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First published online 24 July 2008
doi: 10.1242/dev.023788
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Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, CA 94305, USA.
* Author for correspondence (e-mail: jhelms{at}stanford.edu)
Accepted 30 June 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neural crest, Mesoderm, Periosteum, Osteoblast, Chondrocyte, Graft, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Mammalian bone has such a high turnover rate
that the bulk of skeletal tissues are completely replaced each decade. This
inherent regenerative capacity suggests that the skeleton must harbor a
reservoir of undifferentiated progenitor cells that give rise to new bone. One
source of these skeletal progenitor cells is the periosteum, which harbors
proliferating cells that can differentiate into either chondrocytes or
osteoblasts, which deposit new mineralized matrix. This matrix is continuously
resorbed by osteoclasts, which in turn stimulates new osteoblastogenesis.
Through this mechanism, the skeleton is continually reconstituted throughout
the life of an organism.
Bone formation and resorption occur in all bones, and at a macroscopic
level the skeletal tissues in the body and those in the head are
indistinguishable. There is, however, one unique characteristic: the facial
skeleton is derived exclusively from cranial neural crest, whereas the rest of
the skeleton is derived from mesoderm
(Noden, 1982;
Couly et al., 1993). This
raises a fundamental question: are there two sub-populations of skeletal stem
cells or is there a single population that is responsible for the continual
regeneration of bone?
One way to address this question is to evaluate how mesoderm-derived and
neural crest-derived bones undergo repair because, unlike the life-long
process of remodeling, injury-induced bone regeneration occurs within a
compressed time frame and in a precise location. The cellular and molecular
machinery responsible for adult bone formation in these conditions is,
however, identical (Ferguson et al.,
1998; Colnot et al.,
2003; Gerstenfeld et al.,
2003).
We used a cell-labeling strategy to indelibly mark neural crest-derived cells and mesoderm-derived cells, and then produced skeletal injuries in a neural crest-derived bone, the mandible, and in a mesoderm-derived bone, the tibia. In evaluating the healing phase we uncovered a neural crest stem cell reservoir that contributes cells exclusively to the regeneration of neural crest-derived skeletal elements. We also found that neural crest-derived and mesoderm-derived bones heal through the selective recruitment of cells from their own embryonic origins. We directly tested the extent to which these populations are interchangeable in a clinically relevant model of bone grafting. Our results indicate that skeletal stem cells have `positional memory', which influences how the cells behave when grafted into ectopic locations.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), were
mated to Z/EG mice (Novak et al.,
2000). Prior to expression of Cre recombinase, cells from all
stages of Z/EG mice exhibit constitutive expression of
β-galactosidase, but in the presence of Cre recombinase the expression of
β-galactosidase is replaced by expression of GFP.
Periosteal harvest and surgical implantation
Periosteum was harvested from the mandible and tibia by careful dissection
from the bones followed by rapid washing in cold PBS and transplantation into
drill-hole defects in skeletally mature, male FVB mice. To conduct the tibial
transplantation, an incision was made over the right anterior-proximal tibia
and the tibial surface was exposed while preserving the periosteal surface. A
drill hole was created through a single cortex in the midline of the tibia
distal to the tibial tuberosity with a dental drill (15,000 rpm) using a 1.0
mm drill bit (Drill Bit City, Chicago, IL). The far cortex was neither
penetrated nor touched by the drill. The donor GFP periosteum was then placed
into the host defect. After irrigation, the previously mobilized muscle flap
was placed over the defect and sutured to the skeletal wound using 7-0 Vicryl
(Ethicon, Somerville, NJ). Skin was closed using 7-0 Prolene (Ethicon). In the
mandible, an incision was made horizontally across the right buccinator muscle
and kept open with a retractor. A 1.0 mm drill hole was made into the exposed
ramus of the right mandible without creating root damage to the underlying
teeth. GFP-labeled periosteum was then placed into the defect, the buccinator
muscle approximated and the incision sutured with 7-0 Prolene.
The experimental design allowed for four distinct combinations of transplantation experiments: (1) mandibular GFP-positive periosteum into mandible FVB defect; (2) mandibular GFP-positive periosteum into tibial FVB defect; (3) tibial GFP-positive periosteum into tibial FVB defect; and (4) tibial GFP-positive periosteum into mandible FVB defect.
