Perspectives on Modern Orthopaedics

Recent Developments in the

Biology of Fracture Repair

Francois N. K. Kwong, MD Abstract

Mitchel B. Harris, MD
Perspectives on Modern Orthopaedics
articles provide an objective appraisal of
new or controversial techniques or areas
of investigation in orthopaedic surgery.
Dr. Kwong is Research Fellow, Center
for Molecular Orthopaedics, Brigham
and Women’s Hospital, Harvard Medical
School, Boston, MA. Dr. Harris is
Fracture repair is dependent on local and systemic molecular and
cellular processes. During fracture repair, mesenchymal stem cells
are systemically recruited to the fracture site, and cytokines are
released from the fracture site into the vascular system. In a
significant minority of fractures, healing delays result from adverse
clinical factors that interfere with these processes. Extrinsic
factors, such as aging and smoking, adversely affect the molecular
and cellular processes occurring locally in the fracture site.
Fracture fixation affects healing through local changes in the
biologic signaling within the fracture callus. Current biologic
treatment of fractures includes the local application of
osteoinductive bone morphogenetic proteins (ie, BMP-2, BMP-7)
and cell-based therapies. Although clinical results with bone
morphogenetic proteins have been satisfactory, they have not been
as impressive as those reported in animal studies. Further
understanding of the biology of fracture repair may lead to
improved treatment modalities.

Associate Professor of Orthopaedic
Surgery, Harvard Medical School, and
Chief, Orthopaedic Trauma Service,
Brigham and Women’s Hospital,
Partners Orthopaedic Trauma Service,
Boston, MA.
None of the following authors or a
member of their immediate families has
received anything of value from or owns
stock in a commercial company or
institution related directly or indirectly to
the subject of this article: Dr. Kwong and
Dr. Harris.
Reprint requests: Dr. Kwong, Center for
Molecular Orthopaedics, Room BLI
044, 221 Longwood Avenue, Boston,
MA 02115.
J Am Acad Orthop Surg 2008;16:619-
625
Copyright 2008 by the American
Academy of Orthopaedic Surgeons.
F
racture healing is a highly effi-
cient repair process resulting in
newly formed bone, similar in qual-
ity to the original tissue. However,
in a small but significant number of
instances, adverse conditions impair
this process, causing significant
morbidity. Because fractures are
common in the general population,
delayed healing and nonunion are
significant health care issues. Thus,
there remains a need to develop
treatment methods that enhance
fracture healing and improve out-
comes.
Biologic methods of bone regener-
ation will continue to have an in-
creasing role in the treatment of
fractures. To further develop these
methods, knowledge of the patho-
logic changes in the fracture repair
process that lead to delayed union or
nonunion is important.
Fracture healing may be seen as
both a local and a systemic process
(Figures 1 and 2). There are local mo-
lecular and cellular signaling path-
ways, and evidence is emerging of
the systemic recruitment of mesen-
chymal stem cells (MSCs) to the
fracture site. Extrinsic factors, such
as drugs and aging, influence local
processes to alter the outcome of
fractures, while the biomechanics of
fracture fixation affect the biology of
fracture healing.
Pathophysiology of
Delayed Fracture
Healing and Nonunion
Bone regeneration depends on three
essential elements: progenitor cells,
growth factors (osteoinduction), and
the appropriate milieu (osteoconduc-
tion). Delayed fracture repair and

Volume 16, Number 11, November 2008 619

Recent Developments in the Biology of Fracture Repair
Figure 1
Illustration demonstrating molecular
signaling. Fracture repair depends on
molecular processes occurring both
locally and systemically. BMPs = bone
morphogenetic proteins, IGF-1 =
insulin-like growth factor-1, NSAIDs =
nonsteroidal anti-inflammatory drugs,
TGF-β = transforming growth factor-β,
- = inhibits
Figure 2
Illustrations of cellular signaling and the role of mesenchymal stem cells (MSCs) in fracture healing. A, Source of MSCs.
B, Action of MSCs within fracture callus. BMPs = bone morphogenetic proteins
620
nonunion can result from a lack of
osteoprogenitor cells, insufficient
osteoinductive growth factors, or a
defective milieu, or, more common-
ly, a combination of these factors.
