Injury, Int. J.

Care Injured (2005) 36, 1392—1404

www.elsevier.com/locate/injury

REVIEW

Current concepts of molecular aspects

of bone healing
Rozalia Dimitriou 1, Eleftherios Tsiridis, Peter V. Giannoudis *

Academic Department of Trauma and Orthopaedic Surgery, School of Medicine,

University of Leeds, St James’s University Hospital, Backett Street, LS9 7TF, UK

Accepted 21 July 2005

KEYWORDS Summary Fracture healing is a complex physiological process. It involves the

Molecular; coordinated participation of haematopoietic and immune cells within the bone
Bone; marrow in conjunction with vascular and skeletal cell precursors, including mesench-
Ageing; ymal stem cells (MSCs) that are recruited from the surrounding tissues and the
Fracture healing; circulation. Multiple factors regulate this cascade of molecular events by affecting
Trauma different sites in the osteoblast and chondroblast lineage through various processes
such as migration, proliferation, chemotaxis, differentiation, inhibition, and extra-
cellular protein synthesis. An understanding of the fracture healing cellular and
molecular pathways is not only critical for the future advancement of fracture
treatment, but it may also be informative to our further understanding of the
mechanisms of skeletal growth and repair as well as the mechanisms of aging.
# 2005 Elsevier Ltd. All rights reserved.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393
Historical perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393
Biology of fracture healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393
Direct or primary cortical fracture healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393
Indirect or secondary fracture healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1394
Molecular aspects of fracture healing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1394
Signalling molecules and the role of mesenchymal stem cells (MSCs) . . . . . . . . . . . . . . . . . . . . . . . . . 1394
Pro-inflammatory cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1394
Growth and differentiation factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1394
Metalloproteinases and angiogenic factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1399
The role of mesenchymal stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1399
Sequence of events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1400

* Corresponding author. Present address: Academic Department of Trauma and Orthopaedics, St. James’s University Hospital, Beckett
Street, Leeds LS9 7TF, UK. Tel.: +44 113 20 66460; fax: +44 113 20 65156.
E-mail address: pgiannoudi@aol.com (P.V. Giannoudis).
1
AO Research Fellow.

0020–1383/$ — see front matter # 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.injury.2005.07.019
Current concepts of molecular aspects of bone healing 1393

Intramembranous bone formation . . . . ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1400

Endochondral bone formation . . . . . . . ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1400
Current concepts of systemic enhancement of fracture healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1401
Parathyroid hormone (PTH) . . . . . . . . . . ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1401
Growth hormone (GH) . . . . . . . . . . . . . ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1401
Future directions. . . . . . . . . . . . . . . . . . . ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1401
Tissue engineering . . . . . . . . . . . . . . . . ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1401
Muscle stem cells . . . . . . . . . . . . . . . . ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1401
Clinical applications in trauma . . . . . . . . . . ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1402
Conclusion . . . . . . . . . . . . . . . . . . . . . . . ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1402
Acknowledgements . . . . . . . . . . . . . . . . . ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1402
References. . . . . . . . . . . . . . . . . . . . . . . ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1402

Introduction ongoing to elucidate the unique role of different

Fracture healing remains to a great extent an
unknown cascade of complex biological events. It
involves intracellular and extracellular molecular
signalling for bone induction and conduction. It is
a multistage repair process that follows a definable
temporal and spatial sequence.20,23,63,68
Molecular mechanisms known to regulate skeletal
tissue formation during embryological development
are recapitulated during fracture healing.25 Many
local and systemic regulatory factors, including
growth and differentiation factors, hormones, cyto-
kines, and extracellular matrix, interact with sev-
eral cell types, including bone and cartilage forming
primary cells or even muscle mesenchymal cells,
recruited at the fracture-injury site or from the
circulation.
Cellular and molecular biology provides today
the tolls for the investigation and understanding
of the fracture healing process. Ongoing research
in this field of medicine has improved our under-
standing of fracture healing at the molecular level.
The aim of this review article is to characterise the
chain of events contributing to the healing process
in order to enhance the clinician’s awareness of the
complexity of signalling pathways and molecules
involved.
molecules and their interactions in the healing
cascade.
Biology of fracture healing
Fracture healing is a complex, however, well orche-
strated, regenerative process initiated in response
to injury, resulting in optimal skeletal repair and
restoration of skeletal function. During the repair
process, the pathway of normal embryonic devel-
opment is recapitulated with the coordinated par-
ticipation of several cell types.25 All four
components involved in the injury site, including
the cortex, the periosteum, the bone marrow, and
the external soft tissues, contribute in the healing
process at different extent, depending on multiple
parameters present at the injured tissue such as
growth factors, hormones and nutrients, pH, oxygen
tension, the electrical environment and the
mechanical stability that has been obtained.17,65
In classical histological terms, fracture healing
has been divided into direct (primary) and indirect
(secondary) fracture healing. Integrated cellular
events, and their temporal and spatial character-
istics, have been elucidated by using a model of
experimental fracture healing in the rat.23

