Electron microscopy of the bone ground substance using the pseudo-replica technique

The Mineralization of Bone and Its Analogies with Other Hard Tissues

“…Electron microscope studies have clearly shown that the bone mineral is only partly contained in the collagen fibrils, while it is mostly (about 75% of the total according to Pidaparti et al [157]) located in the extrafibrillar space. This appears to be in contrast with the observation, originally reported in the earliest ultrastructural investigations on bone, that the mineral substance is closely related to the periodic banding of collagen [22,23,158], a relationship later repeatedly confirmed (reviewed by [2,3,38,159,160]. The mineral, in fact, because of its intrinsic electron density, produces a reinforcement of the collagen periodic banding (Figs.…”

“…Specific steric relationships between amino-acid side-chains groups in the collagen fibrils were then considered the possible nucleation centers within the fibrils [21] (see below). A close relationship between the inorganic substance and the collagen fibrils was shown and repeatedly confirmed by electron microscopy, starting with the pioneering studies of Robinson and Watson [22] and Ascenzi and Chiozzotto [23]. Another fundamental observation was obtained by microradiography: Amprino and Engström [24] first showed that the degree of bone mineralization is not constant, because that of primary bone is always higher than that of secondary bone; moreover, they found that osteon calcification occurs in two stages, of which the first is characterized by quick deposition of about 70% of the final mineral content, and the second by the slow completion of this process.…”

Biological Calcification

“…Although carried out using replica techniques (Bradley 1960), which provide poorly resolved images, these studies showed a close relationship between collagen periodic binding and the inorganic substance both in bone (Kellenberger and Rouiller 1950;Rutishauser et al 1950;Ascenzi and Chiozzotto 1955) and in dentin (Rouiller et al 1952). Although carried out using replica techniques (Bradley 1960), which provide poorly resolved images, these studies showed a close relationship between collagen periodic binding and the inorganic substance both in bone (Kellenberger and Rouiller 1950;Rutishauser et al 1950;Ascenzi and Chiozzotto 1955) and in dentin (Rouiller et al 1952).…”

“…found that crystals in trabecular bone are smaller than those in compact bone. The earliest ultrastructural studies had low reliability, because of the rather coarse techniques available at that time -either the pseudo-replica method or the blender dissociation of the calcified matrix (Kellenberger and Rouiller 1950;Rutishauser et al 1950;Rouiller et al 1952a, b;Robinson 1952;Schwarz and Pahlke 1953;Ascenzi and Chiozzotto 1955). They found that crystal length rose with density in calvaria (from about 14.5 to about 16.2 nm), while in long bones it increased to a plateau (from about 13.7 to about 15.0 nm), after which it fell back to its initial values, while crystal width fell with density in calvaria (from about 6.1 to about 5.7 nm) and was almost steady in long bones.…”

The fine structure of developing bone in the embryonic fowl

“…(1951) studied the relationship between the osteocytes and matrix of adult bonew by means of a double replica method; they found that collagen fibrils were arranged ) in a radial manner around the osteocytes, but by this technique it was not possible^ to observe any structural details in the cells. Robinson & Watson (1952, 1955? investigated thin sections of calcified and decalcified post-foetal and adult humant bone, but such preparations only demonstrated the relative place occupied by theu cells and canaliculi and provided no cytological information.…”