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RESEARCH ARTICLE
Polymeric system for dual growth factor delivery
© 2001 Nature Publishing Group http://biotech.nature.com
Thomas P. Richardson1,2, Martin C. Peters1, Alessandra B. Ennett1, and David J. Mooney1–3*
The development of tissues and organs is typically driven by the action of a number of growth factors. However,
efforts to regenerate tissues (e.g., bone, blood vessels) typically rely on the delivery of single factors, and this
may partially explain the limited clinical utility of many current approaches. One constraint on delivering appropriate combinations of factors is a lack of delivery vehicles that allow for a localized and controlled delivery of
more than a single factor. We report a new polymeric system that allows for the tissue-specific delivery of two or
more growth factors, with controlled dose and rate of delivery. The utility of this system was investigated in the
context of therapeutic angiogenesis. We now demonstrate that dual delivery of vascular endothelial growth factor (VEGF)-165 and platelet-derived growth factor (PDGF)-BB, each with distinct kinetics, from a single, structural polymer scaffold results in the rapid formation of a mature vascular network. This is the first report of a
vehicle capable of delivery of multiple angiogenic factors with distinct kinetics, and these results clearly indicate
the importance of multiple growth factor action in tissue regeneration and engineering.
Current therapies to regenerate a variety of tissues (e.g., bone, blood
vessels) in the body rely on the bolus delivery of single growth factors. However, the success of clinical trials has been limited. For
example, in spite of promising early animal studies1,2 and small-scale
clinical studies3,4 of therapeutic angiogenesis resulting from growth
factor delivery, large clinical trials have not demonstrated an effect
that is as significant5. The limited success of current efforts may be
related to both the mode of growth factor delivery and the requirements for multiple signals to drive the regeneration process to completion. Typically, single proteins have been delivered as either bolus
injections into the site of disease or by systemic administration. This
strategy is limited because the inherent instability of many proteins
in vivo requires very high levels of protein for a measurable effect,
and the potential exists for uncontrolled activities at distant sites6.
One approach to bypass limitations of bolus drug delivery is the
localized and sustained delivery of growth factors at the desired site
of action from polymer systems. Biodegradable polymer systems,
which have been developed to provide localized and sustained
growth factor release7–13, can be used to deliver plasmid DNA encoding the factors14,15. However, growth factor delivery systems to date
have not demonstrated the ability to deliver multiple factors with
distinct kinetics, a likely requirement to drive tissue development to
completion. In this report we describe a system fabricated from
biodegradable polymers that allows for the delivery of two or more
growth factors with distinct kinetics. The utility of this system was
investigated using a well-characterized developmental model, the
formation of new blood vessels (angiogenesis). Angiogenesis is
required for most tissues to develop, is deficient in a variety of
ischemic pathologies, and is a critical component of virtually all
tissue-engineering strategies. The process of angiogenesis, which has
been extensively studied, results from a complex cascade of events
involving endothelial cell activation, migration and proliferation,
organization into immature vessels, association of mural cells (pericytes and smooth muscle cells) with the immature vessels, and
matrix deposition as the vessels mature6,16. The molecular mechanisms controlling each of these steps are being delineated, and it is
clear that different factors act at distinct stages of vascular develop-
1Departments
ment6,17–19. For example, although VEGF is a well-established initiator of angiogenesis, its presence is often not sufficient for the formation of a complex, mature vascular network6. PDGF is distinct as it
promotes the maturation of blood vessels by the recruitment of
smooth muscle cells to the endothelial lining of nascent vasculature16,20.
In this study, we tested the hypothesis that dual delivery of VEGF
and PDGF can direct the formation of a mature vasculature, as compared to the delivery of VEGF or PDGF delivered alone or simultaneously. The system developed for these studies to deliver multiple
factors may find broad utility in the regeneration of a variety of other
tissue types (e.g., bone, nerve) as well.
Results
A porous polymer scaffold capable of multiple growth factor delivery was fabricated from poly(lactide-co-glycolide) (PLG) using a
variation of a high-pressure carbon dioxide fabrication process14.
This process allows for sustained protein delivery and maintains the
biological activity of incorporated and released growth factors21.
