Extract
Basic, translational, and clinical research in orthopaedics continues to be
an important part of the mission of the American Academy of Orthopaedic
Surgeons (AAOS). The AAOS Research Committee recently released a report
entitled "Future Directions in Musculoskeletal Research." This
report (available online at
www.aaos.org/wordhtml/research/synthesis/panel_future_directions.pdf)
is a detailed document that summarizes the findings of panels of experts in
seventeen areas of musculoskeletal care. Each panel developed a report that
described the scope of research, including its importance to public health as
well as its clinical importance, recent advances in the field, and future
directions for research. The research areas that were identified in the
seventeen panel reports were sorted into twenty common themes
("Musculoskeletal Research Focus Areas"). The most common research
themes identified by the panels were tissue-engineering, cell biology,
genetics research (including biomarkers and gene therapy), biomechanics and
biophysics, and outcomes research (including assessment tools and clinical
trials). This summary will help to identify fruitful research topics for
orthopaedic clinicians and scientists as well as students and scientists in
training and also will identify areas in need of research support from funding
agencies.
Basic, translational, and clinical research in orthopaedics continues to be
an important part of the mission of the American Academy of Orthopaedic
Surgeons (AAOS). The AAOS Research Committee recently released a report
entitled "Future Directions in Musculoskeletal Research." This
report (available online at
)
is a detailed document that summarizes the findings of panels of experts in
seventeen areas of musculoskeletal care. Each panel developed a report that
described the scope of research, including its importance to public health as
well as its clinical importance, recent advances in the field, and future
directions for research. The research areas that were identified in the
seventeen panel reports were sorted into twenty common themes
("Musculoskeletal Research Focus Areas"). The most common research
themes identified by the panels were tissue-engineering, cell biology,
genetics research (including biomarkers and gene therapy), biomechanics and
biophysics, and outcomes research (including assessment tools and clinical
trials). This summary will help to identify fruitful research topics for
orthopaedic clinicians and scientists as well as students and scientists in
training and also will identify areas in need of research support from funding
agencies.
This review will summarize the latest developments in several areas of
orthopaedic research, including gene therapy, stem cells, tissue-engineering,
and biomarkers. Specific topics that will be reviewed include
fracture-healing, muscle injury, and osteoporosis and bone quality. We will
also summarize information presented at joint symposia of the AAOS and the
Orthopaedic Research Society (ORS) at their recent annual meetings.
The basic biology of bone-healing and methods to augment healing continues
to be an active area of investigation. A combined ORS/AAOS symposium on
biologic factors that can improve fracture-healing was presented at the recent
meetings in Washington, DC. Several new developments will be reviewed here.
Autologous blood concentrates have been approved for marketing by the United
States Food and Drug Administration. This material contains growth factors,
including platelet-derived growth factor (PDGF) and transforming growth
factor-ß (TGF-ß). Several commercial entities now market equipment
that allows separation of platelets and plasma from red blood cells,
concentration of platelets, and formation of a fibrin clot. This clot can then
be used to augment bone-healing. At this time, there is very little clinical
evidence to support its use.
Exogenous cytokines continue to hold promise for the augmentation of
fracture-healing. The angiogenic factor vascular endothelial growth factor
(VEGF) was found to improve healing in a mouse femoral fracture model and a
rabbit radial defect
model1. Furthermore,
inhibition of VEGF with neutralizing VEGF receptor resulted in impaired
healing in a mouse femoral fracture model.
Recombinant human bone morphogenetic proteins (rhBMPs), perhaps the most
powerful osteoinductive factors known, continue to be investigated. Regulatory
agency approval has been provided for the use of BMP-7 (also known as
osteogenic protein-1) for the treatment of long-bone nonunions, and it also
has been provided for the use of BMP-2 for spinal fusion. However, these
applications require implantation of the protein on a collagen-based carrier
material, thus necessitating direct exposure of the fracture site via open
surgery. An important advance in this field has resulted from recent studies
that have demonstrated the potential for percutaneous injection of BMP-2 to
improve fracture-healing. Einhorn et al. created closed femoral fractures in
rats and then percutaneously injected 80 µg of BMP-2 into the fracture site
six hours later2.
They found significant acceleration of fracture-healing according to both
biomechanical and histologic criteria. At four weeks, the strength and
stiffness of the rhBMP-2-treated fracture was equivalent to that of the
contralateral, normal femur, whereas the untreated fractures and the fractures
that had been treated with buffer only remained significantly weaker than the
intact femur. Histologic analysis demonstrated extensive new-bone formation in
the rhBMP-2-treated fractures.
Recombinant human BMP-2 can be detected at the injection site for
approximately seven days after injection with use of an aqueous buffer,
whereas residence time can be extended to several weeks with use of a carrier.
The residence time of rhBMP-2 was adequate to improve healing in the rapidly
healing rat femoral fracture model; however, the next question to be examined
was whether percutaneous injection could accelerate fracture-healing in
slower-healing primate fractures. In a recently published study, Seeherman et
al. found that rhBMP-2 delivered in an injectable calcium phosphate paste
accelerated healing of fibular osteotomies in cynomolgus
monkeys3. The
rhBMP-2/calcium phosphate paste was injected under fluoroscopic guidance into
the osteotomy site three hours after the osteotomy. The investigators found
that the mean callus area, torsional stiffness, and maximum torque were
significantly greater in the treated osteotomy sites as compared with the
paired control osteotomy sites at ten weeks. Histologic analysis confirmed
complete osseous bridging in the rhBMP-2/calcium phosphate paste group at ten
weeks, whereas the paired control osteotomy sites were incompletely healed.
