The dependences of osteocyte network on bone compartment, age, and disease

Abstract

Osteocytes, the most abundant bone cells, form an interconnected network in the lacunar-canalicular pore system (LCS) buried within the mineralized matrix, which allows osteocytes to obtain nutrients from the blood supply, sense external mechanical signals, and communicate among themselves and with other cells on bone surfaces. In this study, we examined key features of the LCS network including the topological parameter and the detailed structure of individual connections and their variations in cortical and cancellous compartments, at different ages, and in two disease conditions with altered mechanosensing (perlecan deficiency and diabetes). LCS network showed both topological stability, in terms of conservation of connectivity among osteocyte lacunae (similar to the "nodes" in a computer network), and considerable variability the pericellular annular fluid gap surrounding lacunae and canaliculi (similar to the "bandwidth" of individual links in a computer network). Age, in the range of our study (15-32 weeks), affected only the pericellular fluid annulus in cortical bone but not in cancellous bone. Diabetes impacted the spacing of the lacunae, while the perlecan deficiency had a profound influence on the pericellular fluid annulus. The LCS network features play important roles in osteocyte signaling and regulation of bone growth and adaptation.

Figure 1

A representative confocal preview image (a) of basic fuchsin stained sagittal section of a murine distal femur, showing the two ROIs located in metaphyseal cortical and cancellous bone compartments. (b&c) Due to larger number of lacunae in ROI1, a grid was overlaid on the image and 30–40 lacunae that fell on the inter-sections of the grid, as outlined by the yellow boundaries, were selected for a quick check of full-depth structures. Ten lacunae with intact 3D structures were chosen for high-resolution 3D imaging. For ROI2, ten lacunae were randomly chosen per animal without the grid.

Figure 2

Representative 3D renderings built from z-stack confocal images of lacunae and associated canaliculi were used to quantify (a) canalicular number in VOLOCITY® and (b) lacunar volume, surface area and major and minor radii in AMIRA®.

Figure 3

The ultrastructural measurements of ostecyte lacunae were obtained from TEM images by (a) tracing the lacunar wall and cell body in Photoshop® for (b) quantifying the shape of the lacuna (width/height), the cross‐sectional areas of lacuna, cell body, and the pericellular annulus in Image J.(c) The mean thickness of the pericellular annular gap was measured using the “bubble” method implemented in the BoneJ plugin of Image J.

Figure 4

Measurements of osteocytic canaliculi were obtained from TEM images following the same procedure as the measurements of lacunae. (a) Traces of canalicular wall and cell process; (b) Quantification of areas of cell process, canalicular wall, and pericellular regions; (c) Measurements of pericellular thickness in canaliculi using the “bubble” method.

Figure 5

Osteoblasts are differentiated into osteocytes, which form an orderly network through the connecting canaliculi. The number of canaliculi is determined mainly by the surface area of lacuna (N = S[μm²]/5) regardless of bone compartment, age, and disease conditions. In cortical bone, aging is associated with larger canalicular annulus area and lower fiber density, while perlecan (Pln) deficiency reduces both annulus area and fiber density compared with wild types (Tables 6 and 7).