Surface modifications at the periosseous region of chick osteoclast as revealed by freeze-substitution.

Improved preservation of osteoclast fine structure can be achieved by quick freezing, freeze-substitution, or detergent extraction. With such techniques the ruffled border mainly contains a disorganized, interconnected meshwork of microfilaments (5-7 nm in diameter), whereas in the clear zone a few ordered arrays of intermediate-type filaments (10-12 nm in diameter) are detectable among the network of microfilaments. In well-frozen samples, well-preserved matrix may have occluded the cytoskeleton; detergent extraction permits visualization of the cytoskeletal components. In fresh-frozen cells an extracellular fuzzy coat overlays the ruffled border. At the site of attachment of the clear zone to the bone surface, extracellular cementing material is detected only after quick freezing. The superiority of quick freezing to preserve ultrastructure is shown in various cytoplasmic organelles. Most vesicles and vacuoles found close to the ruffled border seemed not to make contact with the extracellular matrix. Anhydrous procedures using quick freezing and freeze-substitution stabilize bone mineral in some vacuoles and in the channels of the ruffled border.