1 Department of Orthopaedic Surgery, University of California, San Francisco,
California, 94143-0514
2 Department of Anatomy, University of California, San Francisco, California,
94143-0514
* Author for correspondence (e-mail: helms{at}itsa.ucsf.edu)
Accepted 22 April 2003
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SUMMARY |
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Key words: Matrix metalloproteinase 9, Vascular endothelial growth factor, Endochondral ossification, Cartilage, Bone healing, Mechanical environment, Mouse
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INTRODUCTION |
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There are also notable differences between the adult reparative process and
fetal skeletal development. For example, mechanical forces have not been
implicated in the initiation of chondrogenesis or osteogenesis during fetal
development, but the role of the mechanical environment during adult repair
(Carter et al., 1998;
Probst and Spiegel, 1997
), and
its effect on cartilage and bone formation during fracture healing
(Le et al., 2001
;
Thompson et al., 2002
) has
been well documented. An inflammatory reaction exerts a substantial effect on
adult skeletal repair (Simon et al.,
2002
), whereas the immune system has no known role in fetal
skeletogenesis. Likewise, skeletal progenitor cells abound in the fetus,
whereas they may be limited in number in the adult
(Bruder et al., 1994
;
Ekholm et al., 2002
).
We found the parallels between fetal skeletal development and adult repair particularly intriguing, and pursued this issue in the context of fracture healing. Our investigation focused on the extent to which the functions of one molecule, matrix metalloproteinase 9 (MMP9), were equivalent during skeletal development and fracture healing. We developed several novel models of skeletal repair, and exploited the Mmp9-/- mouse in order to gain the first molecular insights into the regulation of angiogenesis during skeletal tissue regeneration.
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MATERIALS AND METHODS |
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Pin implantation
Mice were anesthetized as described above. Using a percutaneous approach,
stainless steel pins (0.25mm diameter) were inserted through the marrow and
both tibial cortices; pin ends were cut flush with the skin. Mice were
sacrificed 7 and 10 days after surgery, and pins were removed from the tibiae
following decalcification.
Treatment with recombinant vascular endothelial growth factor (VEGF)
protein
Mmp9-/- tibiae were fractured as described above and
recombinant vascular endothelial growth factor (rVEGF) protein (5.0
µg/injection; Genentech) was delivered at four separate time-points (6, 7,
8 and 9 days post-fracture). Following inhalation anesthesia, percutaneous
injections were made directly into the fracture site using a 30 gauge needle.
Extreme care was taken to avoid displacing the bone ends during these
injections. Control Mmp9-/- mice received injections of an
equivalent volume of vehicle (PBS) at the same time-points. PBS and
rVEGF-injected mice were sacrificed at 10 (n=5 PBS-injected,
n=5 rVEGF-injected) and 14 (n=5 PBS-injected, n=5
rVEGF- injected) days post-fracture.
Biomechanical analyses (distraction to failure testing)
Closed tibial fracture tissues were collected at 14 and 19 days post-
fracture. The tibiae were carefully dissected free of surrounding soft tissue,
placed in normal saline and stored overnight at 4°C. The tibiae were
prepared for mechanical testing by placing 0.25 mm transfixion pins (Fine
Science Tools, Foster City, CA) into the bone at locations that were proximal
and distal to the fracture site. The proximal and distal ends of the bone,
including the pins, were embedded in polymethylmethacrylate (PMMA), leaving
the fracture callus exposed. Tissues were kept moist until they were loaded
onto the materials testing system (Bionix 858; MTS, Eden Prairie, MN) with a
precision force transducer (100# [454 N] Load Cell Model 31, Sensotec,
Columbus, OH). The ends of the bone were secured to the machine using a
custom-made clamp. The tibiae were then loaded in distraction at a rate of
0.25 mm/minute with simultaneous force and displacement data recorded. Maximum
force at failure (in Newtons, N) was calculated for each specimen.
