Born Again Bone: Tissue Engineering for Bone
Repair
Martin Braddock,1 Parul Houston,2 Callum Campbell,2 and Patrick Ashcroft3
1
Disease Cell Biology and 2Cardiovascular Systems Units, GlaxoWellcome Medicines Research Centre, Stevenage, Hertfordshire SG1 2NY, England;
and 3Department of Orthopaedics, University of Aberdeen Medical School, Aberdeen Royal Infirmary, Foresterhill, Aberdeen AB9 2ZD, Scotland
Destruction of bone tissue due to disease and inefficient bone healing after traumatic injury may be
addressed by tissue engineering techniques. Growth factor, cytokine protein, and gene therapies
will be developed, which, in conjunction with suitable carriers, will regenerate missing bone
or help in cases of defective healing.
uring the Paleozoic period, evolution produced the skeleton. This 500 million-year-old structure, like the liver and
central nervous system, has the capacity for regeneration, a
term until recently restricted to new tissue formation seen in
hydra, planarians, and salamanders. Why are we thinking
about the regeneration of bone? The stimulation of bone production is often required to treat loss of bone tissue brought
about by trauma, osteonecrosis, and tumors. Currently, bonegrafting procedures are employed to promote healing of fracture nonunion (in which the ends of the bone do not heal
together), in craniofacial reconstruction procedures, and in
fusion techniques for spinal surgery. The clinical gold standard
for bone repair is an autologous graft (grafts from the individuals themselves), which, although effective, is limited by the
availability of sufficient donor tissue and problems with donor
site morbidity. These limitations have led to the development of
both biological agents to induce bone growth and substitute
tissue grafts using different cell types, attached to a variety of
matrices or scaffolds.
Treating nonhealing fractures
There are ~6.5 million fractures per year in the United
States, of which ~15% are difficult to heal. For those fractures
in which the healing is slow (delayed union) or does not occur
(nonunion), there are few effective therapies at present. The
most common method of treatment is insertion of bone from
the individual (autologous) or from alternate sources (autogenous) into the defect. In this procedure, bone is removed from
a variety of sources, most commonly the pelvis following a surgical incision made in the hip area. The donor bone tissue is
inserted at the nonhealing fracture site, and additional support
may be provided by an orthopedic rod or plate. More than
250,000 bone grafts are performed annually in the United
States in an attempt to assist the body in regenerating new
bone lost by trauma or disease. Poor fracture healing is associated with chronic pain and prolonged ambulatory impairment
and must often be treated by surgical intervention. This has
considerable economic implications for healthcare providers.
External fixation devices may stabilize fractures at risk from
poor healing, although a lack of viable bone at the fracture site
may result, at best, in the production of unstable bone that is
prone to refracture. Although bone grafting is generally suc208
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D
cessful, it suffers from the limited amount of donor tissue that
may be available, and the patient may suffer side effects such
as numbness or tingling at the donor site, infection, or prolonged pain. An alternative therapy involves the use of pulsed
electromagnetic fields, which have been shown to have effects
on many aspects of bone formation and healing. This includes
the induction of endothelial and bone cell proliferation, the
formation of capillary sprouts, the stimulation of matrix formation, and calcification. A further recent addition is the use of
low-frequency ultrasound. The role of physical forces in stimulating bone and tissue repair in general will be discussed in a
later section.
What happens when a bone is fractured?
A fracture is a break in a bone that is always associated with
damage to the surrounding tissues. The amount of damage is
related to the energy that created the break. The fracture is
called “open” when the skin envelope around the bone is
breached and “closed” when it remains intact. Soon after a
fracture occurs, a number of events proceed to initiate the
healing and repair process. First, growth and differentiating
factors are activated by the injury process, which in turn activate localized pluripotent osteoprogenitor cells. These cells
produce a class of proteins known as bone morphogenetic
proteins (BMPs), which are intimately bound to collagen.
BMPs belong to the transforming growth factor (TGF)-- superfamily of peptide growth factors (8). The key BMPs found to
have bone-forming activity are listed in Table 1. These osteoinductive proteins, along with other growth factors, cytokines,
and hormones, induce the migration of mesenchymal cells and
their proliferation and differentiation into bone-forming cells.