Direct or primary cortical fracture healing

Historical perspectives
In 1965, Urist revolutionised the current under-
standing of fracture healing by hypothesising the
existence of bone morphogenetic proteins (BMPs)
allocated onto the extracellular collagenous
matrix.76,77 The genetic sequences of BMPs were
first identified by Wozney et al. in 1988,82 and since
then over 40 BMPs have been discovered. Subse-
quently, with the advances made in molecular med-
icine and molecular biology extensive research is
Direct fracture healing occurs only when there is
anatomic reduction of the fracture fragments by
rigid internal fixation and decreased intrafragmen-
tary strain.49 This process involves a direct attempt
by the cortex to re-establish new haversian systems
by the formation of discrete remodelling units
known as ‘cutting cones’, in order to restore
mechanical continuity.49 Vascular endothelial cells
and perivascular mesenchymal cells provide the
osteoprogenitor cells to become osteoblasts. During
1394
this process, little or no periosteal response is noted
(no callus formation).23
Indirect or secondary fracture healing
The majority of fractures heal by indirect fracture
healing. It involves a combination of intramembra-
nous and endochondral ossification with the subse-
quent formation of a callus.23 It is generally enhanced
by motion and inhibited by rigid fixation.49
Intramembranous ossification involves the
formation of bone directly, without first forming
cartilage, from committed osteoprogenitor and
undifferentiated mesenchymal cells that resides
in the periosteum, farther from the fracture site.23
It results in callus formation, described histologi-
cally as ‘hard callus’.23 In this type of healing, the
bone marrow’s contribution to the formation of
bone is during the early phase of healing, when
endothelial cells transform into polymorphic
cells, which subsequently express an osteoblastic
phenotype.12
Endochondral ossification involves the recruit-
ment, proliferation, and differentiation of undiffer-
entiated mesenchymal cells into cartilage, which
becomes calcified and eventually replaced by bone.
Its temporal characteristics include six identifiable
stages including an initial stage of haematoma for-
mation and inflammation, subsequent angiogenesis
and formation of cartilage, cartilage calcification,
cartilage removal, bone formation, and ultimately
bone remodelling.23 This type of fracture healing, is
contributed from the adjacent to the fracture peri-
osteum and the external soft tissues, providing an
early bridging callus, histologically characterized as
‘soft callus’, that stabilizes the fracture frag-
ments.23
The classification of fracture healing in direct and
indirect healing reflects the histological events that
occur during the repair process. However, the
ongoing research in bone regeneration provided a
further understanding of the cellular and molecular
pathways that govern these events, by demonstrat-
ing the existence of various signalling molecules and
elucidating their contribution in the initiation and
control of this physiological process at the molecu-
lar level.
Molecular aspects of fracture healing
Signalling molecules and the role of
mesenchymal stem cells (MSCs)
The signalling molecules can be categorized into
three groups: (1) the pro-inflammatory cytokines,
R. Dimitriou et al.
(2) the transforming growth factor-beta (TGF-b)
superfamily and other growth factors, and (3) the
angiogenic factors (Table 1).33,44
Pro-inflammatory cytokines
Interleukin-1 (IL-1) and Interleukin-6 (IL-6) as well
as tumour necrosis factor-alpha (TNF-a) are shown
to play a role in initiating the repair cascade.24,32
They are secreted not only by macrophages and
inflammatory cells but also by cells of mesenchymal
origin present in the periosteum (Table 1).43 They
carry out central functions in the induction of down-
stream responses to injury by having a chemotactic
effect on other inflammatory cells, enhancing extra-
cellular matrix synthesis, stimulating angiogenesis,
and recruiting endogenous fibrogenic cells to the
injury site.43
They show peak expression within the first 24 h
after fracture, depressed levels during the period of
cartilage formation, and their levels increase again
during bone remodelling (Fig. 1).32,43
Cytokines also regulate endochondral bone for-
mation and remodelling.7 TNF-a promotes the
recruitment of mesenchymal stem cells, induces
apoptosis of hypertrophic chondrocytes during
endochondral ossification and stimulates osteoclas-
tic function. Absence of TNF-a result in delayed
resorption of mineralized cartilage, prohibiting
new bone formation.32 IL-1, IL-6 and TNF-a also
show increased levels of expression during fracture
callus re-shaping later in the process of fracture
healing and remodelling.
Growth and differentiation factors
The transforming growth factor-beta superfami-
ly. The TGF-b superfamily is a large family of
growth and differentiation factors including bone
morphogenetic proteins (BMPs), transforming
growth factor-beta (TGF-b), growth differentiation
factors (GDFs), activins, inhibins and the Mullerian
inhibiting substance. At least 34 members have been
identified in the human genome.75
They originate from high molecular weight pre-
cursors and are activated by proteolytic enzymes.73
They act on serine/threonine kinase membrane
receptor on target cells.48 This ligand—receptor
interaction activates an intracellular signalling path-
way which ultimately affects gene expression in the
nucleus.
Specific members of this superfamily including
bone morphogenetic proteins (BMPs 1—8), growth
and differentiation factors (GDF-1, 5, 8, 10) and
transforming factor beta (TGF-b1, -b2, -b3), pro-
mote the various stages of intramembranous and
endochondral bone ossification during fracture
healing.20
Current concepts of molecular aspects of bone healing 1395

