Corsi Regenerative medicine in orthopaedic surgery 2007

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Regenerative Medicine in Orthopaedic Surgery
Karin A. Corsi,1,2,3 Edward M. Schwarz,4 David J. Mooney,5 Johnny Huard1,2,3,6
1Stem Cell Research Center, Children’s Hospital of Pittsburgh, 4100 Rangos Research Center, 3460 Fifth Avenue,
Pittsburgh, Pennsylvania 15213
2Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania
3Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania
4Center for Musculoskeletal Research, University of Rochester, Rochester, New York
5Division of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts
6Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, Pennsylvania
Received 28 November 2006; accepted 27 March 2007
Published online 12 June 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20432
ABSTRACT: Regenerative medicine holds great promise for orthopaedic surgery. As surgeons
continue to face challenges regarding the healing of diseased or injured musculoskeletal tissues,
regenerative medicine aims to develop novel therapies that will replace, repair, or promote tissue
regeneration. This review article will provide an overview of the different research areas involved in
regenerative medicine, such as stem cells, bioinductive factors, and scaffolds. The potential use of
stem cells for orthopaedic tissue engineering will be addressed by presenting the current progress
with skeletal muscle–derived stem cells. As well, the development of a revascularized massive
allograft will be described and will serve as a prototypic model of orthopaedic tissue engineering.
Lastly, we will describe current approaches used to design cell instructive materials and how they
can be used to promote and regulate the formation of bony tissue. � 2007 Orthopaedic Research
Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1261–1268, 2007
Keywords: orthopaedic surgery; gene therapy; stem cell; allograft; polymer; tissue
engineering
INTRODUCTION
The field of orthopaedics has developed signifi-
cantly in the last century with the emergence of
new products and surgical techniques. Despite
these numerous advances, disease and injury to
the musculoskeletal system continue to occur and
risk increasing with the aging population. To
address such issues, regenerative medicine has
emerged as an important area of research and is
paving the way for new developments in ortho-
paedic surgery. To do so, regenerative medicine
focuses on therapies that will replace, repair, or
promote the regeneration of diseased or damaged
tissue. Research areas that have received parti-
cular attention are those employing stem cells,
scaffolds, and growth factors.
The use of stem cells in regenerative medicine is
a particularly appealing area of research that has
received a great deal of interest in recent years.
This is due, no doubt, to the uncommitted state of
stem cells, which provides them with the ability to
differentiate toward various lineages. Human
embryonic stem cells (hESCs) have been shown to
differentiate toward bone and cartilage lineages.1–3
However, scientific challenges, immunologic issues,
and ethical concerns motivate the examination of
reservoirs of stem cells from postnatal tissues.
These include, but are not limited to, cells isolated
from the bone marrow,4,5 the circulation or blood
vessels,6,7 adipose tissue,8–10 human placenta,11
human umbilical cord,12,13 human amniotic fluid,14
and skeletal muscle.15,16 As a means of demonstrat-
ing the potential of adult stem cell therapy for
regenerative medicine in orthopaedic surgery, the
first part of this review will detail current levels of
progress being made in research utilizing stem cells
isolated from adult skeletal muscle.
Regenerative medicine in orthopaedic surgery
will likely also require the use of scaffolds to
repair large defects. Acellular scaffolds, such as
demineralized bone matrix that have osteoinduc-
tive properties, may be used to promote tissue
regeneration. However, scaffolds may also be
JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2007 1261
Correspondence to: Johnny Huard (Telephone: 412-692-
7807; Fax: 412-692-7095; E-mail: jhuardþ@pitt.edu)
� 2007 Orthopaedic Research Society. Published by Wiley Periodicals,
Inc.
designed to incorporate cells and growth factors, or
cells genetically modified to secrete the growth
factors of interest. Hence, scaffold design is another
important area of regenerative medicine. Although
the field of tissue engineering has steadily pro-
gressed over the last two decades, success of these
products, as measured by approval from the U.S.
