Seminars in Cell & Developmental Biology 19 (2008) 459–466

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Seminars in Cell & Developmental Biology

journal homepage: www.elsevier.com/locate/semcdb

Review

Bone remodeling during fracture repair: The cellular picture

Aaron Schindeler a,b,∗ , Michelle M. McDonald a , Paul Bokko a , David G. Little a,b
a
Department of Orthopaedic Research & Biotechnology, The Children’s Hospital at Westmead, Sydney, Australia
b
Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, Sydney, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Fracture healing is a complex event that involves the coordination of a variety of different processes.
Available online 25 July 2008 Repair is typically characterized by four overlapping stages: the initial inflammatory response, soft callus
formation, hard callus formation, initial bony union and bone remodeling. However, repair can also be
Keywords: seen to represent a juxtaposition of two distinct forces: anabolism or tissue formation, and catabolism or
Bone remodeling remodeling. These anabolic/catabolic concepts are useful for understanding bone repair without giving
Fracture
the false impression of temporally distinct stages that operate independently. They are also relevant when
Fracture healing
considering intervention.
Bone repair
Bisphosphonates
In normal bone development, bone remodeling conventionally refers to the removal of calcified bone
tissue by osteoclasts. However, in the context of bone repair there are two phases of tissue catabolism:
the removal of the initial cartilaginous soft callus, followed by the eventual remodeling of the bony
hard callus. In this review, we have attempted to examine catabolism/remodeling in fractures in a sys-
tematic fashion. The first section briefly summarizes the traditional four-stage view of fracture repair
in a physiological manner. The second section highlights some of the limitations of using a temporal
rather than process-driven model and summarizes the anabolic/catabolic paradigm of fracture repair.
The third section examines the cellular participants in soft callus remodeling and in particular the role
of the osteoclast in endochondral ossification. Finally, the fourth section examines the effects of delaying
osteoclast-dependent hard callus remodeling and also poses questions regarding the crosstalk between
anabolism and catabolism in the latter stages of fracture repair.
© 2008 Elsevier Ltd. All rights reserved.

Contents

1. An overview of fracture repair: the four-stage model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

1.1. Stage 1: inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
1.2. Stage 2: soft callus (fibrocartilage) formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
1.3. Stage 3: hard callus formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
1.4. Stage 4: bone remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
2. The anabolic/catabolic model of fracture repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
3. Soft callus remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
4. Hard callus remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464

1. An overview of fracture repair: the four-stage model

∗ Corresponding author at: Orthopaedic Research & Biotechnology, Research

Fracture healing is a process that recapitulates certain aspects of
Building, The Children’s Hospital at Westmead, Locked Bag 4001, Westmead, NSW
skeletal development and growth involving a complex interplay of
2145, Australia. Tel.: +61 2 98450117; fax: +61 2 98453078. cells, growth factors, and extracellular matrix. Repair is convention-
E-mail address: AaronS@chw.edu.au (A. Schindeler). ally partitioned into four stages, each characterized by a specific set

1084-9521/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.semcdb.2008.07.004
460 A. Schindeler et al. / Seminars in Cell & Developmental Biology 19 (2008) 459–466

