Overview Fracture healing Nickolaou Oct 07

ARTICLE IN PRESS
Current Orthopaedics (2007) 21, 249–257
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/cuor
MINI-SYMPOSIUM: FRACTURE HEALING
(i) Pathways and signalling molecules
V.S. Nikolaou, E. Tsiridis
Academic Department of Trauma & Orthopaedic Surgery, A Floor, Clarendon Wing, Leeds General Infirmary,
Clarendon Way, Leeds LS2 9NS, UK
KEYWORDS
Bone;
Fracture;
Healing;
Enhancement;
Molecules
Summary
Fracture healing is a complex and well orchestrated regenerative process, initiated in
response to injury, resulting in optimal skeletal repair and restoration of skeletal function.
Fracture healing remains to a great extent an unknown cascade, and recent developments
in molecular biology have helped us to better understand the fracture healing cellular and
molecular pathways. In this paper, we summarize the current knowledge of the most
important molecules involved in the sequence of events during fracture healing, and we
also focus on the latest research findings that will play important roles in the near future in
better understanding fracture healing at the molecular level.
& 2007 Published by Elsevier Ltd.
Introduction
Various signalling molecules, including growth and differentiation factors, hormones, and cytokines, interact with
several cell types, including bone and cartilage forming
primary cells or even muscle mesenchymal cells, recruited
at the fracture-injury site or from the circulation. Even
though fracture healing remains to a great extent an
unknown cascade that involves intracellular and extracellular molecular signalling for bone induction and conduction, today cellular and molecular biology provides the tools
for the investigation and understanding of this complex
biological process that results from the recapitulation of
events which take place during embryonic bone formation.
In this paper, we summarize the current knowledge of the
most important molecules involved in the sequence of
events during fracture healing. We also outline recent
Corresponding author.
E-mail address: [email protected] (E. Tsiridis).
0268-0890/$ - see front matter & 2007 Published by Elsevier Ltd.
doi:10.1016/j.cuor.2007.08.001
research findings that may play an important role in better
understanding the molecular pathways of fracture healing.
Classical histological approach
In classical histological terms, fracture healing has been
divided into direct (primary) and indirect (secondary)
fracture healing.
Direct (primary) cortical fracture healing
Direct fracture healing occurs when rigid internal fixation
anatomically reduces the fracture fragments, thereby
reducing inter-fragmentary distances.1 During this process,
little or no periosteal response is noted (no callus formation).2 There is a direct attempt by the cortex to reestablish new Haversian systems by forming discrete
remodelling units known as ‘‘cutting cones’’, a process
aimed at restoring mechanical continuity.1
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Indirect or secondary fracture healing
Direct fracture healing is a rare process in nature and is
usually present after medical intervention by rigid internal
fixation. The majority of fractures heal by indirect fracture
healing, which involves callus formation through a combination of intramembranous and endochondral ossification. This
process is generally enhanced by micro-motion and inhibited
by rigid fixation.2
Intramembranous ossification involves the formation of
bone directly, without first forming cartilage, from committed osteoprogenitor and undifferentiated mesenchymal
cells that reside in the periosteum, farther from the fracture
site. It results in callus formation, described histologically as
‘hard callus’.2 Endochondral ossification involves the recruitment, proliferation and differentiation of undifferentiated mesenchymal cells into cartilage, which becomes
calcified and eventually replaced by bone. This type of
fracture healing is contributed by the periosteum and the
external soft tissues adjacent to the fracture providing an
early bridging callus, histologically characterized as ‘soft
callus’, which stabilizes the fracture fragments. At least five
discrete stages of secondary fracture healing have been
identified: (a) initial haematoma formation and inflammation, (b) angiogenesis and cartilage formation, (c) cartilage
calcification, (d) cartilage removal and bone formation, and
(e) bone remodelling.2
The classification of fracture healing into direct and
indirect healing reflects the histological events that occur
during the repair process. However, ongoing research into
bone regeneration has provided further understanding of
the cellular and molecular pathways that govern these
events.3
Important agents in the fracture healing
process
The agents that interfere with the healing process at the
molecular level can be separated into two general categories: (1) local molecules, and (2) systemic agents
promoting fracture healing. These factors interact with
various pathways in order to effectively enhance or inhibit
the healing process.
