Abstract
Recent progress in human embryonic and adult stem cell research is a cause
for much enthusiasm in bone and joint surgery. Stem cells have therapeutic
potential in the realm of orthopaedic surgery because of their capacity to
self-renew and differentiate into various types of mature cells and tissues,
including bone. Because nonunions remain a clinically important problem, there
is interest in the use of cell-based strategies to augment fracture repair.
Such strategies are being investigated with variations in the model systems,
sources of stem cells, and methods for the application and enhancement of
osseous healing, including genetic modifications and tissue-engineering. This
review highlights the recent progress in the utilization of stem cells and
cell-based gene therapy in promoting fracture-healing and its potential
utility in the clinical setting.
Although new technologies and advances in orthopaedic surgery have
substantially enhanced fracture-healing and surgical outcomes, there is a
subset of fractures that continue to be deficient in bone repair and culminate
in nonunion. Four elements are integral to bone repair: an osteoconductive
matrix, osteoinductive signals, osteogenic cells capable of responding to
these signals, and a sufficient blood supply. Autologous bone-graft provides
an osteoconductive matrix, osteoinductive factors, and osteogenic cells,
resulting in a reliable approach for managing most nonunions and bone defects.
However, there are associated morbidities with the harvesting procedure, a
limited supply, and varying osteoinductive potential of the graft depending on
the patient. As a result, new strategies to optimize nonunion repair are being
developed. This includes the use of stem cells, which has garnered great
interest recently because of the potential shown by stem cells in the
development and regeneration of tissues, including bone. The concept of the
use of stem cells in therapeutic applications is an appealing one, as stem
cells already have a natural role in tissue repair and regeneration. The aim
of this article is to review recent progress in the realm of nonunions and the
potential utility of stem cells and lineage-directing morphogens, such as bone
morphogenetic proteins, in their treatment.
The nonunion of fractures remains a challenging and clinically important
problem in orthopaedic surgery. During normal fracture-healing,
undifferentiated mesenchymal progenitor cells, with the aid of bone
morphogenetic proteins (BMPs) and regulatory cytokines, proliferate,
differentiate into chondrocytes and osteoblasts, and form bone, thereby
repairing the injury. However, some fractures fail to heal and become
nonunions, which may lead to morbidity and functional limitation for patients.
The diagnosis of nonunion is based on a combination of clinical symptoms and
physical findings, including pain and motion at the fracture site with
radiographic evidence of failure of union. Although there is no universally
accepted definition of nonunion, it can be considered the failure of a
fracture to heal in six to eight months without any further progressive
changes toward healing. The incidence of nonunion varies by fracture site but
can be as high as 5% to
20%1-3.
The causes of nonunion are multifactorial and may be categorized by fracture,
host, and surgical factors. These may include soft-tissue damage, loss of
vascularity, distraction of fracture fragments, soft-tissue interposition,
malnutrition, infection, instability, periosteal stripping, and systemic
disease.
Nonunions have traditionally been classified as hypertrophic, oligotrophic,
and atrophic. Hypertrophic nonunions are caused by inadequate mechanical
stabilization. There is adequate vascularity resulting in abundant callus
formation, but there is a persistent gap. Since hypertrophic nonunions
maintain their biologic activity, they require only adequate stabilization
with external or internal fixation to heal. In oligotrophic nonunions, there
is minimal callus formation; however, some biologic viability is usually
maintained. This commonly occurs when the fracture fragments are not properly
apposed, either from displacement during injury or inaccurate internal
fixation. In atrophic nonunions, there is a failed attempt by the body to make
new callus and to bridge the fracture gap. The bone ends are avascular, with
poor healing potential, and in some cases they demonstrate resorption.
Although segmental or critical-size osseous defects are sometimes included as
atrophic nonunions, it is important to discern that the failure in the repair
of these large defects is due to an inability to bridge a physical gap of
substantial size and not from an existing pathologic process or lack of
biologic capability. Over time, the gaps eventually fill with avascular scar
tissue. It is the oligotrophic and atrophic nonunions, which have impaired
biologic potential to unite, that require a treatment directed toward
improving the osteogenic ability and biology of fracture-healing.
Numerous therapeutic strategies have been evaluated in the treatment of
nonunions, including autogenous bone-grafting, demineralized bone
preparations, and, more recently, recombinant
proteins4-6.