Growth factors may be incorporated into scaffolds by two approaches. The first approach involves simply mixing lyophilized VEGF with
polymer particles before processing the polymer into a porous scaffold14 (Fig. 1A) and results in a rapid release (e.g., days to weeks in
duration) of VEGF. The second approach involves pre-encapsulating
a factor in PLG microspheres, and then fabricating scaffolds from
these particles (Fig. 1A). The two approaches may be combined by
mixing particulate polymer and one factor with microspheres containing a pre-encapsulated second factor to provide multiple growth
factor delivery with a distinct release rate for each factor. This
approach was utilized to incorporate VEGF and PDGF into scaffolds
in the current study. The particulate and microsphere PLG fused to
form a continuous, homogeneous matrix with an open pore structure (Fig. 1B), and the scaffolds exhibited mechanical characteristics
similar to those fabricated with particulate PLG alone21 (T.P.
Richardson et al., unpublished observations). These scaffolds
released VEGF in vitro at a rate of 1.7 pmol/day (79 ng/day) for the
first seven days, followed by a slower delivery rate for the remainder
of Biomedical Engineering, 2Biologic and Materials Sciences, and 3Chemical Engineering, University of Michigan, Ann Arbor, MI 48109.
∗Corresponding author (mooneyd@umich.edu).
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A
B
However, there was no statistically significant difference
between any of the three
experimental conditions and
the control (P > 0.05) at four
weeks. This suggests that the
amount of VEGF was insufficient to sustain angiogenesis,
and supports previous reports
indicating that if VEGF levels
fall below critical levels, the
unstable vessels are subjected
to pruning and remodeling22.
We next investigated the
D
C
ability of sustained and localized delivery of an appropriate
combination of VEGF and
PDGF to promote the rapid
formation of a mature vasculature in subcutaneous pockets of Lewis rats. Delivery of
VEGF alone led to a sharp
increase in angiogenesis after
two weeks (Fig. 3A). However,
the newly formed blood vessels were typically small, with
little apparent matrix or mural
Figure 1. Schematic of scaffold fabrication process and growth factor release kinetics. (A) Growth factors were
cell association. Sustained
incorporated into polymer scaffolds by either mixing with polymer particles before processing into scaffolds (VEGF),
delivery of PDGF alone did
or pre-encapsulating the factor (PDGF) into polymer microspheres used to form scaffolds. The VEGF incorporation
approach results in the factor being largely associated with the surface of the polymer, and subject to rapid release. In not significantly alter the density of blood vessels in the tiscontrast, the PDGF incorporation approach is predicted to result in a more even distribution of factor throughout the
polymer, with release regulated by the degradation of the polymer used to form microspheres. The two growth factors
sue (Fig. 3C), although the
were incorporated together into the same scaffolds by mixing polymer microspheres containing pre-encapsulated
existing vessels appeared largPDGF with lyophilized VEGF before processing into scaffolds. (B) Scanning electron micrograph of a typical scaffold
er. In contrast, the dual-release
utilized for dual growth factor release. (C) In vitro release kinetics of VEGF from scaffolds fabricated from PLG (85:15,
scaffolds led to an elevated
lactide:glycolide), measured using 125I-labeled tracers. (D) In vitro release kinetics of PDGF pre-encapsulated in PLG
microspheres ( 75:25, intrinsic viscosity = 0.69 dl/g; 75:25, intrinsic viscosity = 0.2 dl/g), before scaffold
density of vessels, similar to
fabrication. Data represent the mean (n = 5), and error bars represent standard deviation (error bars not visible are
VEGF delivery, and resulted in
smaller than the symbol).