Fracture healing promoting molecules
During fracture healing, a number of signalling molecules
initiate and progress the chain of events of endochondral
and intramembranous bone formation by recruiting undifferentiated mesenchymal stem cells (MSCs) and osteoprogenitors cells, and inducing them to proliferate and
differentiate into osteoblasts.4
The signalling molecules can be categorized into three
groups: (a) the pro-inflammatory cytokines, (b) the TGF-b
superfamily and other growth factors, and (c) the metalloproteinases and angiogenic factors.4
Pro-inflammatory cytokines
Cytokines are known to be secreted not only by macrophages and inflammatory cells but also by cells of
mesenchymal origin present in the periosteum.5 Interleu-
V.S. Nikolaou, E. Tsiridis
kin-1 (IL-1), interleukin-6 (IL-6), and tumour necrosis factora (TNF-a) have been found to participate in the initial phase
of the repair process (Table 1).6 They show peak expression
within the first 24 h following fracture, and then their levels
are seen to reduce during the period of cartilage formation.
They increase for a second time during the bone remodelling
phase. The important central role of these molecules is
their chemotactic effect on other inflammatory cells that
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 day 1 to day 3 and during bone
remodeling
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
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Pathways and signalling molecules
Table 1. (continued )
a-FGF mainly effects chondrocyte proliferation, b-FGF
(more potent) involved in chondrocyte maturation and
bone resorption
Expressed from the early stages until osteoblast
formation
IGFs
Source: Bone matrix, endothelial and mesenchymal
cells (in granulation stage) and osteoblasts and nonhyperthrophic 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
colateral branches from existing vessels
Expressed from the early stages throughout fracture
healing
enhances extracellular matrix synthesis, stimulates angiogenesis, and recruits endogenous fibrogenic cells to the
injury site.5
Recent studies have demonstrated that TNF-a signalling
contributes to the regulation of chondrocyte apoptosis and a
lack of TNF-a signalling leads to a persistence of cartilaginous callus and delayed resorption of mineralized cartilage
by osteoclasts.7 Similarly, absence of TNF-a has been proven
to alter the control of angiogenesis and cartilage turnover.7
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.
The transforming growth factor-b (TGF-b) superfamily and
other growth factors
The TGF-b superfamily is a large family of growth and
differentiation factors that includes 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.8 Specific
251
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), promote various stages of intramembranous and
endochondral bone ossification during fracture healing.9
The role of BMPs
More specifically, 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.10 BMPs
elicit their cellular effects through activating specific
combinations of type I and type II serine/threonine kinase
receptors and their downstream effector proteins, which
are termed Smads.11 Their role in fracture healing has been
proved to be crucial as they induce a cascade of events for
chondro-osteogenesis, including chemotaxis, mesenchymal
and osteoprogenitor cell proliferation and differentiation,
angiogenesis, and controlled synthesis of extracellular
matrix.10 BMPs are divided into at least four separate
subgroups depending on their primary amino acid sequence.