of cellular and molecular events [1–3]. But in practical terms there
is rarely such a clear delineation of events; there is often signif-
icant overlap between the different stages during fracture repair.
The four-stage model can still be a useful system for describing the
basic events that take place.
The four-stage model originated as the result of histological
observations of healing fractures in both human patients and ani-
mal models (Fig. 1A). However, research of the past several decades
has explored both the cellular and molecular forces that drive the
underlying processes [4–6]. At the cellular level, inflammatory cells,
vascular cells, osteochondral progenitors, and osteoclasts are key
players in the repair process. At the molecular level, fracture repair
is driven by the three main classes of factors: pro-inflammatory
cytokines and growth factors, pro-osteogenic factors, and angio-
genic factors [7]. These help establish the relevant morphogenetic
fields by recruiting cells and stimulating growth and/or differ-
entiation. Thereafter, the damaged soft tissues are repaired and
the fracture is bridged by soft callus and later hard callus. The
bridging hard callus is eventually remodeled to re-establish the
original geometry and function of the damaged tissue. In certain
circumstances where there is absolute rigidity, or in well-apposed
metaphyseal fractures, intramembranous hard callus formation
may dominate without the clear presence of an intermediate car-
tilaginous soft callus.
activation of non-specific wound healing pathways that accom-
pany non-skeletal injuries. The extravasation (bleeding) within the
fracture site is contained by the surrounding tissue and devel-
ops into a hematoma. Degranulating platelets, macrophages, and
other inflammatory cells (granulocytes, lymphocytes, and mono-
cytes) infiltrate the hematoma between the fractured fragments
and combat infection, secrete cytokines and growth factors, and
advance clotting into a fibrinous thrombus [1,2]. Over time, cap-
illaries grow into the clot, which is reorganized into granulation
tissue. Macrophages, giant cells and other phagocytic cells clear
degenerated cells and other debris.
This cellular response is coordinated by and involves the
secretion of a range of cytokines and growth factors including
transforming growth factor-␤ (TGF-␤), platelet-derived growth fac-
tor (PDGF), fibroblast growth factor-2 (FGF-2), vascular endothelial
growth factor (VEGF), macrophage colony stimulating factor (M-
CSF), interleukins-1 and -6 (IL-1 and -6), bone morphogenetic
proteins (BMPs), and tumor necrosis factor-␣ (TNF-␣) [1–3]. This
factors facilitate the recruitment of additional inflammatory cells
in a positive feedback loop, and also the migration and invasion
of multipotent mesenchymal stem cells [8,9]. Stem cells originat-
ing from the periosteum [10], bone marrow [11,12], circulation [13],
and the surrounding soft tissues [14], have been implicated in bone
formation and repair.

1.1. Stage 1: inflammation 1.2. Stage 2: soft callus (fibrocartilage) formation

A fracture is typically associated with disruption of the local Most fractures possess some level of mechanical instability and
soft tissue integrity, interruption to normal vascular function, and heal by the process of endochondral ossification—bony callus for-
a distortion of the marrow architecture. This damage leads to mation is preceded by a cartilaginous template [1]. This stage is