larger and apparently more
mature vessels, similar to
of the study (Fig. 1C). The release rate of PDGF from scaffolds was
PDGF delivery (Fig. 3E). The differences between individual versus
varied from 4.2 pmol/day to 0.10 pmol/day by altering the degradadual growth factor delivery were more notable after four weeks
tion rate of the polymer using various polymer formulations and
implantation, in which the dual-release scaffolds (Fig. 3F) led to a
molecular weights (Fig. 1D). The magnitude of the release rate is
higher blood vessel density than either factor alone (Fig. 3B, D), and
readily adjusted in this system by simply altering the amount of facthe vessels appeared quite large and mature. The qualitatively distor incorporated into scaffolds (2 and 3 µg/scaffold of VEGF and
tinct blood vessel densities were confirmed by quantitative analysis
PDGF, respectively, in this study). The kinetics of factor release can
(Fig. 3G). No significant inflammatory and immune response was
be altered by varying the degradation time of the PLG. Both the
observed, similar to previous reports using this type of polymeric
PDGF and VEGF incorporated and released from scaffolds over the
scaffold14. We confirmed that these results are due to localized delivery of human VEGF, versus systemic delivery. ELISA assays perfirst three weeks were biologically active in vitro using cell proliferaformed on the rat blood plasma documented no detectable human
tion assays with smooth muscle cells and endothelial cells, respecVEGF (not shown).
tively (not shown).
It is critical in angiogenesis to promote vessel maturation, as the
To verify the importance of sustained delivery of VEGF and PDGF
stability of an induced vasculature is dependent on the mural cell
in the promotion of angiogenesis, we first investigated blood vessel
association to prevent regression. Before maturation, vessels have
development by bolus, simultaneous administration of VEGF and
been shown to be dependent on the continued presence of VEGF to
PDGF with blank scaffolds (no incorporated growth factors)
prevent vessel regression and endothelial cell apoptosis20,22,23. To
implanted into the subcutaneous tissue of Lewis rats. Growth factors
assess more fully the maturity of blood vessels formed, we stained
were injected either alone or together into the scaffolds at the time of
tissue sections for the presence of α-smooth muscle actin (α-SMA).
implantation, leading to short-term, unsustained presence of growth
This marker is expressed in both pericytes and smooth muscle cells
factor at the implant site. Histological sections revealed relatively few
associated with endothelial cells in larger, mature blood vessels20. A
mature blood vessels at the two- and four-week time points for all
small number of blood vessels were positively stained following
four conditions (Fig. 2). Quantification of the blood vessel densities
implantation of blank scaffolds for two weeks (Fig. 4A, B). Delivery
confirmed the results from histological examination, as delivery of
of VEGF, while increasing the overall number of vessels, led to a low
VEGF, PDGF, or both together resulted in a small increase in blood
percentage of positively stained vessels (Fig. 4C, D). While PDGF
vessel density at two weeks, as compared with control (Fig. 2I).
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A
B
A
B
C
D
C
D
E
F
E
F
G
H
G
I
Figure 2. Bolus delivery is not sufficient for stable vessel formation.
(A–H) Hematoxylin and eosin staining of tissue sections of
subcutaneously implanted blank scaffolds (n = 4) after two weeks (A) and
four weeks (B); scaffolds injected with VEGF only after two weeks (C),
and four weeks (D); scaffolds injected with PDGF only after two weeks (E)
and four weeks (F); and scaffolds with injections of both VEGF and PDGF
at two weeks (G) and four weeks (H). (I) The vascular density within tissue
sections was quantified for each condition. * indicates statistical
significance relative to blank at same time point (P < 0.05); ** indicates
statistical significance relative to VEGF and PDGF (P < 0.05).
Magnification for all photomicrographs was 400×.
delivery did not initiate a significant increase in blood vessel density,
vessels present exhibited a relatively mature phenotype (Fig. 4E, F).
Dual delivery of VEGF and PDGF, however, led to both a high density of vessels and the formation of thicker and larger vessels (Fig. 4G,
H). The relative proportion of smooth muscle cell–positive vessels in
all conditions was also determined. While VEGF induced high numbers of blood vessels, they remained relatively immature (Table 1).
The presence of PDGF, either alone or in combination with VEGF,
resulted in a significantly higher proportion of mature vessels.
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Figure 3. Sustained, dual delivery of VEGF and PDGF rapidly forms
dense vasculature. Scaffolds incorporating VEGF alone, PDGF alone,
and both VEGF/PDGF were implanted as described earlier. Scaffolds
that rapidly release VEGF (Fig. 1C) with a slower release of PDGF
(Fig. 1D, lower curve) were utilized in these experiments. Hematoxylin
and eosin staining of tissue sections from subcutaneous implants (n = 4)
of scaffolds containing VEGF only, after two weeks (A) and four weeks
(B); scaffolds containing PDGF only, after two weeks (C) and four weeks
(D); and scaffolds releasing both VEGF and PDGF, at two weeks (E) and
four weeks (F). The vascular density within tissue sections was quantified
in each condition (G). * indicates statistical significance relative to blank
at the same time point (P < 0.05); ** indicates statistical significance
relative to VEGF and PDGF (P < 0.05). Magnification for all
photomicrographs was 400×.