Group 1 consists of BMP-2 and BMP-4, and group 2 includes
BMP-5, BMP-6, and BMP-7. The third group includes GDF-5
(or BMP-14), GDF-6 (or BMP-13) and GDF-7 (or BMP-12), and
finally group 4 includes BMP-3 (or osteogenin) and GDF-10
(or BMP-3b).10 The extracellular matrix comprises the main
source of BMPs being produced by osteoprogenitors, mesenchymal cells, osteoblasts and chondrocytes (Table 1). 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 (Figure 1). Studies
of the role of BMPs in fracture healing in the mouse and rat
have shown a variety of osteogenic effects, temporal
expressions and mitogenic capacities (Tables 2 and 3).3,9,12
Since 1965, when Urist revolutionized the current understanding of fracture healing by hypothesizing the existence
of bone morphogenetic proteins (BMPs),13 extensive research has been ongoing to further clarify the role of BMPs in
fracture healing. Cheng in 2003,14 analysing the osteogenic
activity of 14 types of BMPs in osteoblastic progenitor cells,
suggested that an osteogenic hierarchical model exists, in
which BMP-2, 6, and 9 may play an important role in
inducing osteoblast differentiation of mesenchymal stem
cells. In contrast, most BMPs are able to stimulate
osteogenesis in mature osteoblasts.14 BMPs may also
stimulate the synthesis and secretion of other bone and
angiogenic growth factors such as insulin-like growth factor
(IGF) and vascular-endothelial growth factor (VEGF).15
Furthermore, it has been shown that BMPs may also
stimulate bone formation by directly activating endothelial
cells to stimulate angiogenesis.11 Studies investigating the
role of BMPs in fracture healing in animals have shown a
variety of osteogenic effects, temporal expressions, and
mitogenic capacities.9,12,16 In humans, Riedel and ValentinOpran were the first to report preliminary results from the
use of BMP-2 to augment the treatment of open tibial
fractures.17 Later, the use of recombinant (rh) BMP-2 was
investigated in the treatment of open tibial fractures,18 and
a faster and higher union rate in the group of patients
treated with rhBMP-2 was reported. Another member of the
BMP family, BMP-7, has also been introduced in clinical
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V.S. Nikolaou, E. Tsiridis
IL-1, IL-6
TNF-α
PDGF
TGF-β1
TGF-β2
TGF-β3
GDF-8
BMP-2
BMP-3,8
*BMP-4
*BMP-7
GDF-10
BMP-5,6
GDF-5
Angiopoietin 1
VEGFs
IGF-I
IGF-II
IGFs
Fracture
Day 1
Day 3
Day 7
Day 14
Day 21
Bone Remodelling
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).
practice. In 1999, Greesink et al.19 in a prospective,
randomized double-blind study in 24 patients undergoing
high tibial osteotomy, evaluating the use of BMP-7 (or human
recombinant osteogenic protein [OP-1]) showed the high
osteogenic activity of BMP-7 in a validated critically sized
human defect. Similarly, Friedlaender et al.20 in a large
prospective randomized study, 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 BMP-7 was a safe and effective alternative to bone
graft in the treatment of tibial non-unions.
The role of transforming growth factor beta
It is well documented that transforming growth factor beta
(TGF-b) stimulates the formation of new bone both in vitro
and in vivo.21–23 Five isoforms of TGF-b have been
isolated.24 Transforming growth factor beta one (TGF-b1)
affects osteogenesis and chondrogenesis by stimulating
different cell types and plays an important role in repair
and remodelling of mesenchymal tissue.25 Recently, it has
been suggested that TGF-b2 and possibly TGF-b3 may play
more important roles in fracture healing than TGF-b1, as
their expression peaks during chondrogenesis.9 It is believed that TGF-b acts as a potent chemotactic stimulator
of mesenchymal stem cells (MSCs) and additionally stimulates proliferation of MSCs, pre-osteoblasts, chondrocytes and osteoblasts.26 Its main role is thought to be
during chondrogenesis and endochondral bone formation
(Table 2).27,28 Additionally, TGF-b might initiate signalling
for synthesis of BMPs, thus indirectly promoting the fracture
healing process.16
The role of the platelet-derived growth factor (PDGF)
Platelet-derived growth factor is a homo- or hetero-dimeric
protein composed of the A- and B-polypeptide chain (PDGFAA, PDGF-BB, and PDGF-AB forms).29 There are two
different receptors: the ‘a’ receptor binds all three dimeric
forms equipotentially, whereas the ‘b’ receptor binds PDGFBB with the highest affinity.30 PDGF is reported to be an
essential factor in bone repair inducing new bone formation
in vivo.31 PDGF is released by platelets during the early
phases of fracture healing and it is a potent chemotactic
stimulator for inflammatory cells and a major proliferative
and migratory stimulus for MSCs and osteoblasts (Figure 1).26
Although it is established that bone cells produce and
respond to PDGF, its roles in fracture repair have not been
fully defined. Nash et al.32 showed an increased callus
density and volume in tibial osteotomies in rabbits treated
with PDGF. In another animal study, Fujji et al.33 suggested
that PDGF contributed in part to the promotion of the
chondrogenic and osteogenic changes of mesenchymal cells
from the early to the mid-phase of fracture healing.