Following surgery, mice received adequate analgesia and were allowed to
ambulate freely (Clifford,
1984). Five mice of each of the four transplant combinations were
sacrificed at 7, 10, 15 and 21 days post-surgery. Additionally, five mice with
mandibular and tibial defects were sacrificed at each time point.
Representative samples were selected for analysis.
Tissue processing, histology and immunohistochemistry
Limbs and mandibles were dissected and processed for paraffin embedding.
Sections (8 µm) were collected on Superfrost-plus slides (Fisher
Scientific, Pittsburgh, PA) for histology using a modification of Movat's
Pentachrome staining (blue to black, nuclei; red, cytoplasm; red, elastic
fibers; yellow, collagen and mineralized bone matrix; blue-green, osteoid or
mineralized cartilage; light-blue, cartilage)
(Sheehan and Hrapchak, 1980).
Immunostaining was performed as described
(Colnot et al., 2003;
Leucht et al., 2007).
Antibodies included green fluorescent protein (GFP; Abcam, Cambridge, MA),
platelet endothelial cell adhesion molecule (PECAM; Pharmingen, San Jose, CA),
proliferating cell nuclear antigen (PCNA; Zymed, San Francisco, CA) and Hoxa11
(Abcam). TRAP staining of osteoclasts was performed as described
(Colnot et al., 2005). For
X-Gal staining, tissues were embedded in OCT, cryosectioned and then stained
with X-Gal overnight at 37°C (Kim et
al., 2007b).
Histomorphometric measurements
The 1.0 mm circular mono-cortical defect was represented across 
100
tissue sections, each of which was 10 µm thick. Out of those 100 sections,
we used a minimum of 20 sections to quantify the amount of new bone. Sections
were stained with Aniline Blue to label osteoid matrix. Digital images were
analyzed with Adobe Photoshop CS2 software. In a fixed region of interest
(ROI), Aniline Blue-positive pixels were selected using the Magic Wand tool
set to a color tolerance of 60 that highlighted pixels with a range of blue
that corresponded precisely with the histological appearance of osseous
tissue. Cortical surfaces or bone fragments were manually deselected. The
total number of Aniline Blue-positive pixels was recorded, then pixel counts
from individual sections were averaged for each sample and differences within
and among treatment groups were calculated based on these averages.
In situ hybridization
Hybridization was performed using digoxigenin-labeled probes complementary
to mouse cDNAs for Wnt1, Hoxa11 and Hoxa13 (Open Biosystems,
Huntsville, AL) as described (Albrecht et
al., 1997).
In vivo image analyses
Bioluminescence imaging was performed using an IVIS 200 (Caliper Life
Sciences, Alameda, CA). Light outputs were quantified using LivingImage
software version 2.5 (Xenogen, Alameda, CA) as an overlay on Igor Pro imaging
analysis software (WaveMetrics, Portland, OR). The final light output, in
photons/second/cm2/steradian, was normalized to the integration
time, the distance from the camera to the animal, the instrument gain, and the
solid angle of measurement to provide for cross-platform comparison.
Bioluminescence imaging was quantified by creation of circular ROIs over the
right proximal tibia.
Periosteum harvest for in vitro experiments
For the in vivo experiments, periosteal cells were harvested from the
mandible and tibia of skeletally mature wild-type or β-actin GFP mice.
Mice were euthanized, then soft tissues surrounding the bones were removed.
Bones were washed in PBS twice, followed by five 10-minute digestions in 2
mg/ml collagenase (Roche, Indianapolis, IN) in DMEM (Gibco and Invitrogen,
Carlsbad, CA). Cells from the sixth digestion were resuspended in DMEM
containing 10% FBS (Omega Scientific, Tarzana, CA), 1%
penicillin/streptomycin, 0.1% gentamycin, followed by plating at equal
density. Media were replenished every 2 days. Approximately 1 week from tissue
harvest, cells were passaged by trypsinization then used for the following
assays.
Cellular proliferation assays
Cells were seeded in 96-well plates at a density of 1000 cells per well and
on days 3, 5 and 7 BrdU assays were performed according to the manufacturer's
instructions (Roche). Means and standard deviations were calculated. Neural
crest-derived periosteal cells were also co-cultured with mesoderm-derived
periosteal cells from β-actin GFP mice. Cells were seeded at equal
density on chamber slides (Nalge Nunc International, Naperville, IL). After 0,
3, 5 and 7 days, slides were imaged under fluorescent light, digital images
were imported into Adobe Photoshop CS2 and GFP-positive and GFP-negative cells
were counted by two independent researchers in 10 random ROIs for each time
point.