Fig. 1. Models of fracture repair and the cellular participants. (A) A representative series of images of the four-stage model of fracture healing. Between the classical stages
2 and 3, the soft callus is systematically remodeled. (B) A more elaborate version of the anabolic/catabolic model of fracture repair [36] that incorporates the concepts of
non-specific anabolism (the early wound repair processes) and non-specific catabolism (soft callus remodeling). (C) A schematic showing the cellular contributors to the
fracture repair processes. The source(s) of mesenchymal progenitors that are able to differentiate into osteoblasts remain ambiguous.
 A. Schindeler et al. / Seminars in Cell & Developmental Biology 19 (2008) 459–466
dominated on a cellular level by chondrocytes and fibroblasts,
although the relative proportions of the different cell types can vary
between fractures. These cells produce a semi-rigid soft callus that
is able to provide mechanical support to the fracture, as well as act
as a template for the bony callus that will later supersede it. Car-
tilaginous callus is principally avascular, although its subsequent
replacement with woven bone involves vascular invasion.
Chondrocytes derived from mesenchymal progenitors prolif-
erate and synthesize cartilaginous matrix until all the fibri-
nous/granulation tissue is replaced by cartilage. Where cartilage
production is deficient, fibroblasts replace the region with gener-
alized fibrous tissue. Discrete cartilaginous regions progressively
grow and merge to produce a central fibrocartilaginous plug
between the fractured fragments that splints the fracture [7]. In
the final stages of soft callus production, the chondrocytes undergo
hypertrophy and mineralize the cartilaginous matrix before under-
going apoptosis.
Fibroblast proliferation and chondrocyte prolifera-
tion/differentiation are stimulated by the coordinated expression
of growth factors including TGF-␤2 and -␤3, PDGF, fibroblast
growth factor-1 (FGF-1), and insulin-like growth factor (IGF)
[1,2]. Additionally, members of the BMP family (BMP-2, -4, -5
and -6) assist in promoting cell proliferation and chondrogenesis.
In response to these factors, chondrocytes are able to generate
considerable amounts of extracellular matrix proteins, particu-
larly collagen II (or collagen X for hypertrophic chondrocytes)
[4].
Invasion of the soft callus by vascular endothelial cells, angio-
genesis, and capillary in-growth is stimulated by pro-angiogenic
factors including VEGF, BMPs, FGF-1 and TGF-␤ [1,15,16]. VEGF
expression by osteogenic cells is dependent on the master bone
and cartilage regulatory factor Cbfa1/Runx2 [17], and is not highly
expressed by the early mesenchymal/inflammatory tissue at the
fracture site [18]. In addition, angiopoietin I and II regulate vascu-
lar morphogenesis of larger vessels and development of collateral
branches from existent vessels [1,4].
1.3. Stage 3: hard callus formation
Also known as primary bone formation [1], this stage represents
most active period of osteogenesis. It is characterized by high lev-
els of osteoblast activity and the formation of mineralized bone
matrix, which arises directly in the peripheral callus in areas of sta-
bility. In order for bridging new hard callus to form, the insecure
soft callus is gradually removed, concomitant with revasculariza-
tion. The new bone is known as the hard callus and it is typically
irregular and under-remodeled. Notably, hard callus can form in
the absence of a cartilaginous template in intramembranous bone
formation (during conditions of high mechanical stability) or in
appositional bone growth, where bone forms directly adjacent
to an existing mineralized surface. However, in the majority of
orthopaedic instances, some level of endochondral ossification is
present.
The initial woven bone matrix contains a combination of pro-
teinaceous and mineralized extracellular matrix tissue. This is
synthesized by mature osteoblasts, which differentiate from osteo-
progenitors in the presence of osteogenic factors. Members of
the BMP family are critical mediators of this process, and have
been shown to be sufficient for de novo bone formation [19,20].
Other growth factors are expressed during this time, although it
is unclear whether their impact on bone healing predominantly
affects osteoprogenitor migration/proliferation versus osteogenic
differentiation [1].
The source(s) of osteoprogenitors responsible for fracture heal-
ing remains ambiguous. The periosteum is a rich source of stem
461
cells able to produce bone and bone healing is often delayed when
the periosteum is damaged or stripped [10,21]. The bone marrow
also contains mesenchymal stem cells capable of contributing to
bone formation during repair [11,12]. However, bones are able to
mount an effort at healing, including hard callus formation, even in
the absence of periosteal and/or marrow osteoprogenitors. Thus it is
possible that osteoprogenitors may originate from multiple sources
including the circulation [13], the vasculature [22], and surrounding