Table 1 The essential signalling molecules during fracture healing; their source and targeted cells, and their major
functions and expression patterns
Cytokines (IL-1, IL-6, TNF-a)
Source: macrophages and other inflammatory cells, cells of mesenchymal origin
Chemotactic effect on other inflammatory cells, stimulation of extracellular matrix synthesis,
angiogenesis, recruitment of endogenous fibrogenic cells to the injury site, and
at later stages bone resorption
Increased levels from days 1 to 3 and during bone remodelling

TGF-b
Source: degranulating platelets, inflammatory cells, endothelium, extracellular matrix,
chondrocytes, osteoblasts
Targeted cells: MSCs, osteoprogenitors cells, osteoblasts, chondrocytes
Potent mitogenic and chemotactic for bone forming cells, chemotactic for macrophages
Expressed from very early stages throughout fracture healing

PDGF
Source: degranulating platelets, macrophages, monocytes (during the granulation stage)
and endothelial cells, osteoblasts (at later stages)
Targeted cells: mesenchymal and inflammatory cells, osteoblasts
Mitogenic for mesenchymal cells and osteoblasts, chemotactic for inflammatory and mesenchymal cells
Released at very early stages of fracture healing

BMPs
Source: osteoprogenitors and mesenchymal cells, osteoblasts, bone extracellular matrix and chondrocytes
Targeted cells: mesenchymal and osteoprogenitor cells, osteoblasts
Differentiation of undifferentiated mesenchymal cells into chondrocytes and osteoblasts
and osteoprogenitors into osteoblasts
Various temporal expression patterns (Table 2)

FGFs
Source: monocytes, macrophages, mesenchymal cells, osteoblasts, chondrocytes
Targeted cells: mesenchymal and epithelial cells, osteoblasts and chondrocytes
Angiogenic and mitogenic for mesenchymal and epithelial cells, osteoblasts, chondrocytes
a-FGF mainly effects chondrocyte proliferation, b-FGF (more potent) involved in chondrocytes
maturation and bone resorption
Expressed from the early stages until osteoblasts formation

IGFs
Source: bone matrix, endothelial and mesenchymal cells (in granulation stage) and osteoblasts
and non-hyperthrophic chondrocytes (in bone and cartilage formation)
Targeted cells: MSCs, endothelial cells, osteoblasts, chondrocytes
IGF-I: mesenchymal and osteoprogenitor cells recruitment and proliferation, expressed
throughout fracture healing
IGF-II: cell proliferation and protein synthesis during endochondral ossification

Metalloproteinases
Source: the extracellular matrix
Degradation of the cartilage and bone allowing the invasion of blood vessels during
the final stages of endochondral ossification and bone remodelling

VEGFs
Potent stimulators of endothelial cell proliferation
Expressed during endochondral formation and bone formation

Angiopoietin (1 and 2)
Formation of larger vessel structures, development of co-lateral branches from existing vessels
Expressed from the early stages throughout fracture healing
1396
Figure 1 Schematic summary of the temporal expression patterns of the signalling molecules during fracture healing.
(The dashed line represents a difference of opinion amongst scientists in terms of the timing of expression.)
Bone morphogenetic proteins. Members of the
BMP family are divided into at least four separate
subgroups depending on their primary amino acid
sequence. Group one consists of BMP-2 BMP-4, and
group two includes BMP-5, -6, and -7. The third
group includes GDF-5 (or BMP-14), GDF-6 (or BMP-
13) and GDF-7 (or BMP-12), and finally group four
includes BMP-3 (or osteogenin) and GDF-10 (or BMP-
3b).67 BMP-1 is not a member of the TGF-b super-
family and it may play a role in modulating BMP
actions by the proteolysis of BMP antagonists/bind-
ing proteins, such as noggin and chondrin.63
BMPs bind to type II serine/threonine kinase
receptors which transphosphorylate type-I recep-
tors.28,57 Subsequently, the Smad intracellular sig-
nalling cascade is initiated. The Smad family
includes eight members which can be subdivided
into three groups: the signal-transducing receptor-
regulated Smads (R-Smads 1, 2, 3, 5, 8), the com-
mon mediator Smad (co-Smad, such as Smad-4), and
the inhibitory Smads (I-Smads, such as Smad-6 and
Smad-7).37,50 R-Smad 1, 5 and 8 are substrates for
BMP receptors and when activated they interact
with Smad-4. These heteromeric complexes trans-
R. Dimitriou et al.
locate into the nucleus and regulate the transcrip-
tion of target genes (Fig. 2).75 BMPs are pleiotropic
morphogens and play a critical role in regulating
growth, differentiation, and apoptosis of various
cell types, including osteoblasts, chondroblasts,
neural cells, and epithelial cells.67
Furthermore, it has been shown that BMP hetero-
dimers, such as BMP-4/-7 and BMP-2/-7 have an
enhanced osteoinductive activity regulating more
efficiently differentiation and proliferation of
mesenchymal cells to osteoblasts in vitro and in
vivo.38
The extracellular matrix comprises the main
source of BMPs being produced by osteoprogenitors,
mesenchymal cells, osteoblasts, and chondrocytes
(Table 1). BMPs induce a sequential cascade of
events for chondro-osteogenesis, including chemo-
taxis, mesenchymal and osteoprogenitor cells pro-
liferation and differentiation, angiogenesis, and
controlled synthesis of extracellular matrix.63,67
Their regulatory effect depends upon the type of
the targeted cell, its differentiation stage, the local
concentration of the ligand as well as the interac-
tion with other circulating factors.35 Interestingly,
Current concepts of molecular aspects of bone healing 1397