Osteogenic differentiation and assessments
Cells were plated in 24-well plates at a density of 5000 cells per well.
After overnight attachment, cells were treated with osteogenic differentiation
medium (ODM) containing DMEM, 10% FBS, 100 µg/ml ascorbic acid, 10 mM
β-glycerophosphate and 100 IU/ml penicillin/streptomycin. Media were
replenished every 3 days. After 10, 12 and 14 days, Alizarin Red staining was
performed.
RNA isolation and quantitative real-time PCR
RNA was isolated (RNeasy Kit, Qiagen, MD), genomic DNA removed (DNA-Free
Kit, Ambion, Austin, TX) and the RNA reverse-transcribed (TaqMan Reverse
Transcription Reagents, Applied Biosystems, Foster City, CA). Quantitative (q)
real-time PCR was carried out using the Applied Biosystems Prism 7900HT
Sequence Detection System and Power SYBR Green PCR Master Mix (Applied
Biosystems). Specific primers were designed based on PrimerBank
(http://pga.mgh.harvard.edu/primerbank/)
sequence. PCR products were run on a 2% agarose gel to confirm the appropriate
size and specificity. Levels of gene expression were determined by normalizing
to their Gapdh values. All reactions were performed in triplicate;
means and standard deviations were calculated.
Statistical analysis
ANOVA two-factor test with replication was used when more than two groups
were compared. Welch's two-tailed t-test was used when standard
deviations between groups were unequal. P
0.01 was considered to
be significant.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Jiang et al.,
2000). When crossed with Z/EG reporter mice
(Novak et al., 2000), the
result is GFP labeling of Wnt1-expressing neural crest derivatives.
Other cell types, including mesodermal derivatives, express
β-galactosidase. We examined a variety of adult tissues to verify that
this labeling strategy worked.
First, mandibles and tibiae from skeletally mature Wnt1Cre::Z/EG
mice were examined. Mandibular osteoblasts and osteocytes exhibited robust GFP
immunostaining, indicating their derivation from the neural crest
(Fig. 1A). β-galactosidase
activity was absent from mandibular osteoblasts and osteocytes, although the
adjacent mesoderm-derived muscle and endothelial cells were positive
(Fig. 1B). In secondary
cartilages that underlie the skull bones
(Le Douarin and Dupin, 1993),
neural crest-derived chondrocytes were GFP-positive
(Fig. 1C), whereas
mesoderm-derived hematopoietic cells
(Cumano and Godin, 2007)
exhibited β-galactosidase activity
(Fig. 1D). In the limbs, neural
crest-derived Schwann cells
(D'Amico-Martel and Noden,
1983) were GFP-positive, whereas the surrounding mesoderm-derived
perineurium (Joseph et al.,
2004) was not (Fig.
1E). All tibial osteocytes and periosteum exhibited robust
β-galactosidase activity but no GFP immunostaining
(Fig. 1E,F). In some tibial
sections, we observed β-galactosidase-positive cells adjacent to a
morphological structure that ran longitudinally through the bone marrow.
Histologically, this structure resembled a nerve with surrounding Schwann
cells (data not shown). We only observed 10-20 β-galactosidase-positive
cells per longitudinal section of the tibia, however, which was significantly
fewer than has been reported elsewhere
(Nagoshi et al., 2008).
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A reservoir of neural crest-derived skeletal progenitor cells exists in adult bones
To ascertain whether neural crest-derived and mesoderm-derived bones use
the same skeletal progenitor cell population for their renewal, we generated
injuries in the mandible and tibia. These injuries heal through
intramembranous ossification and the repair callus has a stereotypical
organization (Colnot et al.,
2005; Kim et al.,
2007a; Kim et al.,
2007b; Leucht et al.,
2008), which facilitated our subsequent histological, molecular
and histomorphometric analyses.
