Mechanical factors—including repetitive manual lifting tasks,
vibration exposure, and acute overloading—have long been implicated in
the etiology of intervertebral disc degeneration. However, recent studies have
demonstrated that these whole-body mechanical factors may play a minor role in
disc degeneration, highlighting a dominant genetic
predisposition1.
This paradox underscores the difficulty of understanding, preventing, and
treating disc degeneration.
An understanding of the role of both mechanical and genetic factors in disc
degeneration requires the mechanobiologic knowledge of how the cells interact
with the extracellular matrix. The intervertebral disc is altered at multiple
scales biologically, biochemically, and mechanically with degeneration. In
vivo and in vitro mechanobiologic studies clearly demonstrate that mechanical
factors can influence the biosynthetic activity of disc cells, altering the
expression of key extracellular matrix
molecules2-4.
Essential to elucidating the mechanotransduction mechanisms is a detailed
understanding of the in situ environment of disc cells. Yet, little is known,
with regard to either healthy or degenerated tissue, about the in situ
environment of the cells that are responsible for maintaining and repairing
the disc. In particular, what mechanical loads do the intervertebral disc
cells experience in situ?
Regardless of the initiating cause, the degenerated disc is physically
altered. Given its inherent mechanical function, an understanding of how
structural changes to the extracellular matrix might alter cellular metabolism
is essential. Therefore, the purpose of this research was to explore the in
situ anatomic and mechanical environment of disc cells. In this investigation,
laser scanning confocal microscopy was used to characterize the
three-dimensional morphology of intervertebral disc cells, the micromechanical
deformation and interaction with the extracellular matrix, and the functional
intercellular communication.
Coccygeal discs from twelve to twenty-four-month-old steers were harvested
within two to four hours after slaughter for use in both the anatomic and
micromechanical investigations. All investigations were conducted with an
inverted laser scanning confocal microscope with 63× magnification and a
1.2 numerical aperture water-immersion objective lens (LSM 510; Carl Zeiss,
Oberkochen, Germany), which enabled three-dimensional visualization of both
cell morphology and motion in situ.
A systematic investigation with transverse, tangential, and oblique
histologic sections was conducted with use of antibodies to cytoskeleton
proteins (actin, vimentin) to examine the three-dimensional morphology of
anular cells5. Live
cell labeling with calcein-AM was also used to examine the full
three-dimensional morphology of the cells. Anticonnexin 43 was used to
identify a gap-junction protein, and anticollagen type VI was used to examine
the pericellular matrix. Propidium iodide was used as a nuclear counterstain
as needed. Electron microscopy was also used to verify observations.
A custom-built load apparatus was mounted on the stage of the inverted
confocal microscope so that the anulus fibrosus specimens could be loaded in
biaxial tension6 and
the intact intervertebral discs could be loaded in
flexion7. Cell
nuclei were labeled with use of nucleic acid stain (SYTO 13 or 83; Molecular
Probes, Eugene, Oregon), and their in situ motion was tracked with the
confocal microscope during loading. The extracellular matrix of the intact
discs was also labeled with 5-(4,6-dichlorotriazinyl) aminofluorescein
(5-DTAF) to fluorescently label the collagenous matrix, and three parallel
lines were then photobleached perpendicular to the collagen fibrils and used
as in situ strain gauges.
To assess the presence of functional gap junctions, specimens were
incubated with calcein-AM in Ca2+-free phosphate-buffered saline
solution and the diffusion between cells was observed with use of fluorescence
recovery after
photobleaching8.
Cells were photobleached with the laser scanning at 100% intensity for ten to
fifteen seconds. Confocal images were then made initially (ti),
immediately following photobleaching (t0), after ten minutes of
recovery (t10), and after twenty minutes of recovery
(t20). Some specimens were placed in a bath containing the
gap-junction blocker 1-octanol for at least fifteen minutes prior to and
during imaging.
In a series of anatomic studies, the complex cellular morphology of anulus
fibrosus cells was illustrated with extensive, sinuous processes interwoven
through the collagenous fibers of the outer aspect of the anulus
(Fig. 1, A). A radial
variation of morphology was observed as the length of the processes decreased
toward the inner aspect of the anulus with larger central cell bodies,
although processes were still readily apparent. Cells were further localized
to lamellar and interlamellar regions, each with distinct morphologies and
environments. The morphology of the cells was illustrated by the actin
cytoskeleton, but the extensiveness of the processes was even more apparent
with live cell cytoplasmic labeling (Fig.
1, B). The lamellar cells were demonstrated to reside in
"linear cell arrays" interconnected by type-VI collagen, forming
an extensive pericellular environment frequently enveloping more than ten
cells (Fig. 1, C). The
lamellar cells were aligned with the collagen fibers in these arrays, with the
cell nuclei aligned like pearls on a string. These arrays, with as many as ten
to twelve cells, could be extracted from the extracellular matrix with the
pericellular matrix intact, much like a chondron, and successfully
cultured.
