U.S. patent application number 17/667189 was filed with the patent office on 2022-08-25 for enhancement of skeletal muscle stem cell engraftment by dual delivery of vegf and igf-1.
The applicant listed for this patent is Brown University, President and Fellows of Harvard College. Invention is credited to Cristina Borselli, Jeff Lichtman, David J. Mooney, Dimitry Shvartsman, Eduardo Alexandre Barros E Silva, Hannah Storrie, Herman H. Vandenburgh, Lin Wang.
Application Number | 20220265779 17/667189 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-25 |
United States Patent
Application |
20220265779 |
Kind Code |
A1 |
Mooney; David J. ; et
al. |
August 25, 2022 |
ENHANCEMENT OF SKELETAL MUSCLE STEM CELL ENGRAFTMENT BY DUAL
DELIVERY OF VEGF AND IGF-1
Abstract
An improved device and method for extended repair and
regeneration of muscle tissue. An exemplary device comprises (a) a
scaffold comprising an ECM component; (b) a combination of growth
factors such as VEGF and IGF; and (c) a population of myogenic
cells. Implantation of the device leads to muscle regeneration and
repair over an extended period of time.
Inventors: |
Mooney; David J.; (Sudbury,
MA) ; Borselli; Cristina; (Napoli, IT) ;
Vandenburgh; Herman H.; (Providence, RI) ; Silva;
Eduardo Alexandre Barros E; (Davis, CA) ; Wang;
Lin; (Wuhan, CN) ; Shvartsman; Dimitry;
(Belmont, MA) ; Storrie; Hannah; (Chapel Hill,
NC) ; Lichtman; Jeff; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College
Brown University |
Cambridge
Providence |
MA
RI |
US
US |
|
|
Appl. No.: |
17/667189 |
Filed: |
February 8, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15436358 |
Feb 17, 2017 |
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17667189 |
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13582900 |
Mar 15, 2013 |
9610328 |
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PCT/US11/27446 |
Mar 7, 2011 |
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15436358 |
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61339526 |
Mar 5, 2010 |
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International
Class: |
A61K 38/30 20060101
A61K038/30; A61L 27/36 20060101 A61L027/36; A61L 27/38 20060101
A61L027/38; A61L 27/52 20060101 A61L027/52; A61L 27/54 20060101
A61L027/54; A61L 27/56 20060101 A61L027/56; A61K 35/34 20060101
A61K035/34; A61K 38/18 20060101 A61K038/18; A61K 35/12 20060101
A61K035/12; A61K 9/06 20060101 A61K009/06; A61K 9/19 20060101
A61K009/19; A61L 27/20 20060101 A61L027/20 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under
DE013349 and AG029705 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A hydrogel comprising a combination of vascular endothelial
growth factor 165 (VEGF.sub.165) and insulin-like growth factor-1
(IGF-1).
2. The hydrogel of claim 1, further comprising a population of
myogenic cells.
3. (canceled)
4. The hydrogel of claim 1, wherein said hydrogel comprises
macropores.
5. (canceled)
6. The hydrogel of claim 1, wherein said hydrogel is lyophilized
and compressed prior to administration to a subject.
7. The hydrogel of claim 6, wherein said hydrogel comprises
shape-memory.
8.-10. (canceled)
11. The hydrogel of claim 1, wherein said hydrogel comprises an
open, interconnected porous structure.
12. The hydrogel of claim 1, wherein said hydrogel comprises
macropores of about 5-500 microns in diameter.
13. (canceled)
14. (canceled)
15. A method of enhancing reinnervation of muscle tissue,
comprising introducing into a muscle tissue of a human subject a
device comprising the hydrogel of claim 1, wherein the VEGF.sub.165
and IGF-1 are released from the device into the muscle tissue, and
wherein introduction of the device into the muscle tissue provides
a synergistic effect on muscle reinnervation in the human
subject.
16. The method of claim 15, wherein said muscle tissue comprises
skeletal muscle tissue.
17. The method of claim 15, wherein said muscle tissue comprises
smooth muscle tissue.
18. The method of claim 15, wherein said muscle tissue comprises
cardiac tissue.
19. The method of claim 15, wherein the device is introduced into a
muscle tissue of the human subject using a needle or an
angiocatheter.
20. The method of claim 15, wherein the muscle reinnervation occurs
at the neuromuscular junction.
21. The method of claim 15, wherein the muscle tissue is
ischemic.
22. The hydrogel of claim 1, wherein the hydrogel comprises
alginate.
23. The hydrogel of claim 22, wherein the alginate comprises low
molecular weight alginate and high molecular weight alginate.
24. The method of claim 15, wherein the muscle tissue is a limb
tissue.
25. The method of claim 15, wherein the subject suffers from
ischemia.
26. The method of claim 15, wherein the subject suffers from a
muscle injury.
27. The method of claim 15, wherein the VEGF.sub.165 is released
from the device in a sustained manner over time, and wherein the
IGF-1 is released from the device at a faster rate than the
VEGF.sub.165.
Description
RELATED APPLICATIONS
[0001] This application is continuation of U.S. patent application
Ser. No. 15/436,358, filed Feb. 17, 2017, which is a continuation
of U.S. patent application Ser. No. 13/582,900, filed Mar. 15,
2013, which is a national stage application, filed under 35 U.S.C.
.sctn. 371, of International Application No. PCT/US2011/027446,
filed Mar. 7, 2011, which claims the benefit of U.S. Provisional
Application No. 61/339,526, filed Mar. 5, 2010. The entire contents
of each of the foregoing applications are incorporated herein by
reference.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 17, 2017, is named 117823_08403_ST25.txt and is 24,576
bytes in size.
BACKGROUND OF THE INVENTION
[0004] Musculoskeletal disorders and diseases are the leading cause
of disability in the United States and account for more that
one-half of all chronic conditions in people over 50 years of age
in developed countries.
[0005] Among various musculoskeletal injuries, soft tissue skeletal
muscle injuries often cause a significant loss of flexibility and
strength. Incomplete healing of these injuries could lead to a
frequent reinjury of skeletal muscles. This scenario is especially
common for athletes and military personnel, for whom the risks of
traumatic skeletal muscle injuries are common.
SUMMARY OF THE INVENTION
[0006] The invention features an improved device and method for
extended repair and regeneration of muscle tissue due to injury
such as combat injury, lacerations, traumatic physical accidents
(e.g., major surgeries, car accidents, work-related accidents) or
disease such as muscular dystrophies, multiple sclerosis, heart
disorders, lung disorders, and urinary tract disorders such as
incontinence. The device is used as injectable delivery vehicle for
the regeneration of muscle tissue and comprises a hydrogel. The
hydrogel comprises a vascular endothelial growth factor (VEGF) or a
combination of VEGF and Insulin-like growth factor (IGF), e.g.
Insulin-like growth factor-1 (IGF-1), for use as injectable
delivery vehicle for the regeneration or innervation of muscle
tissue. The VEGF+IGF combination leads to a synergistic
regeneration effect on muscle tissue.
[0007] The hydrogel optionally further comprises a population of
myogenic cells, e.g., satellite cells or myoblasts. Such cells are
obtained by biopsy from mature muscle tissue of the individual to
be treated. The myogenic cells are seeded into or onto the hydrogel
ex vivo. Alternatively, the cells are seeded into or onto the
hydrogel in vivo following insertion of the hydrogel into the
subject. The cells are expanded ex vivo or used directly, i.e.,
without expansion in culture prior to seeding the hydrogel.
[0008] The hydrogel, if to be used to transplant cells, comprises
pores to permit the structure to be seeded with cells and to allow
the cells to proliferate and migrate out to the structure to
relocate to bodily tissues such as the injured or diseased muscle
in need of repair or regeneration. For example, cells are seeded at
a concentration of about 1.times.10.sup.4 to 1.times.10.sup.7
cells/ml and are administered dropwise onto a dried hydrogel
device. The dose of the gel/device to be delivered to the subject
is scaled depending on the magnitude of the injury or diseased
area, e.g., one milliliter of gel for a relatively small defect and
up to 50 mls of gel for a large wound.
[0009] The hydrogel composition permits cell movement throughout
the structure. Cells move through a structure by virtue porosity
(e.g., pores that are at least one micron in size); by virtue of
their ability to deform the material, e.g., squeeze through the
material or push their way out of the material; or by virtue of the
cell's ability to degrade the material. The scaffold preferably
comprises pores, e.g., nanopores (0.1-100 micron diameter),
micropores (1-50 micron diameter), or macropores (50-500 micron
diameter). For example, the hydrogel comprises macropores that are
characterized by a diameter of 400-500 microns. The gel delivery
devices are suitable for treatment of human beings, as well as
animals such as horses, cats, or dogs.
[0010] In some embodiments, the hydrogel is characterized by
shape-memory. The polymer chains of the hydrogel are covalently
crosslinked and/or oxidized. Such hydrogels are suitable for
minimally-invasive delivery. Prior to delivery into the human body,
such a hydrogel is lyophyllized and compressed prior to
administration to a subject for the regeneration of muscle tissue.
Minimally-invasive delivery is characterized by making only a small
incision into the body. For example, the hydrogel is administered
to a muscle of a subject using a needle or angiocatheter.
Alternatively, the hydrogel delivery vehicles are administered to
the body using conventional surgical techniques.
[0011] An exemplary device is characterized by the following
components. The device comprises (a) a scaffold comprising an ECM
component; (b) a combination of growth factors, said combination
comprising VEGF and IGF; and (c) a population of myogenic cells,
e.g., such as satellite cells. The growth factors are incorporated
into or coated onto said scaffold composition and are released from
the scaffold at approximately the same rate or at different rates.
For example, VEGF is released from the scaffold composition at a
first rate and IGF is released from the scaffold composition at a
second rate. The scaffold may comprise nanopores, micropores, or
macropores. For example, the scaffold comprises an open,
interconnected macroporous structure.