As in the skin and other tissues, bone repair is a continuous
process that sets a cascade of events into motion. These events
are shown in Figs. 1 and 2. The healing of bone includes an initial rapid inflammatory response (minutes to hours), chemotaxis and mitosis (hours to days), production of extracellular
matrix, remodeling of the injury site, and localized angiogenesis (days to weeks). Bone remodeling is ultimately brought
about by primary intramembranous bone formation, chondrogenesis, and endochondral ossification, with production of
bone matrix leading to reconstitution of bone continuity. In
addition to the BMPs, a number of other growth factors, listed
0886-1714/99 5.00 © 2001 Int. Union Physiol. Sci./Am.Physiol. Soc.
TABLE 1. Overview of key bone morphogenetic proteins and their roles in osteoinduction
Protein
Common Name
Role in Bone Induction
BMP-2
BMP2A
Subcutaneous pockets in rats
BMP-3
Osteogenin
Subcutaneous pockets and craniotomy defects in rats
BMP-4
BMP2B
Subcutaneous pockets in rats
BMP-6
Vgr-1
Subcutaneous pockets in nude mice
BMP-7
OP-1
Subcutaneous pockets in rats
BMP-13
CDMP-2, GDF-6
No report of osteoinductivity; ectopic induction of tendon and ligament in rats
BMP-14
CDMP-1, GDF-5
No report of osteoinductivity; ectopic induction of tendon and ligament in rats
BMP, bone morphogenetic protein; Vgr, vegital-related protein; OP, osteogenic protein; CDMP, cartilage-derived morphgenetic protein; GDF, growth and
differentiation factor.
Tissue repair: common mechanisms in injury processes?
When a bone is fractured, skin is torn, blood vessels are ruptured, or ligaments or muscles are damaged, the tissue
acquires a “wounded phenotype.” Tissue repair results from a
number of temporally coordinated processes driven by locally
released mediators. The first event is immediate and consists of
the activation of the coagulation cascade and the formation of
a blood clot. Shortly afterward there follows an acute inflammatory response resulting in tissue edema and cytokine and
growth factor release. Then follows the first stage of collagen
repair, involving deposition and the formation of granulation
tissue, which becomes a new and temporary weak tissue. The
third and final process is the second phase of collagen repair,
resulting in extracellular matrix remodeling, angiogenesis, and
the reproduction of full-strength tissue. Much of the normal
healing process is driven by growth factors and cytokines (20).
In addition to their role in blood clot formation, platelets and
mesenchymal cells generate a number of growth factors that
are found in wound fluid, including TGF-,, platelet-derived
growth factor (PDGF), epidermal growth factor, vascular
endothelial growth factor, TGF--, and insulin-like growth factor-I (IGF-I). In this acute inflammatory response, neutrophil
migration is induced by PDGF, interleukin-1, and -8, tumor
necrosis factor (TNF)-,, granulocyte macrophage-colony stimulating factor, and granulocyte-colony stimulating factor.
Numerous growth factors have been implicated in the repair
of fracture healing, which include acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), PDGF, and
TGF--. Thus multiple growth factors and cytokines play a
major role in tissue repair. The BMPs that are produced by
osteoblasts and mesenchymal cells merely represent one level
of tissue specificity among a plethora of ubiquitous stimulatory
proteins. Thus the wounded phenotype “tags” injured tissue
separately from uninjured healthy tissue. This may provide a
potential therapeutic route to enhancing the healing process or
slowing down the proliferative response in diseases such as
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in Table 2, are produced after injury that may, in their own
right, serve to stimulate osteogenesis and repair bone. These
factors will be discussed in a later section, are not specific to
bone, and are an integral part of the platform that supports tissue repair in both hard and soft tissues.
diabetic retinopathy and dermal scarring in soft tissue and the
production of extraneous bone in patients with fibrodysplasia
ossificans progressiva (FOP).
Mechanical forces and bone repair
The concept that bone formation and remodeling is controlled by both physical and biochemical factors was first proposed over 100 years ago. It has been suggested that bone contains sensor cells, possibly osteocytes, that monitor mechanical stress and activate adaptative biological processes to
remodel the bone. Osteocytes, which are distributed throughout the bone, produce a signal through electrical potentials,
activation of ion channels, flow of interstitial fluid, and activation of signaling cascades that is in direct proportion to the
amount of mechanical loading (15). The initial mechanotransduction event in cells contained in bone is very similar to that
found in endothelial cells that line the cardiovascular system.