Dual growth factor delivery resulted in a dramatic increase in vessel maturity, as determined by quantifying cross-sectional area and
distribution of the vessels (Fig. 5). VEGF-releasing and blank scaffolds resulted in vessel area distributions largely in the immature,
nascent capillary range (<13 µm2). PDGF delivery resulted in a small
increase in maturity. In contrast, dual growth factor delivery resulted
in a markedly different distribution, with a trend toward larger,
mature blood vessels. Dual delivery also resulted in the appearance
of multilayered vessels and sinusoids (>1,000 µm2) in the tissue,
absent in the other conditions.
We extended our study to test the importance of dual delivery
using an in vivo model of therapeutic angiogenesis. We measured
induced collateral vascularization in a hindlimb ischemia model
using a non-obese diabetic (NOD) mouse model13,24 subjected to
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A
B
C
D
E
F
G
H
Figure 4. Dual delivery of VEGF and PDGF induces mural cell
association. α-Smooth muscle actin staining of tissue sections of
subcutaneous implants of blank scaffolds after two weeks (A, B); scaffolds
containing VEGF only (C, D), PDGF only (E, F), and dual release of VEGF
and PDGF (G, H). Magnification for photomicrographs (A, C, E, G) 400×;
(B, D, F, H) 1,000×.
Figure 5. Dual delivery of VEGF and PDGF induces formation of larger
vessels. Cross-sectional areas of blood vessels at two weeks and four
weeks were measured from hematoxylin and eosin–stained tissue
sections. For each condition, at least 150 blood vessels from 10 tissue
sections were counted.
developed to allow controlled, dual release of two distinct proteins
could provide a powerful tool to study a wide array of other developmental processes important in biology and medicine.
The results of this study indicate that therapeutic angiogenesis may
benefit from the sustained and localized action of dually delivered
growth factors. It is widely accepted that the molecular mechanisms
controlling the formation of mature vasculature involve several
sequential factors, each playing a distinct role6,25 . Bolus delivery of
VEGF and/or PDGF did not induce stable increases in blood vessel
density in the present studies. Sustained delivery of PDGF led to vessel maturation, but the increase in vessel density was insignificant.
Discussion
Sustained delivery of VEGF increased blood vessel density, but the
This study documents that dual delivery of proteins involved in disvessels remained small and immature, and the tissue was marked by
tinct aspects of vascular development is critical to the rapid formaedema. These data indicate that sustained delivery of VEGF or PDGF
tion of a mature vasculature. Sustained, localized delivery of two
alone is insufficient for the promotion of a stable, dense vasculature.
growth factors both initiates formation of a significant number of
In contrast, dual delivery of the two molecules induced the formation
blood vessels and induces their maturation. The polymeric scaffold
of mature vessels. Therapeutic angiogenesis would undoubtedly benefit from the actions of both types of
molecules: a rapid initiation of blood
Table 1. Percentage of mature vessels as compared with total vessels assessed by α-smooth
vessels, as provided by VEGF in our
muscle actin staininga
studies or perhaps other stimulators
Scaffold at two weeks
Scaffold at four weeks
(e.g., angiopoietin-2; ref. 26), and the
maturation functions of PDGF or
Blank
VEGF
PDGF
Dual
Blank
VEGF PDGF
Dual
other factors (e.g., angiopoietin-1;
ref. 27). Further, the localized delivery
Ratio (%)
65
43
78
77
69
64
84
88
of factors employed in this study
P (relative to blank)
–
*
NS
NS
–
NS
*
**
P (relative to dual)
NS
**
NS
–
**
*
NS
–
allows for tight control over the doses
required for therapeutic angiogenesis,
aα-Smooth muscle actin staining of tissue sections of subcutaneous implants was quantified to determine the relative
proportion of mature blood vessels in the induced vasculature. *Statistically significant at P < 0.05; **statistically signif- and demonstrates that low doses can
icant at P < 0.01; NS, P > 0.05.