Food and Drug Administration, has, unfortu-
nately, been limited. In the musculoskeletal field,
this is largely due to the technical difficulties in
generating highly efficacious engineered tissues,
but it is also due to a lack of outcome measures to
rigorously evaluate a novel construct in animals,
and the lack of a favorable patient population for a
cost-effective clinical trial. Tissue engineering of
articular and meniscal cartilage provides an excel-
lent example of these limitations for the following
reasons: (1) little is known about what factors, if
any, can truly restore damaged tissue to its native
form; (2) large animals (i.e., goats, sheep) appear to
be the only rigorous models; (3) while enormous,
the patient population is young and healthy, and
thus the risk:benefit assessment of a clinical trial is
very unfavorable for highly innovative technolo-
gies (i.e., genetically modified stem cells, viral gene
therapy); and (4) there are no quantitative out-
come measures to scientifically prove that tissue-
engineered cartilage restores defects to their native
form in humans. In contrast, tissue engineering of
bone has many advantages including the following:
(1) a wealth of available knowledge17; (2) the ability
to utilize acellular constructs that can remodel into
live tissue in vivo; (3) a potential patient population
with a favorable risk:benefit assessment (meta-
static cancer patients undergoing limb-sparing
surgery); and (4) definitive quantitative outcome
measures in animals and humans. Thus, as a
prototypic model of orthopaedic tissue engineering,
and, as a means of demonstrating the importance
of using scaffolds for bone healing, we will also
describe the current progress being made toward
the development of a revascularized massive
allograft.
The delivery of osteogenic and angiogenic sig-
nals will also be of utmost importance for bone
tissue engineering. While systemic or local delivery
of these potent actors via injection of an aqueous
solution is the simplest approach, the short half-
lives of most of these factors require the adminis-
tration of large doses, which increases the cost of
such therapies. Furthermore, the utility of this
approach is seriously limited due to the inherent
broad tissue exposure, and to the potential of
growth factors to drive undesirable responses
at other sites in the body.18 To overcome such
limitations, gene therapy can be used to modify
cells for injection, so that they can produce the
desired growth factors in vivo. This is more cost
effective than direct administration of growth
factors, but may have limitations in terms of the
duration of expression, and the amount of growth
factor secreted. As another alternative to direct
administration of inductive factors, a variety of
external signals or cues may be exploited to direct
cells, including the presentation of immobilized
ligands that engage cellular adhesion receptors. As
well, growth factors can be incorporated into
polymeric scaffolds and be released in a controlled
manner. Hence, in the last section of this review, we
will describe how materials can provide a useful
platform to regulate the presentation of cues for
cells and to instructtransplanted and host cell
populations in the formation of new tissue struc-
tures or the repair of existing tissues.
STEM CELLS IN REGENERATIVE MEDICINE
Current Progress with Muscle-Derived Stem Cells
Skeletal muscle is a readily available tissue source
from which stem cells can be isolated in a mini-
mally invasive manner. In fact, murine skeletal
muscle contains a population of stem cells termed
muscle-derived stem cells (MDSCs), which demon-
strate a capacity for long-term proliferation, self-
renewal, and immune-privileged behavior.16,19
Furthermore, they can undergo multilineage dif-
ferentiation toward skeletal muscle, bone, carti-
lage, and neural, endothelial, and hematopoietic
tissues.15,16,20–26 They are found in murine skele-
tal muscle at a ratio of 1 in 100,000 cells,16 a ratio
similar to that of adult mesenchymal stem cells
(MSCs) isolated from bone marrow aspirates.5
Although this suggests that a large muscle biopsy
will be required to isolate sufficient cells for
therapeutic applications, this limitation is over-
come by the fact that MDSCs and other stem cells
are easily expanded in vitro without loss of
progenitor characteristics.27,28
Like many stem cell studies for orthopaedic
applications, MDSCs require inductive factors
such as bone morphogenetic proteins (BMPs) to
undergo osteogenic or chondrogenic differentia-
tion. When transduced to express BMP4, they are
capable of undergoing chondrogenic differentia-
tion in vitro and in vivo.21 MDSCs have also led to
bone formation and healed critical-sized calvarial
and long bone defects when genetically engineered
to express BMP2 or BMP4.15,22–26 They were
found within the newly formed bone and some
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JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2007 DOI 10.1002/jor
co-localized with osteocalcin, suggesting that the
cells differentiated into osteogenic cells.15
Enhancement of Bone Formation
In addition to BMPs, vascular endothelial growth
factor (VEGF) is also a key component in bone
formation.29 Therapies that increase the blood
supply to the site of injury will lead to the recruit-
ment of progenitor cells and further promote bone
regeneration. The synergistic effect of VEGF and
BMP was tested with MDSCs by transducing them
to express VEGF and either BMP2 or BMP4.23,24
Co-implantation of BMP4- and VEGF-secreting
cells into the muscle pockets or calvarial defects in
mice produced a greater amount of bone formation
than observed after implantation of only BMP4-
expressing MDSCs.24 VEGF in combination with
BMP2 also demonstrated this synergistic effect
between the two growth factors.23 Interestingly,
the dosage of BMP4- to VEGF-expressing cells
became a major determinant in the amount and
quality of bone formation. In both studies, increas-
ing the amount of VEGF expression was detri-
mental to bone formation, which indicates that
controlling the amount of growth factors that is
released will be a necessary optimization to any
therapy for the regeneration of musculoskeletal
tissues.
