Attachment of osteocyte cell processes to the bone matrix

Abstract

In order for osteocytes to perceive mechanical information and regulate bone remodeling accordingly they must be anchored to their extracellular matrix (ECM). To date the nature of this attachment is not understood. Osteocytes are embedded in mineralized bone matrix, but maintain a pericellular space (50-80 nm) to facilitate fluid flow and transport of metabolites. This provides a spatial limit for their attachment to bone matrix. Integrins are cell adhesion proteins that may play a role in osteocyte attachment. However, integrin attachments require proximity between the ECM, cell membrane, and cytoskeleton, which conflicts with the osteocytes requirement for a pericellular fluid space. In this study, we hypothesize that the challenge for osteocytes to attach to surrounding bone matrix, while also maintaining fluid-filled pericellular space, requires different "engineering" solutions than in other tissues that are not similarly constrained. Using novel rapid fixation techniques, to improve cell membrane and matrix protein preservation, and transmission electron microscopy, the attachment of osteocyte processes to their canalicular boundaries are quantified. We report that the canalicular wall is wave-like with periodic conical protrusions extending into the pericellular space. By immunohistochemistry we identify that the integrin alphavbeta3 may play a role in attachment at these complexes; a punctate pattern of staining of beta3 along the canalicular wall was consistent with observations of periodic protrusions extending into the pericellular space. We propose that during osteocyte attachment the pericellular space is periodically interrupted by underlying collagen fibrils that attach directly to the cell process membrane via integrin-attachments.

Figure 1

TEM images showing the results of the fixation using the traditional Karnovsky’s approach. The cell (OC) has significantly shrunken back from the lacunar wall (LW), cell membranes and pericellular matrix organization are poorly preserved.

Figure 2

TEM images showing the results of osteocyte fixation using the Acrolein-based fixative, demonstrating markedly improved osteocyte cell membrane and bone matrix protein preservation.

Figure 3

TEM images showing the results of osteocyte fixation using the Acrolein-based fixative. Arrow in the enlargement at right shows the pericellular matrix that occupies the space between the cell body and the lacunar wall

Figure 4

TEM photomicrograph of osteoctye (A) shows osteocyte and enlarged longitudinal (B) and cross-sections (C) of cell process showing that the bony wall of the canaliculus has protrusions projecting from the wall completely across the pericellular space to contact the cell membrane of the osteocyte process. These protrusions were internally composed of collagen fibrils, identical in size and appearance to other collagen fibrils observed in the bone. That these protrusions have similar profiles in longitudinal and transverse orientations indicates a axisymmetrical structure like a small hillock.

Figure 5

TEM image of osteocyte in osteoid (Acrolein-based fixation) showing that in newly formed bone matrix, collagen fibrils are in intimate contact with the cell membrane along the entire osteocyte cell body and process.

Figure 6

Fluorescence photomicrographs showing the different distributions of β1 and β3 integrin staining in osteocytes and canaliculi in cortical bone. Image A1 demonstrates staining for β1 integrin at osteocyte cell bodies and lacunae (arrows; A2 shows non-immune serum negative control). Images B1 and C show intense staining for β3 integrin along osteocyte processes throughout the bone (double arrows), with the staining in a punctate, “string of pearls” distribution (B2 shows non-immune serum negative control).

Figure 7

TEM image of cell process in osteocyte canaliculus; enlargements demonstrate the collagen “hillocks” versus tethering fibers that occupy the space between the cell process and the lacunar wall