During normal fetal development, a variety of cell-signaling pathways are
regulated in a coordinated manner so that cells can proliferate, move, and
even die off in an organized fashion that allows organs to develop. Over the
past decade, there have been tremendous advances in understanding how the
musculoskeletal system develops. Knowledge about these pathways and how they
regulate cell behavior can be applied to musculoskeletal pathologic conditions
and repair processes. Many of the pathways that are important in development
can be targeted therapeutically; interestingly, many such agents have been
identified by their teratologic potential. Identifying the role of these key
signaling pathways in musculoskeletal disorders carries strong potential to
rapidly identify new therapeutic approaches. Genetically modified organisms,
such as transgenic mice, are important tools in this work. Because these
organisms have genetic abnormalities from the start of development, they can
be used to study the role of genes or cell-signaling pathways both in
development and in pathologic and repair processes that occur in maturity. A
symposium sponsored by the American Academy of Orthopaedic Surgeons, the
Orthopaedic Research and Education Foundation, the Orthopaedic Research
Society, the National Institutes of Health, the Canadian Institutes of Health
Research, the Shriners Hospitals for Children, Kyphon, and Stryker, on
developmental biology in orthopaedics, was held in Toronto in October 2006 to
review the state of the art in developmental biology of the musculoskeletal
system, determine how this information could be applied to disorders treated
by orthopaedists, and foster greater collaboration between orthopaedic
investigators and developmental biology researchers. The participants also
identified several areas of focus for future research that they thought would
have particularly fruitful potential.
Development begins with fertilization of the ovum; this fertilized cell is
called a zygote. The zygote undergoes rapid mitotic divisions and cellular
differentiation, leading to the development of a sphere of approximately 100
cells called a blastula. The blastula then undergoes gastrulation, during
which cells migrate to form three germ layers known as the ectoderm, mesoderm,
and endoderm. After the germ layers are defined, organogenesis begins. During
this phase, the neural plate folds to form the neural tube and somites
develop. Each somite eventually gives rise to a single vertebra and the
tissues associated with the adjacent nerve root. Somite formation is driven by
a segmentation clock, which regulates expression of genes in a periodic manner
to create the boundary for each somite.
Since the somites form individual vertebrae, it is easy to understand how
dysregulation of somite formation might cause spinal problems such as
congenital scoliosis. Several investigators are working to use information
about somite development to understand spine malformations. For example, it
was shown that a mutation in one of the genes that regulates the segmentation
clock and results in abnormal somite segmentation, delta-like 3, causes
spondylocostal dysostosis, in which multilevel congenital scoliosis occurs.
Understanding the genes that regulate the somite clock has a strong potential
to identify the cause of congenital scoliosis. In addition, knowledge about
somite and vertebral development can also be applied in a more general way to
a whole host of congenital and acquired spinal disorders. Knowledge from
genetically modified mice and data from studies that identify genes that
regulate the somite clock have particularly good potential to be applied to
patients with scoliosis. One approach to identifying these genes is to
undertake screening for genes that are regulated in a periodic fashion during
somite development. Another approach is to screen mice that have been
subjected to a chemical mutagen for congenital scoliosis and determine the
causative gene. Comparison of mouse and human phenotypes can be used to focus
on particular genes that have a high probability of causing a particular human
disorder. This approach has already proved successful. Such work carries
strong potential to identify key pathways in vertebral formation and to
provide data that can be used for prenatal diagnosis and prognostic
information. Ultimately, such information can be applied to other forms of
spinal deformity, by identifying key pathways that could be targeted
therapeutically.