Regulation of Bone Formation
While bone formation remains a very desirable
outcome of regenerative medicine for orthopaedic
surgery, regulating the bone that is formed and
preventing its overgrowth is also essential. As
well, another clinical problem faced by orthopaedic
surgeons is heterotopic ossification of muscles,
tendons, and ligaments.30 To overcome these
issues, it will be necessary to develop methods to
regulate or inhibit bone formation. Noggin, a BMP
antagonist, may be a likely candidate to address
both these issues. The ability of noggin to inhibit
heterotopic bone formation was evaluated by
transducing MDSCs to express noggin and co-
implanting them with MDSCs transduced to
secrete BMP4 into the hind limbs of mice.31 It
was found that noggin inhibited the amount of
bone formation in a dose-dependent manner, with
a greater ratio of noggin to BMP4 leading to less
bone.31 In an attempt to regulate bone formation,
MDSCs expressing inducible BMP4 were co-
implanted into critical-sized calvarial defects with
MDSCs expressing noggin.32 At specific dosages,
this combinatorial therapy led not only to regula-
tion of bone formation, but the bone that was
formed in the BMP4 with noggin group was
histologically similar to normal bone. Both BMP4
and noggin have been shown to be present at the
fracture repair site during healing,33 suggesting
that mimicking the physiological environment is
an important part of developing new approaches to
enhance and regulate tissue repair, regeneration,
or replacement. Interestingly, noggin’s inhibition
of BMP is a phenomenon that has, to date, been
studied almost exclusively in the murine model.
Its effect on human cells is still unclear, although
it has recently been reported that noggin acted as
an inductive factor in the osteogenic differentia-
tion of human MSCs.34 This suggests that noggin
may act differently in human than in murine cells,
and that future studies on the use of noggin
for regulation of bone formed by human skeletal
muscle cells are warranted.
The studies with MDSCs for orthopaedic tissue
engineering demonstrate the importance of the cell
source but also the need to investigate different
growth factors and the combination of these
factors for optimal regeneration and regulation of
bone and articular cartilage formation. Cell thera-
pies will likely also require scaffolds to promote
regeneration. The following sections will address
these issues by providing an overview of the work
done with allografts and cell instructive materials.
REVASCULARIZING MASSIVE
ALLOGRAFTS: PROTOTYPIC
MODEL OF TISSUE ENGINEERING
Massive Allografts
Large defects in bones that occur from traumatic
injury or tumors must be replaced by a structural
graft or prosthesis in order to save the limb.