Currently, there is increasing interest in cell-based strategies for the
augmentation of fracture-healing, including the use of stem cells to improve
fracture repair.
The potential of stem cells for therapeutic clinical use arises from their
normal role in tissue repair and their presence in essentially every tissue
and organ in the body, including bone and hematopoietic marrow. Stem cells are
primitive, unspecialized cells with two main characteristics: the ability for
self-renewal indefinitely and the ability to differentiate into distinct
lineages of mature cells. The developmental potential of stem cells can be
described as totipotent, pluripotent, or multipotent. A totipotent cell has
the ability to transform itself into every type of cell in the adult body and
into the cells of the extra-embryonic tissues required for fetal development.
The only true totipotent cells include the fertilized egg and the cells
produced by cleavage from the division cycles immediately following
fertilization. As the cells undergo further division cycles, they become
pluripotent, maintaining the capacity to divide into the three main types of
body tissue—endoderm, mesoderm, and ectoderm—and then giving rise
to any specialized cell type in the body except those required for fetal
development. As the cells become more specialized, they are considered
multipotent. They maintain their ability to self-renew but are limited in the
types of differentiated cells and tissues that they can become, usually to the
particular tissue or physiological system of origin.
There are two major classes of stem cells based on their
origin—embryonic and adult stem cells. Embryonic stem cells are
pluripotent cells isolated from the inner cell mass of the blastocyst that can
be propagated indefinitely in an undifferentiated state. Adult stem cells are
multipotent cells found in specific tissue compartments of the body with more
limited regenerative capability. However, adult stem cells may have more
plasticity than previously believed. A more pluripotent adult stem cell, the
multipotent adult progenitor cell, has been isolated from rat bone marrow and
has been shown to be able to differentiate into cells of the three different
germ layers7. The
two main types of adult stem cells, hematopoietic and mesenchymal, both have
clinical potential. Hematopoietic stem cells are capable of differentiating
into cells of the circulating blood and immune system and are already used
clinically in the treatment of leukemia, lymphoma, multiple myeloma, and
anemias. Mesenchymal stem cells are found in the nonhematopoietic bone-marrow
stroma but have also been found in muscle, synovial membrane, amniotic fluid,
placenta, dermis, periosteum, peripheral blood, and adipose
tissue7-11.
Because of their ubiquity in the body, their tolerance for in vitro expansion,
and their ability to differentiate into many types of tissue, including bone,
tendon, cartilage, ligament, and other tissues of mesenchymal origin, stem
cells hold infinite potential for clinical use in orthopaedic surgery,
especially in the repair and regeneration of
bone12-17.
Many studies have affirmed that stem cells have the capacity for bone
regeneration in vitro as well as in vivo
ectopically15,18-24.
As these efforts have been encouraging, their efficacy in animal
fracture-healing models has also been studied in preparation for the eventual
use of stem cells in clinical practice. These results can be influenced by the
experimental model chosen, which should simulate the clinical human situation
so that it is analogous in its pathophysiology and potential
treament25. Models
used in bone repair research include representations of normal
fracture-healing, segmental bone defects, and fracture nonunions in which
normal healing is prevented without a critical-size defect. Critical-size
segmental defect models require the removal of enough bone so that even in a
normal biological environment, the bridging of the defect will not occur. In
contrast, true nonunions do not heal because of a deficiency of signals or
response of stem cells. Critical-defect animal models often used in bone
regeneration studies include rat calvaria and femora, rabbit calvaria and
ulnae, and dog and sheep
femora26-29.
Noncritical defect models of nonunion require mechanical or metabolic
manipulation to cause failure of osseous union. One such model in the rat
femur simulates a clinical trauma scenario in which soft-tissue damage and
periosteal disruption may eventually cause nonunion. The fractures in this
model were created closed with a drop weight, and then the fracture site was
opened to cauterize the periosteum on each side of the fracture. The
cauterized femora did not heal over eight weeks, while the closed femoral
fracture controls had 100%
healing30. In this
model of a rat atrophic nonunion, there was downregulation of its BMP
expression31 and
the nonunion could be prevented with the application of recombinant human
BMP-74,32.