local tissues [14].
The vasculature is known to be critical for formation of the
hard callus, with increased oxygen tension in the local region
necessary for osteoblast differentiation. Stimulation of vessel for-
mation using angiogenic factors can augment bone formation
and fracture healing in model systems [23,24]. However, recent
studies have shown that delayed union can occur even after the
vasculature has been re-established, indicating that the blood
supply alone is not the only determinant of fracture healing
success [25–27].
1.4. Stage 4: bone remodeling
The final stage of fracture repair encompasses the remodeling
of the woven bone hard callus into the original cortical and/or
trabecular bone configuration. This phase can also be referred to
as secondary bone formation [1]. Initially, this involves converting
the irregular woven bone callus into lamellar bone, although the
standard cortical structure is eventually restored. The remodeling
process is driven by a coupled process of orderly bone resorption
followed by the formation of lamellar bone. Osteoblast-like cells
may also play a minor role in proteolysis of osteoid elements, prior
to new bone formation [28]. The process of neo-vascularization is
maintained.
The key cell type involved with the resorption of mineralized
bone is the osteoclast. The osteoclast is a large multinucleated cell
that is formed by the differentiation and fusion of haematopoietic
precursors [29]. To effect remodeling, osteoclasts become polarized
and adhere to a mineralized surface. They form a ruffled border,
which is sealed off and acid and proteinases are pumped into
the resorption domain. The acid environment demineralizes the
matrix, while proteinases degrade the organic components, such as
collagen. The degradation products are removed through a vesic-
ular pathway from the ruffled border to the functional secretory
domain and the osteoclasts are either apoptosed or return to the
non-resorbing form. Bone resorption by osteoclasts creates erosive
pits on the bone surface known as “Howship’s lacuna”. Once com-
pleted, osteoblasts are able to lay down new bone on the eroded
surface.
Two principal cytokines that are secreted by osteoblasts are
critical for the induction, survival and competency of osteoclasts:
M-CSF and Receptor Activator of NF␬B Ligand (RANKL). M-CSF is
important for the primary induction of haematopoietic stem cell
differentiation towards an osteoclast lineage [30]. RANKL is a factor
produced by mature osteoblasts that is responsible for the coordi-
nation of bone formation and bone resorption [31]. Osteoprotegrin
(OPG) is a secreted decoy receptor that is an important regulator
of RANKL signaling and antagonizes osteoclast differentiation [32].
In addition to these specialized pro-osteoclastic factors, a range
of growth factors and cytokines present in a healing fracture are
known to promote osteoclastogenesis. These include interleukins,
TNF-␣, BMPs, and TGF-␤ [33–35].
The molecular pathways involved in osteoclast differentiation
and function are becoming increasingly well described. Many of
these molecules have been linked to genetic diseases affecting
osteoclasts, and/or identified as putative drug targets for osteoporo-
sis therapies.
462 A. Schindeler et al. / Seminars in Cell & Developmental Biology 19 (2008) 459–466
2. The anabolic/catabolic model of fracture repair
Although the sequential four-stage model describes the funda-
mental events that occur over the timeline of a healing fracture,
there are often significant overlaps between stages. For example,
replacement of peripheral callus with lamellar bone remodeling
occurs concurrently with cartilage mineralisation, vascular inva-
sion and woven bone formation. Moreover, numerous fundamental
events like cell proliferation, differentiation, matrix synthesis at
cellular level is not limited to any one stage [1].
Applying the concepts of anabolism and catabolism may provide
a useful alternative system for understanding the fracture repair
process [36]. In this system, the outcome of the fracture repair pro-
cess exists as a balance between the anabolic (bone forming) and
catabolic (bone resorbing) responses. We have previously used this
approach to describe hard callus remodeling [36]. However, as we
will go on to describe, anabolic/catabolic processes also occur dur-
ing the formation and removal of the fibrocartilage soft callus. Many
of the cell types recruited to the early stage fracture site and the
associated regulatory growth factors and cytokines are seen in a
generalized response to tissue injury and are thus non-specific to
bone. Consequently, we propose that these processes be referred to
as non-specific anabolism and non-specific catabolism (Fig. 1B). We
speculate that the speed of fracture healing may be determined by
the processes of non-specific anabolism and catabolism (recruiting
cells, revascularisation), while the strength of repair relates to the
mechanically driven balance between bone-specific anabolism and
catabolism.