[0012] Methods of muscle repair and regeneration comprise
introducing into a tissue the device described above. Implantation
of the device leads to muscle regeneration and repair over an
extended period of time, e.g., 2, 4, 6, 8, 10, 12, 16, weeks or
more post implantation. Although the methods and devices are
applicable to many different tissue types, a preferred tissue
comprises primarily skeletal muscle tissue, cardiac muscle, or
smooth muscle tissue.
[0013] The devices and methods are particularly useful for
treatment of aged subjects, because the naturally-occurring
regeneration of muscle tissue decreases dramatically with the age
of an individual. Children or teenager comprise a basal level of
muscle regeneration after injury or disease, and the devices and
methods of the invention enhance that level of regeneration.
However, aged subjects (e.g., 20-30 years of age, and more
particularly 35, 40, 50, 60, 70, 80, 90 or more years of age) are
characterized by minimal or no basal regenerative activity. In such
individuals, the hydrogel delivery vehicles comprising VEGF+IGF led
to significant muscle regeneration, a surprisingly beneficial
effect.
[0014] The growth factors used in therapeutic applications are
purified. A purified composition such as a protein or peptide is at
least 60%, by weight, free from proteins and naturally occurring
organic molecules with which it is naturally associated.
Preferably, the preparation is at least 75%, more preferably 90%,
and most preferably at least 99%, by weight, the desired
composition. A purified protein or polypeptide may be obtained, for
example, by affinity chromatography. A purified nucleic acid,
polypeptide, or other molecule is one that has been separated from
the components that naturally accompany it. Typically, the
polypeptide is substantially pure when it is at least 60%, 70%,
80%, 90%, 95%, or even 99%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. For example, a substantially pure polypeptide may be
obtained by extraction from a natural source, by expression of a
recombinant nucleic acid in a cell that does not normally express
that protein, or by chemical synthesis.
[0015] Publications, U.S. patents and applications, GENBANK'/NCBI
accession numbers, and all other references cited herein, are herby
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a diagram of the study protocol. Primary GFP
myoblasts (green), isolated from transgenic Tg(ACTbEGFP)1Osb,
constitutively expressing GFP in all the cells, were seeded in a
macroporous RGD-modified alginate gel (blue) encapsulating VEGF
(red) and IGF-1 (yellow). Wild type C57BL/6J mice were injured with
a myotoxin injection first and an ischemia damage was further
induced after 6 days by a femoral artery and vein ligation before
the treatment. (a). Photograph of the macroporous square-shaped
alginate scaffold (5.times.5.times.2 mm) in a lyophilized form.
(b). Scaffold implant after 2 weeks from treatment. (c-d). Tibialis
cross-sections, H&E stained before (c) and after (d) muscle
injury. (e). Complete loss of locomotion of the injured hindlimb
after injuries.
[0017] FIG. 2 is a series of photomicrographs showing
identification of donor myoblasts engraftment. Representative
images showing GFP expression (green) by immunofluorescence on
transverse sections of muscle harvested 6 weeks after transplantion
and treatment.
[0018] FIGS. 3A-B are a bar graph and photograph showing muscle
weight and size, respectively. (FIG. 3A) The weight of the
uninjured tibialis muscles (Control) at 3 days, 2 weeks and 6 weeks
is compared with the muscles after myotoxin/ischemia injury and
treatment with blank alginate gel (Alginate Gel), alginate gel
delivering VEGF and IGF-1 (gel/VEGF+IGF), alginate gel delivering
cells and VEGF and IGF-1 (Gel/VEGF/IGF-1+cells) and, bolus delivery
of cells and VEGF and IGF-1 in PBS (Bolus). Values represent
mean.+-.SD (n=6) in all graphs. p<0.05 level the means are
significantly different (FIG. 3B) Photographs of explanted tibialis
anterior at 3 days following treatment with blank alginate gel and
alginate gel delivering cells and VEGF and IGF-1
(Gel/VEGF/IGF-1+cells). Size bars are shown on the
photomicrographs.
[0019] FIGS. 4A-B are a bar graph and a series of photomicrographs
showing analysis of muscle regeneration. (FIG. 4A) The number of
centrally located nuclei of regenerating fibers at 3 days and 6
weeks after the induction of myotoxin/ischemia injury was
quantified. (FIG. 4B) Representative photomicrographs of tibialis
tissue sections from injured hindlimbs of C57 mice at postoperative
3 days and 6 weeks, stained with H&E. Cross and longitudinal
section respectively of injured muscles treated with blank alginate
gel (Alginate Gel), alginate gel delivering VEGF and IGF-1
(gel/VEGF+IGF), alginate gel delivering cells and VEGF and IGF-1
(Gel/VEGF/IGF-1+cells) and, bolus delivery of cells and VEGF and
IGF-1 in PBS (Bolus). High power magnification are shown on the
side. ANOVA statistical tests were performed on all data sets. At
p<0.05 level the means are significantly different.
[0020] FIGS. 5A-B are a bar graph and a series of photomicrographs
showing quantification of blood vessel densities. (FIG. 5A)
Quantification of blood vessel densities in tibialis muscles at 3
days and 6 weeks after induction of myotoxin/ischemia injury and
treatment with blank alginate gel (alginate gel), alginate gel
delivering VEGF and IGF-1 (gel/VEGF+IGF), alginate gel delivering
cells and VEGF and IGF-1 (Gel/VEGF/IGF-1+cells), bolus delivery of
cells and VEGF and IGF-1 in PBS (Bolus) and, control (non-operated)
limb. Values are mean.+-.SD. p<0.05. (FIG. 5B) Photomicrographs
of the entire tibialis section were obtained postoperative 6 wks,
and immunostained for the endothelial marker CD-31.
[0021] FIGS. 6A-B are a series of laser Doppler images and a line
graph showing blood perfusion. (FIG. 6A) Representative color-coded
laser Doppler perfusion imaging (LDPI) images at various time
points (after surgery, at 3 days, 2, 4 and 6 weeks post-operation)
of mice for all the conditions analyzed. (FIG. 6B) LDPI blood
perfusion analysis of C57 mice hindlimbs treated with (black
square) blank alginate gel, (gray circle) alginate gel delivering
VEGF and IGF-1, (gray triangle) alginate gel delivering cells and
VEGF and IGF-1 and, (black diamond) bolus delivery of VEGF and
IGF-1 in PBS. p<0.05; mean values are presented with SD.
[0022] FIGS. 7A-B are a bar graph and a series of photomicrographs
showing functional properties of skeletal muscles and interstitial
fibrotic collagen deposition (slow & fast myofibers). (FIG. 7A)
Tetanic force of the anterior tibialis muscles of mice were
measured at 3 days, 2 and 6 weeks after treatment. Tetanic force
was normalized to each muscle's weight to obtain weight-corrected
specific force. Stimulation was evoked via parallel wire electrodes
with 2.0 ms pulse width and 1 sec train duration, and the maximal
stimulation was measured at 15V-300 Hz. Mean values are presented
with SD; p<0.05 (FIG. 7B) Representative photomicrographs show
the deposition of interstitial fibrotic collagen of tissue sections
stained with Masson's trichromic in tibialis muscles from uninjured
hindlimbs (control) and hindlimbs of mice at postoperative 3 days,
2 weeks and 6 weeks treated with blank alginate gel (Alginate Gel),
alginate gel delivering VEGF and IGF-1 (gel/VEGF+IGF), alginate gel
delivering cells and VEGF and IGF-1 (Gel/VEGF/IGF-1+cells) and,
bolus delivery of cells and VEGF and IGF-1 in PBS (Bolus). Images
are representative of 5 independent experiments.
[0023] FIG. 8 is a line graph showing regional blood flow following
gel delivery of VEGF and/or IGF.
[0024] FIG. 9 is a line graph showing ischemic grade/score
following gel delivery of VEGF and/or IGF.
[0025] FIG. 10 is a bar graph showing the ability of animals to
locomote and bear body weight following gel delivery of VEGF and/or
IGF.
[0026] FIG. 11 is a bar graph showing the results of a muscle
function test following gel delivery of VEGF and/or IGF.
[0027] FIGS. 12A-B are line graphs, and FIGS. 12 C-E are
photomicrographs showing that local delivery of VEGF promotes nerve
regeneration and facilitates recovery following nerve damage in
ischemic and healthy muscle.
[0028] FIGS. 13A-C are photomicrographs and FIG. 13D is a line
graph showing that neural regeneration by exogenous VEGF is time
and dose-dependent.
[0029] FIGS. 14A-D are photomicrographs and FIGS. 14 E-F are line
graphs showing that local delivery of VEGF promotes the maturation
of motor axons in motor endplates and neuromuscular junction
remodeling after the ischemic injury and neural crush.
[0030] FIGS. 15A-C are a series of photomicrographs of stained
cryosections showing that injection of VEGF-loaded hydrogels into
ischemic tibialis anterior muscles elevates the expression of
neurotrophic factors within 7 days after the injury.
[0031] FIG. 16A is a series of photomicrographs, FIG. 16B is a bar
graph showing that injection of alginate hydrogels supplemented
with VEGF and Netrin-1 significantly elevates levels of
neuromuscular junction innervation within 7 days of ischemic injury
in TA muscle of mouse hindlimb.
[0032] FIGS. 17A-C are photomicrographs. SEM image of the
lyophilized porous 5% 1LMW:1HMW scaffold is shown in FIG. 17A, and
confocal imaging of PMMGFP cells growing on the RGD modified 5%
1LMW:1HMW scaffold 30 mins after seeding and after 2 weeks in
culture are shown in FIGS. 17B-C. The average pore size, calculated
from the SEM images, was 412.+-.17 p.m. Data represent mean.+-.SEM
(n=4). Low molecular weight (LMW) range from 5000-50,000, while
high molecular weight (HWM) range from 100,000-500,000.