Disease or other adverse factors may
delay fracture healing by affecting
one or more of these elements.
For example, a critical-sized bone
defect (ie, one that does not heal
spontaneously because of its size)
would result in the absence of hu-
man MSCs (hMSCs) within the de-
fect, a lack of osteoinductive signals,
and a milieu that is nonconducive to
healing. Excess motion at the frac-
ture site impairs both the molecular
and cellular processes within the de-
fect. Hypoxia affects osteogenic dif-
ferentiation of hMSCs by reducing
the number of viable cells and alter-
ing the molecular signals pro-
duced.1 Blood supply is important to
fracture repair as blood provides nu-
trients and oxygen for cell survival,
and blood vessels are the route for
inflammatory and osteoprogenitor
cells that are recruited to the frac-
ture site. Systemic factors may affect
fracture repair by reducing the num-
ber of osteoprogenitor cells recruited
Journal of the American Academy of Orthopaedic Surgeons
to the fracture site and/or by affect-
ing the local osteoinductive signals.
Nonunions are commonly classi-
fied on radiographs as hypertrophic
or atrophic. Hypotrophic nonunion
is generally thought to be the result
of mechanical instability and is
treated by restoring stability, usu-
ally by skeletal fixation. Atrophic
nonunion is thought to result from
biologic causes, principally poor vas-
cularization, and is treated by resto-
ration of the osteogenic potential,
with resection of fibrous tissue and
bone grafting or some other method
of osteoinduction. Both groups of
nonunion contain fibrous tissue, fi-
brocartilage, and adipose tissue,
which are not normally present in
healing fractures.2 The hypertrophic
group also has hyaline cartilage and
bone in varying proportions.
Local Molecular
Signaling
Osteoinductive Molecules
Fracture repair is regulated by
several growth factors with varying
osteogenic potential, such as trans-
forming growth factor-β, platelet-

derived growth factor, insulin-like
growth factor-1, and bone morphoge-
netic protein (BMP). Of these, BMP
appears to be among the most conse-
quential. BMP was discovered and
named by Urist3 in 1965; he also first
described the phenomenon of os-
teoinduction. Urist observed new
bone formation occurring locally in
rodents after they were given intra-
muscular implantation of bone cyl-
inders decalcified with hydrochloric
acid. This phenomenon was attrib-
uted to the presence of a protein,
BMP, in bone matrix. Since that dis-
covery, at least 16 different human
BMPs have been identified. These
proteins affect cells and tissues in-
volved in the repair process in a
number of ways, including the re-
cruitment of MSCs from surround-
ing tissues to the fracture site, fol-
lowed by their proliferation and
differentiation into chondrocytes
and osteoblasts, invasion of blood
vessels, and, ultimately, bone forma-
tion. All of these effects are mediat-
ed by the binding of BMPs to specif-
ic transmembrane receptors on
hMSCs, active osteoblasts, and ma-
ture chondrocytes as well as to the
subsequent activation of various in-
tracellular messenger systems.4
Therapeutic Application
of BMPs
Extensive animal data have dem-
onstrated the potential of BMPs to
induce healing of critical-sized (ie,
large) bone defects.5-7 In animal mod-
els, BMPs alone (with their carrier
matrix) have been shown to induce
rapid bone bridging of a defect. The
quality of the repair tissue was
equivalent to or better than that ob-
tained with autologous bone graft-
ing, the standard treatment for bone
defects and nonunions in clinical
practice.6,7 At present, only BMP-2
(Infuse; Medtronic Sofamor Danek,
Memphis, TN) and BMP-7 (OP-1 Im-
plant; Stryker Biotech, Hopkinton,
MA) have been approved by the US
Food and Drug Administration for
clinical use. Several clinical studies
Volume 16, Number 11, November 2008
Francois N. K. Kwong, MD, and Mitchel B. Harris, MD
have already demonstrated the pos-
itive effect of the application of
BMPs on the outcome of fractures
and nonunions. Because a review of
all available evidence is beyond the
scope of this article, we will focus on
the treatment of segmental bone de-
fects in patients because this allows
a direct comparison with the studies
of BMPs in animals.