The published studies to date on stem cells for nonunion repair have been
concentrated in critical-defect models
(Table I) and can be
categorized into small animal, large animal, and gene therapy studies. Adult
stem cells have been combined with different structural carriers to induce
osseous repair in vivo in small animal models of critical-size defects.
Culture-expanded autologous bone marrow-derived mesenchymal stem cells loaded
on ceramic cylinders were implanted in 8-mm segmental defects in rat femora
with successful bone formation. By eight weeks, the mesenchymal stem
cell-loaded implants had substantially more bone fill than the carriers loaded
with fresh bone marrow or the cell-free carriers. They also had substantial
new-bone formation at the host-implant interface that led to bone growth
across the
defect33. This same
group then used human bone marrow-derived mesenchymal stem cells to heal the
segmental femoral defect in adult athymic rats with a ceramic carrier for
implantation. Once again, substantially more bone was found in the mesenchymal
stem cell-loaded cylinders, and the percentage of bone fill was equivalent to
that in the euthymic rats treated with syngenic mesenchymal stem cells in
their previous work. The mesenchymal stem cell-loaded specimens were also
biomechanically
stronger18. At
Stanford University, both adipose-derived adult stromal cells and bone-marrow
stromal cells were found to heal critical-size mouse calvarial defects when
implanted with apatite-coated, polylactic-co-glycolic acid (PLGA) scaffolds
without the addition of exogenous growth
factors34. More
recently, 8-mm calvarial defects in rats were treated with human
adipose-derived stem cells cultured in osteogenic media on PLGA in one
study35, and with
autologous rat human adipose-derived stem cells cultured in osteogenic media
on a biodegradable biphasic calcium phosphate nanocomposite in
another36. Both
studies showed that constructs imbibed with differentiated human
adipose-derived stem cells had more bone regeneration than did constructs with
undifferentiated human adipose-derived stem
cells35,36.
Autologous bone marrow-derived mesenchymal stem cells loaded on ceramic
constructs and implanted in critical-size segmental defects in large animals
such as
dogs37,38
and sheep39 have
also resulted in bone regeneration. Porous ceramic scaffolds seeded or not
seeded with in vitro expanded autologous bone marrow-derived mesenchymal stem
cells were compared in the healing of 21-mm femoral segmental defects in dogs.
After four months, bone had filled the pores of the implants seeded with bone
marrow-derived mesenchymal stem cells and union was seen radiographically,
while nonunion was observed in the control
femurs38. A coral
scaffold was used for the implantation of in vitro expanded bone-marrow
stromal cells in a sheep metatarsal segmental defect, which led to the
formation of an osseous cortex and medullary canal in the most favorable
cases. Clinical union was obtained in three of the seven limbs treated with
the stromal cells and coral
scaffold40. More
recently, this group also showed that enhanced bone formation would occur when
autologous mesenchymal stem cells, expanded in vitro onto coral scaffold
granules instead of one massive construct, were transplanted into the same
metatarsal defect in
sheep41. In a
critical-size defect of sheep tibial shafts, ceramic cylinders implanted with
and without autologous expanded bone marrow-derived mesenchymal stem cells
demonstrated orthotopic bone formation in both groups after two months,
although the bone marrow-derived mesenchymal stem cell-supplemented group
resulted in more extensive callus formation, osseous integration, and
increased
stiffness39.
However, because of the poor resorbability of the 100% hydroxyapatite porous
ceramic used in that pilot study, a subsequent study was performed with use of
a 5-cm tibial gap in a sheep model that had ceramic implants of 100% synthetic
calcium phosphate multiphase biomaterial containing 67% silicon-stabilized
tricalcium phosphate and 33% hydroxyapatite/beta-tricalcium
phosphate42. Good
integration between the ceramic implants and the bone ends was observed. A
progressive increase in new bone formation was seen over time, along with
progressive resorption of the ceramic scaffold. At the one-year time-point,
approximately 10% to 20% of the initial scaffold remained, and after two years
it was almost completely resorbed. Osteogenically induced bone marrow-derived
mesenchymal stem cells were seeded on coral scaffolds to treat 25-mm-long
defects in goat femora. Osseous union was seen at four months and further
remodeling and formation of cortical bone was observed at eight months. The
engineered bone also had bend-load strength and bend rigidity similar to the
contralateral normal
femur43.