On post-injury day 7, mandibular injuries were filled with new bone (Fig. 2A) and the majority of osteoblasts were immunopositive for GFP (n=6; Fig. 2B). β-galactosidase activity was only detectable in adjacent muscle (Fig. 2C). Tibial injuries showed a comparable amount of new bone on day 7 (n=5; Fig. 2D), but in contrast to the mandibular defects, this bone was not GFP-positive (Fig. 2E). Instead, tibial osteoblasts exhibited β-galactosidase activity (n=5; Fig. 2F). These results indicate that there are two sources of skeletal progenitor cells in an adult animal, and that they can be distinguished from one another based on their embryonic origin. In the tibia, this progenitor pool is derived from β-galactosidase-expressing cells. In the mandible, as well as other neural crest-derived bones of the head, skeletal progenitor cells are derived from GFP-expressing neural crest.
Are neural crest-derived and mesoderm-derived skeletal progenitor cells interchangeable?
The mandible and the tibia form from separate embryonic lineages and our
data show that they remodel and heal via these same separate populations. But
are these cell populations functionally different? One way to directly test
whether they were interchangeable was to perform heterotopic grafts, in which
mesoderm-derived skeletal progenitor cells were implanted into a mandibular
injury site and neural crest-derived skeletal progenitor cells were implanted
into a tibial defect. Before undertaking this experiment, however, we had to
ascertain that cells could survive the transplantation procedure and engraft
at the injury site. We used bone marrow stromal cells because of their
abundance and relative ease of harvesting, and isolated them from
L2G85 transgenic mice. Cells from L2G85 mice constitutively
express firefly luciferase and GFP, which allowed us to visualize their
location using both in vivo imaging and GFP immunostaining
(Sheikh et al., 2007). Cells
were transplanted into tibial injuries created in wild-type syngeneic mice.
Control mice sustained a tibial injury but received no cell transplant. At
various times after grafting, host mice were presented with the luciferin
substrate, which is metabolized by luciferase-expressing cells to produce
oxyluciferin and energy in the form of light
(Shinde et al., 2006). Control
mice exhibited no bioluminescence at the injury site
(Fig. 3A). By contrast, mice
that received an L2G85 transplant showed a robust bioluminescent
signal, which was detectable within 48 hours of cell transplantation and
increased with time, peaking at day 9 (Fig.
3B, and data not shown). The signal gradually tapered off but was
still detectable at post-surgery day 23
(Fig. 3C). Between
post-surgical days 2 and 9, there was a 7-fold increase in bioluminescence,
indicating that luciferase-expressing cells proliferated soon after
transplantation.
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We were now in a position to test whether neural crest-derived and
mesoderm-derived osteoprogenitor cells were functionally equivalent. We used
periosteum as a source of skeletal progenitor cells and harvested the tissue
from tibiae and transplanted it into the mandibular injury sites; we also
harvested periosteum from mandibles and transplanted it into tibial injury
sites. The transplantation procedures always involved syngeneic hosts and
genetically labeled periostea [β-actin GFP
(Okabe et al., 1997)] in order
to distinguish transplanted cells from host cells. We also performed two
additional experiments: one in which tibial periosteum was transplanted into a
tibial defect, and one in which mandibular periosteum was transplanted into a
mandibular defect. We refer to these as homotopic grafts, because the
embryonic origin of the graft matched the origin of the injury site. Homotopic
grafts allowed us to evaluate the extent to which transplanted cells
contributed to the bony regenerate, as well as the rate and mechanism of
healing (i.e. endochondral, intramembranous) following a grafting
procedure.
We observed robust bone regeneration in the homotopic grafting experiments. For example, in the tibia (n=7; Fig. 4A) and the mandible (n=7; Fig. 4C), new bone bridged the defect by day 10. We confirmed that the new bone was derived from the grafted periosteum by GFP immunostaining (Fig. 4B,D). We also determined the volume of new bone by histomorphometry and found that homotopic grafts produced almost equivalent amounts of bone in mandibular and tibial injury sites (Fig. 4I). This latter information demonstrated that both tibial and mandibular injury sites fully supported the differentiation of transplanted progenitor cells into osteoblasts.