low concentrations of BMPs in vitro favour the dif-
ferentiation of mesenchymal stem cells into adipo-
cytes.80
BMPs are closely structurally and functionally
related, however, each has a unique role as well a
distinct temporal expression pattern during the
fracture repair process (Fig. 1). Studies of the role
of BMPs in fracture healing in the mouse and rat
have shown a variety of osteogenic effects, tem-
poral expressions, and mitogenic capacities
(Tables 2 and 3).9,10,20
In a comprehensive analysis of the osteogenic
activity of 14 types of BMPs, Cheng et al. suggested
an osteogenic hierarchical model of BMPs. BMP-2,

Table 2 Temporal and functional characteristics of members of the TGF-b superfamily observed during fracture
healing in animal models
Member of the TGF-b Time of expression Specific responses in vivo and in vitro
superfamily
GDF-8 Restricted to day 1 20 Potential function as a negative
regulator of skeletal muscle growth 20
BMP-2 Days 1—2110,20 Recruitment of mesenchymal cells
(the earliest gene to be induced and Chondrogenesis
second elevation during osteogenesis) May initiate the fracture healing
cascade and regulate the expression of
other BMPs
BMP-2, -6, -9 may be the most potent to
induce osteoblast lineage-specific
differentiation of MSCs 19
BMP-3, -8 Days 14—2120 (restricted expression Temporal data suggest a role in the
during osteogenesis) regulation of osteogenesis
BMP-4 Transient increased expression in the Involvement in the formation of callus
surrounding soft tissues 6 h to day 5 9 at a very early stage in the healing
process
Days 14—21 20 In vitro: BMP-3 and -4 stimulate the
migration of human blood monocytes 63
Through out fracture healing 10
BMP-7 Days 14—21 20 Regulatory role in both types of
ossification
From the early stages In vitro: stimulation of relative mature
of fracture healing 9 osteoblasts 19
GDF-10, BMP-5, -6 Days 3—21 20 Regulatory role in both types of
ossification
BMP-6 may initiate chondrocyte
maturation 20
GDF-5, 1 Day 7 (maximal) to day 1420 GDF-5 an exclusive involvement in
(restricted expression chondrogenesis is suggested
during chondrogenic phase)
GDF-1 at extremely low levels Stimulation of mesenchymal aggregation
and induction of angiogenesis through
chemotaxis of endothelial cells and
degradation of matrix proteins
GDF-3, GDF-6, 9 No detectable levels within GDF-6 may be expressed only in articular
the fracture callus 20 cartilage20 and with GDF-5, 7 more
efficiently induce cartilage and
tendon-like structures in vivo 28
TGF-b1, -b2, -b3 Days 1—21 20 Potent chemotactic for bone forming
cells and macrophages
Days 3—14 20 Proliferation of undifferentiated
mesenchymal and osteoprogenitor cells,
osteoblasts, chondrocytes
Days 3—21 20
1398 R. Dimitriou et al.