The clinical use of culture-expanded stem cells has been reported in the
treatment of four patients with diaphyseal segmental defects ranging in size
from 3.0 to 28.3 cm3 in a tibia, a humerus, and two separate ulnar
fractures44,45.
Autologous bone marrow-derived mesenchymal stem cells from the four patients
were expanded ex vivo and were loaded on 100% hydroxyapatite macroporous
ceramic scaffolds. The grafts and defects were stabilized with external
fixation. Initial integration at the bone-implant interface was evident one
month after surgery and was completely consolidated five to seven months after
surgery. The patients all recovered limb function between six and twelve
months. During the long-term follow-up of these patients, no complications
were reported. However, the graft was not resorbed and remained almost
unchanged even after seven years. Because of the high density of the mineral
and its relatively low porosity (50% to 60%), the authors reported difficulty
following new bone formation with radiographs, thereby making it difficult to
establish the timing of the external fixator removal. Although this study has
been the only published report, to our knowledge, in which culture-isolated
and expanded stem cells were used clinically, the critical role of stem cells
has been further emphasized by the work of Hernigou et
al.46. In the
treatment of sixty tibial atrophic nonunions with use of percutaneous
injection of a concentrated buffy coat from an autologous iliac crest
bone-marrow aspirate, there was positive correlation between the volume of the
mineralized callus and the number and concentration of stem cells in the
aspirate. In the seven patients who did not achieve union, both the
concentration and the total number of stem cells injected were significantly
lower than in the patients with osseous union (p = 0.001 and p < 0.01). The
authors noted that the concentrated buffy coat layer, obtained from
centrifugation of the bone-marrow aspirate, contained stem cells as well as
other mononuclear cells, which may have had osteogenic or angiogenic effects
influencing the clinical results. However, although there were no control
groups, the success of the treatment of nonunion with percutaneous bone-marrow
grafting did appear to be dependent on the number and concentration of stem
cells available for injection.
As the eventual goal is to be able to use stem cells to heal nonunions in
patients, their use has already entered the realm of clinical trials.
Thirty-six patients with long-bone atrophic nonunions from type-IIIA or IIIB
fractures with fracture gaps of <6 cm that had failed previous surgical
intervention were enrolled in a multicenter, nonrandomized, open-label,
uncontrolled, single-group phase-I-II clinical trial (NCT 00424567), in which
they were treated with open reduction and internal fixation and an allograft
bone matrix graft extender with the addition of a bone marrow-derived stem,
stromal, and progenitor cell mixture that was expanded from an iliac crest
bone-marrow aspirate previously collected in an outpatient setting. Another
randomized, open-label, single-group phase-I-II clinical trial (NCT 00250302)
is waiting to begin. It involves the autologous implantation of mesenchymal
stem cells for the treatment of distal tibial fractures. Twenty-four patients
will be enrolled to examine the safety of the use of mesenchymal stem cells to
treat patients with distal tibial shaft fractures. The cells will be isolated
from the bone marrow of the patient, loaded onto a carrier, and implanted
locally at the fracture site.
Stem cells have also been genetically engineered to express transgenes of
osteogenic
proteins18,24,37-39,42,45,47.
This would decrease the number of mesenchymal stem cells needed for
implantation and possibly eliminate the need for in vitro culture and
expansion. The secreted protein exerts autocrine and paracrine effects,
leading to the differentiation of the engineered stem cell and to the
recruitment of host stem cells in vivo. Most often these studies have involved
BMPs. Human adipose-derived mesenchymal stem cells have been transduced with
BMP-2 and applied to a collagen-ceramic carrier to heal critical-size femoral
defects in athymic rats. Eleven of the twelve femora in the group treated with
the BMP-2-engineered stem cells healed by eight weeks after implantation.
Placing adipose-derived mesenchymal stem cells alone on the carrier did not
result in significant bone
formation48.
Muscle-derived stem cells from mice were engineered to express recombinant
human BMP-2, recombinant human BMP-4, or vascular endothelial growth factor to
heal calvarial defects in mice. Vascular endothelial growth factor acted
synergistically with BMP-4 to increase recruitment of mesenchymal stem cells
and endochondral cartilage formation, resulting in a substantial enhancement
of bone formation and
healing49-52.