Histology and immunohistochemistry
Under RNase-free conditions, callus tissues were fixed overnight at 4°C
in 4% paraformaldehyde. Callus tissues were decalcified at 4°C in 19% EDTA
(pH 7.4) for 10-14 days, then dehydrated in a graded ethanol series and
embedded in paraffin. The entire callus was sectioned (10 µm thick), and
adjacent sections were analyzed using a variety of histological and cellular
analyses. Safranin-O/Fast Green (SO) staining was performed as described
(Thompson et al., 2002).
Trichrome staining was performed to analyze bone formation in the fracture
callus, the Aniline Blue (AB) component of the trichrome stain was selected
for image analyses. Tartrate resistant acid phosphatase (TRAP) staining was
performed using a leukocyte acid phosphatase kit (Sigma, St. Louis, MO). For
platelet endothelial cell adhesion molecule (PECAM)- and MMP9-antibody
staining, sections were de-paraffinized, immersed in 5%
H2O2/PBS for 5 minutes, washed in PBS, treated with 0.1
M glycine for 30 seconds, washed in PBS, and then incubated in the following
blocking solutions followed by intermediate PBS washes: 5% powdered milk for
10 minutes; 1.0 mg/ml ovalbumin for 10 minutes; and 5% sheep serum for 30
minutes. Sections were incubated overnight at 4°C in monoclonal rat anti-
mouse PECAM1 (BD PharMingen, San Diego, CA) or a polyclonal rabbit anti-mouse
MMP9, washed in PBS, blocked in 5% sheep serum for 30 minutes, and incubated
for 1 hour at room temperature in biotinylated anti-rat IgG (BD PharMingen,
San Diego, CA) or biotinylated anti-rabbit IgG (Jackson Immunoresearch).
Slides were washed in PBS and incubated in horseradish peroxidase-conjugated
streptavidin (Amersham, Cleveland, OH). Signal was revealed by incubation in a
diaminobenzidine solution containing 1% CoCl2 and 1%
Ni3SO4 for PECAM immunostaining
(Vu et al., 1998
). For
MMP9-TRAP double staining, TRAP staining was performed following MMP9
immunostaining.
In situ hybridization
In situ hybridization was performed using mouse cDNAs for Mmp9,
ColIIa (Col2a1 - Mouse Genome Informatics), ColX
(Col10a1 Mouse Genome Informatics), Oc
(Tcirg1 Mouse Genome Informatics), Vegf
(Vegfa Mouse Genome Informatics), and the Vegf receptors
Flk-1, Flt1 (Kdr Mouse Genome Informatics) and
neuropilin 2. Sections were de-waxed, fixed in 4% PFA, treated with 20.0
µg/ml Proteinase K, fixed with 4% PFA, incubated in 0.1% sodium
borohydride/PBS and acetylated in a solution of 0.1 M triethanolamine-HCl.
Sections were then hybridized with 35S-labeled denatured probes
overnight at 45°C. Sections were then washed in 5xSSC containing 20
mM ß-mercaptoethanol at 45°C, then in 50% formamide containing
2xSSC at 45°C, followed by a wash in 2xSSC, and lastly in
0.1xSSC at room temperature. Sections were then dehydrated in a graded
ethanol series. Emulsion coating was performed as described
(Albrecht et al., 1997). Image
analyses were performed as described previously
(Ferguson et al., 1999
).
Histomorphometric measurements
At 10 and 14 days, the fracture callus was composed of cartilage, islands
of bone, and fibrous tissue. To determine the volume of the callus and the
cartilage within each callus, and to circumvent difficulties in assessing a
heterogenous tissue such as the fracture callus, we first sectioned the entire
callus. From the resulting 300 tissue sections (each 10 µm in thickness),
histomorphometric analyses were performed. In our initial studies we analyzed
tissue sections every 30 µm; we later determined that tissue sections taken
at a 300 µm interval produced the same results. Thus, for each callus, an
average of 10-15 tissue sections were used to determine callus and cartilage
volumes. Sections were stained with SO and images of each section were
photographed using a digital camera. Images were imported into Adobe
PhotoShop, and the software was used to quantify the area of the callus and
the area of cartilage (which stained red after SO histological analysis). The
areas of the callus and the cartilage were both determined empirically in a
double-blinded manner, and checked by an independent investigator. These data
were used to calculate the total volume of each callus and the total volume of
cartilage in each callus.