BMPs may also stimulate the synthesis and secre-

Figure 2 BMP-receptor interaction and the intracellular
signalling pathway.
-6, and -9 may be the most potent to induce osteo-
blast differentiation of mesenchymal progenitor
cells, whilst most BMPs (except BMP-3 and -13)
can promote the terminal differentiation of com-
mitted osteoblastic precursors and osteoblasts.19
tion of other bone and angiogenic growth factors
such as insulin-like growth factor (IGF) and vascular-
endothelial growth factor (VEGF), respectively.22
They may also stimulate bone formation by
directly activating endothelial cells to stimulate
angiogenesis.59,79
Recent studies have showed that the expression
of the BMP antagonists, most importantly noggin,72
which blocks BMP-2 interaction with its receptor,29
also play an important role in fracture healing reg-
ulation.36 It has been suggested that the noggin/
BMP-4 balance could be an important factor in the
regulation of callus formation.85
Transforming growth factor beta. Five isoforms
of this group have been isolated.42 Platelets release
TGF-b during the initial inflammatory phase of bone
healing and therefore this factor may be involved in
the initiation of callus formation.8,9 TGF-b is also
produced by osteoblasts and chondrocytes, and is
stored in the bone matrix (Table 1).46 Its effect is
exerted via type-I and type-II serine/threonine
kinase receptors, activating the Smad pathway
(Smad 2 and 3).37
TGF-b is a potent chemotactic stimulator of
mesenchymal stem cells and it enhances prolifera-