The granular formation of the calcium phosphate carrier material was thought
to play a positive role in the efficacy of injected rhBMP-2 in this model as
the rh-BMP-2 binds to the granules and is gradually released over time as the
carrier granules are resorbed. Furthermore, the granular nature of this
carrier material causes the material to be dispersed over a wider area,
allowing infiltration of blood vessels and cells.
A new synthetic peptide drug, TP508 (Chrysalin), has been reported to
enhance bone formation in preclinical models. TP508 is a twenty-three
amino-acid peptide representing the receptor-binding domain of thrombin. At
the site of bone injury, thrombin activates platelets and forms a fibrin clot.
Thrombin is sequestered within the clot and is later released when the clot
dissolves. Thrombin fragments released from the clot activate a cascade of
growth factors and enzymes that initiate and regulate the healing process.
TP508 mimics thrombin effects and appears to act by initiating the body's
natural growth factor cascade at the site of injury. Sheller et al. reported
significantly improved healing, as demonstrated with plain radiographs (p <
0.05), microtomography, and mechanical testing (p < 0.01), in association
with the use of TP508 in biodegradable controlled-release
poly(DL-lactic-co-glycolic acid) (PLGA) microspheres in critically-sized ulnar
defects in rabbits4.
In another study, TP508 improved consolidation of the regenerated bone
following distraction osteogenesis in a rabbit
model5. Quantitative
computed tomography demonstrated significantly greater bone mineral density in
the TP508-treated limbs as compared with controls (p < 0.05). Bone
consolidation and remodeling were more advanced in the TP508-treated limbs
compared with controls. The investigators who performed those studies are now
planning studies designed to evaluate the effect of TP508 in articular
cartilage regeneration.
Although various demineralized bone matrix preparations have been available
for clinical use for several years, there have been very few data comparing
the various products on the market. Demineralized bone matrices are prepared
by acid extraction of allograft bone, resulting in loss of most of the
mineralized component but retention of collagen and noncollagenous proteins,
including growth factors. Demineralized bone matrices have been used to
improve spine fusion, to treat fracture nonunions, and to fill osteolytic
lesions around total joint implants. A recently published study compared three
different commercially available demineralized bone matrix preparations in a
spine fusion model in athymic
rats6. The three
preparations that were tested included Grafton DBM Putty (Osteotech,
Eatontown, New Jersey), DBX Putty (MTF [Musculoskeletal Transplant
Foundation], available through Synthes, West Chester, Pennsylvania), and
AlloMatrix Injectable Putty (Wright Medical Technology, Arlington, Tennessee).
On the basis of radiographs, histological analysis, and manual testing of the
retrieved spines, the animals treated with Grafton Putty had superior rates of
fusion and more new-bone formation. Clinical studies are required to evaluate
the efficacy of these products in patients.
Breakdown products of cartilage matrix molecules and chondrocyte metabolic
products may serve as useful markers of the severity and progress of
osteoarthritis. Most patients with arthritis are identified relatively late in
the disease process, when a host of metabolic events have already occurred and
the process is beyond the point at which pharmacological or surgical
interventions can delay or reverse the process. The ability to detect the
disease in its early stages may open the door for more effective
interventions. The only widely available biomarkers that are in use at this
time are bone markers (such as N-telopeptide) that are used for the evaluation
and treatment of osteoporosis; despite a number of promising markers for
articular cartilage metabolism, none have been validated to the point of being
widely available for clinical use.
A combined ORS/AAOS symposium entitled "Biomarkers of Osteoarthritis:
Need, Challenges, and Potential Use" reviewed the current status of
cartilage biomarkers. The utility of biomarkers will be for the early
identification of patients who are at risk of rapidly progressive
osteoarthritis, allowing for the selection of the most appropriate patients
for pharmacologic or surgical therapy. Accurate biomarkers also will allow
faster clinical trials of arthritis treatments as well as the evaluation of
the response to such treatments. Biomarkers do not have to be only chemicals
that can be measured in joint fluid (or blood or urine) but may also be
indicators of cartilage matrix metabolism that can be detected with imaging
modalities. For example, T2 relaxation time can be measured as an indicator of
collagen organization in articular cartilage, whereas matrix proteoglycan
content is reflected by measurement of the fixed negative-charge density of
cartilage with use of delayed gadolinium-enhanced magnetic resonance imaging
of cartilage (dGEMRIC). Several substantial challenges to biomarker
development exist at this time. Validation of currently available potential
biomarkers is hindered by the absence of effective drugs for the treatment of
osteoarthritis. Systemic (urine and serum) levels of a biomarker will have to
be sensitive to events in a single arthritic joint. Furthermore, more
information is required to understand how these chemicals may be metabolized
in the body prior to their measurement in serum, urine, or even synovial
fluid.
Current biomarkers of interest include molecules that are released during
matrix degradation and molecules that reflect repair processes. Aggrecan is a
cartilage matrix molecule that is cleaved at several specific sites in
arthritis. Antibodies that only recognize the cleavage fragments and identify
the class of enzymes responsible have been produced. Similarly, antibodies
that only recognize cleaved type-II collagen and not the intact molecule have
been developed. As a marker for matrix turnover and repair, newly synthesized
type-II collagen can be detected by propeptides that are released from
procollagen II during collagen fiber formation.
Further development of useful biomarkers will include consideration of the
temporal specificity and process specificity of molecular markers. There is a
temporal pattern of biomarker production and release. Current data suggest
that aggrecan is released early in the arthritic process. The hyaluronan part
of the molecule remains in the tissue and is released at a later point in the
process. Fibromodulin and cartilage oligomeric matrix protein (COMP) are
released at a later stage, and collagen cleavage occurs in the final phase.