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RESULTS |
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Mmp9-/- mice display abnormal fracture
healing
Given the expression of MMP9 throughout the course of bone repair, we
speculated that some aspect of adult skeletal repair would be compromised in
Mmp9-/- mice. The most obvious difference we noted was
during the hard callus phase of repair. Whereas the wild-type cartilage callus
was undergoing rapid degradation and remodeling by 14 days, the
Mmp9-/- callus was apparently resistant to degradation
(Fig. 2A,B). Histomorphometric
analyses confirmed these observations, and revealed that
Mmp9-/- calluses comprised almost three times more
cartilage than wild-type calluses (Fig.
2B). The healing defect in Mmp9-/- mice
persisted into the latter part of the hard callus stage; long after wild-type
cartilage had been replaced by bone, the Mmp9-/- callus
still exhibited residual cartilage islands
(Fig. 2A, 21 days). By the
remodeling phase (e.g. 28 days), Mmp9-/- and wild-type
calluses were both composed primarily of bone (data not shown), indicating
that the Mmp9-/- repair defect was reversible. We had
observed a similar reversal in the Mmp9-/- fetal skeletal
defect around the time of puberty in the mouse
(Vu et al., 1998).
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The Mmp9-/- mutation affects the biomechanical
properties of the fracture callus
A crucial aspect of bone healing is that the regenerated tissue must
provide sufficient strength to the injured limb in order for the animal to
regain function. We sought to determine if the Mmp9-/-
mutation compromised the strength and stability of an injured bone. We first
used a biomechanical test to measure callus strength. We chose the
distraction-to-failure model as a mode of testing because the geometry of a
non-stabilized fracture is highly variable, which renders more standard
biomechanical tests less accurate. Wild-type and Mmp9-/-
calluses were subjected to a gradual distractive force and the maximum force
required to cause rupture of the callus was determined
(White et al., 1977). At 14
days, we found the maximum force at failure was greater in wild-type
[5.19±0.63 Newtons (N), ±s.e.m.; n=8] than in
Mmp9-/- calluses (4.29±1.06 N; n=13;
P=0.045, Student's t-test), indicating that
Mmp9-/- calluses were structurally weaker than wild-type
calluses. By 19 days, the maximum force at failure was equivalent in wild-type
(6.99±1.22 N, n=11) and Mmp9-/- calluses
(6.47±0.97 N; n=10; P=0.41), which supported our
previous observations that the Mmp9-/- defect resolved
around the onset of bony remodeling. Collectively, these data indicate that
MMP9 serves at least two functions during skeletal tissue regeneration. First,
MMP9 is necessary for the efficient degradation and remodeling of the
hypertrophic cartilage callus. Second, MMP9 participates in the regeneration
of osseous tissue in the callus. Ultimately, these data indicate that MMP9
activity is crucial for the regeneration of functional skeletal tissue.
The Mmp9-/- mutation impairs ossification during
fracture healing
Our previous analyses of the cranial and appendicular skeletons of prenatal
and early postnatal Mmp9-/- mice had failed to reveal
defects in intramembranous ossification
(Vu et al., 1998). However, in
our fracture healing model we had observed that intramembranous ossification
was disrupted. Using osteocalcin (Oc) expression as an indicator of
osteogenesis (Lian et al.,
1978
), we ascertained that Oc was expressed at very low
levels, and only at the periphery of the callus, in
Mmp9-/- mice, whereas Oc was expressed throughout
wild-type calluses by day 14 (Fig.
3). This retardation in intramembranous ossification persisted in
Mmp9-/- mice into the remodeling phase of fracture repair
(Fig. 3).