20,33,43
Table 3 Timing of cellular events and expression of signalling molecules during murine fracture healing
Day 1 Haematoma formation, inflammation Cytokines: IL-1, IL-6, TNF-a released
by inflammatory cells
Recruitment of mesenchymal cells PDGF, TFG-beta released from
degranulating platelets
Osteogenic differentiation of MSCs from bone marrow BMP-2 expression and restricted to
day 1 expression of GDF-8
Day 3 MSCs proliferation begins Decline of cytokines levels
Proliferation and differentiation of Expression of TGF-b2, -b3, GDF-10,
preosteoblasts and osteoblasts in regions BMP-5, -6
of intramembranous ossification
Angiogenesis begins Angiopoietin-1 is induced
Day 7 Peak of cell proliferation in intramembranous Peak of TGF-b2 and -b3 expression
ossification between days 7 and 10
Chondrogenesis and endochondral Expression of GDF-5 and probably
ossification begins (days 9—14 maturation GDF-1
of chondrocytes)
Day 14 Cessation of cell proliferation in intramembranous Decreased levels of expression for
ossification, but osteoblastic activity continues TGF-b2, GDF-5, and probably GDF-1
Mineralization of the soft callus, cartilage resorption, Expression of BMP-3, -4, -7, and -8
and woven bone formation
Neo-angiogenesis which infiltrates along VEGFs expression
new mesenchymal cells
Phase of most active osteogenesis until day 21 Second increase of IL-1 and TNF-a
which continues during bone
remodelling
Day 21 Woven bone remodelled and subsequently Decreased expression of TGF-b1
replaced by lamellar bone and TGF-b3, GDF-10, and BMPs (2—8)
Current concepts of molecular aspects of bone healing
tion of MSCs, preosteoblasts, chondrocytes and
osteoblasts.46 It also induces the production of
extracellular proteins such as collagen, proteogly-
cans, osteopontin, osteonectin, and alkaline phos-
phatase.68 Its main role is thought to be during
chondrogenesis and endochondral bone formation
(Table 2).7 TGF-b may also initiate signalling for BMP
synthesis by the osteoprogenitor cells,9 while it may
inhibit osteoclastic activation and promote osteo-
clasts apoptosis.51 Recently, it has been suggested
that TGF-b2 and possibly TGF-b3 play more impor-
tant roles in fracture healing than TGF-b1, as their
expression peak during chondrogenesis. On the
other hand, TGF-b1 has a high basal level of expres-
sion in unfractured diaphyseal bone, and the level of
expression remains constant throughout the frac-
ture healing process, indicating that TGF-b2 and
TGF-b3 may play more important roles in fracture
healing than TGF-b1 (Fig. 1).20
Although various studies showed that TGF-b
enhances cellular proliferation, its osteoinductive
potential seems limited and concern for its unfore-
seen side effects has been expressed.46 Therefore,
its therapeutic potential to enhance bone repair
seems to be limited.
Platelet-derived growth factor (PDGF). PDGF is a
homo- or heterodimeric polypeptide consisting of A
and B chains. PDGF effect is exerted via receptors
that have tyrosine kinase activity. IL-1, TNF-a, and
TGF-b1 affect PDGF’s binding.73 It is synthesized by
platelets, monocytes, macrophages, endothelial
cells, and osteoblasts and it is a potent mitogen
for cells of mesenchymal origin (Table 1).3
PDGF is released by platelets during the early
phases of fracture healing and it is a potent chemo-
tactic stimulator for inflammatory cells and a major
proliferative and migratory stimulus for MSCs and
osteoblasts (Fig. 1).46 Nash et al. showed an
increased callus density and volume in tibial osteo-
tomies in rabbits treated with PDGF.56 However, at
present, its therapeutic potential remains unclear.
Fibroblast growth factor (FGFs). The family of FGFs
consists of nine structurally related polypeptides.
The acidic and basic FGFs are the most abundant
FGFs in normal adult tissue.81 Their action is exerted
by binding to tyrosine kinase receptors.83
During bone healing, FGFs are synthesized by
monocytes, macrophages, mesenchymal cells,
osteoblasts and chondrocytes (Table 1). FGFs pro-
mote growth and differentiation of a variety of cells
such as fibroblasts, myocytes, osteoblasts, and
chondrocytes. FGFs are identified during the early
stages of fracture healing and they play a critical
role in angiogenesis and mesenchymal cell mitogen-
1399
esis (Fig. 1). a-FGF mainly effects chondrocyte
proliferation and is probably important for chondro-
cyte maturation, whilst b-FGF is expressed by osteo-
blasts and is generally more potent than a-FGF.46 In
a canine tibial osteotomy model, a single injection
of FGF-2 was associated with an early increase in
callus size.55
Insulin-like growth factors (IGFs). The sources of
IGF-I (or somatomedin-C) and IGF-II (or skeletal
growth factor) are the bone matrix, endothelial
cells, osteoblasts and chondrocytes. The serum con-
centration of IGF-I is mainly regulated by the growth
hormone (Table 1).46,73 The biological actions of the
IGFs are modulated in a cell-specific manner by IGF-
binding proteins (IGFBPs).71
IGF-I promotes bone matrix formation (type I
collagen and non-collagenous matrix proteins) by
fully differentiated osteoblasts15 and is more potent
than IGF-II.46 IGF-II acts at a later stage of endo-
chondral bone formation and stimulates type I col-
lagen production, cartilage matrix synthesis, and
cellular proliferation (Fig. 1).62 The findings from a
number of animal studies assessing the influence of
IGF on skeletal repair have varied and therefore
further studies are required.7
Metalloproteinases and angiogenic factors
Optimal bone regeneration requires adequate blood
flow. During the final stages of endochondral ossifi-
cation as well as during remodelling phase, specific
matrix metallopoteinases degrade cartilage and
bone, allowing the invasion of blood vessels
(Table 1).33
Two separate pathways are believed to regulate
angiogenesis: a vascular-endothelial growth factor-
dependent pathway and an angiopoietin-dependent
pathway.33 It is speculated that both pathways are
functional during fracture repair. VEGFs are essential
mediators of neo-angiogenesis and endothelial-cell
specific mitogens.26 Whereas, angiopoietin 1 and 2 are
regulatory vascular morphogenetic molecules related
to the formation of larger vessel and development of
co-lateral branches from existing vessels (Fig. 1).
However, their contribution in bone repair is not as
well understood (Table 1).33 Street et al. showed that
fracture repair was enhanced by the exogenous
administration of VEGF.74 Recent studies have also
shown that BMPs stimulate the expression of VEGF
by osteoblasts and osteoblast like cells.22,59,84
The role of mesenchymal stem cells
MSCs are undifferentiated cells capable of extensive