Consequently, recruitment of mesenchymal cells in the
initial step and interaction between osteoclasts and
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Pathways and signalling molecules
253
Table 2 Temporal and functional characteristics of
members of the TGF-beta superfamily observed during
fracture healing in animal models.
Member of Time of expression
the TGF-b
superfamily
Specific responses in
vivo and in vitro
GDF-8
Restricted to day 19
Potential function as
a negative regulator
of skeletal muscle
growth
BMP-2
Day 1 to day 219,12 (the
earliest gene to be
induced and second
elevation during
osteogenesis)
Recruitment of
mesenchymal cells
Chondrogenesis
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
BMP-3, 8
Temporal data
Day 14 to day 219
(restricted expression suggest a role in the
during osteogenesis) regulation of
osteogenesis
BMP-4
Transient increased
expression in the
surrounding soft
tissues 6 h to day 516
Day 14 to day 219
Involvement in the
formation of callus
at a very early stage
in the healing
process
In vitro: BMP-3 and 4 stimulate the
migration of human
blood monocytes
Through out fracture
healing12
BMP-7
Day 14 to day 219
GDF-10
Day 3 to day 219
Regulatory role in
both types of
ossification
BMP-6 may initiate
chondrocyte
maturation
Day 7 (maximal) to day
149 (restricted
expression during
chondrogenic phase)
GDF-5 an exclusive
involvement in
chondrogenesis is
suggested
Regulatory role in
both types of
ossification
From the early stages In vitro: Stimulation
of fracture healing16 of relative mature
osteoblasts
BMP-5, 6
GDF-5, 1
Table 2 (continued )
Member of Time of expression
the TGF-b
superfamily
Specific responses in
vivo and in vitro
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 the fracture
callus9
TGF-b1
Day 1 to day 219
TGF-b2
TGF-b3
Day 3 to day 149
Day 3 to day 219
GDF-6 may be
expressed only in
articular cartilage
and with GDF-5, 7
more efficiently
induce cartilage and
tendon-like
structures in vivo
Potent chemotactic
for bone forming
cells and
macrophages
Proliferation of
undifferentiated
mesenchymal and
osteoprogenitor
cells, osteoblasts,
chondrocytes
osteoblasts in the bone remodelling phase might be
stimulated by the mediation of PDGF. Nevertheless, at the
present time, the exact therapeutic potential of PDGF
remains unclear.