We initially postulated that a delay in intramembranous ossification during
Mmp9-/- fracture healing was due to a primary defect in
cartilage removal, which indirectly delayed subsequent bone formation. An
alternative possibility was that the delay in intramembranous ossification
represented a separate, and primary, defect in bone formation. To resolve this
issue, we developed another model of skeletal repair that allowed us to
evaluate whether Mmp9-/- bones would heal properly if the
primary mode of repair was intramembranous ossification. This repair model was
based on the clinical observation that immobilized fractures heal with little
or no cartilage, and instead form bone through the direct differentiation of
skeletal progenitor cells into osteoblasts
(Carter and Giori, 1991;
Probst and Spiegel, 1997
;
Thompson et al., 2002
). We
stabilized wild-type and Mmp9-/- tibiae with an external
device that immobilized the fractured bone segments
(Thompson et al., 2002
), then
assessed the calluses for the onset of osteoblast differentiation after 7, 10,
14 and 21 days. As expected, the majority of wild-type calluses formed bone
without evidence of chondrogenesis (81%, n=31;
Fig. 4A). By contrast, the
majority of Mmp9-/- mice exhibited late onset, and low
expression, of Oc, indicating that intramembranous ossification was
greatly delayed in the mutant mice (88%, n=16;
Fig. 4A). However, more
surprising was the fact that skeletal progenitor cells in the
Mmp9-/- calluses were not simply delayed in their
differentiation to osteoblasts. Instead, the cells differentiated into
chondrocytes despite the fact that the bone ends were immobilized
(Fig. 4A).
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The pin implant model thus uncovered a potential new role for MMP9 in the
commitment of skeletal progenitor cells to an osteogenic lineage. We reasoned
that if MMP9 was involved in such a cell fate specification, the protein
should be expressed at the time when such decisions are made during fracture
repair. We had already observed that cells within the fracture site express
chondrogenic and osteogenic markers within 3 days of fracture
(Le et al., 2001); using a
functional approach we now demonstrated that cells became specified in their
fate within this same time frame. By permitting motion at the site of a
fracture for 0, 24 or 48 hours, or for 5 days, and subsequently stabilizing
the bone segments until day 10, we were able to show that healing occurred by
endochondral ossification if they were mobile for longer than 48 hours
(Fig. 4C). Fractures unstable
for 24 hours or less healed by intramembranous ossification
(Fig. 4C).
These functional data strongly suggested that skeletal progenitor cells
could commit to a chondrogenic or an osteogenic fate within 48 to 72 hours
following injury. We used immunohistochemistry to show that MMP9 protein was
expressed within that same time frame. MMP9 protein was detected in
TRAP-negative mesenchymal cells located on the endosteal and periosteal
surfaces of the fractured tibia, in the extracellular matrix surrounding the
fracture site (Fig. 4D), and in
TRAP-negative inflammatory cells such as neutrophils
(Fig. 4D). After 5 days, we
also detected MMP9 in TRAP-positive pre-osteoclasts (Figs
1,
5)
(Engsig et al., 2000). Taken
together these data indicate that MMP9 protein is expressed at sites where
skeletal progenitor cells are proposed to exist, and that MMP9 is expressed
during the period when these cells adopt and commit to chondrogenic or
osteogenic fates following injury.
The Mmp9-/- fracture repair defect is caused by
delayed vascularization of the cartilage callus
The delayed repair defect in Mmp9-/- mice resembles a
hypertrophic non-union, a human condition characterized by persistent
hypertrophic cartilage that is attributed to disruptions in the vascular
network of the fracture callus (Einhorn,
1999). Based on the similarities between the mouse phenotype and
the human condition, we investigated the extent to which vascularization was
compromised in the Mmp9-/- callus. Because extracellular
matrix remodeling and angiogenesis are closely linked during skeletal tissue
development, we included molecular markers of both in this analysis. By 10
days post- fracture, the periphery of the wild-type callus was surrounded by
MMP9-expressing, TRAP-positive osteoclasts, but these cells were absent from
the Mmp9-/- callus
(Fig. 5A). By day 14,
osteoclasts had removed most of the wild-type cartilage callus and new bone
occupied the space (Fig. 5A).
