Ultrastructural study of upper surface layer in rat articular cartilage by "in vivo cryotechnique" combined with various treatments

Abstract

The ultrastructures of the upper surface layer of rat articular cartilage were studied with our "in vivo cryotechnique" followed by freeze-substitution method for scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Rat hip or knee articular cartilage was quickly frozen by the in vivo cryotechnique with liquid isopentane-propane cryogen (-193 degrees C), and surface areas of some frozen specimens were freeze-fractured with a scalpel in liquid nitrogen. They were freeze-substituted and freeze-dried, ion-sputtered, and then observed in SEM. Other frozen specimens were routinely freeze-substituted and embedded in epoxy resin for TEM. Many globular structures were detected in the thick upper surface layer that had not been revealed by the conventional fixation methods. Their sizes were reduced by Triton X-100 treatment, and their localization was also detected in synovial fluid, as revealed by SEM. Such globular lipid-like structures in the upper surface layer of hip or knee articular cartilage might contribute to joint lubrication.

Replica immunoelectron microscopic study of the upper surface layer in rat mandibular condylar cartilage by a quick-freezing method

“…Moreover, the present study supports another idea of a mechanical protection barrier at the upper surface layer, which was formed as a bilaminar structure integrated by hyaluronic acid and fibronectin, as already reported (Zea-Aragon et al 2004). By examining the upper surface layer of the articular cartilage at higher resolution with the QF-DE method, we have detected tiny granular structures around filamentous networks, which may probably correspond to phospholipids, as reported in the previous paper (Watanabe et al 2000). These findings also let us speculate on the possibility that the articular surface layer in vivo contained a mixture of several components which function in a group to obtain boundary lubrication of the rat TMJ.…”

“…The present experiment, as reported before (Toriumi et al 1996;Leng et al 1998;Watanabe et al 2000), was performed in accordance with the guidelines governing animal experiments within the University of Yamanashi.…”

“…The articular disks were slightly cut with razor blades exposing the articular surfaces and then immediately frozen in the isopentane-propane mixture (193C) cooled in liquid nitrogen (Fig. 1b), as reported before (Watanabe et al 2000). The…”

“…For observation of the pure three-dimensional morphology, some of the QF-DE replica membranes with the specimens were floated on household bleach solution to dissolve the cartilaginous tissue components, as described before (Toriumi et al 1996;Watanabe et al 2000). The cleaned replica membranes were picked up onto Formvar-filmed grids and observed in a Hitachi H-7500 transmission electron microscope, as described above.…”

Advanced Application of the In Vivo Cryotechnique to Immunohistochemistry for Animal Organs

“…The lack of blood supply into some organs can easily modify their ultrastructure and molecular distributions, presumably induced by anoxia and rapid loss of blood volume and pressure [17,18,26,27,[30][31][32]35]. In addition, such loss of blood supply, inevitable in experimental studies involving conventional chemical fixation or common QF of fresh tissue specimens, sometimes modifies the immunoreactivity of various functional molecules, due to the rapid ischemia, which induces diverse responses in living animal organs [8,29].…”