The term "process specificity" refers to enzymes and molecules
that may be specific to a particular disease. This approach could help to
distinguish inflammatory arthritis (such as rheumatoid arthritis) from
osteoarthritis.
Studies from the laboratory of Huard et al. have contributed greatly to our
understanding of the response of muscle to injury. Those investigators have
studied the basic biology of muscle-healing in mouse models. The healing
process in injured muscle involves degeneration and inflammation,
regeneration, and eventual fibrosis. Studies of healing muscle have
demonstrated that degeneration and inflammation occur in the early repair
process. Muscle regeneration takes place in the first one to two weeks after
the muscle injury, and then fibrosis begins during the second week after the
injury. Scar tissue formation then gradually increases over time.
Huard et al. evaluated the effect of several agents that can inhibit muscle
fibrosis and can improve the number of regenerating myofibers. Those authors
presented several studies at the recent meeting of the Orthopaedic Research
Society. They reported that NS-398, a cyclooxygenase-2 (COX-2)-specific
inhibitor, delayed the normal muscle-healing process by inhibiting muscle
regeneration and promoting the formation of fibrous tissue in a gastrocnemius
muscle laceration model in
mice7. These
findings were present at early time-points, but there were no differences
between the groups by twenty-eight days after the injury, suggesting that the
effect of the COX-2 inhibition is transient. In another study, those authors
studied lacerated tibialis anterior muscles in mice and found that suramin (an
antiparasitic and antitumor drug) inhibited fibrous tissue formation and
improved muscle-healing after
injury8. The
mechanism of action of suramin appears to be inhibition of TGF-ß. There
were more regenerating myofibers at the laceration site in suramin-injected
muscles as compared with control muscles. Importantly, muscle function was
improved in the treatment group, as demonstrated by significantly greater peak
muscle contractile force as compared with that in controls that had been
injected with saline solution only (p < 0.05). Another study from that
group demonstrated that relaxin promoted improved muscle-healing with a
greater number of regenerating myofibers in a mouse muscle-strain injury
model. It was thought that this effect was due to relaxin-induced attenuation
of TGF-ß at the muscle injury site.
Because most clinical scenarios require the treatment of muscle fibrosis
after it has already developed, Huard et al. also evaluated the effect of
injection of matrix metalloproteinase-1 (MMP-1, collagenase) into an area of
fibrosis that had formed following muscle injury in a mouse
model9. The authors
found that the MMP-1-treated limbs contained significantly more regenerating
myofibers in the area of injury than did control limbs (p < 0.001). There
was also significantly less fibrous tissue within the injury zone in the
MMP-1-treated muscles as compared with controls (p < 0.05). These findings
suggest the potential to ultimately treat muscle injury after scar tissue has
formed.
Another area of recent interest in muscle research is the effect of rotator
cuff tendon detachment on the structure and function of the rotator cuff
muscle. Muscle atrophy and fatty infiltration have been found to be important
prognostic factors following rotator cuff repair. Gerber et al. studied the
effect of infraspinatus tendon release and delayed repair in a sheep model to
elucidate the changes in the muscle and to determine whether these changes
could be reversed following tendon
repair10. The
authors found that retraction of the muscle-tendon unit following release was
associated with profound structural and functional changes in the muscle,
including muscle atrophy, fatty infiltration, and an increase in interstitial
connective tissue. An important finding of that study was that there was fatty
infiltration of the muscle rather than fatty degeneration. Muscle fibers did
not appear to degenerate, suggesting the potential for recovery with
appropriate stimulation. The amount of fatty infiltration was proportional to
the degree of muscle retraction. Muscle retraction was associated with loss of
elasticity of the muscle-tendon unit, which is consistent with clinical
experience with the repair of long-standing, retracted rotator cuff tears. The
structural changes in the muscle were not reversed after delayed repair of the
tendon.
The Human Genome Project was very successful in sequencing the human
genome. The next step is to identify the function of the molecules encoded in
the human genome. Extensive research will be necessary to translate
genome-based knowledge into improvements in medical diagnosis and treatment. A
symposium at the recent annual meeting of the American Academy of Orthopaedic
Surgeons entitled "Genomics: What Will Be Its Impact" explored
ways in which information from the Human Genome Project may have profound
effects on medical practice. The obvious application of the genome data is to
develop new approaches to the diagnosis and treatment of many common diseases.
We also may be able to develop genome-based approaches for the early detection
of diseases such as osteoarthritis or osteoporosis and for the prediction of
disease susceptibility. Furthermore, the genome data may allow for the
development of a molecular taxonomy of various diseases. Understanding the
genetic basis of disease ultimately may allow us to define diseases on the
basis of the underlying molecular and genetic mechanism(s) rather than on the
basis of symptoms.
The genome information is also likely to have profound effects on drug
development and how medications are used to treat various conditions. For
example, pharmacologic therapy could be initiated for individuals who are
genetically predisposed to a certain condition before that condition occurs.
Perhaps disease-modifying agents for musculoskeletal conditions could be
initiated prior to the onset of symptoms. Substantial improvements in drug
therapy could be realized by individualized use of medication based on genetic
variations in effects and side effects. Numerous important ethical questions
also will need to be addressed as we consider the consequences of defining the
genetic basis of disease susceptibility. How should this information be used
in nonmedical settings? Policy guidelines will have to be developed to address
this and other important questions. The social, legal, and ethical
implications of genomics research will also require ongoing discussion and
debate.