In both cases, transactivation of target genes involves stimulation of mitogen-activated protein kinase signaling cascades, as
well as protein phosphorylation and dephosphorylation, and
ultimately results in the direct binding of transcription factors
to gene promoters. The mechanical shear stress that is the stimulus in both systems causes transcriptional activation via distinct transcription factor binding sites that function as shear
stress response elements (4). These elements are found in many
promoters such as PDGF-B, TGF--, and FGF (13) that are activated by injury. Transcription factors themselves, such as early
growth response factor-1 (Egr-1) and stimulating protein-1 (Sp1) are also responsive to mechanical stimuli such as shear
stress (18). At a fracture site, it appears that an optimum level
of mechanical stress is a route to fast and effective healing. For
example, distraction osteogenesis is a surgical procedure that
may be used to correct limb length discrepancies or deformities or to treat nonunion fracture following acute loss of functional bone, for instance after a crush injury. In this procedure,
an osteotomy is performed followed by the application of an
external fixator device such as an Ilizarov frame. After a period
of ~1 wk, distraction is performed at controlled rate and the
bone begins to grow and will eventually fill the gap, growing
at a rate of ~1 cm/1
2 mo distraction time. Recent studies in a
rabbit model of distraction osteogenesis have shown that the
expression of BMP-2, -4, and -7 is increased after distraction is
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FIGURE 1. Biochemical responses to bone fracture. Bone fracture and tissue damage activate many biochemical processes. Some occur within minutes, such as
an acute inflammatory response, activation of immediate-early genes, and stimulation of signaling cascades, e.g., mitogen-activated protein kinase signaling. Some
occur within hours, such as release of growth factors and cytokines. Then, within days, development of repair blastema and angiogenesis occur, which enables
full tissue repair within weeks.
initiated and maintained throughout the distraction period
(17). In addition, in vitro studies in rat osteoblasts exposed to
pulsed electromagnetic fields showed that expression of BMP2 and -4 was increased, which was accompanied by an
increase in mineralized bone nodule formation (1). These
observations are consistent with physical forces inducing BMP
expression and subsequent stimulation of new bone formation.
Finally, it has been known for some time that cartilage regeneration may be enhanced by drill hole injury into subchondral
bone. One possible explanation for this regenerative effect
may be mechanotransduction, in which injury-induced stimulation of immediate-early genes (IEGs) drives the production of
growth-stimulating proteins. BMP-4, for example, stimulates
the production of the IEG Egr-1, which may feed back to stimulate BMP-4 expression and which in turn serves to promote
tissue repair by activation of multiple growth factors and
cytokines (see below).
Agents to induce bone repair: preclinical studies
There are many agents known to induce bone formation in
vitro and in animal models of ectopic bone formation or acute
injury, for example osteotomy. Some of these factors are aFGF,
bFGF, TGF--, PDGF, IGF-I, and BMP-2, -4, and -7 [osteogenic
protein (OP)-1], Indian and Sonic hedgehog, and parathyroid
hormone. TGF--1 has received considerable attention in the
promotion of both soft and hard tissue repair. TGF--1 has been
shown to stimulate the three-dimensional cellular development of human bone ex vivo, a process that is prerequisite for
successful tissue-engineered bone (12). Observed effects with
TGF--1 in vivo have, however, been variable, and one study
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reports no beneficial effect of this factor in a rabbit model of
distraction osteogenesis. Of this collection of growth factors,
BMPs have proven to be the most potent stimulators of osteoinduction, and recombinant human (rh) BMP protein has been
used to heal intermediate-sized bone defects in a variety of
animal models, including rats, dogs, rabbits, and nonhuman
primates. It should be borne in mind, however, that an animal
model of acute bone injury may not necessarily reflect the
inability of bone to heal, such as in nonunion fracture, periodontal bone regeneration, spinal fusion, or revision total joint
surgery. One such example relates to the use of BMP-3
(osteogenin), which in humans failed to promote periodontal
regeneration despite showing powerful osteoinductive capacity in animal models (14).