We then tested whether neural crest-derived and mesoderm-derived skeletal progenitor cells were interchangeable. When grafts of mandibular periosteum were transplanted into tibial defects (i.e. tibial heterotopic grafts; n=9), we observed the same osteogenic effect that we had seen in the homotopic grafting scenario: a bony matrix bridged the defect (Fig. 4E,F). The volume of new bone in this heterotopic grafting experiment, however, exceeded that of both homotopic grafts (Fig. 4I). Was this because the tibial injury supported bone formation better than the mandible, or because the mandibular periosteal cells had a more robust osteogenic potential? The former explanation was unlikely, because both the mandibular and tibial injury sites had nearly equivalent amounts of new bone (Fig. 4I). The latter explanation was more feasible, because mandibular homotopic grafts showed more robust osteogenesis overall (Fig. 4I). The mechanism(s) behind this apparent increase in osteogenic potential of mandibular periosteum became the subject of an additional set of experiments.
If one population of skeletal progenitor cells has `increased osteogenic potential', this could be attributable to an enhanced rate of proliferation, or an increased rate of differentiation. To distinguish between these possibilities we co-cultured equivalent numbers of neural crest-derived progenitor cells with mesoderm-derived progenitor cells. Cultures were examined at multiple time points and cell numbers counted. From this visual analysis it was clear that mesoderm-derived cells out-proliferated neural crest-derived cells (Fig. 5A,B). BrdU incorporation confirmed that mesoderm-derived osteoprogenitor cells had a significantly greater proliferative potential than neural crest-derived cells (Fig. 5C).
Was there also a difference in the rate of osteogenic differentiation? Both neural crest and mesoderm populations were cultured in osteogenic differentiation media (ODM) and at all time points examined, neural crest-derived cells showed increased Alizarin Red staining (Fig. 5D-F). qRT-PCR assays confirmed this enhanced osteogenic potential: after 4 days in ODM, neural crest-derived cells showed a significant increase in the expression of early osteoblast markers such as Runx2 and Col1a (Fig. 5G). After 10 days in ODM, neural crest-derived cells showed significantly higher levels of osteogenic markers including Runx2, Col1a, osteopontin (Spp1 - Mouse Genome Informatics) and osteocalcin (Bglap1/2) (Fig. 5H). Thus, mesoderm-derived skeletal progenitor cells proliferate more, but neural crest-derived skeletal progenitor cells differentiate faster. Therefore, our in vivo finding of enhanced osteogenesis in neural crest transplants is explained by the increased osteogenic capacity of neural crest-derived cells.
We returned to our homotopic and heterotopic grafting experiments, and performed one final graft in which tibial periosteum was transplanted into mandibular defects (n=9). These mandibular heterotopic grafts showed a different outcome than all of the previous grafts: we found an abundance of cartilage instead of bone in the injury site (Fig. 4G,H,J). Was the cartilage derived from the grafted cells? We performed GFP immunostaining and confirmed that chondrocytes were derived exclusively from β-actin GFP mesodermal cells (Fig. 4K). This finding also confirmed that the transplanted periosteum contains skeletal progenitor cells, because these cells retain their ability to differentiate into chondrocytes and osteoblasts.
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Neural crest-derived skeletal progenitor cells adopt a skeletogenic lineage appropriate to their locale
Thus far, our data demonstrate that there are at least two populations of
adult skeletal progenitor cells that can be distinguished based on their
embryonic origin. Furthermore, neural crest-derived and mesoderm-derived bones
heal through the preferential recruitment of cells from their own embryonic
origin. Although this might simply be due to the proximity of one population
of progenitor cells, we also demonstrated that in some cases the skeletal
progenitor cells are interchangeable. Namely, neural crest-derived
osteoprogenitor cells form new bone regardless of whether they were placed
into a tibial defect or into another mandibular injury site. Was this because
neural crest-derived progenitor cells were committed to an osteogenic fate,
which precluded them from adopting a chondrogenic one? We tested this by
generating fractures in the mandible, which heal via endochondral
ossification. In these cases, neural crest-derived progenitor cells
differentiated into chondrocytes (data not shown). Therefore, neural
crest-derived progenitor cells exhibit considerable plasticity, even into
adulthood: regardless of where they were transplanted, these cells adopted the
skeletogenic lineage appropriate to their locale.
An association between embryonic lineage and Hox status might influence skeletal progenitor cell fate
We were still left with the observation that mesoderm-derived skeletal
progenitor cells did not appear to exhibit the same plasticity as neural
crest-derived skeletal progenitor cells: when they were transplanted into
mandibular defects, the grafted mesodermal cells differentiated into
chondrocytes even though the environment fully supported osteogenic
differentiation (Fig. 4G).