The role of the fibroblast growth factors (FGFs)
Fibroblast growth factors are present in significant amounts
in bone and several studies have suggested that they may be
involved in normal fracture healing.34 The family of FGFs
consists of nine structurally related polypeptides. The acidic
and basic FGFs are the most abundant FGFs in normal adult
tissue.35 Investigators have found that during normal
fracture repair bFGF is expressed in the cells of the
expanded cambial layer and is associated with a rapid
increase in the population of fibroblast-like mesenchymal
cells.36 In a rat model, multiple local injections of aFGF to a
fractured femur resulted in an increase in the cartilage
portion of the forming callus.37 Similarly, in a canine tibial
osteotomy model, a single injection of b-FGF was associated
with an early increase in callus size.38 When one application
of bFGF was delivered in a fibrin gel, it increased the callus
size, mineral content, and mechanical strength in fractured
fibulae in normal rats and in those with diabetes,39 and
similar effects were observed on intraosseous bone formation after a single injection of bFGF into the femurs of
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V.S. Nikolaou, E. Tsiridis
Table 3 Timing of cellular events and expression of
signalling molecules during murine fracture healing.20,33,43
Cytokines: IL-1, IL-6,
Day Haematoma
1
formation,
TNF-a released by
inflammation
inflammatory cells
Recruitment of
PDGF, TFG-beta
mesenchymal cells
released from
Osteogenic
degranulating
differentiation of MSCs
platelets
BMP-2 expression and
from bone marrow
restricted to day 1
expression of GDF-8
Day MSCs proliferation
begins
3
Proliferation and
differentiation of
preosteoblasts and
osteoblasts in regions
of intramembranous
ossification
Angiogenesis begins
Decline of cytokines
Day Peak of cell
proliferation in
7
intramembranous
ossification between
day 7 and 10
Chondrogenesis and
endochondral
ossification begins
(day 9–14 maturation
of chondrocytes)
Peak of TGF-b2 and
Day Cessation of cell
14
proliferation in
intramembranous
ossification, but
osteoblastic activity
continues
Mineralization of the
soft callus, cartilage
resorption, and woven
bone formation
Neo-angiogenesis
which infiltrates along
new mesenchymal
cells
Phase of most active
osteogenesis until day
21
Decreased levels of
levels
Expression of TGF-b2,
-b3, GDF-10, BMP-5, 6
Angiopoietin-1 is
induced
TGF-b3 expression
Expression of GDF-5
and probably GDF-1
expression for TGF-b2,
GDF-5, and probably
GDF-1
Expression of BMP-3,
4, 7, and 8
VEGFs expression
Second increase of IL-1
and TNF-a which
continues during bone
remodeling
Decreased expression
Day Woven bone
21
remodelled and
of TGF-b1 and TGF-b3,
subsequently replaced
GDF-10, and BMPs
by lamellar bone
(2–8)
normal rabbits and into rabbits that had been ovariectomized.40
The role of the insulin-like growth factors
The sources of IGF-I (or somatomedin-C) and IGF-II (or
skeletal growth factor) are the bone matrix, endothelial
cells, osteoblasts and chondrocytes. IGF-I is a 7.6-kDa
polypeptide that produces in conjunction with the type 1
IGF receptor (IGF1R) a potent proliferative signalling
system, which stimulates growth in various cell types and
blocks apoptosis. In vitro, it mediates stimulatory effects on
osteoblast activity and chemotaxis.41,42 IGF-II acts at a later
stage of endochondral bone formation and stimulates type I
collagen production, cartilage matrix synthesis, and cellular
proliferation (Figure 1).43 In vivo results on new bone
formation using IGF-I have been quite disparate. The
continuous infusion of IGF-I in rats with a femoral osteotomy
did not enhance fracture healing, and the callus weight in
the IGF-I treated animals was even slightly reduced
compared to the untreated controls.44,45 Conversely, new
bone was formed upon subcutaneous administration in rats
leading to a full bridging of 8-mm calvarial defects within 8
weeks.46,47 The variation of the findings of studies assessing
the influence of IGF on skeletal repair indicates the need of
further studies regarding this agent.27
The role of growth differentiation factors (GDFs)
Bone morphogenic protein-14 (BMP-14), also known as
growth and differentiation factor-5 (GDF-5) and cartilage
derived morphogenetic protein-1 (CDMP-1), is best known
for its role in joint formation and tendon healing but has
recently been shown to influence endochondral bone
growth.48 Its involvement in fracture repair was shown in
GDF-5 deficient mice, which showed a delay in fracture
repair. This delay was probably related to impaired cellular
recruitment and chondrocyte differentiation during the
early stages of the repair process.48 Spiro et al.49 performed
spinal fusions in rabbits using GDF-5 in three different
collagen matrices. They found no significant difference
between autograft and at least two of the formulations of
GDF-5 with fusion rates as high as 80% for the mineralized
collagen strips containing 1 mg/cm3 of rhGDF-5. These
results suggest that this molecule has potential as a
supplement in fracture repair but at the moment, there
are no human studies supporting its clinical use.
