The cast imaging of the osteon lacunar-canalicular system and the implications with functional models of intracanalicular flow

Abstract

A casting technique with methyl-methacrylate (MMA) was applied to the study of the osteon lacunar-canalicular network of human and rabbit cortical bone. The MMA monomer infiltration inside the vascular canals and from these into the lacunar-canalicular system was driven by capillarity, helped by evaporation and the resulting negative pressure in a system of small pipes. There was uniform, centrifugal penetration of the resin inside some osteons, but this was limited to a depth of four to five layers of lacunae. Moreover, not all of the osteon population was infiltrated. This failure can be the result of one of two factors: the incomplete removal of organic debris from the canal and canalicular systems, and lack of drainage at the osteon external border. These data suggest that each secondary osteon is a closed system with a peripheral barrier (represented by the reversal line). As the resin advances into the osteon, the air contained inside the canalicula is compressed and its pressure increases until infiltration is stopped. The casts gave a reliable visualization of the lacunar shape, position and connections between the lacunae without the need for manipulations such as cutting or sawing. Two systems of canalicula could be distinguished, the equatorial, which connected the lacunae (therefore the osteocytes) lying on the same concentric level, and the radial, which established connections between different levels. The equatorial canalicula radiated from the lacunar border forming ramifications on a planar surface around the lacuna, whereas the radial canalicula had a predominantly straight direction perpendicular to the equatorial plane. The mean length of the radial canalicula was 40.12 ± 10.26 μm in rabbits and 38.4 ± 7.35 μm in human osteons; their mean diameter was 174.4 ± 71.12 nm and 195.7 ± 79.58 nm, respectively. The mean equatorial canalicula diameter was 237 ± 66.04 nm in rabbit and 249.7 ± 73.78 nm in human bones, both significantly larger (P < 0.001) than the radial. There were no significant differences between the two species. The lacunar surface measured on the equatorial plane was higher in rabbit than in man, but the difference was not statistically significant. The cast of the lacunar-canalicular network obtained with the reported technique allows a direct, 3-D representation of the system architecture and illustrates how the connections between osteocytes are organized. The comparison with models derived by the assumption of the role of hydraulic conductance and other mechanistic functions provides descriptive, morphological data to the ongoing discussion on the Haversian system biology.

Fig. 1

Image of the cast during the processing phase of bone matrix decalcification/maceration. Casts of the Haversian canals cleared from the surrounding matrix come out perpendicularly from the MMA base. Some of them are hollow (arrows) as a result of the slowed polymerization of the resin. The white material between canal casts (asterisks) is as yet undigested bone matrix.

Fig. 2

(A) Vascular canal cast from a mono-layer infiltration depth of the lacunar-canalicular network. The light-blue tinted area corresponds to a segment of the hemi-canal surface used to estimate the density of the lacunar cast first layer (n mm⁻²). (B) Diagram illustrating the method of assessment of the hemi-canal segment surface (nr.h). It was assumed that the transverse section of the canal was a regular circumference.

Fig. 3

Osteocyte lacunar cast from a mono-layer infiltration depth of the lacunar-canalicular network. The light-blue tinted area corresponds to the measured, lacunar bone surface: it includes the spur cones of the equatorial processes: they are numbered in blue and the radial in red.

Fig. 4

(A) Cast of a bare vascular canal where the resin penetrated inside the first tract of the canalicula openings into the canal lumen. The collagen fibril pattern is reproduced by the cast showing the ordered, parallel disposition with different angles with the canal axis. Scattered lacunae have been infiltrated by the progression of the resin within the lacunar-canalicular system. (B) Detail of (A) showing the initial cast of the canalicula connecting the canal lumen with the intra-osteonal system. The oval areas where they are more densely packed correspond to the positions of the osteocyte not yet infiltrated by the resin.

Fig. 5

Multi-layered cast: the canalicular network is formed by a mixture of equatorial and radial processes. Longitudinal shrinkage has stretched the radial canalicula along the axis of the central canal. Between the meshes can be observed the lacunar casts of the underlying layer.

Fig. 6

Detail of multi-layered cast: the canalicular network is formed by a mixture of equatorial and radial processes. The latter have been stretched by longitudinal shrinkage.

Fig. 7

(A) Triangulation of the lacunar network of the mono-layer cast. (B) Triangulation of the lacunar network of multi-layer cast. The triangular meshes are stretched along the central canal axis (longitudinal shrinkage) because the lacunar-canalicular system was not supported and stiffened by the bone matrix scaffold after the decalcification/maceration process.

Fig. 8

(A) Equatorial canalicula cast network: the canicula are formed by processes emerging from a cone, with many ramifications which form a network. (B) Radial canalicula casts are formed by elongated and straight processes with no, or very few, ramifications; their origin is from the convex bone or vascular surface of the lacunar cast or from equatorial ramifications.

Fig. 9

Scheme of the equatorial canalicular network (red) in mono-layer casts (A) and the complex pattern established by equatorial and radial (blue) canalicular network in multi-layer casts (B) deformed by longitudinal shrinkage.

Fig. 10

3-D scheme of the lacunar-canalicular system organization.