replication without differentiation.13 MSCs have the
1400
potential to commit and differentiate along multi-
ple cell lineages. They give rise to those cells that
form mesenchymal tissues, including bone, carti-
lage, tendon, muscle, ligament, and marrow stroma
and fat.13,60
During fracture healing, potential sources of stem
cells are the bone marrow, the granulation tissue,
the deep layer of the periosteum, the endosteum,
and the surroundings soft tissues.23,33,53 Also, peri-
vascular mesenchymal stem cells that exist in blood
vessels walls contribute to fracture healing.11 The
primary tissue source of MSCs is the periosteum53
and studies from Buckwalter et al. showed that the
capacity for fracture callus development is dimin-
ished if the periosteum is removed.14
Sequence of events
Fracture healing, like all other repair responses, is
initiated through the induction of an immune
response.24 During this initial stage, a haematoma
is formed and inflammation occurs. The major
players at this initial inflammatory phase include
cytokines, platelets, BMPs and MSCs.
IL-1, IL-6 and TNF-a secreted by inflammatory
cells have a chemotactic effect on other inflamma-
tory cells and on the recruitment of mesenchymal
cells.43 Cho et al. reported a peak in expression of
IL-1 and -6 one day after fracture followed by a rapid
decline until day 3 to near undetectable levels.20 At
the same time, platelets, activated by thrombin and
subendothelial collagen, release PDGF and TGF-b,
which play a role on the initiation of fracture
repair.8 These factors induce mesenchymal cell
migration, activation and proliferation, angiogen-
esis, chemotaxis of acute inflammatory cells and
further aggregation of platelets. Simultaneously,
BMPs not only are released from the bone matrix
but also are expressed by recruited primary
mesenchymal cells.9 During the subsequent days,
MSCs proliferate and differentiate into a chondro-
genic or osteogenic lineage.69 During this early
phase of events, angiogenesis also takes place
and this is a prerequisite for further progression
of the regeneration cascade. The vascular ingrowth
into the developing callus is regulated by FGF,
VEGF and angiopoietin 1 and 2.33,46 Angiopoietic
1 has been suggested to be induced during the
initial periods of fracture healing whereas VEGF
later on mainly during endochondral and bone
formation.33
Intramembranous bone formation
The source of the cells that contribute to intramem-
branous bone formation appears to be the under-
lying cortical bone, the periosteum within a few
R. Dimitriou et al.
millimetres from the fracture site and the region of
the bone marrow with high cellular density.23 Within
the first 24 h of the fracture, the latter cells begin to
differentiate into an osteoblastic phenotype. By day
3, osteoblasts from the cortex and committed osteo-
progenitors derived from the periosteal cambium
divide and differentiate, forming woven bone (hard
callus). Their proliferation peaks by days 7 and 10,
and it ceases by day 14 while the osteoblastic
activity continues.7 In a rat fracture healing model,
BMP-2, -4, and -7 showed increased levels of expres-
sion during the early stages of intramembranous
ossification.9
Endochondral bone formation
In regions that are mechanically less stable, endo-
chondral bone formation occurs. This type of ossi-
fication mainly occurs at the adjacent to the
fracture site periosteum and enhanced by the soft
tissues around the fracture site. During this process,
MSCs are recruited and begin to proliferate by day 3
after fracture. Their subsequent differentiation into
chondroblasts (chondrogenesis) and the prolifera-
tion of these new chondrocytes occur from days 7 to
21, resulting to soft callus formation. These cells
synthesize and secrete cartilage-specific matrix,
including type II collagen and proteoglycans and
once firm mechanical stability is established, the
cartilage undergoes hypertrophy and mineralization
in a spatially organized manner.7 As vasculature
begins to invade, the calcifying hypertrophic chon-
drocytes are being removed by chondroclasts and
woven bone formation occurs after the recruitment
and osteogenic differentiation of new MSCs.23 Lee
et al. hypothesised that this stage of endochondral
ossification is the final stage of a genetically pro-
grammed process which results to apoptotic chon-
drocyte death.45 Eventually, this soft callus is
replaced by woven marrow-filled bone, which
undergoes significant remodelling to become
weight-bearing bone following the pathway
observed in the growth plate. Recently, Cho et al.
demonstrated the different temporal patterns for
members of the TGF-b superfamily during murine
fracture healing, suggesting a potentially unique
role of each BMP (Table 3).20
The stimulation of undifferentiated MSCs and
osteoprogenitors cells to differentiate into osteo-
blasts (osteoinduction)73 is a morphogenetic cas-
cade involving discrete cellular transitions. It is
predominantly regulated by the complex interac-
tions that take place between the multiple local
(paracrine and autocrine) signals that are pre-
sented to mesenchymal stem cells in their natural
environment (Table 1).31 Further research is
required in order to elucidate the role of each
Current concepts of molecular aspects of bone healing
factor at each discrete stage of fracture healing as
well as their combined functioning to promote its
various stages.
Although the cellular events during fracture heal-
ing seem to be predominantly regulated by local
factors and cytokines, systemic hormones (para-
thyroid/thyroid hormone, the growth hormone,
the 1,25 dihydroxyvitamin D and the sex steroids)
may also modulate these events.51
Current concepts of systemic
enhancement of fracture healing
Parathyroid hormone (PTH)
Contrary to the assumption that PTH has a catabolic
effect on the skeleton, intermittent exposure sti-
mulates osteoblasts and results in increased bone
formation in rats. It has been shown that low-dose
human PTH (1—34) enhances callus formation by
stimulating the early proliferation and differentia-
tion of osteoprogenitor cells, increasing the produc-
tion of bone matrix proteins and enhancing
osteoclastogenesis during callus remodelling. PTH
effect is likely to be mediated by osteoblastic activ-
ity, as PTH receptors are found on osteoblast mem-
branes.1,54
Growth hormone (GH)
Growth hormone is a systemic hormone and its
effect on the skeleton is mediated by IGF-1 (known
as somatomedin-C) which promotes bone matrix
formation (type I collagen and non-collagenous
matrix proteins) by fully differentiated osteoblasts.
Systemically administration of GH in a rat tibial