Because human cortical bone is the ideal replace-
ment material to fill segmental defects >5 cm,
structural allografts have been used for over
50 years for this purpose. Unfortunately, the
limited bone forming and remodeling of structural
allografts is directly associated with the 25% to
35% failure rate within 3 years due to infection,
fracture, and non-union.35,36 For those allografts
that survive, the failure rate at 10 years has been
documented to be as high as 60%.37–39 The
fractures at this late stage are the result of the
accumulation of microcracks that cannot be repair-
ed by the necrotic bone because there is no
vascular supply. As a result of this poor clinical
success, the use of structural allografts has been
restricted to repair segmental defects following
tumor resection in cancer patients. Clinical studies
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DOI 10.1002/jor JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2007
in these patients have provided the field with
definitive outcome objectives because a massive
allograft that (1) stimulates a new bone collar
spanning the entire length of the graft, (2) vascu-
larizes >1 mm from the graft:host junction, or
(3) becomes ‘‘hot’’ on a technetium Tc 99m dipho-
sphonate labeled bone scan has never been
documented. To the end of developing a tissue
engineered structural allograft that achieves these
outcomes, stem cell and gene therapy approaches
have been developed.40–42 Radiological and bio-
mechanical outcome measures in a murine femur
model have also been developed and have demon-
strated the efficacy of these tissue engineered
allografts.43
Tissue Engineered Periosteum
In order to understand the fundamental dif-
ferences between live autograft and processed
allograft healing, both were evaluated in a novel
murine segmental femoral graft model.43 Rosa26
transgenic mice were also incorporated into this
model so that live isografts that express b-
galactosidase could be transplanted into nontrans-
genic littermates, and the donor cells could be
tracked via X-gal stainedhistology.44 These
studies clearly demonstrated that superior healing
observed in the autografts is attributed to the live
periosteal cells that actively participate in the
early angiogenic, osteogenic, and osteoclastogenic
response. Based on this, a tissue engineered
periosteum was developed from bone marrow–
derived MSCs (BM-MSC), which were transduced
with recombinant adenovirus expressing Lac Z
(Ad-LacZ) or BMP2 (Ad-BMP2).45 These cells were
then encased in gel foam and wrapped around
femoral allografts before transplanting them into
mice. The BM-MSC persisted on the allograft up to
28 days, with the BM-MSC expressing BMP2
showing a local expression of the growth factor
up to 21 days.44 While the BM-MSC alone did not
display any significant effects on allograft healing,
the effects of the Ad-BMP2 transduced BM-MSC
were dramatic and included a new bone volume,
new vascular volume, and torsional properties that
were equivalent to the live isograft control. The
only significant difference between the cellular-
ized allograft and the live isograft was the absence
of remodeling in the allograft before 12 weeks.
This lack of remodeling, due to the incorporation
of cells in the allograft, could be considered an
improvement over live autografts, which experi-
ence a period of decreased strength due to
extensive remodeling.
rAAV-Coated Allografts
While stem cells have tremendous potential for
musculoskeletal tissue engineering, as was shown
in the previous sections with MDSCs and the
incorporation of MSCs in allografts, practical
concerns, such as where the cells will come from,
quality control, and cost, remain serious issues.
To overcome these issues, an acellular tissue
engineering approach was also investigated. It is
similar to the gene-activated matrix,46 and aims
to deliver angiogenic, osteogenic, and remodeling
signals to the cortical surface of the allograft via
immobilized recombinant adeno-associated viruses
(rAAV). The advantage of this system is that the
rAAV, which has very high transduction effi-
ciency, can be freeze-dried onto any implant
surface without loosing activity in vitro and
in vivo.40 The efficacy of this approach was
demonstrated using two different strategies. The
first induces a new bone collar around the cortical
surface of the allograft through BMP signals
transduced by a rAAV-caAlk2 coating.41 The
second induces vascular ingrowth and osteoclastic
resorption through transduction of a combination
of rAAV-VEGF and rAAV-RANKL.40 Although
detectable transgene expression only persists for
2 to 3 weeks, the treatment triggers the natural
bone remodeling process that perpetuates until
healing is complete. This includes a new bone
collar around the graft, complete vascular invasion
and new bone marrow. These modifications with
allografts demonstrate the importance of exerting
significant control over the fate of cells. An
alternative to modified allografts, which need to
be harvested, would be to employ polymers that
can be modified to instruct the cells to form bony
tissue. Examples of such modifications are adhe-
sion ligand presentation, the delivery of inductive
factors, and a combination of the two.