Clinical and experimental studies in the use of stem
cells—genetically modified and otherwise—continue to be at the
vanguard of evolving therapies in orthopaedics and other fields. The use of
bone marrow-derived cells, or putative "mesenchymal stem cells"
remains popular, in part, because they are easily harvested and manipulated.
Of note, a clinical trial is being planned to evaluate the ability to
regenerate meniscal tissue with use of allograft mesenchymal stem cells that
are injected into the knee joint. Important new research has begun to
elucidate aspects of the in vivo stem cell niche as well as the biology of
stem cell differentiation. Embryonic stem cells also have been the focus of
many new studies, and several laboratories have reported on the
differentiation of these cells into osteoblasts, chondrocytes, and adipocytes.
Key to the understanding of the genetic regulation of differentiation has been
the study of transcription factors. Gene therapy remains integral to stem cell
research, not only because of the need to control the behavior of stem cells
genetically but also because stem cells promise to be excellent platforms for
gene delivery. A major goal of clinical gene therapy remains the development
of safe methods of permanent gene transfer, although for most musculoskeletal
applications gene transfer needs to last only long enough to have the desired
effect (for example, augmentation of meniscal healing).
The in vitro isolation of single-cell clones with a high proliferative
potential and ability to differentiate into multiple cell types, including
osteoblasts, chondrocytes, and adipocytes, suggests that mesenchymal stem
cells reside in the bone and bone marrow. However, little is known about the
in vivo mesenchymal stem cells niche, such as where exactly in the bone they
may reside, what the characteristics of that microenvironment may be, and also
how the mesenchymal stem cells themselves may proliferate, migrate, and
differentiate in vivo. Recent evidence suggests that interaction with
hematopoietic stem cells is essential for the mesenchymal stem cells niche. A
recent study by Mendes et al. demonstrated colocalization of mesenchymal stem
cells with sites of hematopoiesis throughout embryonic
development11.
Furthermore, recent clinical studies have demonstrated improved grafting of
hematopoietic stem cells when co-infused with mesenchymal stem cells. The
group led by Elaine Fuchs at the Howard Hughes Medical Institute of
Rockefeller University recently reported on a novel method of visualizing and
tracking stem cells within skin in
vivo12,13.
Using genetically modified mice and genes expressed under skin-specific
promoters, researchers in Fuchs' group were able preferentially to label stem
cells in the skin with green fluorescent protein (GFP). Tracking these labeled
cells has yielded a great deal of information regarding the characteristics
and behavior of epidermal stem cells in vivo, including cell surface markers
and specific stimuli that induce their proliferation and differentiation. It
is possible that with the use of bone-specific and/or cartilage-specific
promoters such as the osteocalcin or type-II collagen promoter, this type of
strategy could be used to study cartilage and bone stem cells in vivo.
The way in which mesenchymal stem cells differentiate into osteoblasts,
chondrocytes, and other cell types also remains a central focus in the field
of mesenchymal stem cell biology. Studies by the group led by Rocky Tuan at
the Cartilage Biology and Orthopaedics Branch of the National Institute of
Arthritis and Musculoskeletal and Skin Diseases/National Institutes of Health
suggest that this differentiation is not linear and that transdifferentiation
of one committed cell type to another can occur, at least in vitro. Song and
Tuan reported that trabecular bone-derived mesenchymal stem cells that have
differentiated into osteoblasts (as evidenced by their osteocalcin
promoter-driven GFP expression) can be redifferentiated to express markers of
either the chondrocyte or adipocyte
phenotype14.
Dedifferentiation seems to be an obligate middle process during this
transdifferentiation. That group of investigators also examined the common and
exclusive genes expressed during the differentiation of osteoblasts,
chondrocytes, and adipocytes. The commonalities in these groups, along with
the observation that some subsets of cells can differentiate into two but not
all three of these cell types, suggest that, in addition to tripotent
precursors, distinct osteoblast/adipocyte and osteoblast/chondrocyte bipotent
precursors may exist.
In the quest to understand how stem cell differentiation is genetically
controlled, the study of transcription factors has become particularly
important. Transcription factors regulate gene expression, orchestrating the
turning on and off of a number of genes at once, usually by binding to a
segment of DNA upstream of the coding region of specific genes (i.e., in a
part of the genetic sequence that precedes the part that is actually
transcribed and translated into a protein). Transcription factors often work
together in discrete groups containing cofactors and other transcription
factors. The specific groupings may affect whether a given factor will act to
turn a target gene on or off. Developmental studies have elucidated several
transcription factors that appear to act as "master regulators" of
a specific cell type. Examples include the expression of peroxisome
proliferator-activated receptor-gamma in adipocytes, Sox9 in chondrocytes, and
Runx2 during osteogenesis. As these factors are expressed very early during
the commitment of precursor cells, they are of interest not only in the
discovery of mechanisms of differentiation, but also as markers of a
particular cell type.
Studies on the Runx2 transcription factor (also known as core binding
factor 1 [Cbfa1] or polyoma enhancer binding protein 2A [Pebp2A]) exemplify
the importance of master transcriptional regulators as well as their complex
biology (as reviewed by
Komori15).
