ATTACHMENT OF OSTEOCYTE CELL PROCESSES TO THE BONE MATRIX
+*McNamara, LM, *Majeska, RJ, **Weinbaum, S, *Friedrich, V, *Schaffler, MB
+*Leni and Peter W. May Department of Orthopaedics, Mount Sinai School of Medicine, New York
[email protected]
Introduction: Osteocytes are thought to influence adaptive modeling
and remodeling in response to changes in strain and fluid flow that occur
Protrusion of canalicular
wall touching cell
in bone tissue matrix under physiological loading [1,2]. In order for
membrane
osteocytes to perceive mechanical information they must be anchored to
their extracellular matrix (ECM). However, the precise nature of this
attachment is not understood. Given that osteocytes exist within the
impermeable matrix of mineralized bone, they are surrounded by a
pericellular fluid space, typically 50-80 nm in width, that allows flow
and transport of metabolites, which provides an additional spatial limit
for bone matrix attachment.
Integrins are ubiquitous cell adhesion proteins that couple the
cytoskeleton and bind ECM proteins in almost all tissues. ß1 and ß3
(a)
integrin subunits have previously been shown in bone [3]. Thus,
integrins might be reasonably expected to play a role in osteocyte
attachment. However, integrins attachments typically require close
proximity between the ECM and cell membrane and cytoskeleton, which
conflicts with the osteocytes requirement for a pericellular fluid space.
We hypothesize that the challenge for osteocytes to attach to the
surrounding bone matrix, while also maintaining fluid-filled pericellular
space, requires different “engineering” solutions that in other tissues that
(c)
(b)
are not similarly constrained.
Figure 1: (a) TEM image revealing focal attachments and pericellular
space between the cell process and ECM (b,c) Staining of
Methods: A novel, rapid penetrating fixative was used to optimize
integrin ß3 in canaliculi
preservation of osteocyte cell membranes and surrounding bone matrix
architecture for study using transmission electron microscopy (TEM);
Discussion: In this study, we used a new TEM fixation approach that
this approach was design to overcome some of the well-known
improves osteocyte cell membrane and bone matrix protein preservation.
difficulties in fixation and ultrastructural studies of adult osteocytes. 10Using this approach we report for the first time that the canalicular wall
20 week old mice were perfused with 1% Acrolein, 1% Glutaraldehyde
structure is wave-like and possesses infrequent, but regular protrusions
and 1% Paraformaldehyde in 0.12M phosphate buffer (pH 7.4, 37ºC)
that project across the pericellular space to attach directly to the adjacent
followed by 2% Acrolein, 2% Glutaraldehyde and 2% Paraformaldehyde
cell membrane; these “hillocks” are built upon underlying bone collagen
in the same buffer. Tibiae and femora of were excised and then
fibrils. The exact nature of these structures remain obscure as does their
immersed in secondary fixative for 48 hours. Bones were decalcified in
origin. Recent data [5] suggests that osteocytes use proteolytic enzymes
EDTA in 0.1M TRIS-HCl buffer for 6 weeks and then post-fixed in 1%
to create the canalicular space into which the cell process grows. In this
OsO4. Samples were embedded in Epon 812 and ultrathin sections
context it seems reasonable to speculate that collagen fibrils along the
(20nm) were cut using a diamond knife and mounted on Formvar grids.
new wall that are not removed during the canalicular formation process
Sections were stained with 8% uranyl acetate solution in 50% alcohol
can form typical matrix attachments to the cell membrane if it is nearby.
and lead citrate. Sections were viewed using a Philips 300 transmission
electron microscope. Images of osteocytes were acquired at 80,000The current integrin staining data, showing a punctate pattern of β3
100,000x magnifications.
staining along the canalicular wall, are consistent with this idea.
Immunohistochemistry (IHC) was performed to assess the in situ
The ß1 integrin subunit is found ubiquitously in bone cells and our
presence and localization of selected integrins (i.e., those previously
data support its presence on osteocyte cell bodies. [5,6] However, that
shown in osteoblasts or osteocytes in vitro) in bone. Bones for IHC were
the ß3 subunit is exclusively expressed along the osteocyte cell process
fixed in 10% formalin, decalcified in EDTA, paraffin embedded and
and appears at punctate locations, has not been previously reported. The
sectioned serially at 5 µm. IHC methods: Deparaffinized section were
subunit β3 works in concert with either αIIb and αV subunits. The
quenched in 10% H2O2 to inhibit endogenous peroxidase and then
former complex (αIIbβ3) occurs in platelets, while αvß3 integrin, has
immersed in protein block (DAKO) to block non-specific binding sites.
been identified in human osteocytes. It binds to a wide spectrum of
Sections were incubated with the anti-ß1 integrin monoclonal antibody
matrix ligands found in bone tissue, including vitronectin, fibronectin,
(1:100) or anti-ß3 polyclonal antibody (1:50) (AB1 and AB2,
thrombospondin, osteopontin, tenascin and bone sialoprotein. Of these,
respectively, Santa Cruz). Detection was performed using and Alexaosteopontin is found in abundance along the canalilcular wall [6,7].
Fluor 488 labeled rabbit anti-goat IgG (1:100, Sigma) according to the
manufacturer’s protocol. Normal goat IgG was used as a negative
Conclusion: Osteocyte processes have a complex attachment with their
control. Both integrin ß1 and ß3 subunits are present in platelet cells,
bone walls. The pericellular space that allows fluid flow is periodically
which are found in bone vasculature, thus constituting good positive
interrupted by protrusion of underlying collagen fibrils that bulge across
controls.
this space and attach directly to the membrane of the cell process. The
adhesion of the osteocyte to these sites appears to be integrin-based.
Results TEM studies with this new fixation approach revealed the
Whether these intermittent “spot-welds” between the osteocyte process
expected pericellular space surrounding osteocyte processes. However, it
and the canalicular wall play a role in mechanotransduction or function
was observed that the bony wall had a wave-like structure with periodic
primarily to stabilize the cell against the high levels of fluid shear and
protrusions projecting from the bony wall completely across the
drag forces that occur surrounding osteocytes is not yet known.
pericellular space to contact the cell membrane of the osteocyte process
(Fig 1a). These contact points were frequently asymmetric, appearing on
Acknowledgments: Funded by AR41210 and AR48699
one side of the process but not the other. These protrusions were
References:
composed of collagen fibrils, identical in size and appearance to other
(1) Laynon, 1993, CTI; 53 1:S102 (2) Klein-Nulend, 2003, Curr
collagen fibrils observed in the bone (Fig 1a).
Osteoporos Rep.; 1: 5 (3) Bennett, 2001, Arch Oral Biol.; 46(3): 229-38
Immunohistochemistry: ß1 staining was found at osteocyte cell bodies.
(4) Dalton, 1995, J. Cell Sci. 108, 2083–2092 (5) Karsdal, 2002, JBC,
In contrast, ß3 integrin staining was observed only along osteocyte cell
15; 277(46): 44061-7 (6) Devoll, 1997, CTI, 60(4): 380-6 (7) Noda,
processes. Moreover, ß3 integrin expression was not continuous along
2003, Clin Calcium; 13(4): 464-6. (8) You, 2004, Anat Rec, 278: 505
the process, but rather was in a punctate distributions along the
** Dept. of Biomedical Engineering, The City College of New York,
canalicular wall (Fig 1b and 1c).
New York
52nd Annual Meeting of the Orthopaedic Research Society
Paper No: 0393