Events in fracture healing are considerably influenced by numer-
ous physiological and pharmacological factors in the prevailing
environment that determine the state of the fracture and the pace
of repair. Key features include the nature location and extent of
injury, the biomechanical forces, infection and/or disease, surgi-
cal fixation, adjunctive drug therapies, nutrition and general bone
health, as well as any underlying genetic conditions. All of these fac-
tors can be described in terms of their effects on non-specific and
specific anabolism and/or catabolism. The utility of this approach
is that as pro-anabolic and anti-catabolic bone therapies become
more advanced, treatment approaches can be targeted to produce
the maximal benefit-dependent on the underlying bone healing
deficiency [36].
3. Soft callus remodeling
Soft callus remodeling occurs between stages 2 and 3 of the tra-
ditional four-stage model of fracture repair. This process involves
the gradual removal of the soft callus cartilage/fibrocartilage and its
systematic replacement with woven bone [37]. Failure to remove
this tissue can lead to delay or arrest of the fracture repair process,
thus considerable research has been undertaken to understand the
cellular determinants of the process. It was traditionally believed
that osteoclasts were the key cell type involved with both soft callus
and hard callus remodeling [38]. However, recent evidence suggests
that osteoclasts may be somewhat redundant in the remodeling of
the soft callus.
The suggestion that osteoclasts may be involved with soft cal-
lus remodeling is not without basis. There is histological evidence
that large multinucleated cells are present within the soft cal-
lus together with vascular endothelial cells at the invasion front
[38–40]. These cells are also termed chondroclasts based on their
physical location, although they stain for classic osteoclastic mark-
ers and exhibit a similar morphology [41].
However, if osteoclasts are indeed critical for soft callus remod-
eling, anti-osteoclastic agents could be expected to interfere with
this process and possibly delay fracture repair. This is clinically
relevant as anti-resorptive drugs, including but not limited to
bisphosphonates (BPs), are widely used in the osteoporotic popula-
tion. Should BPs or other interventions delay soft callus remodeling
in osteoporotic fractures, this effect would be of considerable clin-
ical concern [42].
One clinical case study suggested that the delayed or poor bone
healing observed in nine patients with osteoporosis related bone
loss was a result of their chronic treatment with BPs [43]. Although
the findings of this study showed extensive reductions in systemic
bone formation in these patients, the direct link from the BP treat-
ment to the poor bone healing cannot be disproved, fuelling the
concerns of clinicians treating osteoporotic patients with fractures.
A clear and defined role for osteoclast activity during initial endo-
chondral bone union after fracture therefore must be rigorously
evaluated.
With a focus on the clinical questions surrounding BP treatment
during periods of bone healing, our group has recently examined
the effect of the two BPs, zoledronic acid (ZA) and pamidronate
(PAM), on endochondral union in a rat closed fracture model
[44,45]. In these studies, both therapeutic and extremely high doses
of these agents failed to interfere with the achievement of endo-
chondral union. These outcomes suggest that BPs may be safely
administered to patients undergoing periods of bone repair. How-
ever, as healthy growing rats were used, care needs to be made in
translating these results to the treatment of osteoporotic patients.
Osteoporotic animals show impaired fracture repair [46–48], and
thus investigating the influence of BPs in osteoporotic rodent mod-
els would be highly relevant.
Based on this recent data, we propose a new model regarding
osteoclast function and bone repair: that osteoclastic resorptive
function may be redundant during initial endochondral bone union.
In this model, soft callus remodeling is a non-specific catabolic
process that can involve the contribution of multiple cell types.
In support of this hypothesis we have also examined endochon-
dral bone healing in the incisor absent (ia/ia) rat. These rats exhibit
extensive osteopetrosis due to harboring a mutant osteoclast pop-
ulation with reduced functional activity [49]. Within the period
prior to recovery from osteopetrosis, fractures performed in these
rats reached endochondral union within the same time frame as
their normal littermates [50].
This model is further supported by experiments using genet-
ically modified mice undertaken to examine the functional role
of osteoclasts in endochondral bone repair. When fracture heal-
ing was examined in the osteoclast-deficient osteopetrotic (op/op)
mouse, the removal of the cartilage callus was identical between
mutant mice and normal littermates [51]. These investigators went
on to administer recombinant osteoprotegerin (OPG) as a pharma-
