Calcium Enriched Plasma Membrane-Associated Amorphous Deposits in Chondrocytes of Calcifying Cartilage

Abstract

In previous studies on rat and mouse alveolar bone and rat incisor mantle predentin, amorphous-appearing deposits approximately 5 to 35 nm in diameter were seen to be associated with matrix vesicles in each of these mineralized tissues and with the plasma membranes of osteoblasts and young odontoblasts. It was concluded that these membrane-associated amorphous appearing deposits (MAADs) were mineral because of (1) their inherent electron density in gl utaraldehyde-only fixed tissue and (2) their extract-ability from ultrathin sections by neutral solutions of EDTA (ethylene diamine tetraccetic acid) and EGTA (ethylene glycol bis-(β-aminoethyl ether) N,N1-tetracetic acid). Distilled water and 50% ethanol failed to extract the MAADs from ultrathin sections. While these observations are indicative, they do not offer conclusive evidence for the mineral nature the MAADs. It was considered important to demonstrate the presence of Ca in the deposits by non-destructive analytical methods and to show that these deposits occurred in other calcifying tissues. For these purposes, tibial epiphyses of young growing rats were prepared for routine electron microscopy and energy-dispersive x-ray analysis in the STEM mode of a JE0L-100-CX using previously published procedures (Dougherty, 1978). Ultrathin sections of gl utaral dehyde/osmi urn tetroxide fixed tissue samples were viewed unstained. Figure 1 shows a low magnification TEM image of two chondrocytes in the proliferative zone of the tibial epiphyseal plate. MAADs are present. Figure 2 illustrates in a STEM image at higher magnification two MAADs on another chondrocyte in the proliferative zone. The energy dispersive spectra of point C (an MAAD) and point E (plasma membrane lacking an MAAD) are illustrated in figures 3 and 4. It can be seen that the MAAD at point C is enriched in Ca, while point E is lacking in Ca. Other MAADs on other chondrocytes were enriched in Ca also. Thus, it can be concluded that the MAADs are mineral in nature and that they occur in another major calcifying tissue. It is possible that the MAADs represent the initial nucleation centers in calcifying tissues.