. We conclude that mineral sites in cartilage and bone, which exist as platelike structures, are in fact aggregates of small 120–170×50 Å HAP crystals. The consequences of these findings are discussed in view of crystal relationships with collagen and other macromolecules.

A comparative electron microscopic study of apatite crystals in collagen fibrils of rat bone, dentin and calcified turkey leg tendons.

Crystal-collagen relationships in calcified turkey leg tendons and cortical bone and dentin of the rat were studied by bright field and selected-area dark field electron microscopy. The latter imaging technique enables the specific and direct visualization of apatite crystal sizes and their crystallographic orientations within collagen fibrils. Cortical bone possessed the longest mean c-axial length (170 +/- 50 A), then the tendon (142 +/- 43 A) and the smallest was dentin (110 +/- 30 A). Crystallographic orientations of apatite were found to alternate between a,b- and c-axial planes along the axial period of longitudinally sectioned collagen. This distribution of apatite may reflect a crystal alignment with collagen molecules as they spiral in superhelical fashion along the long axis of the collagen fibril. Apatite crystals were localized within both the gap and overlap zones of collagen fibrils even at very early stages of mineralization. The relative amounts of mineral within single collagen periods were determined as a function of electron absorbency. In the tendon at the onset of mineralization 80% of the mineral was located in the gap zone and 20% in the overlap zone; with further mineralization these relative amounts changed to 55% in the gap zone and 45% in the overlap zone. This 55/45% ratio observed in the heavily mineralized tendon was also observed in both cortical bone and dentin. The implications of these findings are discussed in view of collagen molecular ordering and the spread of apatite along collagen fibrils.

Electron microscopic analysis of mineral deposits in the calcifying epiphyseal growth plate

SummaryEarly mineral deposits within calcifying rat epiphyseal growth plates were studied by bright field and selected-area dark field electron microscopy, and X-ray microanalysis. These mineral deposits were preparedin situ by high-pressure freezing, freeze substitution, and low-temperature embedding, and were examined in unstained, stained, and ethyleneglycol tetraacetic acid (EGTA)-treated stained thin sections. On unstained sections mineral rods occur within an amorphous density of calcium and phosphorus (CaP). X-ray microanalysis of stained sections reveals that the location of electron-dense deposits does not always correspond to that of the CaP mineral deposits identified in electron microscopic images. Such an analysis showed a depletion of both Ca and P in stained sections at sites corresponding to high levels of these elements in unstained sections. Staining thus demineralizes early deposition sites of CaP; at the same time lead (Pb) and uranium (U) bind to the organic components of the extracellular matrix formerly associated with Ca and P. This substitution phenomenon alters the overall fine structure of mineral sites by depleting the amorphous density of Ca and P, and by creating isolated rodlike structures that have formerly been interpreted as representing hydroxyapatite (HAP) crystals. Selected-area dark field imaging shows nascent sites of HAP crystals to be associated with the limiting membrane of matrix vesicles, but such crystals were undetectable at these sites with conventional bright field images. Dark field imaging also showed that the typical 30–80 nm crystal rods found in calcified cartilage consist of aggregates of HAP crystals.

An electron microscopic and spectroscopic study of murine epiphyseal cartilage: Analysis of fine structure and matrix vesicles preserved by slam freezing and freeze substitution

Newborn mice epiphyseal growth plates were preserved by slam freezing/freeze substitution and examined by conventional electron microscopy, stereopsis, high voltage electron microscopy, and electron spectroscopic imaging (ESI). To illustrate the improved ultrastructure of this cryogenic procedure, conventional, aqueously fixed growth plates were included showing collapsed hypertrophic chondrocytes surrounded by a depleted and condensed extracellular matrix. In contrast, the cryogenically prepared epiphyses contain chondrocytes and extracellular matrix vesicles both in direct contact with proteoglycan filaments retained in an expanded state. ESI is an electron microscopic technique which enables the direct localization of atomic elements superimposed over fine structural details. This technique was used to examine the colocalization of calcium and phosphorus within matrix vesicles and within their associated extracellular environments. Matrix vesicles appeared in three distinct diameter ranges. The integrity of the matrix vesicles was examined at various stages of mineralization and also within the mineralized zone of provisional calcification.