CELL INSTRUCTIVE POLYMERS
Adhesion Ligand Presentation
Binding of cells to extracellular matrix (ECM)
molecules has long been appreciated to regulate
many aspects of cell phenotype. The identification
of the transmembrane cellular receptors respon-
sible for these binding interactions, and the pepti-
de domains in the large ECM molecules to which
these receptors bind has created the possibility of
developing synthetic ECM that provides certain
biological functions of ECM molecules in the
context of a highly defined, flexible, and reprodu-
cible material platform.47
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JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2007 DOI 10.1002/jor
While the qualitative act of presenting cell
adhesion peptides from biomaterials can clearly
regulate cell function in vitro and in vivo, it has
recently become increasingly clear that the specific
presentation of the peptides is critical to the cell
response. The density of adhesion ligands, similar
to past work with intact ECM molecules, regulates
the adhesion, proliferation, and differentiation of
cell populations of interest in tissue engineering
and regeneration.48–50 However, the nanoscale
organization of these ligands appears to modulate
the cell response as well,51 and may be as important
as the overall density of ligands presented to cells.
Further, the mechanical properties of the synthetic
ECM presenting the ligands is equally important,
and can significantly alter the response of cells to a
specific ligand presentation (type of ligand, density,
and nanoscale organization).52 The large variable
space inherent to adhesion ligand presentation
would require massive experimentation to explore
in full, but the development of models that can
predict the specific range of the relevant variables
likely to alter cell receptor binding and clustering
promises to focus experiments on the relevant
variable ranges.51,53
While a large number of studies indicate that cell
adhesion and phenotype can be regulated in vitro
by peptide presentation, the importance of these
manipulations to in vivo tissue formation or
regeneration is only recently becoming clear.
Appropriate adhesion peptide presentation has
been shown to regulate bone and cartilage forma-
tion by transplanted cells,48,54 and matching of
the degradation rate of the synthetic ECM to the
rate of tissue formation in these systems greatly
increases the extent and rate of tissue formation.55
Co-transplantation of appropriate cell populations
in these materials has also been demonstrated to
lead to the formation of multicomponent tissues,
and in one example led to the formation of tissues
structurally and functionally resembling growth
plates.56 While the materials will most directly
control cells originally placed or recruited to the
surface of the material, a number of reports
indicate long-term control over both gene expres-
sion and tissue formation in vitro and in vivo, even
though a small percentage of the cells is adherent to
the material at those stages.48,57
Inductive Factor Delivery
Signaling between distinct cell populations clearly
regulates many developmental processes, and co-
transplantation of cell populations participating in
this cross-talk can enhance tissue regeneration.58
As an alternative to transplanting multiple cell
populations, one can instead consider replacing
their action by the delivery of the growth factors or
morphogens underlying their bioactivity. The bio-
activity of a multitude of morphogens and growth
factors has been exploited in attempts to regulate
the timing, location, and extent of tissue regenera-
tion.18 Immobilization of biologically active macro-
molecules within polymeric carriers that can
meter their release in a predefined profile to
control their local concentration and duration of
signaling can dramatically improve their effec-
tiveness. A variety of systems have been developed
to encapsulate both recombinant growth factors
and plasmid DNA encoding these factors in bio-
degradable polymer platforms useful for tissue
engineering,59,60 and these systems can be design-
ed to enable combinations or sequences of factors
to be delivered with well controlled kinetics,61,62
which more closely mimics the normal develop-
mental and regenerative processes than delivery
of single factors. The utility of this approach has
recently been demonstrated in the context of
bone and muscle regeneration,54,61,63 and in the
formation of new blood vessel networks that can
alleviate tissue ischemia and enhance the regene-
rative potential of cell transplantation strategies.64
Combining and Integrating Signaling
An advantageous feature of the use of synthetic
ECM todrive tissue regeneration is that they, like
the natural ECM they are intended to mimic, can
be designed to be multifunctional. For example,
these materials can be designed to simultaneously