In most clinical studies of the
treatment of segmental bone de-
fects, BMPs have been used in con-
junction with allograft or autograft
bone. Jones et al8 demonstrated that
a combination of BMP-2 and al-
lograft bone was equivalent to autol-
ogous bone for the treatment of seg-
mental bone defects. In that study,
patients with a tibial diaphyseal
fracture and a residual cortical de-
fect were randomly assigned to re-
ceive either autogenous bone graft
or allograft with an onlay applica-
tion of recombinant human BMP-2
(rhBMP-2). Radiographic and func-
tional outcomes were similar in
both groups. To date, the only pub-
lished clinical study on the treat-
ment of segmental defects with
BMPs alone (with a nonosteocon-
ductive carrier matrix only) in hu-
mans showed healing of critical-
sized fibular defects in patients
undergoing opening wedge high tib-
ial osteotomy with fibulectomy.9
RhBMP-7 bound to collagen type
1 sponge induced bony union in five
of six patients with a critical-sized
fibular defect, whereas there was no
healing in any of the six patients
treated with the type 1 collagen car-
rier only. Despite these favorable re-
sults, no large studies have been
done on the use of BMPs alone in
humans.
The pace of healing of segmental
defects treated with BMPs differs
significantly among species. Seg-
mental defects in large animals
treated with BMPs alone healed in
<3 months,5,6 whereas in humans,
critical tibial defects treated with al-
lograft bone and BMP-2 required ≥6
months to achieve bony union.8 The
cause for this difference remains un-
clear. Clinically, poor fracture heal-
ing in humans may be associated
with adverse factors not present in
animal studies. For example, soft-
tissue coverage of the fracture may
not be adequate. The initial dose of
BMPs given to human subjects (7 mg
of BMP-7 and 2 g of collagen carrier,
or 12 mg of rhBMP-2 and collagen
sponge) was much higher than in the
animal studies. Because the release
of BMP inhibitors depends on the ex-
tracellular level of BMPs, it is postu-
lated that this higher concentration
of BMPs leads to the expression of
several BMP antagonists, which fur-
ther limits their efficacy and reduc-
es the rate of bone healing. It is also
speculated that BMP receptors in an-
imals and humans are different in
their degree of responsiveness to the
BMP molecules.
Role of BMPs and
Their Inhibitors in
Fracture Healing
The activity of BMPs can be lim-
ited by several antagonists, which
bind to them and interfere with their
ability to induce receptor activation.
One of the most characterized BMP
inhibitors is noggin, a protein that
binds to both BMP-2 and BMP-7 and
antagonizes their actions by prevent-
ing binding with their membrane
receptors.10 Its expression by osteo-
blasts is induced by BMP-2,10 imply-
ing that BMP-2 and noggin are in-
volved in a negative feedback loop
during bone formation. This may
provide a physiologic mechanism
that prevents overexposure of osteo-
blasts to BMP signaling.
The balance between BMPs and
their inhibitors is likely to be a crit-
ical determinant of fracture healing,
with a decreased expression of BMPs
and/or a relative increase of BMP an-
tagonists adversely affecting healing.
In a rat model of fracture nonunion,
a downregulation of the gene expres-
sion of BMPs was demonstrated.11 In
an animal model of atrophic non-
union, reversal of this decreased ex-
621
Recent Developments in the Biology of Fracture Repair
pression of osteoinductive factors,
induced by an early local injection of
rhBMP-7, prevented the develop-
ment of nonunion.12 The expression
of chordin, a BMP antagonist with a
mode of action similar to that of
noggin, was upregulated in an ani-
mal model of fracture nonunion,11
suggesting that downregulation of
chordin in a fracture nonunion has
the potential of improving bone
healing.