Metalloproteinases and angiogenic factors
During endochondral bone repair, as with embryonic bone
development, there is an intimate coordination between the
resolution of chondrogenic events with the programmed
death of chondrocytes and the initiation of bone formation.
Central to this transition is the removal of the cartilage and
its matrix in coordination with the ingrowth of new
vasculature to support new bone formation.7 During this
remodelling phase, specific matrix metallopoteinases
(MMPs) degrade cartilage and bone, allowing the invasion
of blood vessels (Table 1).4 Recent studies have demonstrated the requirement specifically for MMP-9 in the
embryonic growth plate, as well as in fracture repair. In an
animal study, mice with targeted deletions of MMP-9
displayed impaired revascularization and hypertrophic
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Pathways and signalling molecules
chondrocyte apoptosis. At the fracture site in these mice,
there were larger amounts of cartilaginous callus, delayed
degradation of cartilage matrix, and non-unions.50 Similarly,
Takahara et al.51 showed that MMP-9 positive cells cooperated with vascular endothelial cells in cartilage angiogenesis in an animal model studying the vascular and cellular
invasion in endochondral ossification in long bones. A further
association between MMP activity and concurrent angiogenic
events is demonstrated by the observation that the healing
of fractures in MMP-9 deficient mice can be rescued with
recombinant vascular-endothelial growth factor (VEGF)
treatment.50 VEGF has been demonstrated to be required
for both normal bone formation and repair.52 Street et al.
showed that fracture repair was enhanced by the exogenous
administration of VEGF.53 Additionally, VEGF appears to have
direct effects on osteogenic cell differentiation independent of its angiogenic influences and VEGF bioavailability
during fracture repair may be dependent on local MMP
activity.7 However, VEGF contribution in bone repair has yet
to be clarified.
Systematic agents promoting fracture healing
Parathyroid hormone (PTH)
Parathyroid hormone is involved in regulating serum calcium
and in bone remodelling. Continuous exposure of the
skeleton to PTH has resulted in increased bone turnover
and a decrease in bone mass, mainly due to the catabolic
effect of PTH through the direct activation of osteoblasts.
Nevertheless, recent studies have found that intermittent
exposure to PTH can have anabolic effects.54 Interestingly,
PTH administration enhanced bone remodelling in the early
stages of fracture healing in parathyroidectomized rats.55
Similarly, in experiments with rats, Andreassen et al.
showed that administration of a high dose of PTH increased
tibial fracture callus volume and ultimate load strength by
75% and 99%, respectively, after 20 days.56 Other animal
studies have also confirmed fracture healing enhancement
through the use of recombinant human parathyroid hormone
(PTH 1–34).57,58 At the moment, PTH has been approved in
several countries for treating postmenopausal osteoporosis,
with good results.59 Clinical trials to test the efficacy of PTH
for enhancing skeletal repair are currently underway.
