Freeze‐substitution of rabbit tibial articular cartilage reveals that radial zone collagen fibres are tubules

Abstract

Investigations of the micromorphology of rabbit tibial articular cartilage using scanning and transmission electron microscopy revealed that the collagenous elements in the tissue form fluid-containing tubular structures. The commonly described radial or deep zone longitudinal fibres were found to be tubular structures with internal diameters of 1-2 microm. The walls of the tubules were composed of tightly packed fibrils of collagen. The tangential zone, close to the tibial plateau, was composed mainly of a spongy arrangement of collagen fibrils, containing bunches of tangentially lying small (< 1 microm) diameter tubules. The application of conventional chemical fixation techniques resulted in the fine detail of this tissue being obscured. When the tissue was frozen, followed by cryo-scanning electron microscopy or freeze-drying, prior to observation in the scanning electron microscope the tubule structures were not obviously present. It was only by applying freeze-substitution techniques, followed by critical point drying or resin embedding, that the structure was revealed clearly. Segregation of water into ice crystals did occur during the freezing process, but the formation of those crystals played no part in creating the tubular morphology observed. A similar structure was still revealed following pre-treatment with glycerol, methanol or Triton X-100, provided that concentration of these additives was not too high. The walls of the tubules in the radial region were composed of straight, longitudinally arranged as well as helically arranged, 30 nm diameter fibrils. The lumen of the tubules appears to be lined by a circumferentially arranged array of approximately 10 nm diameter fibres, spaced at regular intervals of 50-70 nm.

Novel aspects to the structure of rabbit articular cartilage

“…Much of the argument is dealt with, at some length, in our Journal of Microscopy paper (ap Gwynn et al, 2000). Here we show that the tubular structure is preserved even after the addition of cryoprotectants, and that the dimensions of the tubules remain relatively constant even when different freezing procedures are used.…”

“…The fastest freezing method available was rapid plunging into liquid propane at -196°C. The possibility of the observed tubules being artefacts of ice crystal formation, as a result of the application of conditions that would not be expected to lead to vitrification, has already been discounted (ap Gwynn et al, 2000). Although small ice crystals are observed, the tubular arrangement of collagen fibrils is genuine.…”

“…Cores were taken from plateau areas where the femoral condyle applied pressure. The specimen was then frozen rapidly in liquid propane at liquid nitrogen temperature, fractured under liquid nitrogen using a special jig (Figure 3), freezesubstituted in 1:2 acetone:methanol containing 10% acrolein and 0.2% tannic acid -followed by anhydrous acetone, critical point dried in carbon dioxide using a Polaron E3000 apparatus (Agar Scientific, Stansted, UK), sputter coated with 10nm platinum or 8nm gold-palladium (4:1), using a Baltec (Balzers, Liechtenstein) Med 020 unit, and examined in an FESEM (Hitachi, Tokyo, Japan; s-4100 or s-4700) operated at an appropriate accelerating voltage (ap Gwynn et al, 2000). Other samples were treated, instead of applying critical point drying, by transferring through an ethanol series into Lowicryl K11M embedding resin (Agar Scientific).…”

“…In between the chondrons, the radial zone is seen as an array of tubules, whose walls are composed of a compact matrix of tightly packed collagen fibrils (ap Gwynn et al, 2000). The tubular structure appears to confine the chondrocytes to a vertical chondron column arrangement.…”

The ultrastructure of mouse articular cartilage: Collagen orientation and implications for tissue functionality. A polarised light and scanning electron microscope study and review.

“…Since then light microscopy (Fawns and Landells, 1953;Anderson, 1961;Stockwell and Scott, 1967;Poole, 1970;Rosenberg, 1971;Shephard and Mitchell, 1976;Kincaid and Evander, 1985;Olivier et al, 2001;Hyllested et al, review, 2002), polarised light microscopy (Benninghoff, 1925;MacConail, 1951;Arokoski et al, 1996;Módis et al, 1996;Olivier et al, 2001), transmission electron microscopy (Anderson, 1964;Weiss et al, 1968;Minns and Stevens, 1977;Engfeldt et al, 1986;Broom, 1986;Jurvelin et al, 1996;Hunziker et al, 1997) and scanning electron microscopy (Inoue et al, 1969;Clarke, 1971;Clarke, 1974;Redler, 1974;Ghadially et al, 1975;Clark, 1990;Jeffery et al, 1991;Clark and Simonian, 1997;Kääb et al, 1999;Clark et al, 1999;ap Gwynn et al, 2000;, among other techniques, have all been employed in the attempt to determine the structure of AC. The ultrastructure of AC is an area still under debate and several different models of AC structure are described.…”