be highly effective.
femoral artery and vein ligation. Blank scaffolds implanted into the
site of ligation resulted in the formation of few blood vessels staining
positive for α-SMA per mm2 (Fig. 6A, B), while delivery of VEGF led
to a statistically significant increase in density of vessels (P < 0.05)
(Fig. 6C, D). Sustained delivery of PDGF led to an increased density
of blood vessels (Fig. 6E, F), although this difference was statistically
insignificant (P > 0.15; Fig. 6I). The dual delivery of both factors, on
the other hand, led to statistically significant increase in the density
of positively stained vessels (Fig. 6G–I).
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A
B
C
D
E
F
G
H
ciation and by quantifying the size distribution of vessels. Robust
staining for mural cells indicated multilayered, mature blood vessels. The average sizes of blood vessel areas were larger and the distribution exhibited a statistically significant shift toward the
mature phenotype, in parallel with increased staining for mural
cells. Further, the relative proportion of vessels staining positive
for smooth-muscle actin was significantly increased after four
weeks, as compared to the two-week results. This suggests maturation of the initially formed vessels; a process controlled by PDGF.
It is not possible to determine from these studies if the larger vessels noted with PDGF delivery were a result of the formation and
maturation of new vessels, or due to the muscularization of present vessels. However, it is likely that at least some of the large vessels derive from newly formed capillaries due to the overall
increase in blood vessel number. The mature vessels induced by
dual delivery did not appear to regress, as indicated by the sustained numbers of blood vessels at the later time points.
The ability to control tissue development by regulating the local
availability of combinations of growth factors will provide a powerful tool to study and manipulate a wide array of developmental
and regenerative processes important in biology and medicine.
We have demonstrated that this system provides a useful tool to
study mechanisms related to blood vessel destabilization, regression, and remodeling. This system is highly versatile, and can be
readily fabricated in a variety of structures potentially useful in
different therapies (e.g., heart patches to treat cardiac ischemia,
films to treat diabetic ulcers). This system might be relevant to
other biological processes as well (e.g., bone regeneration28).
Further, the differentiation of many stem cell types typically
requires the action of several growth factors at distinct stages29.
Efforts to manipulate this process in vivo, or exploit these cells for
therapeutic purposes in the future, may be greatly enhanced by
temporally and spatially regulating the signals presented to these
cells in vivo.
I
Experimental protocol
Figure 6. Angiogenesis is induced in non-obese diabetic (NOD) mice
subjected to femoral ligation surgery. Sections of tissue adjacent to
delivery scaffolds were stained for α-smooth muscle actin after two weeks
(n = 4). Blank scaffolds (A, B), scaffolds containing VEGF only (C, D),
PDGF only (E, F), and dual release of VEGF and PDGF (G, H). The
vascular density within tissue sections was quantified for each condition
(I). * indicates statistical significance relative to blank at the same time
point (P < 0.05); ** indicates statistical significance relative to VEGF and
PDGF (P < 0.05); NS, not statistically significant (P > 0.05). Magnification
for photomicrographs (A, C, E, G) 400×; (B, D, F, H) 1,000×.
The precise functions of VEGF and PDGF have been shown to
be dependent on the developmental state of the blood vessel, and
it is clear that there is a critical window for action of these molecules, as they can have antagonistic actions. The decreased vessel
number noted here with simultaneous bolus delivery of VEGF and
PDGF supports the concept that high levels of PDGF, before sufficient pericyte recruitment, results in a destabilized vessel and subsequent regression20. In contrast, the system developed for this
study demonstrates that temporally controlling the doses of
growth factors delivered can result in an increased maturation of
the vessels. The maturity of blood vessels in this study was determined both by immunohistochemical staining for mural cell assohttp://biotech.nature.com
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Materials. PLG (Resomer RG858 (85:15, intrinsic viscosity = 1.5 dl/g);
Resomer RG756 (75:25, intrinsic viscosity = 0.8 dl/g); Resomer RG752
(75:25, intrinsic viscosity = 0.2 dl/g)) was from Boehringer Ingleheim
(Petersburg, VA). PGDF-BB and VEGF-165 were from Intergen (Purchase,
NY), and 125I-PDGF and 125I-VEGF were from New England Nuclear
(Boston, MA). Alginate was from ProNova (Oslo, Norway). Lewis rats were
from Charles River Labs (Boston, MA), and NOD mice were from Taconic
Farms, Inc. The α-SMA detection kit used was the ARK (Animal Research
Kit) purchased from DAKO (Carpinteria, CA), consisting of a primary antibody mouse-anti-human, α-SMA (M0851 clone 1A4) and secondary antibody biotinylated anti-mouse IgG.