[0033] FIGS. 18A-H is a series of photographs showing dehydrated
scaffold/delivery vehicle for minimally invasive delivery. The
lyophilized scaffold is compressed from a thickness of around 1 mm
(FIGS. 18A and 18B) to a thickness of 0.1 mm (FIG. 18C), cut to
desired size (13.5.times.2.6 mm2) (FIG. 18D), rolled up into a
tight cylinder around a 10 G needle (FIGS. 18E and 18F), and
delivered through 1.8 mm (FIGS. 18I and 18D) angiocath (FIGS. 18F
and 18G) to achieve minimally invasive delivery through a 2-3 mm
incision in the skin. The scaffold has been rehydrated with DPBS
from the 10 G needle (FIG. 18H), which is also used to deliver a 50
.mu.L suspension of cells and growth factors to rehydrate the
scaffold immediately after insertion next to the injured muscle.
The scaffold absorbs the DPBS solution and rapidly (<30 seconds)
and recovers its original 3D dimensions (FIG. 18G).
[0034] FIG. 19 depicts a scheme for the synthesis and cross-linking
of poly(aldehyde guluronate).
[0035] FIG. 20 depicts a scheme for the preparation of covalently
cross-linked alginate scaffolds.
DETAILED DESCRIPTION
[0036] The two main strategies today in cell therapy consist of the
direct injection of cells into the damaged tissue or their
pre-culture and transplantation on scaffolds that serve as a
template for neo-tissue formation. However, modulation of tissue
regeneration subsequent to injury by cell transplantation requires
the survival of donor cells and their stable incorporation into the
host tissue. Previous approaches have been limited by low survival
and integration rate of injected cells into host tissue. The work
described herein involves the transplantation of progenitor cells
on cell-instructive scaffolds designed to maintain cell viability,
promote cell activation (proliferation) and outward migration from
the scaffold in order to promote repopulation of the host damaged
tissue and regeneration of the myotoxin-injured skeletal muscle
ischemia. The goal was to direct the myogenic cells to bypass their
normal tendency to differentiate and remain in a proliferative
phase until a sufficient number of cells is attained to regenerate
the tissue.
[0037] Bolus delivery of VEGF and/or IGF is ineffective for tissue
regeneration. However, dual delivery of VEGF with IGF-1 from
macro-porous peptide-modified alginate scaffolds enhanced the
engraftment of transplanted myogenic stem cells participating in
subsequent rounds of injury repair, increased the proliferation of
the satellite cells, limited fibrosis and, accelerated the
regenerative process of injured skeletal muscle, resulting in
increased muscle mass and most importantly, improved contractile
function. Together, these results demonstrate the efficacy of
finely controlled differentiated state of myogenic stem cell
transplant for treating muscle degenerative disease or injury to
muscle tissue.
[0038] Exemplary hydrogel delivery vehicles for muscle regeneration
require the following components: (1) a composition to mediate
adhesion of cells' (2) a composition to induce migration of cells
into surrounding tissues; and (3) a composition to induce an
angiogenic response. ECM molecules such as RGD peptides are useful
to mediate cell adhesion and then migration. For muscle cells,
e.g., myogenic cells such as satellite cells or myoblasts, IGF is
useful to induce migration out of the scaffold delivery device and
into surrounding muscular tissue. HGF and FGF2 is also useful for
this purpose. The studies described herein indicate that IGF is as
good as or even better than the combination of HGF and FGF2.
Finally, VEGF is useful to induce the host angiogenic response. The
presence of VEGF in the hydrogel leads to enhanced regeneration
compared to the level in its absence.
Skeletal Muscle and Skeletal Muscle Injury
[0039] Skeletal muscle accounts for half of the total body mass and
is the most abundant tissue of the human body. The major function
of skeletal muscle is to coordinate body movements through
attachment to the skeleton. To maintain its physiological function,
the muscle tissue needs to be vascularized and innervated. A
skeletal muscle is composed of many bundles of myofibers. A single
myofiber is derived from the fusion of numerous myoblasts. Each
myofiber contains many myofibrils, which are composed of repeating
sarcomeres. Adult skeletal muscle has a large population of
quiescent muscle stem cells termed satellite cells (2-3% of the
nuclei in the tissue) that reside just outside the muscle fiber's
plasma membrane.
[0040] When injured or otherwise compromised by disease (e.g., a
degenerative disease), a skeletal muscle has limited ability to
restore morphology and function. The major obstacle for skeletal
muscle regeneration is fibrosis and formation of scar tissue during
the muscle healing process, which leads to incomplete functional
recovery, loss of flexibility, and muscle strength. The
compositions and methods described herein speed up the repair
process of muscle healing and reduce the formation of scar
tissue.
[0041] Direct injection of muscle stem cells has been unsuccessful
due to the rapid loss in viability of the majority of the cells.
Prior to the invention, numerous reports have indicated that
cultured myoblasts demonstrate poor engraftment efficiency when
subsequently transplanted, with little functional impact. In
contrast, the studies described herein demonstrate that delivery of
cultured myoblasts on an appropriate delivery vehicle leads to a
high level of engraftment, and profound functional impact.
[0042] Biodegradable scaffolds loaded with therapeutic molecules
(VEGF and/or IGF), and in some cases loaded with myoblast cells,
led to an enhancement in tissue regeneration. Among various
musculoskeletal injuries, soft tissue skeletal muscle injuries
often cause a significant loss of flexibility and strength.
Incomplete healing of these injuries could lead to a frequent
reinjury of skeletal muscles.
Satellite Cells
[0043] Muscle degeneration is rapidly followed by the activation of
an auto-repairing process. This phase is characterized first by the
activation of adult muscle satellite cells and the subsequent
events: proliferation, differentiation, and fusion of these cells,
leading to new myofiber formation and restoration of the functions
of a contractile apparatus. With minor damage, skeletal muscles can
often repair themselves by regenerating muscle fibers and restoring
muscle strength. As soon as a muscle injury occurs, myogenic
precursor cells are able to initiate rapid and efficient growth and
regeneration. The predominant source of the myogenic precursor
cells is satellite cells. Satellite cells are small mononuclear
progenitor cells that reside between the basement membrane and
sarcolemma of individual skeletal muscle fibers. They are involved
in the normal growth of muscle, as well as regeneration following
injury or disease. Their primary function is to mediate postnatal
muscle growth and repair. They can be triggered to proliferate and
differentiate into myogenic cells, fusing to augment existing
muscle fibers and to form new fibers.
[0044] In undamaged muscle, the majority of satellite cells remain
quiescent; however, in response to mechanical strain or injury,
satellite cells become activated. When muscle cells undergo injury,
quiescent satellite cells respond to the injury and are released
from their niche. Satellite cells can be activated to give rise to
skeletal myoblasts. The myoblasts in turn differentiate and form
post-mitotic myotubes. These myotubes can facilitate muscle
regeneration and repair by fusing into existing neighboring
myofibers. Satellite cells are harvested from an individual to be
treated and loaded into growth factor-containing hydrogels.
Optionally, the harvested satellite cells are cultured ex vivo
prior to being loaded onto the hydrogel (e.g., dehydrated doped
hydrogel described below) and administered to the patient.
[0045] Satellite cells express a number of distinctive molecular
markers that are used to identify and purify the cells (e.g., using
flow cytometry and Fluorescence Activated Cell Sorting (FACS)
analysis). For therapeutic application, the satellite cells need
not be isolated from surrounding muscle tissue but transplanted on
intact muscle fibers or merely gently dissociated prior to applying
the cells to the scaffold/delivery vehicle. In some cases, freshly
harvested cells (e.g., satellite cells on muscle fibers, rather
than isolated, cultured cells) are preferable due to their greater
capacity to generate tissue. In this case, fewer cells are required
than when cultured cells are used. Human satellite cells are
obtained by biopsy from a mature muscle, e.g., quadriceps, gluteus
maximus, bicep, tricep, or any muscle of the individual to be
treated. The cells are multiplied ex vivo or used without expanding
the cells in culture. For example, the cell suspension to be used
to seed the gel comprises muscle fibers and satellite cells.
Alternatively, the suspension is a population of purified satellite
cells. Prior to the invention, cultured myoblasts were not
effective for muscle regeneration; however, cultured myoblasts
delivered in the hydrogels of the invention proliferate and are
induced to migrate due to the structure of the device and the
presence of growth factors in the device leading to clinically
beneficial muscle regeneration.
[0046] Activated quiescent satellite cells express myogenic
transcription factors such as MyoD and/or Myf5. Pax-7 is expressed
in both quiescent/or activated satellite cells. Proliferating
satellite cells express muscle-specific filament proteins such as
desmin as they differentiate to myoblasts. During the muscle repair
process, proliferating myoblasts withdraw from the cell cycle to
become terminally differentiated myocytes that express Myogenin and
MRF4, and subsequently muscle-specific genes such as myosin heavy
chain (MEW) and muscle creatine kinase (MCK) [20]. Finally, myocyte
fusion gives rise to multinucleated myofibers, which then fuse to
each other to form postmitotic muscle fibers or mature skeletal
muscle tissue.
Tissue Remodeling and Fibrosis
[0047] After naturally-occurring satellite cell initiated
regeneration occurs, tissue remodeling starts. Phagocytosis of the
damaged tissue and formation of a connective-tissue scar (fibrosis)
occurs. Fibrosis is a pathological process that impairs post-injury
regeneration of muscle tissue. It starts roughly 2 weeks after
injury and can last for up to 2 weeks. Fibrotic tissue inhibits the
regenerative growth and reinnervation of muscle tissue, which in
turn results in incomplete functional recovery, physical impairment
of neighboring normal tissue structure, loss of strength and
flexibility, and propensity of reinjury and even atrophy. The
compositions and methods described herein speed up the process of
muscle healing and reduce the formation of scar tissue following
muscle damage.
Growth Factors
[0048] Muscle regeneration is regulated by multiple biochemical
pathways in which inflammatory cytokines, and growth factors (Table
1) play important roles. The identification of factors that improve
the process of muscle healing and reduce the formation of scar
tissue is of great importance for restoring the function and
structure of the injured muscle.