diaphyseal fracture model during the first 3 weeks
of healing increased callus formation and enhanced
fracture strength.2 Furthermore, in a fracture heal-
ing and distraction osteogenesis model in Yucatan
micropigs, administration of homologous recombi-
nant porcine GH led to an increase in serum IGF-1,
stimulation of fracture healing and acceleration of
ossification of bone regenerate in distraction osteo-
genesis.5
Future directions
Tissue engineering
Currently, as the molecular and cellular events
during the fracture healing cascade are becoming
gradually more understood, new strategies are
investigated in order to promote or facilitate the
1401
healing process. Bone tissue engineering, combining
the application of the principles of orthopaedic
surgery with basic science and engineering, has
been heralded as an alternative when bone regen-
eration is required to replace or restore the function
of traumatised, damaged, or lost bone.66 In essence,
tissue engineering aims to combine progenitor cells,
such as MSCs, or mature cells (for osteogenesis) with
biocompatible materials or scaffolds (for osteocon-
duction), with appropriate growth factors (for
osteoinduction), in order to generate and maintain
bone.66,78 Although this new strategy is still in its
infancy, its clinical application offers great poten-
tials for the treatment of conditions requiring bone
repair.
A promising technology in the field of bone tissue
engineering is the application of gene therapy as a
method of growth factor delivery for the clinical
management of orthopaedic disorders, including
bone healing.18 It involves the transfer of genetic
material into targeted cell’s genome, and thus
allowing the expression of bioactive factors from
the cells themselves and for longer periods of time.
The gene transfer can be performed using a viral
(transfection) or a non-viral (transduction) vector,
by either an in vivo or ex vivo gene-transfer strat-
egy.18,46 With the in vivo technique, a technically
easier method, the genetic material is transferred
directly into the host. However, there are safety
concerns with this strategy. The indirect ex vivo
technique involves the collection of cells by tissue
harvest and their genetic modification in vitro
before transfer back into the host. It is a technically
more demanding but safer method, as it allows
testing of the cells for any abnormal behaviour
before re-implantation as well as selection of those
with the greatest gene expression.18
Gene therapy has been used to promote fracture
repair through the expression of BMP-26,47 and -470
in animal studies. Although promising, issues of its
biosafety and efficacy need to be answered before
human trials take place.
Muscle stem cells
Since 1965, when Urist first noted the ectopic bone
formation after demineralized bone matrix implan-
tation into skeletal muscle,76,77 subsequent studies
have reported similar observations. This phenom-
enon of osteoinduction has been attributed to the
mitogenic effects of BMP-2 (in vitro)40 and BMP-3 (in
vivo)41 on cells in muscle In skeletal muscle,
researchers have identified adult stem cells that
can differentiate into cells of different line-
ages.16,58 The most well-known muscle progenitor
cells, termed satellite cells, are the primary source
1402
of the myoblasts, but they can also exhibit osteo-
genic and adipogenic differentiation.4 Recently, a
population of stem cells that appears to be distinct
from the satellite cells has also been discovered:
the muscle-derived stem cells (MDSCs). These cells
have the ability to differentiate into multiple
lineages including osteogenic and haematopoietic
lines.21,39
These muscle-based progenitor cells possess a
therapeutic potential for tissue repair and regen-
eration applications in various musculoskeletal as
well as cardiac muscle disorders either as a source of
inducible progenitor cells or as gene delivery vehi-
cles.21 For bone formation and healing in particular,
MDSCs genetically engineered to express BMP-252
and both BMP-4 and VEGF58 have been shown to be
capable of stimulating osteogenesis and angiogen-
esis, respectively.
Clinical applications in trauma
Approximately 5—10%, of the 6.2 million fractures
occurring annually in the United States, are asso-
ciated with impaired healing including delayed
healing or non-union.61
Various animal studies and clinical trials in
humans have been performed and demonstrated
the potential use of several of the biological factors
described in bone regeneration and skeletal repair,
with the BMPs to be the most promising.
Riedel and Valentin-Opran were the first to report
preliminary results from the use of BMP-2 to aug-
ment the treatment of open tibial fractures.64
Govender et al. in a large prospective randomized
controlled multi-center trial evaluated the effects
of recombinant (rh) BMP-2 on the treatment of open
tibial fractures. They reported a higher union rate,
an accelerated time to union, improved wound
healing, reduced infection rate, and fewer second-
ary invasive interventions in the group of patients
treated with rhBMP-2 and IM nail fixation, compared
to the group treated with IM nail fixation alone.34
In 1999, Geesink et al. demonstrated the ability
of BMP-7 to cause repair of a critical-sized fibular
defect in patients undergoing opening wedge high
tibial osteotomy with fibulectomy.30 Friedlaender
et al. in a large prospective randomized controlled
and partially blinded multicenter study, they
assessed the efficacy of rhBMP-7 over iliac crest
bone graft in the treatment of 122 patients with
124 tibial non-unions. The authors concluded that
whilst no statistical difference was noted between
the two groups, BMP-7 was a safe and effective
alternative to bone graft in the treatment of tibial
non-unions.27
R. Dimitriou et al.
Currently, recombinant BMP-2 and -7 are com-
mercially available and, although the research is
ongoing, they are considered adjuncts in the sur-
geon’s armamentarium for the treatment of clini-
cally challenging situations.
Conclusion
Fracture healing is a complex physiological process
which involves a well orchestrated series of biolo-
gical events. Whilst our knowledge has vastly
expanded, with the increasing understanding of
the multiple factors and complex pathways
involved, a lot of new developments are anticipated
in the years to come. It is hoped that many bone
disease processes secondary to trauma, aging and
metabolic disorders will be successfully treated
with novel treatment protocols.
Acknowledgements
Rozalia Dimitriou is a research fellow supported via
a grant (03-943) by the AO foundation.
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