Agents to induce bone repair: clinical studies
To date there have been a number of clinical studies using
BMPs to promote bone repair by using either rhBMP-2 or
rhOP-1 (BMP-7) protein on a collagen scaffold (3, 6). Although
some promising results have been obtained, there appears to
be a varying response from individual to individual in terms of
new bone production. rhBMP-2 protein was used to augment
maxillary sinus and alveolar ridge formation and to form new
bone in tooth extraction pockets. rhBMP-2 has been used in a
small number of patients with hip osteonecrosis; the protein
was implanted at the site of core decompression, a process that
removes the dead bone from inside the femoral head. BMP
protein allografting has also been shown to be effective in the
reconstruction of femoral nonunion in patients with failed fracture healing. OP-1 has been used to treat patients undergoing
Transcription factor gene therapy to promote tissue
repair
When bone is injured, one of the earliest events to occur is
the activation of intracellular signaling cascades. These cascades serve to stimulate the rapid transcription of growth factors and cytokines, and they are initiated, at least in part, by the
stimulation of IEGs. Egr-1 (also called NGFI-A, Krox24, Zif268,
and TIS8) is an IEG coding for an 80-kDa phosphoprotein transcription factor. Egr-1 belongs to the C2H2 class of zinc finger
proteins that includes Egr-2 (also called Krox-20), Egr-3, and
Egr-4 (also called NGFI-C). Egr-1 is naturally produced in
response to acute stimuli, and the Egr-1 promoter comprises
transcription factor binding sites that allow modulation of Egr1 transcription by a number of physiological stimuli. These
stimuli include hypoxia, laminar fluid shear stress generated in
vitro or in vivo, mechanical stretch, bone loading, and acute
tissue injury. Egr-1 transcription may also be induced by nonphysiological stimuli such as electric shock, phorbol esters,
radiation, and urea. The induction of Egr-1 protein stimulates
the production of many genes whose products play a role in
cellular growth, development, and differentiation. These
include genes encoding cytokines (TNF-,), adhesion molecules (intercellular adhesion molecule-1), members of the
coagulation cascade (tissue factor, urokinase-type plasminogen activator), and growth factors such as aFGF, bFGF, TGF--1,
PDGF-A and -B, hepatocyte growth factor, vascular endothelial growth factor, and IGF-II. Gene gun delivery of Egr-1 DNA
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maxillary sinus floor elevation and unstable thoracolumbar
spinal fractures as well as in a randomized study of bone healing after fibula osteotomy. Although deemed to have a successful outcome, large amounts (in some case milligram quantities) of BMP were used in some studies. This may not be commercially viable and could also have an undesirable side effect
profile. Such potential limitations may be obviated by the use
of a better carrier or delivery system, for example, by the use
of polymer matrices, an adenovirus expressing the BMP-2
gene, or an autologous cell therapy in which disaggregated
cells are propagated in vitro to a synthetic scaffold and
implanted at the site of injury. However, the patient-to-patient
variation in response to administration of BMP protein may, in
part, be due to the induction of natural BMP repressor proteins,
such as noggin, follistatin, and chordin. This suggests that a
more subtle modulation of the natural response to tissue healing, in which a cascade of multiple growth-inducing factors is
stimulated, may result in a gradual and sustained production of
new bone.
In addition to the therapeutic use of the BMPs, several
growth factors have been shown to induce bone formation in
the clinic. In studies of 38 patients with moderate-to-severe
periodontal disease aimed at periodontal and peri-implant
bone regeneration, the safety and efficacy of a combination of
rhPDGF-BB and rhIGF-I was evaluated. Patients who received
PDGF/IGF-I responded with 43.5% osseous defect fill,
whereas the control group showed 18.5% osseous defect fill
(10).
FIGURE 2. The bone regeneration, remodeling, and repair process. The injury process stimulates growth factor and cytokine production, whose role is to stimulate osteoprogenitor cells to produce osteoinductive proteins. These proteins recruit mesenchymal stem cells and induce them to differentiate and proliferate into
osteoblasts, the cornerstone of bone-forming cells. IGF, insulin-like growth factor; VEGF, vascular endothelial growth factor; TGF, transforming growth factor; FGF,
fibroblast growth factor; PDGF, platelet-derived growth factor; BMP, bone morphogenetic proteins; TNF, tumor necrosis factor; IL, interleukin.
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TABLE 2. Overview of non-BMP factors with potential osteoinductive capacity
Activity
Bone Induction
Egr-1
Transcription factor
Subcutaneous pockets in rats; bridging of defect following radial osteotomy in rabbits
Fra-I
Transcription factor
Increase in bone mass of entire skeleton in transgenic mice
hPTH1-34
Hormone
Bridging of critical defect in beagle tibia osteotomy model
TGF--
Growth factor
Enhances healing in rabbit and rat tibial fracture models
CGF
Growth factor
Promotes collagen synthesis in human lung fibroblasts
Activin
Growth factor
Increases tibial bone mass in rats after systemic administration and bone mass and lumbar strength
in ovariectomized rats after intramuscular injection
IGF-I
Growth factor
Null mice show 35% reduction in long bone growth; promotes cementogenesis after application to
tooth root surfaces
PDGF-BB
Growth factor
Enhances healing in a rabbit tibial fracture model, bone formation in a rat model of calvarial
defect, and bone regeneration in periodontal disease in combination with IGF-I
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Protein
Egr, early growth response factor; Fra, fas-related antigen; hPTH, human parathyroid hormone; TGF, transforming growth factor; CGF, cementum-derived
growth factor; IGF, insulin-like growth factor; PDGF, platelet-derived growth factor.