Scaffold fabrication and analysis of release kinetics in vitro. Scaffolds were
formed as described30,31. PDGF was pre-encapsulated in PLG microspheres
formed from two different polymers (75:25, intrinsic viscosity = 0.69 dl/g; and
75:25, intrinsic viscosity = 0.2 dl/g) processed by standard double emulsion32.
All scaffolds were formed by an identical process using equal masses of PLG in
the form of microspheres (±PDGF, particle size 5–50 µm in diameter) and
particulate PLG (85:15; particle size sieved to a diameter between 106 µm and
250 µm), containing ±VEGF and lyophilized alginate. Scaffolds were fabricated with either 125I-VEGF or 125I-PDGF as tracers and in vitro release kinetics
performed as described31,33. Scaffolds used as subcutaneous implants were
formed using a total of 20 mg of polymer and 380 mg of salt to final dimensions of 13 mm diameter by 1.5 mm thickness. Scaffolds used in the collateral
vascularization assay were scaled down for the mice while maintaining the
protein loading, and were formed from 4 mg of polymer and 76 mg of salt.
These scaffolds were 4.2 mm in diameter by 2.3 mm thick.
Lewis rat model. The treatment of experimental animals was in accordance
with University of Michigan animal care guidelines, and we observed all
National Institutes of Health (NIH) animal-handling procedures. Scaffolds
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were implanted into the subcutaneous pockets on the dorsal side of Lewis rats
(male, 8–10 weeks old). Briefly, rats were anesthetized with a solution of ketamine (87 mg/ml) and xylazine (2.6 mg/ml), injected peritoneally, using 1 µl
per g of body weight. Four small incisions were made at various points in the
back for insertion of the scaffolds (blank, VEGF alone, PDGF alone, and
VEGF/PDGF). Implants were retrieved at two and four weeks, placed in 3.7%
formaldehyde overnight, and stored in 70% ethanol before sectioning for histology. Blank scaffolds accompanied by bolus injections into the implanted
scaffold of VEGF (2 µg), PDGF (3 µg), or both simultaneously, were also
implanted and analyzed.
Histological analysis. Tissue samples were bisected and subjected to butyl
processing and paraffinization by standard procedures. Using a Nikon
Eclipse E800, blood vessels were counted manually and normalized to
tissue area. Tissue sections were stained with antibodies raised against
α-SMA. The number of positively stained blood vessels was counted and
normalized to tissue area. To measure cross-sectional area of the vessels, a
minimum of 10 individual images were sampled, and at least 150 blood
vessels were analyzed. Vessels were analyzed using Scion Image 1.62c, and
the measured pixel size of each vessel cross section was converted to square
microns.
NOD mouse model. Scaffolds were implanted into the hindlimbs of NOD mice
(male, 8–10 weeks old, one scaffold per animal) subjected to femoral artery and
vein ligation13. One scaffold was inserted into the region to deliver growth factors to the adductors following the ligation. A total of 16 animals were used,
4 each for blank, VEGF alone (2 µg), PDGF alone (3 µg), and dual-release scaffolds. Animals were supplemented with 2 U of insulin every two to three days.
Implants were retrieved at two weeks and processed as described above.
Acknowledgments
The authors acknowledge the National Institute of Dental and Craniofacial
Research for financial support: R01 DE 13033 (D.J.M.), T32 DE 07057
(T.P.R.), T32 GM08353 (A.B.E.), and the Whitaker Foundation for a graduate
student fellowship (M.C.P.).
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nature biotechnology
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VOLUME 19
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Received 2 May 2001; accepted 4 September 2001
NOVEMBER 2001
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http://biotech.nature.com