These results suggested that there was a fundamental difference in the
plasticity of neural crest-derived and mesoderm-derived progenitor cells.
During development, Hox genes are expressed in a nested pattern along the
body axis (Chisaka and Capecchi,
1991; Creuzet et al.,
2002; Wellik and Capecchi,
2003; Le Douarin et al.,
2004), where they provide cells with positional information. For
example, Hoxa11 and Hoxa13 are expressed in limb mesoderm
(Wellik and Capecchi, 2003;
Knosp et al., 2004;
Rinn et al., 2008), where they
regulate patterning and morphogenesis of the fetal appendicular skeleton
(Tabin, 1995). Are adult cells
provided with positional memory through a similar molecular mechanism? Using
in situ hybridization we found that Hoxa13 and Hoxa11
expression persisted in the adult skeleton: Hoxa11 transcripts were
readily detectable in tibial osteocytes
(Fig. 6A,B) and throughout
tibial injuries (Fig. 6C), but
were conspicuously absent from the intact or injured mandible
(Fig. 6D,E). Our subsequent
analyses focused on Hoxa11 because of its exclusive expression in the
tibia and absence from the mandible.
Embryonic cell transplantation experiments have shown that Hox gene
expression is a critical determinant of whether grafted cells integrate into
their new location (reviewed by Le Douarin
et al., 2004). Did a discrepancy in Hox expression underlie the
chondrogenic outcome of the adult heterotopic grafts? We harvested
Hox-positive tibial periosteum and transplanted it into the Hox-negative
mandible, then evaluated the status of Hox gene expression in the injury site.
After 7 days, we found that the grafted cells maintained their
Hoxa11-positive status, and the injury site sustained its
Hoxa11-negative status (Fig.
6F,G). Furthermore, the domain of Hoxa11 expression
coincided precisely with the chondrogenic region in the heterotopic callus
(Fig. 6F,G).
Embryonic studies have shown that when Hox-negative tissues are
transplanted they adopt the Hox status of their new location
(Itasaki et al., 1996). We
examined the tibial heterotopic transplants to see whether the same kind of
change occurred in an adult regenerative context. At the time of harvest,
mandibular periosteum was Hox-negative
(Fig. 6D), but when examined at
post-transplantation day 7 the grafted cells expressed Hoxa11
(Fig. 6H,I). Was this just an
artifact of the transplantation procedure, or were the Hox-positive host cells
responsible for this switch in molecular fate? We co-cultured
Hoxa11-expressing tibial periosteal cells
(Fig. 6J,K) with Hox-negative
mandibular periosteal cells and found that within 5 days the formerly
Hox-negative mandibular periosteal cells began to express Hoxa11
(Fig. 6L,M).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our cellular and molecular analyses belie this histological equivalency. Using Wnt1Cre::Z/EG mice we found that when the neural crest-derived mandible is injured, the callus is composed entirely of neural crest-derived cells. In parallel, we found that when the tibia is damaged, the injury site is occupied entirely by mesoderm-derived cells. Thus, there are at least two populations of adult skeletal progenitor cells that can be distinguished based on their embryonic origins.
Injured skeletal elements normally use cells of their own embryonic origin
for repair. At least in the mandibular repair site, we found no evidence of
cells from the circulation that contributed in a major way to the repair
process. Likewise, we did not observe a major contribution from endothelial
cells, which in the head are derived from mesoderm
(Couly et al., 1995). The
presence of two distinct populations of skeletal stem cells in the adult might
have clinical implications because if bones preferentially heal using cells
that share the same embryonic origin, then reparative strategies may have to
take this variable into account in order to be maximally effective.
But does the fact that mandibular defects heal via neural crest-derived progenitor cells, and tibial defects heal via mesoderm-derived progenitors, indicate a preferential recruitment? Or are cells from the local environment merely conscripted to aid in the reparative process, and it just happens that these cells that contribute to the regenerate share the same embryonic origin as the damaged bone? This latter possibility would suggest that despite differences in embryonic origin, cranial and appendicular bones can heal using any skeletal progenitor cell population.
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;
Ferguson et al., 1999).
