Ultrastructural evaluation of shrinkage artefacts induced by fixatives and embedding resins on osteocyte processes and pericellular space dimensions.

The integrity of the interface between the osteocyte (Ot) process and the canalicular wall was investigated in terms of change in the lateral dimensions of the Ot process in relation to the canalicular width, i.e., widening of the pericellular space. This has been interpreted as shrinkage of the Ot process relative to the canalicular wall during sample preparation stages of fixation, dehydration, and resin embedding. Sprague-Dawley rat tibial cross-sections were prepared for transmission electron microscopy (TEM). Four different fixative preparations: paraformaldehyde (PF), modified Karnovsky's (MK), glutaraldehyde (GRR) with ruthenium red (GRR), and zinc formalin (ZF); and two different embedding resins: LR Gold (LRG) and Epon812 (Epon) were evaluated. It was found that for LRG embedding, formalin-only fixatives (PF and ZF) induced lower shrinkage than GRR-containing fixatives (MK and GRR). In contrast, for Epon embedding, MK showed the highest shrinkage, while no differences were found between the remaining fixatives (PF, ZF, and GRR). All formalin-containing fixatives (MK, PF, and ZF) induced similar shrinkage in both embedding media. The most dramatic difference was for GRR fixation, which in combination with LRG embedding showed ∼ 62% more shrinkage than with Epon embedding, suggesting that the combination of GRR fixation and LRG embedding synergistically amplifies Ot shrinkage. These differences likely suggest a role of the resin in secondarily influencing the tissue structure following fixation. Further, the work confirms LRG as a poor embedding medium for bone specimens, as it causes large variations in shrinkage depending on fixation.