Scanning electron microscopic observations of rat tibia using the HCl-collagenase method.

Abstract

Summary. The metaphysis of rat tibia was examined by scanning electron microscopy (SEM) using the HC1-collagenase method (EVAN et al., 1976) and the three-dimensional structure of osteocytes and the relationship between osteocytes and osteoblasts were demonstrated.After the HC1-collagenase treatment, the bone matrix near the exposed surface was completely removed but the cellular components retained their structural integrity.Young osteocytes located close to osteoblasts showed flattened, spindly shapes. They were connected with adjacent osteoblasts by short, wide and slightly branched processes.On the other hand, the osteocytes located in the deeper portion of trabecular bone were smaller in size, being round or oval and flattened in shape. They were connected with both osteoblasts and neighboring osteocytes by long and more extensively branched processes forming a complicated network.From these results, it is suggested that osteoblasts extend their processes perpendicularly to the bone surface, with these processes branching off and connecting to each other at the osteoid layer.Osteocytes have numerous slender processes which extend some distance into the bone canaliculi. These processes contact with those of neighboring osteocytes or osteoblasts by a gap junction to form an extensive cellular network in the bone.But it is difficult to directly observe the three-dimensional fine structure of the cellular network under the scanning electron microscope (SEM) by routine preparative techniques, because the osteocytes and their processes are buried in calcified bone matrix.A few studies on osteocytes demonstrated by the fracturing method under the SEM (BoYDE, 1972;KESSEL and KARDON, 1979) are available, but this method only makes it possible to find osteocytes occurring at the fractured surface by chance and, moreover, most of the processes and their connections are either destroyed or obscured by bone matrix.The present study was designed to reveal the three-dimensional structure of osteocytes and the relationship between osteocytes and osteoblasts or among osteocytes after removing the bone matrix using the HCl-collagenase method originated by EVAN et al. (1976).

The incidence and size of gap junctions between the bone cells in rat calvaria

“…In this respect, it is worth noting firstly that the removal of like contiguous neighbours by the ingress of osteoclasts to the cell sheet must change the flow of communication and, secondly, that the repair phase of the resorption-repair sequence of bone and other calcified tissues often "overflows" on to the unresorbed surface adjacent to a resorbed area once the original cavity has been filled (Jones and Boyde 1972). Doty (1981, ]988) has suggested that volumes of bone may be regulated by gap junction communication of osteocytes, and the extensive links between bone cells have been illustrated by Ejiri and Ozawa (1982). There is little information concerning how far such functionally connected zones might extend in depth.…”

Overexpression of Bcl2 in Osteoblasts Inhibits Osteoblast Differentiation and Induces Osteocyte Apoptosis

“…Ultrastructural analysis was performed using a transmission electron microscope (H-7100; Hitachi, Tokyo, Japan) as previously described [26]. To observe the three-dimensional ultrastructure of osteocytes, the HCl-collagenase method [27] was applied. The treated specimens were observed under a scanning electron microscope (S-3500N; Hitachi).…”

Scanning electron microscopy of bone: Instrument, specimen, and issues

“…However, it is also possible to see their 3D relationships by removing the bone matrix that encases them. Ejiri and Ozawa (1982) employed HCl and collagenase, Johnson and Highison (1983) used formic acid-sodium citrate and collagenase to expose osteocytes, whilst Abe et al (1992) used an EDTA-KOH Fig. 9. (See page 107.)…”