Haploinsufficiency of the Runx2 gene is the well-documented cause of
cleidocranial dysplasia, and, as expected from this phenotype, Runx2 is
critical for osteoblastic differentiation. Mice lacking Runx2 have
cartilaginous skeletons that do not ossify, pointing to the critical role of
Runx2 in chondrocyte maturation and subsequent ossification. Runx2 also
appears to be important in the early commitment of mesenchymal cells to
chondrocytes; however, as is the case in vitro, Runx2-deficient mesenchymal
stem cells tend to mature into adipocytes unless they are stimulated with
chondrogenic factors such as BMP-2. The Runx2 transcription factor is known to
form heterodimers with transcriptional coactivator core binding factor ß
(Cbfß/polyoma enhancer binding protein 2ß [Pebp2ß]), which
affect the affinity and specificity of their binding to target DNAs. It also
interacts with a number of other transcription factors, such as C/EBPß
and d and also ETS1, as well as with BMP signaling molecules Smad1 and
Smad5, to upregulate the expression of osteoblast-related genes such as
osteocalcin and osteopontin.
Transcription factor studies such as those mentioned above suggest that the
coordinated regulation and subsequent expression of a number of genes may be
possible by harnessing our understanding of the network of genes that are
turned on and/or off by specific transcription factors that function as master
regulators of a specific cell phenotype. In imagining the potential
therapeutic applications of these studies, however, it is important to
remember that much of our understanding of the transcriptional regulation of
differentiation comes from observations made during limb development.
Differentiation by postnatal stem cells is likely to follow a similar course
and to depend on similar genes. However, whether and how developmental
differentiation and postnatal stem cell differentiation may be similar or
different are questions that have yet to be fully explored.
Understanding the genetic basis of stem cell differentiation is a necessary
prerequisite to controlling stem cells by means of gene therapy. The
plasticity and proliferative potential of stem cells require that they be
carefully controlled to prevent undesired transformation and/or overgrowth.
Within their normal niche, stem cells rarely divide. Furthermore, they
proliferate, migrate, and differentiate under a carefully coordinated sequence
of local cues in their microenvironment. This knowledge underscores the
importance of understanding the biology of the stem cell niche when
considering the use of stem cells for clinical therapies. The combination of
gene and stem-cell therapy also presents the possibility of effective
permanent gene replacement, with stem cells being used as a platform for gene
therapy. A modest number of genetically modified stem cells may be able to
proliferate in vivo, potentially repopulating target tissues with a sufficient
number of genetically "corrected" cells to have therapeutic
impact.
One of the most successful clinical gene-therapy trials to date involved
the transfer of a corrective gene to hematopoietic stem cells for the
treatment of X-linked severe combined immunodeficiency (XSCID). However, that
trial also points to specific areas in which gene and stem-cell therapy need
continued development. Whereas successful generation of genetically corrected,
mature, functional T-cells and natural killer (NK) cells was detected in the
blood of patients who were managed with hematopoietic stem cells modified by
retrovirus-mediated gene transfer, at least two of those patients had
development of T-cell leukemias. Whether the insertion of retrovirus genes
into the host cell genome resulting in the overexpression of the
growth-promoting LIM-only 2 (LMO2) genes actually caused the T-cell leukemia
remains conjectural. However, that study and subsequent follow-up studies
supported the concept that insertional mutagenesis is not as random or
improbable as once imagined and that virus-mediated insertions, in fact, favor
transcriptionally active sites, particularly when stem cells are
"forced" to divide using cytokine
stimulation16,17.
Despite the various risk factors, viral gene delivery has dominated
clinical trials primarily because of its in vivo efficiency when compared with
nonviral methods. To improve the efficiency of nonviral gene transfer, methods
are being developed in which viral and other elements are incorporated into
synthetic carriers, such as polycation polymers of polyethylenimine, that bind
DNA on the basis of charge interaction. Elements to enhance these synthetic
carriers may include antibodies that bind receptors on the surface of target
cells as well as viral and other proteins that may improve cell membrane
translocation, intracellular tracking, and/or nuclear targeting. These
modified or "hybrid" vectors have begun to be used in some
clinical trials, particularly for the treatment of cancer. These hybrid
vectors are designed to take advantage of the desirable aspects of viral gene
delivery while eschewing the undesirable ones, including insertional
mutagenesis16,17.
Regardless of whether genes are delivered by viral or other means,
site-specific integration is one way in which permanent gene transfer might be
made safer. Site-specific integration, of course, is now a routinely used
technology in the area of mouse genetics. For example, the Cre/lox system,
adopted from a prokaryotic gene for site-specific integration, has been widely
used to delete genes only in specific tissues. While prokaryotic genes cannot,
of course, be introduced into humans for the purpose of site-specific gene
transfer, studies have shown that endogenous sites that can accommodate
site-specific gene transfer do exist in the human genome. For example, the
bacteriophage integrase F31 has been used successfully to integrate the
human coagulation factor-IX gene into "hot spot" pseudo-sites
(called mpsA and mpsL1) in mice with resultant increases in the circulating
factor-IX level. Current strategies can only insert genes into such sites at
low frequency. However, continued research in this area holds promise for
safe, permanent gene transfer in the
future16.
Therapeutic gene transfer may be particularly important for controlling the
behavior of embryonic stem cells. Compared with mesenchymal stem cells,
embryonic stem cells are more plastic and have a greater capacity for
proliferation. As such, careful control of their phenotype as well as control
of their growth will be critical to their safe use. Recent studies have
demonstrated that genes such as BMPs can direct the differentiation of
embryonic stem cells toward osteoblasts and
chondrocytes18,19.
Other studies are beginning to elucidate the signals, such as those of the
Wnts, which can maintain cells in an undifferentiated or "stem"
state. While embryonic stem cells hold great promise for future therapeutic
applications, ethical considerations still abound. Further studies will be
necessary to show what advantages embryonic stem cells may have over adult
mesenchymal stem cells when considering orthopaedic applications.