TABLE-US-00001 TABLE 1 Growth factors involved in muscle repair
Cell Cell Growth Factor Proliferation Differentiation Hepatocyte
growth factor (HGF) Stimulates Stimulates Basic fibroblast growth
factor (bFGF) Stimulates Stimulates Insulin-like growth factor-1
(IGF-1) Stimulates Stimulates Nerve growth factor (NGF) Stimulates
Stimulates Leukemia Inhibitory factor (LIF) Stimulates Stimulates
Acid fibroblast growth factor (aFGF) Inhibits Stimulates
Platelet-derived growth factor (PDGF-AA) Inhibits Stimulates
Platelet-derived growth factor (PDGF-BB) Stimulates Inhibits
Epidermal growth factor (EGF) Inhibits Inhibits Transforming growth
factor- .alpha. (TGF-.alpha.) Inhibits Inhibits Transforming growth
factor- .beta.1(TGF- .beta.1) Inhibits Inhibits
IGF Compared to a Combination of HGF and FGF2 for Muscle
Regeneration
[0049] Both HGF and members of the FGF family have been widely used
for inducing activation, proliferation and migration of the
myoblast cells. Besides those factors, numerous other factors are
involved as initiators of satellite cell activation showing both
mitogenic and motogenic effects on satellite cells. The role of
growth factors in skeletal muscle regeneration were compared in
order to identify the best candidate to initiate satellite cell
activation, stimulating cells to enter the cell cycle, and inducing
their migration out of scaffolds. In particular, HGF, bFGF, IGF-1
(at concentrations ranging between 5, 100, 250 ng/gel for HGF; 5,
100, 250 ng/gel for bFGF and 2.5, 5, 12.5 ng/gel IGF-1) alone or in
combination were added to a solution of G.sub.4RGDSP-modified
alginate scaffolds, prior to gelation via calcium sulfate. The gels
were cooled to produce macro-porous scaffolds with open
interconnected pores, freeze dried and cell seeded
(2.times.10.sup.6 cell/mL; 50 .mu.l of cell suspension/gel). The
size and morphology of the pores in alginate scaffolds were imaged
utilizing a scanning electron microscope. The SEM characterization
showed 98% porosity with high pore connectivity of ovoidal-shaped
pores with a diameter ranging between 0.009 to 0.130 mm. Scaffold
fabrication/characterization and cell seeding is described below
and in patent application U.S. Ser. No. 12/992,617, hereby
incorporated by reference.
[0050] The release kinetics of the growth factors incorporated into
the modified binary alginate scaffolds was then quantified. After
the gels had completely polymerized, they were cut into 5 mm
squares and placed in 24 well plates, and 1 mL of PBS was added to
each well. At various time points, the PBS was removed and fresh
PBS was added to the scaffolds. The PBS samples were measured for
total factor content via quantitative ELISA and the results were
compared to the initially incorporated growth factor. The
quantification showed that IGF-1, likely due to its smaller size
(7.5 Kda) and its non-heparin binding nature, showed a faster
release; in fact, approximately 80% of the total IGF loaded was
released in the first 24 h, then a sustained release of 0.05% was
observed in the following weeks. Conversely, a sustained release
was observed for the two heparin binding proteins bFGF and HGF, as
previously described (Hill et al., Tissue Eng. 2006).
[0051] Primary myoblasts derived from 4-12 weeks-old C57BL/6 mice
skeletal musculature were seeded into the scaffolds after being
expanded in culture for 7 days and characterized for the expression
of the myogenic protein, desmin. Analysis via light microscopy
fields revealed that the cell cultures purified via Percoll density
gradient fractionation consisted of a 95% desmin-positive
population. The resulting cell viability and migration of myoblasts
from alginate scaffolds incorporating the GFs alone or in
combination was subsequently measured by maintaining scaffolds in
culture for various time points. The cell viability inside the
scaffold was high in all the conditions analyzed. In particular,
the viability of primary myoblasts inside the scaffold was high
(80-90%) with IGF-1, and this was a significant improvement
compared to the control (blank alginate scaffold without any
factors) and IGF at the concentration of 12.5 ng/gel. Conversely, a
less pronounced increase (60-70%) of cell viability was found with
the following combination of factors IGF100 ng/gel/FGF100 ng/gel;
IGF100 ng/gel/HGF100 ng/gel; IGF100 ng/gel/FGF100 ng/gel/HGF100
ng/gel.
[0052] Furthermore, IGF-1 was shown to be more effective in
inducing a sustained outward migration of the myoblasts when
compared with the combination of all factors, both in a wound
healing assay and when examining outward migration from
macro-porous scaffolds. The results showed that IGF-1 release at
all the concentration analysed (IGF 2.5, 5, 12.5 ng/gel) induced
the activation of satellite cells promoting a sustained significant
outward migration for an extended period of time (2 weeks). In
particular, IGF (5 ng/gel) induced respectively a 1.6, 1.5, 1.9,
1.9, 2.9, 4-fold increase respectively at 24 h, 48 h, 72 h, 96 h, 1
week and 2 weeks compared to the control. Moreover, the IGF-1
release alone at the lowest concentrations (2.5 and 5 ng/gel)
induced the activation of satellite cells promoting outward
migration comparable with the combination of factors IGF100
ng/gel/HGF100 ng/gel and multiple release of IGF100 ng/gel/bFGF100
ng/gel/HGF100 ng/gel. Interestingly the combination of IGF/FGF
induced a particular significant outward migration at 48 h followed
by a little increase over time; conversely IGF 2.5 and IGF 5 ng/gel
induced a sustained significant increase of outward migration over
time.
[0053] To determine the speed of primary myoblast migration, cells
were cultured in two-dimensional plates until near confluency. A
scratch, simulating a wound, was generated along the middle line of
the plate and the repair of the wound was recorded. The results
showed that 7.5 h after the injury IGF-1 at 2.5 ng/gel induces a
rapid wound healing when compared with all the other conditions
(IGF 2.5, 5, 12.5 ng/gel; HGF 100 ng/gel; FGF 100 ng/gel; and the
combinations HGF100 ng/gel/IGF100 ng/gel; FGF100 ng/gel/IGF100
ng/gel; FGF100 ng/gel/HGF100 ng/gel/IGF100 ng/gel).
[0054] The combination of factors was not as effective as the
induction of IGF alone at the concentrations of 2.5 and 5 ng/gel.
All these results together lead to the surprising conclusion that
IGF alone is a better candidate to enhance both cell viability
inside the scaffold and outward migration compared with all the
other factors alone or in combination. Along with these findings,
the data showed that macroporous alginate gels delivering IGF in
combination with VEGF was shown to increase the speed of outward
migration and the persistence time of migrating cells, and led to
an improved design of a macroporous vehicle for primary myoblast
delivery with potential utility for (ischemic) skeletal muscle
tissue engineering able to maintain cell viability and promote a
prolonged and sustained migration outward the vehicle.
[0055] Alginate modification and scaffold fabrication was carried
out as follows. A solution of non-irradiated high-molecular-weight
(2%, wt/vol) RGD-modified alginate was prepared in DMEM. HGF, bFGF,
IGF-1 and VEGF165 were added to the alginate solution alone or in
combination (at concentrations ranging between 5, 100, 250 ng/gel
for HGF; 5, 100, 250 ng/gel for bFGF and 2.5, 5, 12.5 ng/gel
IGF-1). A calcium sulfate slurry (0.41 g CaSO.sub.4/ml dd H2O) was
added at a ratio of 40 .mu.l of CaSO.sub.4 for 1 mL of alginate and
vigorously mixed. The resulting solution was immediately expressed
into the molds 2 mm depth. A sterile glass plate was placed over
the mold and, after the alginate had completely gelled for 30 min,
square of 5 mm.times.5 mm were cut using a blazer. To produce
macro-porous scaffolds with open interconnected pores, the gels
were cooled to -80.degree. C., and the gels were lyophilized and
stored at -20.degree. C. until cell seeding. Fifty .mu.l (100,000
cells/gel) of a cell suspension (2.times.10.sup.6 cells/ml) was
gently poured onto modified open-pore polymer scaffolds. The gel
were incubated for about 20 min before adding a 500 .mu.l of
complete culture medium. The experiments were done in 12 well
plates.
[0056] Growth factors incorporation and release were evaluated as
follows. To determine the release kinetics of the growth factors
incorporated into modified binary alginate scaffolds, a
quantitative sandwich enzyme immunoassay technique (ELISA) was
employed. Recombinant proteins (Santa Cruz Biotechnology) was
incorporated into alginate solutions prior to gelling and gels were
cast as previously described. After the gels had completely
polymerized, they were cut into 5 mm squares and placed in 24 well
plates, and 1 mL of PBS was added to each well. At various time
points, the PBS was removed and stored at -80.degree. C. and fresh
PBS was added to the scaffolds. The PBS samples were measured for
total GF content via quantitative ELISA (Quantikine, Minneapolis,
Minn.), and the results were compared to the initially incorporated
GF.
[0057] Myoblast purification culture, and characterization were
carried out as follows. Primary myoblasts and both C2C12 and
GFP-PMM23.8 cell lines were used. Myoblasts were derived from 4-12
weeks-old C57BL/6 and transgenic Tg(ACTbEGFP)1Osb, constitutively
express GFP in all the cells, mice skeletal musculature. Under
sterile conditions, the all musculature was surgically excised,
finely minced, and disassociated in 0.02% Trypsin
(Gibco/Invitrogen) and 2% collagenase type 4 (Worthington
Biochemical, Lakewood, N.J.) for 60 min at 37.degree. C./5%
CO.sub.2 while agitating on an orbital shaker. Disassociated cells
were strained through a 70 .mu.m sieve, centrifuged at 1600 rpm for
5 min, and re-suspended in high-glucose DMEM, with added pyruvate
(Gibco). The medium was further supplemented with 10% fetal bovine
serum (FBS) and 10% penicillin/streptomycin (P/S, Gibco) and this
was used in all cell culture studies (for both primary and cell
line). Cells were plated and cultured at 37.degree. C./5% CO.sub.2
for 72 h before media change. After 72 h in culture, the media were
changed every 48 h until cells were 80% confluent (about 7 days).