In normally healing fractures, the
balance between BMPs and their in-
hibitors can also be manipulated to
hasten repair. This would involve
the addition of the osteoinductive
factors (eg, BMP-2), inhibition of the
activity of BMP inhibitors, or a com-
bination of both methods. So far, bi-
ologic methods of enhancing bone
regeneration have centered on the
promotion of osteoinduction via the
delivery of BMPs. However, it was
recently demonstrated that noggin
or chordin suppression can acceler-
ate osteogenesis in vitro13,14 and that
noggin knockdown increased the
rate of intramembranous ossifica-
tion in an animal model.14 We be-
lieve that these findings will be ex-
tended to fracture healing and that
blockade of the activity of BMP in-
hibitors may provide a novel strate-
gy for expediting fracture repair.
Local Cellular Signaling
Complete fracture healing requires
that a sufficient number of hMSCs
differentiate into chondrocytes and
osteoblasts, as well as other cells of
the mesenchymal lineage, such as
adipocytes, and stromal and endo-
thelial cells. In addition to differen-
tiation, the trophic, or nutritional,
activity of MSCs in the repair of oth-
er tissues is now well established.15
This refers to the capacity of MSCs
to secrete growth factors, which
stimulate blood vessel formation
and the proliferation of other local
MSCs.15 It is postulated that MSCs
exert a trophic activity in the early
stages of fracture repair, although
622
this has not yet been specifically
demonstrated in fractures. MSCs are
thought to be recruited locally from
the cortex, bone marrow, perios-
teum, and external soft tissues (Fig-
ure 2). The relative contribution of
MSCs from each tissue is uncertain
but is thought to depend on the local
parameters present at the injured tis-
sue, such as growth factors, oxygen
gradient, and mechanical stability.
The clinical relevance of muscle as a
source of progenitor cells during
fracture repair has been the subject
of several recent studies.16,17
The presence in muscle of a pop-
ulation of adult stem cells that can
differentiate into cells of different
lineages has been suspected for some
time based on two observations.
First, muscle has the potential to
turn into bone, as occurs during het-
erotopic ossification. Second, the
original description of osteoinduc-
tion by Urist3 has been attributed to
the effects of BMPs on progenitor
cells within muscle tissue. Howev-
er, the isolation of the relevant MSC
population from this tissue is rela-
tively recent.16 Clinically, the impor-
tance of muscle as a source of os-
teoprogenitor cells is underlined by
the poor outcome of fractures in
which muscle has been devitalized,
although this poor outcome is often
attributed to the coexisting damage
to the periosteal blood supply. Mus-
cle resection significantly reduces
callus formation and the biome-
chanical properties of the healed
bone, while a muscle crush does not
significantly affect bone healing.18
Conversely, heterotopic ossification
in acetabular fractures has been re-
duced as surgeons have become
more aggressive in débriding injured
and necrotic muscle from the surgi-
cal field.19
Systemic Recruitment
of Cells and Molecular
Signaling
Traditionally, it was thought that
the cells involved in fracture repair
Journal of the American Academy of Orthopaedic Surgeons
were recruited only locally. Howev-
er, a systemic mobilization and re-
cruitment of osteoblastic precursors
to the fracture site from the periph-
eral circulation have now been dem-
onstrated in several recent studies.
In a rabbit ulnar osteotomy model, it
was demonstrated that some osteo-
blasts involved in fracture healing
were systemically mobilized and re-
cruited to the fracture from remote
bone marrow sites.20 Shen et al21
demonstrated in a murine model
that, following systemic injection of
MSCs, osteoprogenitor cells local-
ized to the fracture callus.
These studies have implications
for the development of future cell-
based therapies for fracture healing.
Cell-based therapies are needed
when insufficient cells are present
within a fracture callus (eg, segmen-
tal defect). In such a situation, even
when all of the osteoprogenitor cells
at the site of fracture are working to
the maximum, there will be no bony
union, nor will any osteoinductive
agents be effective because maximal
osteogenesis per cell is already oc-
curring. In a level III study (case-
control), Hernigou et al22 demon-
strated the clinical effectiveness of
local percutaneous injection of bone
marrow aspirate in treating tibial
nonunions. We believe that the stud-
ies mentioned here suggest that it
might be possible to develop cell-
based therapies in which cells are
systemically administered and local-
ized to the site of injury.