Growth hormone (GH)
As early as 1959, it had been proposed that growth hormone
has positive effects in fracture healing.60 Since then several
studies have shown conflicting results.61,62 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 noncollagenous matrix proteins) by fully differentiated osteoblasts.3 In a recent study, Raschke et al.63 looked at
recombinant porcine growth hormone (r-pGH) in the repair
of experimentally induced tibial fractures in pigs. The
investigators found that the treated animals had a significant increase in bone mineral content while no difference was seen in bone mineral density, in comparison with
the control group treated with normal saline. Additionally,
the strength of the fracture callus was significantly greater
in the animals treated with r-pGH. In another study, in a
255
fracture healing and distraction osteogenesis model in pigs,
administration of homologous recombinant porcine GH led
to an increase in serum IGF-1, stimulation of fracture
healing and acceleration of ossification of bone regenerate
in distraction osteogenesis.64
HMG-CoA reductase inhibitors
There is evidence that HMG-CoA reductase inhibitors
(statins) can affect bone mineral density in humans and
reduce the risk of fracture,65,66 HMG-CoA reductase inhibitors are lipid-lowering drugs that inhibit cholesterol synthesis by blocking mevalonic acid production. Skoglund et al.
tested Simvastatin in a mouse model of femur fracture.67
This study showed that at day 14, the mechanical strength of
the Simvastatin group was 63% greater than in the control
mice and the callus was 53% larger. Nevertheless, at day 21,
this increase did not continue. It seems that statins enhance
osteoblast activity through increased expression of BMP-2,
nevertheless there are not enough data at the moment to
support the use of this drug category for fracture healing
enhancement.
Most recent advances and future directions
Gene therapy
Gene therapy involves the transfer of genetic material into a
targeted cell’s genome, thus allowing the expression of
bioactive factors from the cell itself for long 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 strategy.68 Gene therapy has been
used to promote fracture repair through the expression of
BMP-2 and BMP-4 in animal studies.69,70 For example,
Lieberman et al.69 devised a delivery system using BMP-2
and bone marrow cells. They implanted the transfected cells
into critical sized defects in rat femora and compared bone
regeneration at 2 months with rhBMP-2 or vehicle-treated
bone marrow cells. Results showed that each of the defects
treated with BMP-2 transfected bone marrow cells and
rhBMP had healed radiographically, while control animals
had failed to heal. Although promising, issues of its biosafety and efficacy need to be answered before gene
therapy application in humans takes place.
Muscle derived stem cells (MDSC)
Muscle derived stem cells have the ability to differentiate
into multiple lineages, including osteogenic and haematopoietic lines.71 These muscle-based progenitor cells possess
a therapeutic potential for tissue repair and regeneration
applications in various musculoskeletal as well as cardiac
muscle disorders, either as a source of inducible progenitor
cells or as gene delivery vehicles. Peng et al.72 used MDSCs
to study the effects of VEGF in BMP-4 induced bone
regeneration. MDSCs were transfected with either BMP-4
or VEGF. The investigators then impregnated Gelfoam disks
with cells expressing BMP-4 alone or cells expressing both
BMP-4 and VEGF. The disks were then placed in cortical
defects in the parietal bones of mice. The results showed
that with the addition of VEGF, there was a significant
ARTICLE IN PRESS
256
increase in the density of the bone formed. In a more recent
study the same investigators used MDSCs that were
transfected with noggin, a specific BMP antagonist, to
regulate the bone formation induced by locally implanted
BMP-4. In this way, they were able to control the amount of
bone formed reducing thus the heterotropic or overabundant bone formation otherwise seen with unopposed BMP-4
transfection.73
Conclusions
Our knowledge regarding fracture healing has significantly
improved following increased understanding of the multiple
and complex molecular pathways involved, and is expected
to improve further in the near future. These advances may
potentially facilitate the development of drugs or techniques to accelerate repair, heal non-unions, and prevent
delayed unions. Nevertheless, whilst intensive research is
conducted in the laboratories, more well designed clinical
trials are needed to investigate the efficacy and most
importantly the safety of the new treatment methods. The
molecular understanding of fracture healing extends beyond
fracture repair to organogenesis and differentiation during
embryonic development. It is of great importance to
identify the molecular signalling of these events in order
to predict and potentially to treat musculoskeletal and
other related connective disorders at the embryonic stage.
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