cological means of inhibiting osteoclast formation. OPG acts as a
decoy receptor for receptor activator of NF␬B (RANK) signaling, and
antagonizes the differentiation of osteoclast progenitors. Despite
very low osteoclast number, OPG-treated mice showed compara-
ble union rates to untreated control mice [51]. Furthermore, neither
of two recent studies examining fracture repair in rats using OPG
treatment reported a delay in the achievement of initial endochon-
dral union [52,53].
One of the few examples of impaired initial union in the absence
of osteoclasts involved Rank−/− knockout mice [51]. This mouse line
completely lacks osteoclasts, leading to a grossly osteopetrotic phe-
notype such that tibial fractures could not be fixated as per the
control mice. Unstabilized Rank−/− mouse fractures formed abun-
dant soft callus, but hard callus formation was significantly delayed.
While the authors suggested that this could be due to reduced bone
formation and/or revascularization, it is unclear whether cartilage
removal could have also been delayed. More likely, the magnitude of
soft callus that formed in the absence of any fixation overwhelmed
 A. Schindeler et al. / Seminars in Cell & Developmental Biology 19 (2008) 459–466
the normal soft callus remodeling process. Certainly, when osteo-
clast formation was blocked in wild type mice using an anti-RANK
ligand antibody, this did not effect soft callus formation or remod-
eling, or the union rates of stabilized fractures [51].
If osteoclast function is redundant during endochondral fracture
repair then the role of other cell lineages must be considered. Such a
cell type must (i) be present in the soft callus during the remodeling
process and (ii) secrete factors able to break down collagen and
other extracellular matrix proteins found in soft callus cartilage.
A group of proteases known as matrix metalloproteinases
(MMPs) have been implicated as a fundamental class of col-
lagenases and gelatinases responsible for extracellular matrix
degradation. Secretion of MMPs has been demonstrated by a num-
ber of cell types involved in or localized to the chondro-osseous
junction (the boundary between cartilage and bone tissue) dur-
ing endochondral bone repair. MMP-9 co-localizes to cells of
the chondro-osseous junction, and has been shown to co-stain
with both inflammatory cells and osteoclasts [54,55]. MMP-13 co-
expresses with markers for pre-osteoblasts, mature osteoblasts and
hypertrophic chondrocytes. Unlike MMP-9 and MMP-13 has not
been localized in osteoclasts. The absence of either MMP-9 or MMP-
13 impedes soft callus removal, causing a delay in union during
endochondral fracture repair [55,56]. MMP-14 is another abundant
metalloproteinase that plays a critical role in growth plate chondro-
cytes and may therefore also be relevant to endochondral fracture
repair [57]. These studies support the concept that multiple MMP-
expressing cell types, not only osteoclasts, may be able to contribute
to soft callus remodeling.
MMPs have also been strongly implicated in driving angiogene-
sis during tumor invasion and bone development [58]. The invasion
of new blood vessels into the avascular hypertrophic cartilage of the
soft callus is a critical step in the process of endochondral bone
repair. Structural and immunohistochemical staining of healing
fractures showed capillaries lined with laminin positive basement
membranes containing endothelial cells adjacent to the hyper-
trophic chondrocyte matrix at the invasion front [59]. Perivascular
cells were commonly observed lining these vessels, where they
were suggested to participate in degrading the chondral matrix.
Significantly, both vascular endothelial cells and perivascular cells
are able to secrete MMPs.
Angiogenesis has been deemed to be of critical importance
to skeletal repair. TNP-470 is potent inhibitor of angiogenesis;
it inhibits a specific isoform of methionine aminopeptidase and
blocks the proliferation of vascular cells. Rat femoral fractures
treated with TNP-470 showed severely impaired healing [60].
Intramembranous and endochondral ossification were disturbed
by the lack of angiogenesis. The early formation of fracture blastema
was also effected, with TNP-470-treated rats producing dense
fibrous tissue instead of a normal blastema. Similarly, inhibition
of VEGF signaling using a soluble, neutralizing VEGF receptor (Flt-
IgG) resulted in a delay in the vascular invasion and replacement
of cartilaginous callus with bone responsible for the transition
from soft callus to hard callus [61]. This is highly relevant to clini-
cal situations where patients receiving drugs with anti-angiogenic
effects sustain fractures. A recent bone healing study examined
the effects of Rapamycin, immunosuppressive agent with known
anti-angiogenic properties. Rapamycin-treated mice exhibited a
significant delay in endochondral repair. Calluses in treated mice
were significantly smaller and exhibited increased fibrocartilagi-
nous tissue compared to untreated controls [62].