The mechanical environment also influences how bones heal and we wondered
whether this might have contributed to the chondrogenic differentiation of
mesoderm-derived progenitors placed into a mandibular defect. Irrespective of
the embryonic origin of a bone, however, a stabilized environment favors
intramembranous ossification, whereas a non-stabilized environment heals
through endochondral ossification (reviewed by
Carter et al., 1998). In our
injury model, the mechanical environment was equivalent. Therefore, we cannot
attribute the disparity in healing to differences in the mechanical
environment.
We sought a molecular explanation for this phenomenon, and examined the injury sites for alterations in gene expression. Once cells had begun the process of osteoblast differentiation, however, there was no discernible difference in their expression patterns. What we searched for was a difference in `molecular identity' between the two populations of osteoprogenitor cells, which led us to consider Hox gene expression.
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),
and we found that Hoxa11 expression persisted in adult tibial
osteoblasts and osteocytes (Fig.
6). Hoxa11 was not expressed in the adult mandible
(Fig. 6), in keeping with its
Hox-negative status during embryonic development
(Creuzet et al., 2002).
The absence of Hoxa11 expression in the mandible was especially
important in our next experiments, in which we examined the heterotopic grafts
for their Hox status. Tibial grafts maintained their Hox-positive status even
after transplantation into a Hox-negative mandibular environment. Hox-negative
neural crest-derived progenitors, by contrast, adopted Hox expression when
transplanted into a Hox-positive location. Our in vitro data parallel these in
vivo findings: Hox-negative neural crest-derived cells adopted a Hox code
after co-culture with Hox-expressing mesoderm-derived cells, and these
mesodermal cells did not alter their Hox-positive status. Thus, adult skeletal
stem cells are equipped with a type of positional memory that is retained even
after transplantation. This conclusion is not without precedent: when
embryonic Hox-negative cranial neural crest cells are transplanted into a
Hox-positive domain within the neural tube, the cells adopt the Hox status in
their new environment; conversely, Hox-positive cells maintain their Hox
status when transplanted into a Hox-negative environment
(Grapin-Botton et al., 1995;
Couly et al., 1998).
These and additional experiments indicate that the Hox status of a cell
confers upon it a sense of positional identity, and this identity is unchanged
when cells are placed into a new environment
(Le Douarin et al., 2004).
There is a `flip side' to this: mandibular neural crest cells normally lack
Hox gene expression and this `Hox-free' condition has been strongly associated
with their remarkable plasticity in development and evolution (reviewed by
Helms et al., 2005).
Other groups have evaluated the correlation between Hox expression and the
skeletogenic capacity of a cell (Abzhanov
et al., 2003), but these experiments are difficult to compare with
our findings. First, our experiments were largely conducted in vivo, whereas
the majority of those analyses were carried out in vitro. Second, our data do
not implicate Hox status in influencing whether a cell population has
skeletogenic capacity or not. Instead, we show that Hox expression seems to
confer a positional memory on adult cells, which closely parallels the
function of Hox genes during embryonic development.
We found that Hox-negative adult neural crest cells begin to express posterior Hox genes when placed into a Hox-positive environment. This is a very intriguing finding because it implies that injury sites have specific Hox codes, and a synchrony between the cells occupying the injury site and the injury environment itself might be a crucial component of normal healing. How is such a Hox code established in a wound? Future experiments will directly test whether a disparity in Hox gene expression underlies the ability of any grafted cell to heal wounds more efficiently.
Other adult cells use a Hox code to distinguish their position in the body:
dermal fibroblasts retain the Hox code appropriate to their site of initial
derivation despite numerous passages in culture
(Chang et al., 2002;
Rinn et al., 2008).
Furthermore, Hox-positive fibroblasts do not alter their regional Hox code
even when cells from different locales are cultured together
(Rinn et al., 2008). Those
experiments, however, did not evaluate fibroblasts from the head. Are cranial
neural crest-derived dermal fibroblasts, like neural crest-derived skeletal
progenitor cells, Hox-negative? If so, do they readily adopt the Hox status
when co-cultured or transplanted as we have shown here for skeletal progenitor
cells?
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).
Clearly, further studies are needed to directly test whether the molecular
differences between a graft and the recipient site are critical determinants
of successful tissue repair and regeneration.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/17/2845/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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