Evidence is emerging that distant
skeletal sites can be affected in re-
sponse to a local bone injury.23 An
increased osteogenic response has
been detected in sites distant from
the fracture in animal models.23 This
may result from the release of
growth factors (eg, transforming
growth factor-β, insulin-like growth
factor-1) from the fracture site into
the systemic circulation, as shown
in a clinical study.24 It is not known
whether the level of these factors in
serum reflects the repair activity of
the fracture.

Systemic Factors and
Local Fracture Healing
It is widely accepted that extrinsic
factors have an influence on the out-
come of fracture healing. However, it
is often difficult to isolate the role of
a particular systemic factor in clini-
cal situations. For example, impaired
fracture healing in the elderly may be
related to age, osteoporosis, drugs,
malnutrition, and/or anemia. Evi-
dence gained from animal models as
well as recently uncovered cellular
and molecular processes have led to
better understanding of the role of
systemic factors.
Nonsteroidal
Anti-inflammatory Drugs
During fracture repair, the en-
zyme cyclooxygenase-2 (COX-2) is
activated to produce prostaglandins,
which are needed during inflamma-
tion and are critical for starting the
osteogenic response.25 Nonsteroidal
anti-inflammatory drugs (NSAIDs)
inhibit COX-2, dramatically reduc-
ing prostaglandin production, and
therefore have the potential to nega-
tively affect fracture repair. Al-
though results in various animal
studies have been conflicting, it is
generally accepted that NSAIDs im-
pair fracture healing in animal mod-
els in which a high dose has been ad-
ministered.26 This inhibitory effect
is most potent during the early phase
of healing, thereby underlining the
importance of the initial inflamma-
tory reaction.26
Although it is not known in
which clinical situations NSAIDs in
high local concentrations will affect
early-phase fracture healing, admin-
istration of NSAIDs has been associ-
ated with delayed union and pseud-
arthrosis.27 The inhibitory effect of
COX-2 blockade in vitro has been
shown to be reversed by the admin-
istration of BMP-2.28
Age
Aging is an independent factor
that negatively affects fracture re-
Volume 16, Number 11, November 2008
Francois N. K. Kwong, MD, and Mitchel B. Harris, MD
pair. Delayed fracture healing in the
elderly may be caused by differences
in molecular signaling locally with-
in the fracture callus as well as to
systemic factors. Meyer et al28 re-
ported a decreased expression of
BMP-2 and Indian hedgehog (a factor
related to endochondral callus for-
mation) in fracture calluses of older
rats. Noggin expression was not
changed with age. However, the de-
crease in expression of BMP-2 im-
plies that the BMP inhibitor predom-
inated over BMP-2 in older rats.
Hormonal differences with aging
may also be a factor. Sera obtained
from aged donors are less potent in-
ducers of osteoblast differentiation of
hMSC than are sera obtained from
young donors.29 These effects were
specific for osteoblast differentiation
because no donor age differences in
the ability to support differentiation
of other cell types were observed.
Short-term bone marrow cultures es-
tablished from young and old donors
contain similar numbers of hMSCs
and exhibit similar proliferation
rates.30 In addition, the capacity of
hMSC to differentiate into osteo-
blasts and adipocytes was main-
tained irrespective of donor age.31
These studies suggest that there are
no intrinsic defects in hMSCs with
aging and that extrinsic factors
present in the aging environment of
hMSCs may be responsible for the
impaired osteoblast functions seen
with aging.
These observations need further
investigation. If poor fracture repair
in the elderly is related to impaired
osteoinductive signals rather than to
differences in the cellular compo-
nent of the healing fractures, then
the elderly patient with trauma is
more likely to benefit from an os-
teoinductive agent, such as BMP-2,
than from cell-based therapies.
Smoking
Smoking has an adverse effect on
fracture healing. In one study, the
time to union for tibial fractures
among smokers was significantly
(P < 0.05) longer by 4 weeks than in
nonsmokers.32 This effect may be
mediated by either nicotine or some
other, yet undefined components in
cigarette smoke,33 or both.
In animal models, nicotine has
been shown to delay cellular differen-
tiation into chondrocytes and to slow
the physiologic transition from car-
tilaginous callus to bone.34 It is also
highly likely that the compromise of
microcirculation secondary to nico-
tine causes a delay in fracture repair.