Nevertheless, the precise role of angiogenic cells in soft callus
remodeling remains ambiguous. While vascular endothelial cells
and perivascular cells may secrete MMPs that degrade cartilage,
like osteoclasts they may be redundant to the process. The process
of vascular invasion causes a change in oxygen tension that itself
463
can also affect a variety of cell signaling responses and cell fate
decisions [63]. Finally, the formation of new vessels may allow the
invasion of additional cells from the circulation to contribute to soft
callus remodeling.
4. Hard callus remodeling
While osteoclasts may not be essential in the initial stages of
endochondral bone repair, remodeling of the hard callus is an
osteoclast-dependent process. This is most clearly seen in fracture
experiments where osteoclast activity is abolished following treat-
ment with a bisphosphonate. While there are conflicting reports
regarding the overall benefit versus risk of BPs, all reports unequiv-
ocally show an inhibition of hard callus remodeling.
The literature contains numerous animal fracture models
involving BP treatment that can vary in their selection of surgi-
cal site, fixation, and BP dosing regimen. Nevertheless, a number
of common themes emerge. Many of these studies found little or
no effect on the ultimate radiographic union rates or mechanical
strength, but that remodeling of the hard callus to a normal lamel-
lar bone structure was delayed [64–66]. High dose local application
can ablate the bone formation response, but this is not through
an osteoclastic mechanism, with decreased angiogenesis and pri-
mary bone formation being observed [67]. Two clinical studies have
indicated that BP treatment might inhibit initial union [43,68].
However, these papers refer to examples of chronic and/or high
dose BP treatment that are likely to result in an overall decrease in
bone turnover, rather than a specific inhibition of osteoclast func-
tion.
One of the first studies to show a potential benefit for delay-
ing hard callus remodeling used a single dose of PAM to treat
a rat femoral open fracture model [69]. In this study, BP was
administered via systemic injection (3 mg/kg s.c.) or locally by a
poly-l-lactide-coated K-wire (0.1 or 1.0 mg) used for intramedullary
fixation. Both delivery systems resulted in significant increases in
callus volume and bone mineral density (BMD), as well as improve-
ments in mechanical strength at early time points. A subsequent
study focusing on timing of anti-catabolic intervention was per-
formed using ZA in a rat femoral-closed fracture model [70]. This
study found that hard callus retention was maximized when ZA
was given at 2 weeks following fracture. When the drug was local-
ized using 14C-labelled ZA, more was present in the callus of the
2-week post-fracture dosed samples, supporting the concept that
BPs bind to areas of active bone formation/resorption [70].
Although BPs can delay hard callus remodeling, this delay does
not necessarily lead to negative functional outcomes. A remodeled
lamellar callus may have superior properties per unit area of bone,
but a larger woven bone callus may provide a similar mechanical
advantage. In a rat closed femoral fracture study where rats were
either pre-dosed or pre- and post-dosed with daily incadronate,
two different results were observed. Pre-dosing alone led to a
greater initial volume of hard callus, however this then remod-
eled to form a normal cortical shell over 6–16 weeks. In contrast,
continuous dosing maintained a disorganized woven bone cal-
lus. While disordered, the large callus observed in the high-dose
continuous group showed greater mechanical strength (ultimate
load) in 3-point bending tests compared to any of the remodeled
lamellar calluses [65]. In an analogous study using a similar frac-
ture model, rats were dosed with a single bolus dose of ZA versus
multiple intermittent ZA doses, both led to an increase in bone
strength at 6 weeks [44]. Again, the callus properties of the two
dosing groups were divergent: the bolus dosing group had par-
tially remodeled and possessed a cortex while the intermittent
dosing group exhibited a substantial woven bone callus. Critically,
464 A. Schindeler et al. / Seminars in Cell & Developmental Biology 19 (2008) 459–466
the mechanical strength of both treatment groups did not differ sig-
nificantly, showing that delayed remodeling did not compromise
strength.
While BPs act to suppress the bone resorptive ability of osteo-
clasts, they do not prevent osteoclast formation or signaling
crosstalk between osteoclasts and other cell types. However, in
mice treated with 10 mg/kg human osteoprotegerin (hOPG), osteo-
clastogenesis was inhibited and osteoclast numbers were reduced
in healing tibial fractures by 93% at 3 weeks (P < 0.001) and 92% at
8 weeks (P < 0.001) [52]. OPG treatment did not affect the strength
of the fractures, but decreased the material properties of the callus.
This reinforces the long-held concept that osteoclast-dependent
bone remodeling is responsible for transforming a sizable woven