By contrast, very few osteoclasts were detectable in the
Mmp9-/- unremodeled cartilage callus
(Fig. 5A). This failure to
recruit TRAP-positive cells to the Mmp9-/- callus was
paralleled by a delay in vascular invasion. Whereas PECAM-positive endothelial
cells had accumulated at the periphery of the wild- type hypertrophic
cartilage callus, very few endothelial cells were found adjacent to the
Mmp9-/- hypertrophic cartilage
(Fig. 5A). By day 14,
endothelial cells invaded the wild-type callus cartilage, whereas in the
Mmp9-/- callus the endothelial cells remained restricted
to the periphery (Fig. 5A).
A delay in endothelial cell recruitment could indicate a delay in cartilage
calcification. We performed a series of molecular and histological analyses to
test this possibility, but failed to detect any defect in cartilage matrix
mineralization (Fig. 3, and
data not shown). Another explanation for the
Mmp9-/--dependent delay in vascular invasion might be the
lack, or decreased expression, of an angiogenic signal or some part of the
angiogenic signaling machinery. We examined Mmp9-/-
calluses at multiple time points during fracture repair for changes in the
expression of Vegf and the VEGF receptors Flt1, Flk1 and
neuropilin 1 and 2. All of these molecules were expressed within the
Mmp9-/- callus (Fig.
5B, and data not shown). The only difference we detected was in
levels of expression: the number of cells expressing Vegf was
actually higher in the Mmp9-/- callus, which was due to
the persistence of Vegf-positive hypertrophic cartilage
(Fig. 5B) (see
Colnot and Helms, 2001). We
also noted reduced Flk1 expression, reflecting the paucity of
endothelial cells in the Mmp9-/- callus
(Fig. 5B). Collectively, these
data indicated that the Mmp9-/- angiogenic defect was not
caused by the failure of expression of a potent angiogenic stimulator or its
receptors during skeletal repair. Another possible explanation was that MMP9
might be regulating the bioavailability of VEGF, as it does during some
disease processes (Bergers et al.,
2000
).
Exogenous VEGF rescues the Mmp9-dependent skeletal repair
defect
We reasoned that if functional VEGF is limiting during
Mmp9-/- skeletal repair, then exogenously applied VEGF
should rescue the Mmp9-/- defect. We injected recombinant
human VEGF protein (rVEGF), or PBS as a control, into the fracture site of
Mmp9-/- mice, beginning on day 6 (the time at which
Vegf is normally expressed in the callus), then daily for three more
days. We then compared the callus tissues of Mmp9-/- mice
that received rVEGF with those that received PBS. By 14 days, we noted a clear
difference between the two groups: Mmp9-/- calluses
treated with rVEGF had significantly less cartilage than the controls
(Fig. 6A,B). Not only did rVEGF
injection reduce the amount of hypertrophic cartilage (ColX;
Fig. 6C), it also resulted in
an increased synthesis of bone matrix (Fig.
6C, boxed area in red, AB). rVEGF treatment also resulted in an
increase in TRAP activity (Fig.
6C) and in the numbers of endothelial cells that invaded the
Mmp9-/- callus (Fig.
6C, arrows). These observations demonstrated that rVEGF
compensated for the lack of Mmp9 by stimulating the recruitment/and
or differentiation of the three cell types that express the VEGF receptor:
chondroclasts/osteoclasts that remodel hypertrophic cartilage, endothelial
cells that form vascular channels, and osteoblasts that generate new bone at
the injury site.
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DISCUSSION |
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MMP9 regulates hypertrophic cartilage angiogenesis
Our data indicate that MMP9 stimulates angiogenesis of the hypertrophic
cartilage callus (Fig. 5). One
model that may account for the Mmp9-/- repair defect is
that the loss of MMP9 affects the bioavailability of a potent angiogenic
molecule, VEGF. The fact that VEGF cannot accelerate healing in wild- type
animals (data not shown) but can rescue the phenotype of Mmp9-/-
mutants indicates that VEGF is a limiting factor in the absence of MMP9, which
suggests an interaction between the two molecules. MMP9 may regulate vascular
invasion by releasing VEGF that is bound to the hypertrophic cartilage matrix.