Efforts to grow replacement tissues in vitro with mechanical, histological,
and biochemical properties similar to those of the native tissue that they are
intended to replace continues to be a focus of orthopaedic research. Variables
during the preimplantation incubation period include the type of cell used
(predifferentiated mesenchymal stem cells, undifferentiated mesenchymal stem
cells, chondrocytes, osteoblasts), the material into which the cells are
seeded (alginate, polycaprolactone, ceramics), the factors to which they are
exposed (tumor-like growth factor, bone morphogenetic proteins), and the
loading conditions to which they are subjected (no loading, hydro-dynamic
loading via bioreactors).
Recently, these parameters have been varied in an attempt to engineer
bilayered composite tissues. For example, Tanaka et al. described a bioceramic
ß-tricalcium phosphate block over-laid with chondrocytes seeded in a
collagen gel for the repair of osteochondral
defects20. Even
though ß-tricalcium has been used to support bone-tissue formation since
the 1970s, its use in the engineering of a composite tissue is novel.
Thirty-week follow-up in a rabbit trochlear osteochondral defect model
demonstrated resorption of the osseous substitute in tandem with osseous
growth and synthesis of a cartilaginous-like matrix. However, the superficial
zone was comprised mainly of fibrous tissue and the newly formed cartilaginous
tissue did not integrate with the surrounding cartilage.
Schek et al. also described a "biphasic scaffold" for repairing
osteochondral
defects21. The
osseous layer consisted of porous hydroxyapatite seeded with fibroblasts that
had been infected with a BMP-7-expressing adenovirus. The cartilaginous layer
consisted of chondrocytes seeded into a porous poly-L-lactic acid polymer. The
two layers were separated by a thin film of polyglycolic acid. Implantation
into a subcutaneous mouse model after four weeks revealed the concurrent
formation of cartilaginous material and bone, with a mineralized tissue
interface in between. The newly formed cartilage-like material stained only
slightly for proteoglycans, and osteogenesis occurred in the polymeric phase,
possibly as a result of migration of the transfected cells.
In all of these studies, and in many others, the engineered tissue is
predominantly characterized with use of histological or biochemical
techniques—for example, the proteoglycan content of the
tissue-engineered cartilage is frequently quantified. The swelling pressure
generated in the cartilage matrix by proteoglycans can resist compressive
loads. Thus, although a measure of proteoglycan content can help us to infer
the mechanical properties of the tissue, it is the interaction of the
proteoglycans with the collagen network and the permeability of the tissue
that truly determine the tissue's response to load. The only way to truly
assess the functional performance of the tissue is to measure it mechanically.
Rarely are the mechanical properties of the engineered constructs
characterized, which makes understanding their true functional performance
impossible.
Many research groups are using mesenchymal stem cells for
tissue-engineering applications. Alhadlaq et al. used a single population of
mesenchymal stem cells in combination with a novel mold design and polymers to
engineer a femoral
condyle22. Bone
marrow-derived mesenchymal stem cells were predifferentiated down an
osteogenic or chondrogenic lineage and were encapsulated in stratified
polyethylene glycol-based hydrogel layers, which were shaped to have a
geometry similar to that of a femoral condyle. The gels were photopolymerized
and implanted subcutaneously in a rat model. Both in vivo and in vitro data
showed the expression of chondrogenic and osteogenic markers within these
layers as well as the respective progression of tissue formation. That study
suggested that a single population of mesenchymal stem cells implanted in vivo
into a suitable scaffold can lead to the in vivo production of a
cartilage/bone composite.
Tissues for the replacement of the complete intervertebral disc, consisting
of the anulus fibrosus and nucleus pulposus, also have been formed and grown
in culture as composite tissues. In the study by Mizuno et al., cells isolated
from the anulus fibrosus were seeded into a porous mesh of polyglycolic
acid/polylactic acid (PGA/PLA) and cells isolated from the nucleus pulposus
were suspended in alginate and injected into the center of the PGA/PLA
mesh23. The
constructs were implanted into a subcutaneous mouse model for periods of as
long as twelve weeks. Histological and biochemical analyses revealed collagen
and proteoglycan quantities similar to those of native discs. However, the
newly formed tissue in the anulus fibrosus was not directionally organized as
was the case with native tissue; this finding was possibly attributable to the
non-physiological loading to which the constructs were subjected in the
subcutaneous model. Furthermore, the nucleus pulposus had low levels of
collagen (<10% of that of the native tissue). Again, although the
mechanical characteristics of the newly formed tissue were not characterized,
that study nonetheless represented a novel approach to the composite
engineering of a replacement disc.
It has been estimated that ten million Americans over the age of fifty
years have osteoporosis, while another thirty-four million are at risk for
development of the
disease24. To more
effectively treat osteoporosis and to help to identify the disease at an
earlier stage, it is vital that we understand more about the factors that
contribute to overall bone properties. New technologies that more closely
elucidate the molecular and crystalline structure of mineralized tissues are
being developed; two such techniques are Fourier transform infrared
spectroscopy (FTIR) and 31P solid-state nuclear magnetic resonance imaging.
FTIR can be used to measure the absorption of various infrared light
wave-lengths, and the absorption bands can identify specific molecular
components and structures. Analysis of calcified tissues with use of FTIR
allows one to measure the relative amount of mineral and the arrangement of
apatite and organic matrix. 31P solid-state nuclear magnetic resonance imaging
can be used to quantify the mass of hydroxyapatite in the tissue. It is
envisaged that these combined tools will lead to a more complete indicator of
the risk of bone fracture than is provided by bone mineral density
measurements alone.