During limb development, pluripotential cells differentiate into various
types, each at a precise location that facilitates the proper patterning of
the limb. Mesenchymal cells are of particular interest in musculoskeletal
development and in the postnatal musculoskeletal system because these cells
differentiate to become the cells that form the main structures of the
limb—osteocytes, chondrocytes, and fibroblasts. Long bones initially
form from a condensation of these pluripotential mesenchymal cells, which then
differentiate into chondrocytes that form a cartilaginous template of the
future bone. Joints form from a process in which cell death occurs within the
precursor tissues. In recent years, investigators have used genetically
modified mice to identify the mature tissues that select populations of
developing cells ultimately become. Such mice are engineered to express
proteins that can be easily detected when a certain regulatory element of a
gene is activated. Such studies have shown that chondrocytes differentiate to
one population that becomes the joint-lining structures and another population
that becomes the growth plate. The differentiation of pluripotential cells
into mature mesenchymal cell types is regulated by a number of cell-signaling
pathways and genes, several of which were discussed at the symposium,
including sex-determining region Y-box family (SOX), runt-related
transcription factor (RUNX), bone morphogenetic protein (BMP), transforming
growth factor-ß (TGF-ß), fibroblast growth factor (FGF), and
wingless-type MMTV (mouse mammary tumor virus) integration site family (WNT).
At each step in differentiation, precise regulation of these signaling
pathways is required to allow proper cell differentiation. Further work in
this area will allow for a precise understanding of how each of these genes
regulates cell differentiation, and such knowledge can be readily applied to
musculoskeletal repair processes.
Mature articular chondrocytes have a limited potential for repair, and
knowledge about how these cells initially develop from undifferentiated
precursors could be used to unlock the mechanisms that will allow mature cells
to behave in an appropriate way in order to heal articular defects. At the
present time, investigators are only just beginning to understand this
process. This is an area in which additional research is needed; such work can
rapidly be applied to the fields of cartilage repair, tissue engineering of
cartilage replacement, and arthritis. The use of genetically engineered mice
is particularly useful in this regard, as the role of particular genes and
pathways can be rigorously assessed. Several investigators are developing
techniques to induce arthritis in mice. Osteoarthritis can be surgically
induced in mice, and there are a variety of methods by which inflammatory
arthritis can be induced, including the injection of antigens.
As the long bones develop, the cartilaginous template is replaced by
osteoblasts—first as a primary ossification center in the middle of the
bone and then as secondary ossification centers at the ends of the bone. The
cartilage near the primary ossification center becomes the growth plate, which
provides longitudinal growth to the bone during both fetal and postnatal
growth. The past decade has seen tremendous advances in the understanding of
fetal growth-plate development. Roles for hedgehog signaling, parathyroid
hormone-related protein (PTHrP), FGF, SOX9, TGF-ß, BMPs, WNTs, and matrix
metalloproteinases (MMPs) have been elucidated. Genetically modified mice, in
which genes can be conditionally knocked out or expressed just in
chondrocytes, have been particularly useful in this regard. The interpretation
of data from genetically modified mice is confounded by the fact they not only
produce alterations in the targeted gene but also may alter the populations of
cells that develop. New methods of analyzing growth plates, such as detailed
studies of blood flow, are providing novel insight into how these genes alter
growth-plate function; some of these challenge conventional concepts. For
example, such data suggest that growth plates may be much more vascular than
once thought, and this vascularity may be responsible for the transmission of
local growth-plate regulatory factors over long distances as well as for the
response of the growth plate to trauma. The development of hypoxia in
growth-plate chondrocytes, mediated by genes such as hypoxia-induced factor
(HIF), plays a role in regulating growth-plate chondrocyte differentiation.
Imaging techniques may be applied to both mice and humans to correlate
findings in genetically modified mice with human pathologic states. New
magnetic resonance imaging techniques show excellent potential in this regard.
Despite the wealth of data in fetal growth-plate function, little is available
on growth-plate function in the growing child; this is an area in which
research is needed. Such work can be applied to conditions such as limb-length
inequality and short-stature disorders. Indeed, short-stature conditions are
known to be associated with mutations disturbing growth-plate function. For
instance, a mutation in an FGF-receptor causes achondroplasia. Research into
pharmacologic methods to target growth-factor signaling pathways in the growth
plate has strong potential to lead to pharmacologic treatments for these
disorders.