Once released, VEGF could bind to its receptors on endothelial cells,
osteoclasts and osteoblasts (Nakagawa et
al., 2000; Risau,
1997
), stimulating their migration and activity at the fracture
site. Precisely how MMP9 and VEGF coordinate the activity of three cell types
(osteoblasts, osteoclasts/chondroclasts and endothelial cells) during the
process of skeletal repair is still a puzzle. Other angiogenic factors are
clearly involved in skeletal repair, as the Mmp9-/- defect
is transient (Fig. 2). Likewise, other proteases may mediate angiogenic activity
(Yamagiwa et al., 1999
), as is
suggested by skeletal defects in Mt1-Mmp-/- mice
(Holmbeck et al., 1999
).
Understanding the identities and contributions of other angiogenic regulators
and proteases will undoubtedly provide a more complete view of how
extracellular matrix remodeling and angiogenesis are synchronized during
skeletal tissue regeneration.
MMP9 mediates an angiogenic switch during bone regeneration
The transition of an anti-angiogenic tissue, cartilage, to an angiogenic
one, bone, is a crucial feature of adult bone regeneration
(Fig. 5). Tumors undergo a
similar angiogenic switch, heralding their progression to a more aggressive
form of the disease (Bergers et al.,
2000; Huss et al.,
2001
; Semenza,
2000
). Angiogenic switches also occur during the development of
other tissues, such as the lung and the mammary gland
(Muratore et al., 2000
;
Pepper et al., 2000
).
During fracture healing, a failure of cartilage to undergo an angiogenic
switch is associated with a pathological skeletal condition known as
hypertrophic non-union. The Mmp9-/- fracture healing
phenotype is a prime example of how a genetic mutation can impair adult
healing in a phenotypically normal individual. As illustrated by the
Mmp9-/- mouse, a complete loss of MMP9 can be compatible
with life and normal reproduction; humans with mutations in MMP2 are also
viable (Martignetti et al.,
2001). It will be interesting to ascertain if a genetic
predisposition underlies human skeletal healing defects that are associated
with perturbations in vascular remodeling, and if biologically based therapies
can be developed for the treatment of such recalcitrant skeletal injuries.
MMP9 and cell fate specification
MMP9 plays another role during the early phase of stabilized skeletal
repair. Although stabilized skeletal injuries heal through intramembranous
ossification in wild-type mice, the same injuries heal through endochondral
ossification in Mmp9-/- mice
(Fig. 4). There are a number of
possible explanations for this curious phenotype. For example, MMP9 may
participate in the mobilization of osteoprogenitor cells from the periosteum
and/or from the bone marrow. In the bone marrow, MMP9 is involved in the
mobilization and activation of hematopoietic and endothelial stem cells, and
MMP9- expessing cells may in fact share a common lineage with these cells
(Heissig et al., 2002). The
Mmp9-/- intramembranous ossification defect may therefore
be cause by a failure to release and/or activate osteoprogenitor cells from
either the periosteum or the bone marrow cavity.
Alternatively, the differentiation of skeletal progenitor cells may be
delayed in Mmp9-/- stabilized injuries. MMP9 appears to
release VEGF from its extracellular matrix stores
(Bergers et al., 2000), and a
failure to activate one of the VEGF receptors on osteoprogenitor cells
(Deckers et al., 2000
;
Midy and Plouët, 1994
)
may ultimately affect the rate at which these cells differentiate. As the
Mmp9-/- ossification defect is transient, it is highly
likely that other signals also participate in osteoblast differentiation
during repair. The nature of these compensatory signals is, at present,
unknown.
Another possibility is that the MMP9 mutation may disrupt the formation of
an intact vascular network during the initial stages of stabilized repair. A
stable environment has long been thought to favor the formation of an intact
vascular network (Claes et al.,
2002; Glowacki,
1998
), and this oxygen-rich environment appears to support the
differentiation of skeletal progenitor cells into osteoblasts. One possibility
is that a vascular network fails to form around the
Mmp9-/- stabilized fracture or the
Mmp9-/- implant site
(Fig. 3). However, we think
this is the least likely explanation, as rVEGF failed to rescue the
Mmp9-/- stabilized healing defect. Other angiogenic
molecules, such as CTGF, the angiopoietins
(Street et al., 2002
;
Thurston et al., 1999
) and the
fibroblast growth factors (Kawaguchi et
al., 1994
), may regulate the establishment of a vascular network
during early stages of fracture healing.