Efforts also are being directed toward the development of bone-tissue
substitutes. Xu et al. used calcium phosphate cement that hardens in situ to
form solid
hydroxyapatite25.
The strength and toughness of calcium phosphate cement was increased with use
of chitosan and mesh reinforcement, and macropores were created for bone
ingrowth. With excellent biocompatibility combined with mechanical properties
that can be tailored to mimic that of bone, the material is a suitable
candidate for orthopaedic applications requiring bone reconstruction.
Bioresponsive materials respond and remodel in response to biological
signals from newly forming tissue. Their particular advantage for use as
scaffolds in tissue-engineering applications is that they allow for the
generation of new tissue in tandem with scaffold resorption. Wang et al.
expanded on this concept by linking a phosphoester to a polyethylene
glycol-based bioresponsive material in order to enhance in situ
mineralization26.
Mesenchymal stem cells that had been incubated in osteogenic medium were
encapsulated and incubated for six weeks. Expression of osteogenic-relevant
markers (osteonectin and alkaline phosphatase) was found; the constructs
became calcified and appeared to be differentiating toward osteogenesis. While
preliminary, that study emphasized the capabilities of further enhancing
bioresponsive materials for the purposes of bone-tissue engineering.
In response to the challenges of regulating the clinical introduction of
tissue-engineered-cell-based constructs, an advisory committee known as the
Cellular, Tissue, and Gene Therapies Advisory Committee was convened by the
United States Food and Drug Administration in March 2005. In the first of many
such meetings, the advisory committee (formerly known as the Biological
Response Modifiers Advisory Committee) started to discuss specific concerns
from industry and academia about ways to ensure the safe and effective
clinical use of cellular, tissue, gene-transfer, and other biological
products. Transcripts from this meeting are available online at
.
An additional challenge is to accurately assess the progressive growth and
integration of the implanted tissue over time. While postoperative biopsies
provide useful information, surgically removing tissue from a repair site is
far from ideal. Enhanced imaging techniques such as phase-contrast magnetic
resonance imaging have enabled the structure and content of repair tissue to
be examined in a noninvasive manner. Delayed gadolinium-enhanced magnetic
resonance imaging of cartilage (dGEMRIC) is a relatively new imaging technique
that has been used to estimate joint cartilage glycosaminoglycan content. A
hydrophilic negatively charged contrast agent, Gd-DTPA(2-), is injected into
the joint and distributes in inverse relation to the concentration of
negatively charged proteoglycans. A magnetic resonance imaging scan, followed
by measurement of T1 relaxation time, allows the Gd-DTPA(2-) concentration in
the tissue to be computed. Short T1 relaxation times in dGEMRIC correlate with
low proteoglycan content, and prolonged T2 relaxation times correlate with
decreased collagen
organization27.
The ability to add a tracking agent to tissue-engineered constructs for the
purposes of tracking their performance with use of imaging is a powerful
capability. For example, Bull et al. described novel self-assembled peptide
amphiphile nanofibers that were conjugated to contrast agents to enable
tracking of gel scaffolds with use of magnetic resonance
imaging28.
The development of so-called "combination products" that
combine traditionally implanted materials (such as cobalt alloys, titanium, or
stainless steel) with biological factors that are added to enhance osseous
fixation or bacterial resistance has become a more active area of orthopaedic
research. For example, Lu et al. developed titanium-alloy discs coated with a
biomimetically coprecipitated layer of calcium phosphate and
BMP-229. After five
weeks of implantation in a rat model, the coatings induced bone formation at
an ectopic site and sustained this activity for a considerable period of time.
Nablo et al. described nitric oxide-releasing gel films as antibacterial
coatings for orthopaedic
implants30. The
gels consisted of 40% N-aminohexyl-N-aminopropyltrimethoxysilane and 60%
isobutyltrimethoxysilane. The coated surfaces had statistically significant
less bacterial adhesion of Pseudomonas aeruginosa, Staphylococcus
aureus, and Staphylococcus epidermidis as compared with uncoated
steel and steel coated with the gel alone.
Alumina nanofibers have also been used to enhance implant
fixation31, with
the hypothesis that the nanostructure is more suited to controlling the
adhesion of nanoscaled biological structures (such as proteins and ligands) as
compared with the µm-scaled structure of the metallic alloys traditionally
used. Various crystalline-structured nanofiber pieces of alumina were
manufactured, and, after fourteen days of culture with osteoblasts, alkaline
phosphatase activity and calcium deposition were increased as compared with
uncoated surfaces. The results were highly dependent on crystalline
structure.
Highly crosslinked ultra-high molecular weight polyethylene (hereafter
called crosslinked polyethylene) was approved for clinical use in the United
States in 2000. Digas et al. used radiostereometric analysis in a prospective,
randomized clinical study on patients who received either highly crosslinked
or conventional polyethylene
liners32. At two
years, a 62% reduction in femoral head penetration was found when crosslinked
liners were compared with conventional liners. Researchers are already
developing a second generation of crosslinked polyethylene in an effort to
improve baseline material properties, such as fracture toughness and strength.
The main difference between the first and second generation of materials is
the method of manufacture. The first generation of crosslinked polyethylene is
irradiated to produce molecular cross-linking and then typically is
heat-treated or annealed to eliminate free radicals, whereas the new
generation of materials is manufactured with use of repeated bursts of
radiation, each of which is followed by successive heat treatment. In early
2005, Biomet received marketing approval from the United States Food and Drug
Administration for its second-generation highly crosslinked polyethylene,
ArComXL.