Mechanically, collagen fibril sliding was demonstrated to govern cell
mechanics and strain transfer in the anulus fibrosus during loading
activities. Lamellar sheets of anulus fibrosus were subjected to biaxial
tension and intact discs to flexion; however, the measured in situ cellular
strains differed from the matrix strains and were nonuniform. Lamellar cells
were largely protected from direct tensile strains in the matrix with minimal
intercellular strains. However, intercellular strains between lamellar cells
in adjacent arrays were large, illustrating shearing between linear cell
arrays. This shearing was further highlighted through fluorescent labeling on
the extracellular matrix (Fig. 1,
D). A gradual shearing between fibrils was observed as
the lines that were photobleached into the fluorescently labeled matrix were
distorted. In the outer aspect of the anulus, with a denser type-I collagenous
matrix, the shearing was very gradual, while in the inner aspect, distinct
shear dislocations could be observed in the looser woven matrix; however,
these dislocations rarely occurred at a cell-matrix interface. Appreciable
shear was observed across the lamellar cell bodies as well as to the extensive
cellular processes woven into the matrix.
The gap-junction protein connexin 43 was found within cells throughout the
anulus fibrosus in both the cell bodies and along the cellular processes,
including cells with no physical contact with other cells. With use of
fluorescence recovery after photobleaching, the cells of the outer aspect of
the anulus were demonstrated to be interconnected, with functional gap
junctions forming a network through which cells could communicate
(Fig. 1, E). Although
interlamellar cells were also found to form a network through functional gap
junctions, cells in the inner aspect of the anulus did not.
These findings demonstrate the morphologic and micromechanical complexity
of anulus fibrosus cells and their interaction with the extracellular matrix
(Fig. 2). These findings are
essential to understanding how whole-body mechanical loads may be transferred
to the disc cells in situ. Furthermore, characterizing the in situ
micromechanical environment of the cells is crucial to understanding how
genetic factors might predispose an individual to disc degeneration because it
is the cells that must maintain and repair the extracellular matrix to give
the disc its structural integrity.
This research has four broad implications. First, the transduction of
mechanical loads in the extracellular matrix to the anular cells is complex
and will require extensive research to fully discern. In the anulus fibrosus,
the lamellar cells are largely isolated from the tensile strains in the
extracellular matrix. The cells are aligned in linear cell arrays within an
extensive pericellular matrix with very small intercellular strains observed
in both biaxial and flexion loading. Loading of the intervertebral disc
results in shearing between collagen fibrils of the lamellae and rotation of
adjacent lamellae, thereby absorbing the mechanical strains in the
extracellular matrix. On the other hand, the extensive cellular processes
woven into the collagen fibers are subjected to these shear strains; large,
nonuniform motions of the processes have been observed, illustrating how they
interact with the sliding matrix. The morphologic complexity of the anular
cells and their interwoven attachment to the collagenous matrix ensure that
the load transfer from matrix to cell membrane to cytoskeleton is not a simple
path.
Second, the isolation of intervertebral cells from their matrix and the
study of their mechanobiologic behavior in culture must be conducted with
caution. Anular cells in situ reside in linear cell arrays in an extensive
pericellular matrix, with processes woven into the collagen fibrils as well as
forming a network of communication in outer anular and interlamellar cells.
The aggressive digestion of the extracellular matrix to extract cells
completely disrupts this in situ environment, generally yielding isolated
spherical cells. These cell cultures are often subjected to simple mechanical
stimulation, such as hydrostatic pressure or tensile strain, which differs
significantly from the mechanical signals the cells experience in situ. While
these investigations enable very specific questions to be answered, it may be
very difficult to extrapolate such results back to the intact disc in vivo.
The current findings should help to bridge the gap between in vitro and in
vivo mechanobiologic research.
Third, the extensive changes to the extracellular matrix with degeneration
will greatly alter the in situ environment of the cells. While my colleagues
and I have focused our investigations to date on the healthy disc, the
observed complexity of the cell morphology and its micromechanical behavior
will undoubtedly be impacted in a degenerated disc. Radial tears and
delamination alter the physical integrity of the anulus fibrosus, altering its
load-bearing capacity and likely altering how the cells interact with matrix.
The micromechanical environment of the cells, with processes woven into the
degenerating matrix, will change; whether or not the cells have the capacity
to adapt is unknown. The tools and knowledge gained from work with healthy
tissue must now be extended to degenerated discs to determine possible changes
in both cell morphology and micromechanics.
Fourth, the development of scaffolds to support the growth of anulus
fibrosus cells should be conducted with an awareness of the complexity of the
cellular environment. Because such studies often begin with isolated cells
that are then cultured in various gels, the cell environment differs greatly
from that in situ. The cells typically no longer have processes and are
isolated from each other with no pericellular matrix, cultured in gels that
may not enable sliding at the cellular level; therefore, the cells could be
subjected to entirely different loads than those in the intact tissues. An
appreciation of the morphologic and micromechanical environment of the cells
is essential in biomaterial development.
Knowledge of the in situ environment of disc cells will provide a base for
investigating the mechanical implications of disc degeneration on the cellular
environment and a better understanding of how mechanical and genetic risk
factors can impact the cells that are essential to maintaining the
intervertebral disc. Future research will incorporate this base knowledge and
these tools to understand how changes in the structural integrity of the
matrix associated with human disc degeneration may impact the in situ
mechanical environment and potentially the biosynthetic response of cells.
Prevention and treatment of disc degeneration will require an understanding of
how the cells respond to changes in the matrix with degeneration (and the
possible genetic links to these changes) and the development of biologic
interventions based on a knowledge of the cell-matrix interactions. The
results summarized here, which attempt to bridge the biology with the
micromechanics, will hopefully lay the foundation for improved treatment of
disc degeneration. ?