Cells were collected via centrifugation and overlaid on a Percoll
gradient (Amersham Biosciences, Uppsala, Sweden) in a 15 mL Falcon
tube. The gradient consisted of 3 mL of 20% Percoll diluted in DMEM
(Invitrogen), 3 mL of 30% Percoll diluted in PBS (Gibco), and 3 mL
of 50% Percoll diluted in DMEM (Invitrogen). Cells were immediately
centrifuged at 1600 rpm for 20 min at 25.degree. C. The cells from
the 30% fraction were collected and re-suspended in high-glucose
DMEM.
[0058] Immunohistochemistry analysis was carried out as follows. To
characterize myoblast cultures for the expression of myogenic
proteins, Percoll purified primary myoblasts were plated on sterile
cover slips overnight and fixed in 0.2% paraformaldehyde for 20
min. Cover slips were rinsed in phosphate-buffered saline with 0.5%
Triton-X (PBS-X) and incubated in Hoechst nuclear dye (1:1000).
Cover slips were also incubated in an anti-desmin (1/100)
monoclonal antibody (Chemicon, Temecula, Calif.) followed by
immunofluorescent secondary antibody (1:1000) (FITC, Jackson Labs,
West Grove, Pa.). After secondary antibody binding, cover slips
were mounted on glass slides with aqueous mounting medium and
sealed with clear nail polish. Slides were viewed with a
conventional fluorescent light microscope (Nikon Eclipse E-800,
Tokyo, Japan) or stored in total darkness for later analysis.
Images were captured utilizing NIH imaging software (Bethesda,
Md.), Spot digital camera (Sterling Heights, Mich.), and Adobe
Photoshop (San Jose, Calif.).
[0059] Viability and proliferation were evaluated. To analyze the
cell viability and proliferation within the scaffolds, the
scaffolds were finely minced and treated with 1 mL of trypsin for 1
min at 37.degree. C. and 7 mL 50 mM EDTA for 15 min at 37.degree.
C. All the volume (8 mL) of dissolved alginate and suspended cells
were then counted under Coulter Cell (using as a blank the 50 mM
EDTA), and 1 mL of this solution was then analysed for cell
viability with V Cell via Trypan Blue exclusion (dead cells appear
blue due to their inability to exclude Trypan Blue from their
nucleus).
[0060] To measure the cell ability to migrate outward the scaffold,
myoblasts were seeded in three-dimensional alginate scaffolds
(2.times.10.sup.6 cell/mL) in 24 well plates. In particular, a
solution of cells in medium (30 .mu.L) was pipetted into each
lyophilized scaffold; the medium was rapidly absorbed. The
resulting viability and migration of myoblasts from alginate
scaffolds incorporating several GFs alone or in combination was
subsequently measured by maintaining scaffolds in culture for
various time points. To measure the outward migration of myoblasts,
scaffolds were placed in new plates (24 well plates) every 24
hours, and the cells that had colonized the plates over the
previous 24 h were removed via trypsinization and counted in a
Coulter counter (Beckman) using as a blank the isotonic solution.
The total number of cells that migrated out of the scaffold was
normalized to the total number of cells initially seeded into the
alginate scaffolds. Cells from separate isolations were used to
generate each data set, and duplication of experiments with cells
from a second, independent isolation was performed to confirm
results.
[0061] To determine the speed of wound healing purified primary
myoblasts were cultured in two-dimensional plates (2.times.10.sup.6
cell/mL) until a nearly confluency (95%). A straight width limited
scratch, simulating a wound, was generated with a 0.5-10 .mu.l
pipet tip under an angle of 30.degree. along the middle line of the
plate. The plate was then transferred to a miscroscope stage in a
incubator maintained at 37.degree. C. and 5% CO.sub.2, and the
front of cells migrating into the wounded area was recorded.
Acquisitions were taken every 10 min for 18 hours.
[0062] Scanning electron microscopy (SEM) was also used to
characterize the scaffold delivery vehicles. The size and
morphology of the pores in alginate scaffolds were imaged utilizing
a scanning electron microscope (ISIDS 130, Topcon Techn. CA, Tokyo,
Japan). All samples were dried and sputter coated (Desk II, Denton
Vacuum, Moorestown, N.J.) prior to analysis. All statistical
analysis was done using ANOVA test. Differences between conditions
were considered significant if p<0.05.
Vascular Endothelial Growth Factor (VEGF, Also Known as VEGF-A)
[0063] The term "VEGF" broadly encompasses two families of proteins
that result from the alternate splicing of a single gene, VEGF,
composed of 8 exons. The alternate splice sites reside in the exons
6, 7, and 8. However, the alternate splice site in the terminal
exon 8 is functionally important. One family of proteins arise from
the proximal splice site and are denoted (VEGF.sub.xxx). Proteins
produced by alternate splicing at this proximal location are
PRO-angiogenic and are expressed conditionally (for instance, when
tissues are hypoxic and secreted signals induce angiogenesis). The
other family of proteins arise from the distal splice site and are
denoted (VEGF.sub.xxxb). Proteins produced by alternate splicing at
this distal location are ANTI-angiogenic and are expressed in
healthy tissues under normal conditions.
[0064] VEGF exons 6 and 7 contain splice sites (result in the
inclusion or exclusion of exons 6 and 7) that affect heparin
binding affinity and amino acid number. Humans comprise
VEGF.sub.121, VEGF.sub.121b, VEGF.sub.145, VEGF.sub.165,
VEGF.sub.165b, VEGF.sub.189, and VEGF.sub.206. Heparin binding
affinity, interactions with heparin surface proteoglycans (HSPGs)
and neuropilin co-receptors on the cell surface mediated by amino
acid sequences in exons 6 and 7 enhance the ability of VEGF
variants to activate VEGF signaling receptors (VEGFRs).
[0065] Endogenous VEGF splice variants are released from cells as
glycosylated disulfide-bonded dimers. Structurally VEGF belongs to
the PDGF family of cysteine-knot growth factors comprising Placenta
growth factor (PlGF), VEGF-B, VEGF-C and VEGF-D (the VEGF
sub-family of growth factors). VEGF is sometimes referred to as
VEGF-A to differentiate it from these related growth factors. The
term "VEGF" used herein to describe the present invention is meant
to refer to VEGF-A.
[0066] Members of the VEGF family stimulate cellular responses by
binding to cell-surface tyrosine kinase receptors (the VEGFRs).
VEGF-A binds to VEGFR-1 (also known as Flt-1) and VEGFR-2 (also
known as KDR/Flk-1). VEGFR-2 is the predominant receptor for VEGF-A
mediating almost all of the known cellular responses to this growth
factor. The function of VEGFR-1 is unclear, although it is thought
to modulate VEGFR-2 signaling. VEGFR-1 may also sequester VEGF from
VEGFR-2 binding (which may be important during development).
[0067] Compositions, methods, and devices of the present invention
comprise all VEGF polypeptides generated from alternative splicing
including pro- and anti-angiogenic forms. Devices of the present
invention administered to a subject contain only pro-angiogenic
VEGF polypeptide splice forms. Alternatively, or in addition,
devices of the present invention administered to a subject contain
a mixture of pro- and anti-angiogenic VEGF polypeptide splice
forms. Pro- and anti-angiogenic VEGF polypeptide splice forms are
released by the scaffold composition of the device simultaneously
or sequentially. For example, the opposing splice forms are
released together in order to achieve a precise level of
stimulation. Alternatively, the opposing splice forms are released
sequentially to stimulate angiogenesis and subsequently attenuate
the signal when the desired result has been achieved. In another
embodiment, devices comprising pro-angiogenic VEGF polypeptide
splice forms are placed at the target tissue site while devices
comprising anti-angiogenic VEGF polypeptide splice forms are placed
in surrounding tissues in order to prevent pro-angiogenic signals
from disseminating into and stimulating non-target tissue.
[0068] Exemplary VEGF polypeptide splice forms comprised by the
compositions, methods, and devices of the present invention
include, but are not limited to, the polypeptides described by the
following sequences and SED ID NOs. VEGF polypeptide splice forms
are released from compositions, scaffolds, or devices of the
present invention as naked, or glycosylated polypeptides.
Alternatively, or in addition, VEGF polypeptide splice forms are
monomers or disulfide-bonded dimers. In a preferred embodiment,
VEGF polypeptide splice forms are released into target tissues from
compositions, scaffolds, and/or devices of the present invention as
glysosylated disulfide-bonded dimers.
TABLE-US-00002 Human VEGF.sub.148 comprises the following amino
acid sequence (NCBI Accession No. NP_001020540 and SEQ ID NO: 1): 1
mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg
61 csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr
lgarkpgswt 121 geaavcadsa paarapqala rasgrggrva rrgaeesgpp
hspsrrgsas ragpgraset 181 mnfllswvhw slalllylhh akwsqaapma
egggqnhhev vkfmdvyqrs ychpietlvd 241 ifqeypdeie yifkpscvpl
mrcggccnde glecvptees nitmqimrik phqgqhigem 301 sflqhnkcec
rpkkdrarqe npcgpcserr khlfvgdpqt ckcsckntds rckm Human VEGF.sub.165
comprises the following amino acid sequence (NCBI Accession No.
NP_001020539 and SEQ ID NO: 2): 1 mtdrqtdtap spsyhllpgr rrtvdaaasr
gqgpepapgg gvegvgargv alklfvqllg 61 csrfggavvr ageaepsgaa
rsassgreep qpeegeeeee keeergpqwr lgarkpgswt 121 geaavcadsa
paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181
mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd
241 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik
phqgqhigem 301 sflqhnkcec rpkkdrarqe npcgpcserr khlfvqdpqt
ckcsckntds rckarqleln 361 ertcrcdkpr r Human VEGF.sub.165b
comprises the following amino acid sequence (NCBI Accession No.