During development, bone forms in the middle of the cartilaginous bone
template through a process that is associated with matrix degradation and
vascular ingrowth. Pluripotential mesenchymal cells differentiate to become
osteoblasts, regulated by a variety of genes and signaling pathways, such as
RUNX2, BMP, and WNT. The source of these cells has not been completely
elucidated, but recent evidence suggests that periosteal cells play a role in
this process. Factors that regulate blood vessel formation and matrix
degradation (such as vascular endothelial growth factor [VEGF] and select
MMPs) are critical for normal bone development. During postnatal bone repair
and regeneration (fracture-healing and distraction osteogenesis), many of
these same pathways are also used. For instance, studies on mice have found
that the same factors that play a crucial role in regulating vascularity and
hypoxia in bone formation (HIF or VEGF) also play a role in the response to
osteonecrosis and during distraction osteogenesis. As such, research into
pathways that regulate osteoblast development and how they are used in
regenerative processes has good potential to result in therapies to improve
fracture-healing, speed the rate of regenerative bone formation, improve
fusion rates, and improve bone ingrowth in joint arthroplasty.
Stem cells are pluripotential cells with the characteristic that, when
dividing, one or both of the daughter cells maintains the ability to act as a
pluripotential stem cell while the other can differentiate down various
lineages. A small population of cells with stem-cell characteristics seems to
be present in mature animals in many tissues, and it is thought that such
cells act to maintain these tissues throughout the life of the organism. Stem
cells during early development do not behave the same way as these mature stem
cells. Recent studies of mesenchymal stem cells show this different behavior
and suggest that, during repair, stem cells also express factors that induce
other cells to behave in a way to promote healing. Additional research into
the biologic function of stem cells during repair processes is needed in order
to develop better methods of using these pluripotential cells to improve bone
and cartilage repair. There is a variety of sources for such cells; one source
is in muscle, in which precursor cells exist as satellite cells. Such cells
can be used not only to alter muscle behavior but also to deliver factors to
select tissues, with use of a form of gene therapy. Since age and gender
differences in pluripotential cell behavior may influence factors such as
muscle strength and bone density, research into this area has particularly
important applications in a variety of orthopaedic conditions associated with
aging.
Cells in musculoskeletal tumors often utilize the same cell pathways that
are important in development. Cell-signaling pathways that are important in
the growth plate, such as those activated by hypoxia, also play a role in
cartilage tumors. Cancers may be maintained by a population of stem cells,
just like other mature tissues. Targeting these cancer stem cells can be
developed into a more effective therapy for sarcomas. Additional research into
cancer stem cells in sarcomas can identify these cells and the pathways that
can be targeted therapeutically in order to eradicate these lesions.
Although there has been a wealth of work on bone and joint development,
relatively little has been undertaken to study ligament and tendon
development. Recent studies have found that ligaments arise from periarticular
cells expressing unique transcription factors during development. They are
initially quite cellular structures, whose ultimate extracellular matrix
composition and strength are regulated by a variety of genes. Transcriptional
regulation of genes in these tendons, their mechanical environment, and the
rate of expression of factors regulating this extracellular matrix composition
all play roles in repair and long-term tendon and ligament function. Work in
this area has the potential to identify pathways that can be exploited to
improve ligament repair or to allow cells to behave with the precise
regulation necessary for optimal behavior of tissue-engineered ligament
replacements.
Presentations at this symposium highlighted the tremendous advances of the
past few decades in our understanding of the development of the
musculoskeletal system. In the coming decades, in addition to research to
improve our understanding of the fundamental control of development, research
into the use of genetically modified organisms, e.g., mice and other species
such as fish, will allow for the study of these pathways in musculoskeletal
pathologic disorders as well as in repair and regeneration. The development of
targeted viral vectors and cell-based therapies to deliver proteins to
specific cell types will aid in the ultimate translation of this line of
research to improve patient care. The use of pharmacologic agents that target
developmentally important signaling pathways, some of which were initially
identified as teratogens, can be rapidly applied to patient care. Research in
developmental biology of the musculoskeletal system is at the threshold of
effective translation into improved treatments for patients with orthopaedic
disorders. The participants in this symposium identified targeted areas for
future work that will bring such research in developmental biology into the
realm of patient care.