More recently, the use of autologous adipose-derived stem cells transduced
with BMP-2 was shown to repair critical-size canine ulnar defects. Twenty-four
2.5-cm defect sites were implanted in one of four ways: beta-tricalcium
phosphate granules alone, tricalcium phosphate and adipose-derived stem cells,
tricalcium phosphate and osteogenic-induced adipose-derived stem cells, or
tricalcium phosphate and BMP-2-transduced adipose-derived stem cells. The
transduced adipose-derived stem-cell group showed substantial new bone
formation, and all six ulnae had a healed cortex on at least one side. The
osteogenic-induced adipose-derived stem cells also created new bone, but less
than the transduced group, and three of the six study animals continued to
have an evident defect at sixteen
weeks53. The
results also show that adipose-derived stem cells alone, without modification
or induction, are unable to heal critical-size bone defects.
The explosive advances in stem cell biology augur well for advances in the
realm of nonunion treatment and fracture-healing. The rapid progress with
embryonic stem cells will immensely aid potential applications in the
treatment of nonunions and in optimizing fracture-healing. Recent research
points to the reprogramming of normal skin cells to an embryonic state in
mice. Concurrent work in several laboratories all over the world is focused on
the application of the findings in mice to human cells. It is inevitable that
such research advances will lead to stem cells genetically matched to
individual patients. In fact, recent work points to obtaining inducible
pluripotent cells from fibroblasts by transfection with four transcription
factors: Oct-3/4, Sox 2, c-Myc, and
KLF454.
The inducible pluripotent cells can be stimulated to form skeletogenic cells
in the nonunion site to promote healing.
However, although scientific progress continues to further our knowledge of
stem cells and their potential for nonunion repair, unresolved questions that
require further study remain. We need additional expertise in the
characterization, isolation, differentiation, and expansion of these cells.
The identification of cell-specific markers is needed to characterize the stem
cells and their stages of differentiation to aid in the selection, detection,
and testing of stem cells and their treatments. Standard procedures for
isolating stem cells, techniques to propagate an adequate quantity,
optimization of cell-culture conditions, and maintenance of cell efficacy can
be achieved when there is further understanding of the ideal environment for
stem-cell growth and differentiation. New sources of stem cells that may lead
to enriched populations requiring less culture time are being identified. An
unambiguous relationship between these stem cells and the repaired or
regenerated bone will have to be demonstrated. In addition to establishing
criteria to confirm that function has been restored by the repaired tissue,
the bone must be fully integrated and immunologically compatible with the
host. As the research into stem cells and tissue regeneration is so recent,
there is still insufficient knowledge about the long-term stability of the
repair tissue, and it will be a period of time before long-term outcome and
safety can be assessed.
There are also issues specific to embryonic stem cells and genetically
engineered stem cells that will need to be addressed before they are applied
therapeutically in patients. There is the obstacle of opposition to embryonic
stem-cell research on ethical and moral grounds. It will also be necessary to
minimize or eliminate the risks of tumorigenicity and immune rejection.
Genetically engineered stem cells expressing osteogenic proteins could
decrease the number of stem cells needed for implantation and possibly
eliminate the need for in vitro culture and expansion by providing factors to
augment the healing process. However, most methods of gene delivery have
involved viral vectors, which have risks such as evoked immune reactions,
insertional mutagenesis, and uncontrolled transgene expression. Further
research is needed to improve the transfer of transgenes with safer vectors
and to find effective means of controlling their expression. There is also the
question of which osteogenic and angiogenic factors should be expressed in
stem cells to be most advantageous for impaired fracture-healing.
With a more established foundation of knowledge, investigators will be able
to answer questions about therapeutic approaches such as whether
undifferentiated or differentiated cells should be implanted and what the
optimal sources, proportion, and methods of application of stem cells for
fracture-healing will be. There is already potential for the systemic
administration of stem cells to potentiate
fracture-healing55.
Comprehensive and reproducible scientific investigations are needed before
claims about the full therapeutic capabilities of stem cells can be made. It
will be important to continue studies to understand the basic biology of stem
cells and their clinical applicability because their true potential can only
be realized when these issues have been completely comprehended. With the
progress that has been achieved recently, the promise of stem cells in
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