NP_001028928 and SEQ ID NO: 3): 1 mtdrqtdtap spsyhllpgr rrtvdaaasr
gqgpepapgg gvegvgargv alklfvqllg 61 csrfggavvr ageaepsgaa
rsassgreep qpeegeeeee keeergpqwr lgarkpgswt 121 geaavcadsa
paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181
mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd
241 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik
phqgqhigem 301 sflqhnkcec rpkkdrarqe npcgpcserr khlfvgdpqt
ckcsckntds rckarqleln 361 ertcrsltrk d Human VEGF.sub.183 comprises
the following amino acid sequence (NCBI Accession No. NP_001020538
and SEQ ID NO: 4): 1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg
gvegvgargv alklfvqllg 61 csrfggavvr ageaepsgaa rsassgreep
qpeegeeeee keeergpqwr lgarkpgswt 121 geaavcadsa paarapqala
rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181 mnfllswvhw
slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd 241
ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
301 sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksrp cgpcserrkh
lfvqdpqtck 361 csckntdsrc karqlelner tcrcdkprr Human VEGF.sub.189
comprises the following amino acid sequence (NCBI Accession No.
NP_003367 and SEQ ID NO: 5): 1 mtdrqtdtap spsyhllpgr rrtvdaaasr
gqgpepapgg gvegvgargv alklfvqllg 61 csrfggavvr ageaepsgaa
rsassgreep qpeegeeeee keeergpqwr lgarkpgswt 121 geaavcadsa
paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181
mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd
241 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik
phqgqhigem 301 sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksry
kswsvpcgpc serrkhlfvq 361 dpqtckcsck ntdsrckarq lelnertcrc dkprr
Human VEGF.sub.206 comprises the following amino acid sequence
(NCBI Accession No. NP_001020537 and SEQ ID NO: 6): 1 mtdrqtdtap
spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg 61
csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt
121 geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas
ragpgraset 181 mnfllswvhw slalllylhh akwsqaapma egggqnhhev
vkfmdvyqrs ychpietlvd 241 ifqeypdeie yifkpscvpl mrcggccnde
glecvptees nitmqimrik phqgqhigem 301 sflqhnkcec rpkkdrarqe
kksvrgkgkg qkrkrkksry kswsvyvgar cclmpwslpg 361 phpcgpcser
rkhlfvqdpq tckcsckntd srckarglel nertcrcdkp rr
Insulin-Like Growth Factor (IGF-1)
[0069] IGF-1 is a single chain polypeptide of 70 amino acids
crosslinked by three disulfide bridges. (Rinderknecht et al., 1978,
J. Biol. Chem. 253:2768-2776; sequence on p. 2771, hereby
incorporated by reference). Human IGF-1 comprises the following
amino acid sequence (GenBank: CAA01954.1 and SEQ ID NO: 7). Human
IGF-1 can be purchased from R&D Systems (614 McKinley Place
Nebr. Minneapolis, Minn. 55413)
TABLE-US-00003 1 mgpetlcgae lvdalqfvcg drgfyfnkpt gygsssrrap
qtgmvdeccf rscdlrrlem 61 ycaplkpaks a
[0070] Human IGF-1B isoform comprises the following sequence
(GenBank: CAA40093.1; SEQ ID NO: 8). The mature peptide comprises
residues 49-118.
TABLE-US-00004 1 mgkisslptq lfkccfcdfl kvkmhtmsss hlfylalcll
tftssatagp etlcgaelvd 61 alqfvcgdrg fyfnkptgyg sssrrapqtg
ivdeccfrsc dlrrlemyca plkpaksars 121 vraqrhtdmp ktqkyqppst
nkntksqrrk gwpkthpgge qkegteaslq irgkkkeqrr 181 eigsrnaecr
gkkgk
Myoblast Transfer Therapy (MTT)
[0071] The descendents of satellite cells--myoblasts--have been
considered as powerful candidates for cell-based therapies to treat
muscle injury, muscular dystrophies, and other neuromuscular
diseases. Myoblast transfer therapy (MTT) involves the
intramuscular injection into host muscle of cultured muscle
precursor cells--myoblasts are isolated from normal donor skeletal
muscles, expanded in vitro, and injected to the muscle injury site
of the recipient. However prior to the invention, this treatment
was limited by the rapid and massive death of donor myoblasts
following injection into the host muscle. The failure of MTT was
due to a number of different reasons: host immune rejection to the
injected myoblasts, poor migration of the cells, reduced cell
myogenic potential after in vitro culture, mechanical stress,
limited availability of oxygen and/or nutrient supply, and delayed
clearance of metabolites. The invention solves these problems of
earlier approaches in three significant ways: (1) the hydrogel
delivery device provides temporary housing by virtue of its
porosity and presence of ECM compositions (e.g., RGD-containing
peptides) to mediate temporary adhesion of cells; (2) the presence
of IGF in or on the delivery device induces migration of the cells
(e.g., satellite cells or myoblasts) out of the scaffold device and
into the subject's muscular tissue; and (3) VEGF in or on the
device promotes a host angiogenic response. As a result,
implantation of the VEGF-containing, IGF-containing, cell-seeded,
ECM-derivitized hydrogel leads to enhanced muscle generation that
is superior to previous approaches.
Scaffold Compositions and Architecture
[0072] Components of the scaffolds are organized in a variety of
geometric shapes (e.g., beads, pellets), niches, planar layers
(e.g., sheets). For example, sheetlike are used in bandages or
wound dressings. The device is placed on or administered into a
target tissue. Devices are introduced into or onto a bodily tissue
using a variety of known methods and tools, e.g., spoon, tweezers
or graspers, hypodermic needle, endoscopic manipulator, endo- or
trans-vascular-catheter, stereotaxic needle, snake device,
organ-surface-crawling robot (United States Patent Application
20050154376; Ota et al., 2006, Innovations 1:227-231), minimally
invasive surgical devices, surgical implantation tools, and
transdermal patches.
[0073] A scaffold or scaffold device is the physical structure upon
which or into which cells associate or attach, and a scaffold
composition is the material from which the structure is made. For
example, scaffold compositions include biodegradable or permanent
materials such as those listed below. The mechanical
characteristics of the scaffold vary according to the application
or tissue type for which regeneration is sought. It is
biodegradable (e.g., collagen, alginates, polysaccharides,
polyethylene glycol (PEG), poly(glycolide) (PGA), poly(L-lactide)
(PLA), or poly(lactide-co-glycolide) (PLGA) or permanent (e.g.,
silk). In the case of biodegradable structures, the composition is
degraded by physical or chemical action, e.g., level of hydration,
heat or ion exchange or by cellular action, e.g., elaboration of
enzyme, peptides, or other compounds by nearby or resident cells.
The consistency varies from a soft/pliable (e.g., a gel) to glassy,
rubbery, brittle, tough, elastic, stiff. The structures contain
pores, which are nanoporous, microporous, or macroporous, and the
pattern of the pores is optionally homogeneous, heterogenous,
aligned, repeating, or random.
[0074] Alginates are versatile polysaccharide based polymers that
may be formulated for specific applications by controlling the
molecular weight, rate of degradation and method of scaffold
formation. Coupling reactions can be used to covalently attach
bioactive epitopes, such as the cell adhesion sequence RGD to the
polymer backbone. Alginate polymers are formed into a variety of
scaffold types. Injectable hydrogels can be formed from low MW
alginate solutions upon addition of a cross-linking agents, such as
calcium ions, while macroporous scaffolds are formed by
lyophilization of high MW alginate discs. The cross-linking is
ionic or covalent (as in the case of shape-memory delivery
devices).
[0075] Differences in scaffold formulation control the kinetics of
scaffold degradation. Release rates of morphogens or other
bioactive substances from alginate scaffolds is controlled by
scaffold formulation to present morphogens in a spatially and
temporally controlled manner. This controlled release not only
eliminates systemic side effects and the need for multiple
injections, but can be used to create a microenvironment that
activates host cells at the implant site and transplanted cells
seeded onto a scaffold.
##STR00001##
[0076] The scaffold comprises a biocompatible polymer matrix that
is optionally biodegradable in whole or in part. A hydrogel is one
example of a suitable polymer matrix material. Examples of
materials which can form hydrogels include polylactic acid,
polyglycolic acid, PLGA polymers, alginates and alginate
derivatives, gelatin, collagen, agarose, natural and synthetic
polysaccharides, polyamino acids such as polypeptides particularly
poly(lysine), polyesters such as polyhydroxybutyrate and
poly-epsilon.-caprolactone, polyanhydrides; polyphosphazines,
poly(vinyl alcohols), poly(alkylene oxides) particularly
poly(ethylene oxides), poly(allylamines)(PAM), poly(acrylates),
modified styrene polymers such as poly(4-aminomethyl styrene),
pluronic polyols, polyoxamers, poly(uronic acids),
poly(vinylpyrrolidone) and copolymers of the above, including graft
copolymers.
[0077] The scaffolds are fabricated from a variety of synthetic
polymers and naturally-occurring polymers such as, but not limited
to, collagen, fibrin, hyaluronic acid, agarose, and laminin-rich
gels. One preferred material for the hydrogel is alginate or
modified alginate material. Alginate molecules are comprised of
(1-4)-linked .beta.-D-mannuronic acid (M units) and .alpha.
L-guluronic acid (G units) monomers, which can vary in proportion
and sequential distribution along the polymer chain. Alginate
polysaccharides are polyelectrolyte systems which have a strong
affinity for divalent cations (e.g. Ca.sup.+2, Mg.sup.+2,
Ba.sup.+2) and form stable hydrogels when exposed to these
molecules. See Martinsen A., et al., Biotech. & Bioeng., 33
(1989) 79-89.) For example, calcium cross-linked alginate hydrogels
are useful for dental applications, wound dressings chondrocyte
transplantation and as a matrix for other cell types.
[0078] An exemplary device utilizes an alginate or other
polysaccharide of a relatively low molecular weight, preferably of
size which, after dissolution, is at the renal threshold for
clearance by humans, e.g., the alginate or polysaccharide is
reduced to a molecular weight of 1000 to 80,000 daltons.