provide osteoconductive surfaces to promote bone
regeneration, while releasing angiogenic factors
to drive neovascularization in the regenerating
tissue.65,66 In this context, it is important to
recognize that an interplay between these mate-
rial cues and other environmental signals will
regulate the net cell and tissue response. The
mechanical properties of synthetic ECM, for
example, will regulate the ability of cells to
rearrange adhesion peptides presented from the
material,52 and through their control of cell
proliferation will regulate transfection of adherent
cell populations.67 Externally applied mechanical
signals will also regulate the response of cells to
the synthetic ECM68 and the release of growth
factors and subsequent tissue formation.69
CONCLUSIONS
We have reviewed different approaches to pro-
mote, enhance, and regulate musculoskeletal
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tissue formation. Although we have focused on
strategies that can be directly placed into the
patient, other areas of research, such as the use of
cultivation systems, are also making significant
contributions to the advancement of regenerative
medicine in orthopaedic surgery. In these cases,
the goal is to produce a neo-formed tissue in vitro
for later implantation in vivo.70,71 Regardless of
the method used to regenerate diseased or
damaged musculoskeletal tissue, a common ele-
ment in all of these cases is the use of inductive
factors. Cells that are implanted either directly
into the defect or first within a scaffold, as well as
cells from the host that are recruited to the site of
injury, all require stimulation by growth factors or
morphogens to help in the formation of newly
formed tissue. This was particularly evident in
this review of cell-based therapies, allografts, and
cell instructive materials (Fig. 1). It has been
demonstrated that inductive factors may be
delivered by means of gene therapy as with
MDSCs transduced to express BMP, VEGF, or
noggin, and BM-MSCs genetically modified to
express BMP2 in allografts. Alternatively, they
may be delivered in a more controlled manner by
immobilizing rAAV on allografts or by incorporat-
ing the growth factors in cell instructive materials.
Because the complexity of options renders the
optimal solution, as yet, undetermined, the inves-
tigation of growth factors, their particular indivi-
dual effects on various stem cells, their potential
combinatory effects on new tissue formation, and
their most advantageous mode of delivery should
be further pursued.
In conclusion, we have demonstrated that
regenerative medicine in orthopaedic surgery con-
sists of a wide range of research areas. While some
laboratories focus on isolating the ideal stem cell
and studying the different growth factors required
to develop cell therapies, others direct their efforts
towards investigating different scaffolds and
improving their design and function. A collabora-
tive effort between these different research areas
should be encouraged, as it will facilitate the
development of new treatment options in regen-
erative medicine for orthopaedic surgery.
ACKNOWLEDGMENTS
EMS acknowledges the financial support from the
National Institutes of Health (NIH; NIAMS AR51469)
and The Musculoskeletal Transplant Foundation. EMS
is the Founder of LAGeT Inc. DJM acknowledges the
financial support from the NIH (NHLBI, NIDCR) and
the Army Research Office (ARO/DOD). JH acknowl-
edges the financial support from the NIH (1R01 AR
47973, 1RO1 AR49684, 1RO1 DE13420), the Depart-
ment of Defense, the William F. and Jean W. Donaldson
Figure 1. Importance of inductive factors in regenerative medicine for orthopaedic surgery. The
incorporation of inductive factors in regenerative medicine is necessary to promote the healing of the
diseased or damaged tissue by the implanted and host cells. Cell-based therapies, such as the use of
MDSCs, require gene therapy to deliver growth factors (BMP, VEGF, and noggin) to the site of
injury. It was shown that the combination of the different growth factors was important for bone
formation and regulation. Massive allografts were modified with either BM-MSC expressing BMP2
or coated with rAAV to deliver the necessary growth factors or inductive elements within the bone
defect. Cell instructive materials were also designed to incorporate growth factors to promote
healing. As well, they could contain cells or adhesion ligands that could also regulate cell function.
1266 CORSI ET AL.
JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2007 DOI 10.1002/jor
Chair at Children’s Hospital of Pittsburgh, and the
Henry J. Mankin Endowed Chair for Orthopaedic
Research at the University of Pittsburgh. The authors
wish to thank David Humiston for his editorial assis-
tance with the manuscript.
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