It remains to be seen whether the
deleterious effects of smoking are re-
versible with smoking cessation and
whether BMPs can improve healing
in smokers. These questions should
be investigated using animal models
of fracture healing and smoking.
Influence of Fracture
Fixation
The local mechanical forces on a
fracture resulting in movement at
the fracture site are critical factors in
the success of fracture repair. Excess
motion can delay healing; casting
and fracture fixation aim to provide
a mechanical environment in which
strains are decreased to avoid de-
layed union or nonunion. However,
some movement, referred to as mi-
cromotion, is beneficial to fracture
healing. Different fractures and dif-
ferent areas of the skeleton respond
differently to mechanical forces and
resultant strains. Yet precisely how
these changes in local mechanical
loading result in a cartilaginous cal-
lus or intramembranous ossification
remains speculative. The influence
of the mechanical environment on
the fracture repair can be viewed at
three levels: tissue, cellular, and mo-
lecular.
Tissue differentiation requires
mechanical stability. In an animal
model, Claes et al35 demonstrated a
strong association between fracture
stability and the spatial distribution
of newly formed blood vessels and
specific tissue formation. It was also
independently demonstrated in an
623
Recent Developments in the Biology of Fracture Repair
animal model that increased stabil-
ity affected endochondral ossifica-
tion by decreasing the overall
amount of cartilage that formed at
the fracture site.36 This was thought
to be secondary to an increase in the
rate of maturation of chondrocytes
during the endochondral ossification
stage of fracture healing. Conversely,
orthopaedic surgeons often observe
an increase in the size of fracture
callus with increased movement
around the fracture. Endochondral
ossification is also affected by the
type of intermittent forces applied to
the fracture site. In a rat model of
healing osteotomy, intermittent ten-
sile strains stimulated endochondral
ossification, as opposed to compres-
sive strains, which favored direct in-
tramembranous ossification.37
The method of fracture fixation
also has an effect on the biology of
the healing callus. In a murine mod-
el of femoral fracture healing, the
fracture callus was significantly larg-
er with intramedullary nail fixation
than with plate fixation.38 The
changes in gene expression follow-
ing each fixation procedure were
similar and occurred after the same
postoperative time interval. Howev-
er, the intramedullary group had sig-
nificantly greater expression of genes
related to cartilage, cell division, and
inflammation (P < 0.05 for all), and
there was greater expression of genes
related to macrophage activity in the
plate group than in the nail group
(P < 0.001).
Osteoprogenitor cells have the ca-
pacity to detect their mechanical en-
vironment and modify their rate of
differentiation, a process mediated
via effects on the BMP signal-
ing pathway. Cyclic stretching of
hMSCs or osteoblastic precursor cells
in a collagen matrix increased their
proliferation and osteogenic differen-
tiation,39,40 a phenomenon associated
with an increase in BMP-2 pro-
duction. This increase in osteogenic
differentiation may also be mediated
by the downregulation of peroxi-
some proliferator–activated receptor
624
gamma in bone marrow stromal
cells, a mediator that favors adipo-
genesis over osteogenesis.41 However,
compression can also stimulate BMP
production and decrease noggin pro-
duction, thereby stimulating the in
vitro differentiation of human osteo-
blastic cells.42 These studies indicate
that the same changes in molecular
signaling can be induced by different
types of forces acting on the progen-
itor cells in various circumstances.
Summary
Significant advances have been
made in the understanding of the bi-
ology of fracture healing. In particu-
lar, it is now understood that frac-
ture repair is not only a local
phenomenon but is itself under the
influence of extrinsic factors. Ad-
verse local and systemic clinical fac-
tors can affect the molecular and cel-
lular processes involved and can lead
to delayed fracture repair and non-
union. The management of these
healing problems remains challeng-
ing, despite the introduction of ther-
apeutic BMPs and other biologic
methods of bone regeneration. Fur-
ther understanding of the patho-
physiology of fracture repair is need-
ed to develop improved treatment
strategies targeted to the molecular
and cellular processes affected in
specific clinical conditions.
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