Preferably, the molecular mass is 1000 to 60,000 daltons,
particularly preferably 1000 to 50,000 daltons. It is also useful
to use an alginate material of high guluronate content since the
guluronate units, as opposed to the mannuronate units, provide
sites for ionic crosslinking through divalent cations to gel the
polymer. U.S. Pat. No. 6,642,363, incorporated herein by reference
discloses methods for making and using polymers containing
polysaccharides such as alginates or modified alginates that are
particularly useful for cell transplantation and tissue engineering
applications.
[0079] Useful polysaccharides other than alginates include agarose
and microbial polysaccharides such as those listed in the table
below.
TABLE-US-00005 Polysaccharide Scaffold Compositions Polymers.sup.a
Structure Fungal Pullulan (N) 1,4-; 1,6-.alpha.-D-Glucan
Scleroglucan (N) 1,3; 1,6- .alpha. -D-Glucan Chitin (N)
1,4-.beta.-D-Acetyl Glucosamine Chitosan (C) 1,4-
.beta..-D-N-Glucosamine Elsinan (N) 1,4-; 1,3- .alpha. -D-Glucan
Bacterial Xanthan gum (A) 1,4- .beta..-D-Glucan with D-mannose;
D-glucuronic Acid as side groups Curdlan (N) 1,3- .beta..-D-Glucan
(with branching) Dextran (N) 1,6- .alpha. -D-Glucan with some 1,2;
1,3- 1,4-.alpha. - linkages Gellan (A) 1,4- .beta..-D-Glucan with
rhamose, D-glucuronic acid Levan (N) 2,6- .beta. -D-Fructan with
some .beta. -2,1-branching Emulsan (A) Lipoheteropolysaccharide
Cellulose (N) 1,4- .beta.-D-Glucan
Alginate Hydrogels as Cell/Growth Factor Delivery Vehicles
[0080] Hydrogels are made from water-soluble polymers and are a
class of three-dimensional, water-swollen cross-linked homopolymers
or copolymers. Alginates are a class of hydrogel-forming material
that has been widely utilized in tissue engineering and drug
delivery applications. They are a naturally derived polysaccharide,
extracted from brown algae. Their chemical structure shows that
they are copolymers of (1,4)-linked b-D-mannuronic acid (M) and
a-L-guluronic acid (G). Alginate hydrogels are highly hydrated
three-dimensional networks and their structure resembles the native
ECM of tissues.
[0081] Alginate hydrogels have been used in the food industry and
medicine due to their high biocompatibility and advantageous
physical and chemical properties. Alginate hydrogels are easy to
fabricate and process, and can be readily formed into defined
structures and form three-dimensional matrices in a hydrated state.
High water content is another unique property that makes alginate
hydrogels resemble native tissues and thus makes them a good
candidate material for cell culture matrix for tissue repair. In
addition, they have material properties that are readily tunable by
varying the type and degree of cross linking in the polymer network
and other chemical or physical modifications.
Example 1: Functional Muscle Regeneration with Combined Delivery of
Angiogenesis and Myogenesis Factors
[0082] Studies were carried out to investigate an interplay between
VEGF and IGF-1 in ischemic muscle regeneration, and the possibility
that dual sustained delivery of these two critical morphogens could
induce the regeneration of functional muscle in ischemic hindlimbs.
The impact of the distance of the muscle from the factor delivery
site on the regeneration process was also examined by analysing
distinct muscles in the hindlimbs. As targets for these
experiments, we chose the gracilis and tibialis muscles,
respectively corresponding to the muscle site of injection and a
muscle distant to the site of polymer placement. The ultimate goal
of this approach is to preserve the local progenitor cells from
apoptosis and necrosis during the degeneration process, and instead
to activate the progenitor cells to enter the proliferative phase
and differentiate into contractile muscle fibers to regenerate
functional tissue.
[0083] Regenerative efforts typically focus on the delivery of
single factors, but it is likely that multiple factors regulating
distinct aspects of the regenerative process (e.g., vascularization
and stem cell activation) can be utilized in parallel to affect
regeneration of functional tissues. This possibility was addressed
in the context of ischemic muscle injury, which typically leads to
necrosis and loss of tissue and function. The role of sustained
delivery, via injectable gel, of a combination of vascular
endothelial growth factor (VEGF) to promote angiogenesis and
insulin growth factor-1 (IGF-1) to directly promote muscle
regeneration and the return of muscle function in ischemic rodent
hindlimbs was investigated. Sustained VEGF delivery alone led to
neo-angiogenesis in ischemic limbs with complete return of tissue
perfusion to normal levels by 3 weeks, as well as protection from
hypoxia and tissue necrosis, leading to an improvement in muscle
contractility. Sustained IGF-1 delivery alone was found to enhance
muscle fiber regeneration and, protected cells from apoptosis.
However, the combined delivery of VEGF and IGF-1 led to parallel
angiogenesis, reinnervation and myogenesis, as satellite cell
activation and proliferation was stimulated, cells were protected
from apoptosis, the inflammatory response was muted, and highly
functional muscle tissue was formed. In contrast, bolus delivery of
factors did not have any benefit in neoangiogenesis and perfusion,
and minimal effect on muscle regeneration. These results support
the utility of simultaneously targeting distinct aspects of the
regenerative process.
[0084] The following materials and methods were used to generate
the data described in Example 1.
Growth Factor Incorporation and Release Kinetics
[0085] Ultrapure MVG alginate was purchased from ProNova Biomedical
(Norway). Biodegradable gels were formed from a combination of
polymer molecular weights. Alginates were reconstituted in EBM-2
(Cambrex Corporation, Walkersville, Md., USA) to obtain a 2% w/v
solution prior to gelation, and cross-linked with aqueous slurries
of a calcium sulphate solution (0.21 g CaSO4/mL dH2O) at a ratio of
25:1 (40 pl of CaSO4 per 1 mL of 2% w/v alginate solution).
Alginates were pre-mixed with recombinant human .sub.VEGF165
protein (generously provided by Biological Resources Branch of the
National Cancer Institute) and/or with recombinant human IGF1
(R&D System), at a final concentration of 60 pg/mL for each
protein; in vitro release kinetics were measured using ELISA.
Mixtures were allowed to gel for 30 min, and maintained at
4.degree. C. prior to animal injections.
Animals and Surgical Procedures
[0086] Female C57BL/6J mice (6-7 weeks; Jackson Laboratories, Bar
Harbour, Me., USA) were anesthetized with an intraperitoneal
injection of a mixture of ketamine 80 mg/kg and xylazine 5 mg/kg
prior to all surgical procedures. Hindlimb ischemia was induced by
unilateral external iliac and femoral artery and vein ligation.
After the vessel ligation, mice were injected with a total volume
of 50 pl of alginate gel containing 3 pg of .sub.VEGF165 and/or 3
pg of IGF1, gel containing 3 pg of IGF-1, gel with no GFs, or a PBS
solution containing 3 pg of .sub.VEGF165 and 3 pg of IGF-1 (bolus
delivery). Injections were performed using a 25 G needle (Becton
Dickinson, Franklin Lakes, N.J., USA), directly into the gracilis
muscle (1-3 mm inside the muscle) at the site of vessel ligation.
The incision was surgically closed, and animals monitored over
time.
[0087] For analysis of reinnervation, hindlimb ischemia and gel
delivery were carried out as described in transgenic C57BL/6 mice
selectively expressing yellow fluorescent protein (YFP) under
control of a thy-1 promoter in motoneurons.
Ischemia, Perfusion and Hypoxia Analysis
[0088] Measurements of the ischemic/normal limb blood flow ratio
were performed on anesthetized animals (n=10) using a LDPI analyzer
(Perimed AB, Stockholm, Sweden). Perfusion measurements were
obtained by scanning entire hindlimbs under basal conditions and
then weekly after surgery, and the ratio of perfusion of the
ischemic to non-ischemic limb of the same animal was calculated.
Tissue hypoxia was visualized in tissue sections using hydroxyprobe
reagent, as per supplier instructions (Chemicon).
Histological Assessment of Skeletal Muscle
[0089] Mice were sacrificed and hindlimb muscle tissues (n=10 per
time point per experimental condition) were processed for
histological analyses. For regeneration metrics, the samples were
stained with H&E, and fiber diameter and the number of
centrally located nuclei were analyzed. Vascular ECs were
identified by immunostaining for mouse CD31 (BD Biosciences
Pharmingen, San Diego, Calif., USA). For measurement of capillary
densities, histological analysis was performed in a blinded fashion
as described. Immunostaining for Ki-67 (Ki-67 mouse IgG1, Dako,
Carpinteria, Calif.) was performed to identify cell proliferation.
Qualitative analysis of apoptosis was assessed by TUNEL assay
(Roche). Interstitial fibrosis was morphometrically assessed in
Masson Trichrome (Sigma Aldrich) stained sections.
Analysis of Reinnervation
[0090] Mice were anesthetized by an intraperitoneal injection of
ketamine/xylazine and fixed by transcardial injection of 4%
paraformaldehyde. The tibialis muscle was explanted, and stained
with Alexa594-bungarotoxin (Invitrogen, Frederick, Md.) to
visualize acetylcholine receptors. Innervation at the neuromuscular
junction was imaged using a Zeiss Pascal 5 LSM upright laser
scanning confocal microscope using an Ar laser to excite YFP at 488
nm, and a He/Ne laser to excite Alexa594 bungarotoxin at 543 nm.
All images were processed using Zeiss software and images are
displayed as Z-maximum intensity projections. Reinnervation was
quantified by counting sites of overlap of motor neuron axon
(yellow) and endplate (red) as a site of reinnervation. At least 50
NMJs were counted for each condition. Statistical significance was
determined using unpaired ANOVA analysis.