Since 2003, the following designs have secured Food and Drug Administration
approval for use in the United States: Reflection hip (Smith and Nephew,
Memphis, Tennessee), Transcend Acetabular System (Wright Medical Technology),
Trident Ceramic Hip System (Stryker Orthopaedics, Mahwah, New Jersey), and the
Keramos Acetabular System (Encore Medical, Austin, Texas). As the United
States' experience with these implants continues to grow, the prevalence of
clinical fracture appears low. However, it remains unclear as to how resistant
these devices are to repeated cyclic impingement.
Zirconium oxide, or Zirconia, has been used since the 1980s in the
ceramic-polyethylene bearing combination of total hip replacements. Zirconia
can exist in three different phases, or states of atomic bonding, depending on
its temperature. The strongest and toughest form is called the tetragonal
phase, which is naturally stable at temperatures of >1000°C. This phase
can be artificially stabilized through the addition of yttria (5% by weight).
However, if the phase becomes unstable, the material (or parts of the
material) reverts to a weaker monoclinic phase. It was the uncontrollable
transformation of Zirconia in vivo from its stronger to its weaker phase that
led to the 2002 recall of ceramic heads manufactured by Saint Gobain Advanced
Ceramics Demarquest (Monreuil, France). Efforts by Deville et al. to identify
tools that can be used to determine the tendency of yttria-stabilized Zirconia
to undergo phase transformations will ideally lead to preclinical tests for
the screening of Zirconia heads under simulated in vivo
conditions33.
Oxinium, the Smith and Nephew trade name for oxidized zirconium, is a
metallic alloy, the surface of which has been oxidized to form a ceramic
substrate. The concept of the material is that the ceramic surface layer will
provide improved wear resistance without the brittleness normally associated
with ceramics. Despite showing a 42% reduction in wear when compared with
cobalt-chromium alloys during in vitro knee-simulator
tests34, the
cementless versions of the Oxinium knee implants (Genesis II and Profix II
knee replacement systems) were recalled in 2003. It appears that a lack of
osseous growth into the implant followed by implant loosening led to the
recall.
The SB Charité III total disc replacement design (DePuy
Spine/Johnson and Johnson, Raynham, Massachusetts) was approved for clinical
use in the United States in 2004. The SB Charité III consists of two
end plates that are made of cast cobalt-chromium-molybdenum alloy. Each end
plate has teeth that facilitate fixation into the adjacent vertebral bodies.
The end plates are separated by a spacer made from ultra-high molecular weight
polyethylene. Earlier concerns about osteolysis caused by the presence of wear
debris in the spine appear to be unfounded, although it remains unclear where
the debris will migrate and what effect it will have at other anatomical
locations. The ProDisc system (Spine Solutions/Synthes) also consists of two
cobalt-chromium-alloy end plates and a polyethylene inlay. ProDisc is the only
artificial disc undergoing Food and Drug Administration trials for the
treatment of multiple-level lumbar disc disease. Other total disc replacement
designs undergoing Food and Drug Administration review include the Maverick
metal-on-metal articulation system (Medtronic Sofamor Danek, Minneapolis,
Minnesota), the FlexiCore system (SpineCore/Stryker Spine, Allendale, New
Jersey), and the Bryan Cervical Disc System (Spinal Dynamics/Medtronic Sofamor
Danek, Mercer Island, Washington).
For earlier stage spine problems in which the surrounding soft tissues are
structurally intact, implants that do not replace the entire disc but that
replace the nucleus pulposus alone are being developed and tested
preclinically35.
Unlike the total disc replacements, implant stability relies heavily on the
surrounding soft tissues and necessitates minimally disruptive surgical
techniques to insert the implant and to ensure continuing functionality of the
ligamentous structures.
The ORS is the premier organization in our field for dissemination of
research findings. The annual meeting is held each year just prior to the AAOS
Annual Meeting. The fifty-second annual meeting of the ORS will be held on
March 5 through 8, 2006, in New Orleans, Louisiana. The ORS web site contains
a listing of members who are willing to serve as mentors for young scientists.
These individuals have expressed willingness to serve in various capacities,
such as providing brief telephone consultations, performing confidential
review of manuscripts and/or grants, or hosting a young scientist for a short
period of time to teach a new research technique. The ORS also sponsors a
Career Development Fellowship that provides as much as $7500 of funding for an
individual to spend time at another institution. The goals of this program are
to provide education for the recipient, to foster interdisciplinary
collaborations, and to promote the interchange of ideas. The ORS also sponsors
various research awards, and information is available on the ORS web site.
The AAOS web site is another valuable source of up-to-date information on
upcoming AAOS-sponsored symposia, scientific meetings, and reports from AAOS
research committees. The web site also lists the various committees under the
Council on Research. Individuals interested in research are encouraged to
consult this web site and to consider applying for committee membership.
Information is available on research awards, research funding, and the
American Academy of Orthopaedic Surgeons/Orthopaedic Research and Education
Foundation (AAOS/OREF) fellowship. Another important way for clinicians to
become involved in research is to serve as reviewers on National Institutes of
Health study sections. There is currently a critical need for orthopaedic
surgeons to serve on review panels.
In summary, orthopaedic research continues to advance at a rapid pace as
new techniques are applied to musculoskeletal tissues. The discovery of
biologic solutions to important problems such as fracture-healing, soft-tissue
repair, osteoporosis, and osteoarthritis continues to be an important research
focus. At the same time, research in biomaterials and biomechanics is critical
to advances in current areas such as tissue-engineering and cytokine
delivery.
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