bone callus into a compact, mechanically efficient lamellar struc-
ture. As an alternative to pharmaceutical intervention, genetic
models of osteoclast dysfunction have been pursued. Bone repair
experiments in these mouse and rat models have yielded compara-
ble results to BP treatment. For example, following a closed fracture
callus size was enhanced in the osteopetrotic ia/ia rats and hard
callus remodeling was also delayed [44,71,72].
Although hard callus remodeling is primarily driven by osteo-
clasts, it is essential that a parallel anabolic activity is maintained
by osteoblasts. In the remodeling phase, the process of anabolism
is coupled to bone resorption. This is in contrast to the early
anabolic response that forms the initial callus, which is primarily
cytokine/growth factor-driven and occurs in the absence of bone
resorptive forces.
Coupling between anabolic and catabolic forces is intrinsic to
normal bone anabolism. When osteoclast formation is altered,
some compensatory anabolic change is typically observed. For
instance, Opg−/− mice develop excess osteoclasts but this is par-
tially offset by increased bone formation [73]. Conversely, bone
formation is reduced in c-Fos mutant mice that lack osteoclasts
[74]. Due to the complexity of accurately measuring bone forma-
tion and resorption in a healing fracture, there are few examples
of anabolic/catabolic coupling in fracture repair. In osteogenesis
imperfecta patients on long-term PAM treatment, bone catabolism
is reduced and the anabolic response in bone healing is simi-
larly impaired [68]. In the osteoclast-deficient op/op mice, both
anabolic and catabolic responses are reduced in the growth plate,
a site of endochondral ossification analogous to a healing fracture
[75].
Nevertheless, the precise mechanism underlying the coupling
of anabolic and catabolic responses during remodeling remains
ambiguous [76]. Several models have been suggested to explain
the coupling process during remodeling. Active bone resorption is
capable of releasing numerous growth factors and osteoid break-
down products including BMPs, TGF␤, and collagen peptides. These
products may be responsible for stimulating local anabolism in
regions of high bone resorption [77]. However, rodent models have
revealed that anabolism can remain coupled in the presence of non-
functional osteoclasts, suggesting that the release of factors from
the bone matrix may not be critical [78,79]. Thus it is possible that
signaling molecules directly secreted by osteoclasts, such as osteo-
clast inhibitory lectin (OCIL) [80], may be important for stimulating
osteoblast function.
In addition to anabolic/catabolic coupling, the process of remod-
eling is modulated by mechanical forces. This tenet of orthopaedics
(Wolff’s law) stipulates that a healthy bone adapts to the load that it
is placed under. This principle can manifest in many ways in fracture
repair. For instance, bone healing is impaired in unloaded situations
such as disuse and/or rigid fixation. The osteocyte is a key cellu-
lar player involved in mechanosensing in bone and signals from
the osteocyte may be transduced to control both anabolism and
catabolism [81].
The secretion of sclerostin (the SOST gene product) by osteo-
cytes has emerged as an important regulator of bone anabolism
[82,83]. SOST expression in osteocytes is upregulated in regions of
disuse, which would be expected to reduce bone anabolism [84].
While sclerostin is important for the systemic regulation of bone
homeostasis it is unclear whether it plays a specific role in fracture
remodeling. SOST is expressed by cell types present in the remod-
eling callus (although not the early phases of bone repair) [85].
Thus sclerostin may important for the normal downregulation of
specific anabolism in the callus, and may also represent a poten-
tial pharmacological target for modulating the anabolic response
during fracture remodeling in orthopaedic medicine.
5. Concluding remarks
Fracture repair is a complex and highly regulated process that
is influenced by physiological, cellular, and molecular/genetic fac-
tors. Remodeling of the fracture callus is fundamental to the process
of repair. This includes remodeling of the soft callus (non-specific
catabolism), which is a largely osteoclast-independent process, and
remodeling of the hard callus, which is an osteoclast-dependent
process (specific catabolism). Future work is required to better
define the cell types capable of remodeling soft callus in the absence
of functional osteoclasts. The ability of anti-osteoclastic agents
to modulate the catabolic response may be of medical utility in
orthopaedics if a delay in remodeling may lead to stronger initial
union. Finally, the coordinated regulation of hard callus remodeling
by osteoblasts has been highlighted as an area for further study.
Acknowledgment
Our research into the mechanisms underlying fracture repair has
been supported by the National Health & Medical Research Council
(NHMRC).
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