The mechanical environment influences progenitor cell fate
decisions
Until recently (Henderson and Carter,
2002), it was thought that the most prominent difference between
fetal and adult bone formation was the fact that fetal endochondral
ossification takes place independently of mechanical stimuli, whereas the fate
of progenitor cells in the fracture callus depends upon the mechanical
environment. Clinical examination, biomechanical data
(Carter and Giori, 1991
;
Carter et al., 1998
) and
biological observations (Claes et al.,
1998
; Park et al.,
2003
; Thompson et al.,
2002
) clearly indicate that stabilization favors the
differentiation of cells into osteoblasts, whereas the lack of stabilization
leads initially to the production of chondrocytes. In
Mmp9-/- mice, the majority of progenitor cells in the
fracture callus adopt a chondrogenic fate regardless of whether the fracture
is stabilized or not. Precisely how the mechanical environment and MMP9
function are coordinated is still open to speculation. The differences in
stabilized and non-stabilized fracture healing may be attributable to the
extent of tissue disruption, and therefore may not be directly comparable. For
example, stabilized fractures and implant models may be characterized by
minimal tissue disruption following acute injury, whereas non-stabilized
fractures may be subjected to continued tissue disruption. These differences
might trigger signaling pathways that are not induced in a stable mechanical
environment and therefore healing between the two models may be difficult to
compare. However, there is no current data to support or refute the hypothesis
that the extent of mechanical disruption leads to the activation of different
molecular pathways.
In vitro studies show that stretch and compression forces applied to
tissues from a fracture site upregulate the expression of some MMPs
(Haas et al., 1999;
Rubin et al., 1999
), and this
upregulation occurs through cell adhesion molecules, such as integrins
(Spessotto et al., 2002
;
Sugiura and Berditchevski,
1999
). Thus it is possible that different MMP-induced responses to
varying mechanical stimuli may modulate the activity of different sets of
growth factors, analogous to the case of hematopoietic reconstitution
(Heissig et al., 2002
).
Probable candidate growth and differentiation factors include VEGF
(Villars et al., 2000
), and
members of the transforming growth factor ß
(Alliston et al., 2001
;
Bonewald and Dallas, 1994
;
Centrella et al., 1994
), bone
morphogenetic protein (Fromigue et al.,
1998
), hedgehog protein (Pola
et al., 2001
; Spinella-Jaegle
et al., 2001
) and fibroblast growth factor
(Liu et al., 2002
;
Scutt and Bertram, 1999
)
families. It is equally probable that subtle shifts in the balance of these
and other growth factors subsequently affect the differentiation, or survival
and proliferation, of osteo- or chondroprogenitor cells that populate the
fracture site.
Inflammation and skeletal repair
Inflammation plays an important role in bone repair
(Altman et al., 1995;
Banovac et al., 1995
;
Zhang et al., 2002
). The
mechanical environment influences the inflammatory response
(Hankemeier et al., 2001
),
although the mechanisms by which this is achieved are unclear. MMP9
participates in the inflammatory response associated with skeletal injury, as
demonstrated by the fact that neutrophils and macrophages strongly express the
protein in the early fracture callus and in the implant site. The function(s)
of MMP9 in the inflammatory response associated with the early stages of
skeletal repair is still unclear. MMP9 released from neutrophils, mast cells
and macrophages may be involved in remodeling the early fracture callus.
Inflammatory cells expressing MMP9 may preferentially accumulate in a stable
mechanical environment, which would result in the delivery of more cytokines
to these sites of injury. In turn, these cytokines may stimulate osteogenesis
to a greater degree than is observed in a non- stabilized fracture.
In conclusion, these data reveal new roles for MMP9 in fracture healing. The continued close scrutiny of the parallels, and differences, between fetal and adult programs of cell differentiation, extracellular matrix remodeling and angiogenesis will surely yield new insights into these crucial events in tissue regeneration.
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ACKNOWLEDGMENTS |
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