has been shown to promote healing following dermal wounding in healthy rodents, in which it was shown to promote
angiogenesis in vitro and in vivo, enhance reepithelialization,
increase collagen production, and accelerate wound closure
(5). In addition, Egr-1 is capable of inducing bone formation in
a rodent model of ectopic osteogenesis. These studies demonstrate that Egr-1 can accelerate the normal healing process and
raise the potential use of this pleiotropic transcription factor as
a therapy for any aspect of tissue repair.
Recent studies have shown that another transcription factor,
fos-related antigen-I (Fra-I), may also increase bone formation
(11). Fra-I is a c-Fos-related protein belonging to the activator
protein-1 (AP-1) family of transcription factors. In transgenic
mice overexpressing Fra-I, there was an increase in the number of mature osteoblasts and a progressive increase in bone
mass of the entire skeleton. This study suggests that Fra-I stimulates bone formation by promoting osteoblast differentiation
and may represent a further pathway for therapeutic intervention to increase bone mass after acute injury or during later life
in patients with osteoporosis.
Delivery of osteogenic substances
So far we have described a number of potential protein or
gene therapies that may be used to induce bone repair. However, both the safety and efficacy of biological therapies will be
considerably enhanced by the use of an efficient delivery vehicle or matrix. Vehicles for gene delivery are numerous and
include viral constructs such as retrovirus, adenovirus, adenoassociated virus vectors, and nonviral delivery approaches
such as cationic lipids and liposomal formulations. Matrices
for gene or protein delivery that provide a stable and sustained
release of the agent have been the subject of research for many
years and are now beginning to show promise. Some studies
have used allogenic cortical bone, whereas others have investigated the use of synthetic substances. These matrices or scaffolds are biocomposites of either natural or synthetic macromolecules such as those constructed from bone substitutes,
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copolymers, or bovine collagen (2) and represent the cornerstone of the first generation of biodegradable implants. Other
means of inducing both soft and hard tissue repair involve the
use of genetically engineered pluripotent mesenchymal cells
(7) or marrow stromal cells (MSC), a subset of stem-like cells
that can now be extended almost without limit in vitro without
losing their potential for differentiation. MSC when mixed with
coral implants have been shown to stimulate bone regeneration and achieve clinical union in a animal model of bone
defect (16). A preliminary clinical trial shows promise for the
treatment of osteogenesis imperfecta, a bone disease characterized by a mutation in the type I collagen gene (9). In this
study, allogenic bone marrow transplantation was shown to
lead to engraftment of functional mesenchymal progenitor
cells (MSC) that, three months after osteoblast engraftment,
stimulated total body bone mineral content.
Limiting the repair process
The ability of osteoinductive proteins to stimulate bone healing offers considerable promise to patients who present with
recalcitrant bone repair. However, what limits bone production? In patients with the rare autosomal dominant disease
FOP, bone formation occurs in the muscle, tendons, ligaments,
and other surrounding connective tissue, resulting in partial or
complete immobility. Patients with FOP have constitutively
high levels of BMP-4 expression (19), which may explain their
increase in heterotopic bone formation. As alluded to earlier,
BMP-4 expression may, in part, be downregulated by the natural antagonists follistatin, chordin, and noggin. The use of
noggin gene therapy to counteract unwanted bone formation
in patients with FOP is being explored. Thus it may be necessary to carefully control the level of BMP-4 expression such
that its activity is sufficient to promote local bone formation
but is insufficient to promote undesirable bone formation. It is
also paramount that our natural repressor proteins retain the
ability to act as a “brake” on this potent osteoinductive
cytokine.
In summary, the next few years will bring together exciting
and rapidly developing areas of biomedical research, which
will involve the collaborative disciplines of bioengineers,
molecular biologists, physiologists, and drug and gene delivery
specialists. The constant discovery of new genes and the
exploitation of gene targets with previously characterized functions will help spur on the evolving science of regenerative
medicine.
Present address for M. Braddock: Disease Sciences Section, Discovery Biosciences Division, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire LE11 5RH, England.
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