Mechanical Measurements
[0091] Intact gracilis and tibialis muscles were dissected
(n=5/condition), mounted vertically midway between two fine
cylindrical parallel steel wire electrodes (1.6 mm diameter, 21 mm
long), attached by their tendons to microclips connected to a force
transducer (FORT 25, WPII, Sarasota, Fla., USA) and bathed in a
physiological saline solution in a chamber oxygenated with 95%
02-5% CO.sub.2 at 25.degree. C. Muscle length was adjusted until
maximum twitch force was achieved (100-300 Hz). A wave pulse was
initiated from a computer using a custom-written Lab VIEW program
and delivered to the stimulation electrodes via a purpose-built
power amplifier (QSC USA 1310). A switch on the amplifier permitted
stimulation via wire electrodes. Contractions were continuously
monitored on a LabView chart recorder, and contractions saved on a
PC. Contractions were evoked every 5 min. Tetani were usually
evoked at 300 Hz-15-20V with constant pulse width and train
duration of 2 ms and 1 s, respectively. These stimulation
frequencies and voltages were required to generate maximum force
but exceed the naturally occurring median firing frequencies of
100-200 Hz in tibialis and gracilis. After force measurements were
completed, the muscles were removed from the bath and weighed. Peak
tetanic force was determined as the difference between the maximum
force during a contraction and the baseline level, and specific
force calculated by normalization by muscle weight.
Statistical Analyses
[0092] All results are expressed as mean.+-.standard deviation
(SD). Multivariate repeated-measures ANOVA was performed to test
for interactions between conditions. Differences between conditions
were considered significant if p value<0.05.
Dual Sustained Delivery of VEGF and IGF-1
[0093] Dual sustained delivery of these two critical morphogens
induces the regeneration of functional muscle in ischemic
hindlimbs. The impact of the distance of the muscle from the factor
delivery site on the regeneration process was examined by analysing
distinct muscles in the hindlimbs. The gracilis and tibialis
muscles were chosen for the muscle site of injection and a muscle
distant to the site of polymer placement, respectively. The goal of
this approach was to preserve the local progenitor cells from
apoptosis and necrosis during the degeneration process, and instead
to activate the progenitor cells to enter the proliferative phase
and differentiate into contractile muscle fibers to regenerate
functional tissue.
Sustained VEGF&IGF-1 Presentation Enhance Muscle Size and Limb
Vascularization
[0094] An ischemia injury was selected for these studies following
analysis of the spontaneous recovery of muscle mechanical function
subsequent to various types of injuries, including partial
laceration, cryoinjury, and notexin injection. Ischemia led to the
greatest loss of muscle function, as compared to the other injury
models, and the least spontaneous return of function. Further,
analysis of tissue sections 2 wks following injury revealed a
largely necrotic defect with diffusely disorganized and
disrupted/broken myofibers in the ischemic condition, supporting
the stringency of this model.
[0095] Mice were treated at the time of induction of severe
hindlimb ischemia with an injectable, degradable alginate gel. In
vitro, after an initial burst, VEGF was released in a sustained
manner over time, while IGF, due to its smaller size (7.5 Kda) and
its non-heparin binding nature showed a faster release,
approximately 80% of the total IGF loaded was released in the first
24 h. The following five interventions were analysed: (i) blank
alginate gel, (ii) alginate gel delivering VEGF (3 ug), (iii)
alginate gel delivering VEGF and IGF-1 (3 ug each), (iv) alginate
gel delivering IGF-1 (3 ug), and (v) bolus delivery of VEGF and
IGF-1 (3 ug each) in PBS.
[0096] Significant muscle loss was noted at seven weeks
post-surgery with blank gel treatment, while injured muscles
treated with gel containing both GFs were grossly larger.
Quantification of the weight of these muscles revealed
insignificant changes with gel releasing either VEGF or IGF alone,
or with the saline bolus treatment, whereas statistically
significant increases of 26%.+-.11 and 30%.+-.22 occurred for the
tibialis (distant to gel injection) and gracilis muscles (site of
gel injection) respectively, receiving gel releasing both GFs as
compared with the blank treatment. The large standard deviations in
the gracilis muscle analysis were due to the difficulty in
isolating the gracilis muscle from the other tightly associated
muscles.
[0097] As the effects of the VEGF delivery on muscle regeneration
were likely mediated by its effects on angiogenesis, the level of
muscle hypoxia, perfusion of ischemic tissues, and tissue necrosis
were next analyzed. Immunohistochemical analysis of tibialis and
gracilis muscle tissues revealed that VEGF-delivering alginate gels
(alginate/VEGF, and alginate VEGF/IGF-1) increased muscle blood
vessel densities, as compared with injection of a blank vehicle or
bolus delivery of VEGF/IGF. In particular, at 7 wks, VEGF delivery
from the gels resulted in an approximately 2-fold increase in
vessel density in tibialis muscle and 3-fold increase in the
gracilis muscle, as compared to the ischemic hindlimb treated with
the blank alginate. IGF delivery alone had no significant effect on
vascularization in the gracilis muscle, and a modest effect in the
tibialis. The bolus delivery had no effect on blood vessel
densities, as compared to the controls.
[0098] A Laser Doppler Perfusion Imaging (LDPI) system was used to
quantify perfusion. The regional blood flow was reduced immediately
after surgery to approximately 20% of normal in all conditions, as
expected. Alginate gel only treatment led to a slow increase in
reperfusion over time, and the ischemic limbs for the most part
remained necrotic. Bolus delivery resulted in little difference
from the no-treatment control or blank alginate injection. In
contrast, VEGF and dual GF delivery from the vehicle led to a final
recovery of respectively 80% and 95% of normal limbs. In
particular, animals treated with alginate gels delivering
VEGF/IGF-1 showed a marked increase in blood flow starting around
the 4.sup.th week after the injury, and an additional 20% increase
at 7 weeks compared with the control. The level of tissue necrosis
was also quantified by visual observation. Hindlimb ischemia led to
severe toe or foot gangrene in control animals, but treatment with
alginate gel with VEGF and VEGF/IGF largely spared the limbs from
necrosis. Protection of myofibers from hypoxia was also observed
with alginate gel VEGF and VEGF/IGF delivery, as based on hypoxia
immunostaining.
VEGF and IGF-1 Induce Myoblast Proliferation and Protect Against
Apoptosis
[0099] Immunostaining of tissue sections against the
proliferation-associated protein Ki67 was performed to determine
cell proliferation activity at early (2 weeks) and late (7 weeks)
times. Abundant expression of Ki67 was detected in muscle tissues
receiving alginate gels releasing VEGF alone and VEGF/IGF-1 in both
tibialis and gracilis muscles at 2 weeks and 7 weeks. A less
pronounced increase was observed with alginate gel delivering IGF,
while no proliferation was observed in muscles treated with the
blank vehicle. Furthermore, triple immunofluorescence for CD31,
Ki67, and Dapi for nuclear staining suggested that both myoblasts
and ECs proliferated at early stages of the reparative process.
TUNEL analysis was performed to measure apoptosis in the
regenerating muscles at 2 weeks post-ischemia. While significant
apoptosis was observed in the blank vehicle group, apoptosis was
reduced in the muscles treated with alginate delivering VEGF, and
was significantly lower with vehicles delivering IGF alone. The
combination of the two GFs was particularly effective in combating
ischemia-induced apoptosis. Apoptosis was virtually absent in
contra-lateral normoperfused muscles, as expected. Similar results
were seen in five independent experiments.
Muscle Regeneration Enhanced by VEGF and IGF-1, Along with Reduced
Fibrosis
[0100] To directly analyze muscle regeneration, the mean diameter
of regenerated myofibers and number of centrally located nuclei in
the resolving muscle tissue were quantified. The mean diameter of
muscle fibers were quantitatively greater in muscles treated with
alginate delivering both growth factors, as compared with alginate
delivering only VEGF or IGF-1 or the two growth factors in bolus
saline, in both tibialis and gracilis muscles. The tibialis muscles
treated with alginate delivering VEGF or IGF-1 alone showed an
approximately 10% increase in average diameter, while co-delivery
of both GFs led to a 25% increase in the diameter of regenerating
fibers, compared to the blank alginate gel, and a 19% increase
compared to gel/VEGF (p<0.05). An increase was also observed in
gracilis muscle with VEGF/IGF delivery from the alginate gels. At 2
wks post-injury the tibialis muscle fibers in the injury group
treated with VEGF or IGF-1 alone also showed an approximately 40%
increase in centrally located nuclei, versus a lesser increase of
30% with bolus factors delivery, as compared with the blank. The
two factors in combination with alginate delivery led to a 53% and
a 39% increase in centrally located nuclei, as compared with the
blank alginate or alginate delivering VEGF alone. The number of
centrally located nuclei in the gracilis fibers treated with
alginate delivering both GFs increased .about.70% and 20% increase,
respectively, when compared with either the blank alginate or with
alginate delivering VEGF only. Representative cross and
longitudinal micro sections of tibialis tissue highlight the
increase in centrally located myonuclei in the ischemic muscles
treated with alginate delivering both GFs. Analysis of the muscle
fiber types confirmed an active regenerative process induced by
growth factor delivery. Type IIC fibers were noted at early times
(3 days) following injury with delivery of growth factor from the
gel, but were not present in uninjured control muscles or uninjured
muscles treated with gel/growth factor. Further, analysis of
injured muscle treated with gel delivering VEGF revealed a
significant increase in myogenin positive cells, which contrasts
with few myogenin-positive cells in control, uninjured muscle),
also supporting an active muscle regeneration process.
[0101] Injured muscle tissue treated with blank alginate
demonstrated significant interstitial fibrotic tissue. Control
(non-operated) limbs demonstrated little fibrosis, as expected.
However, limbs treated with alginate gel delivery of both GFs
exhibited a significant decrease in fibrosis. A less pronounced
reduction of fibrosis was observed with the two GFs delivered
alone. Conversely, in the bolus injection condition a large content
of fibrotic tissue was formed.
Growth Factor Delivery Promotes Earlier Regeneration of Damaged
Neuromuscular Junctions
[0102] Induction of ischemia in the hindlimb and treatment with a
blank hydrogel led to a significant loss of innervation at the
neuromuscular junction (NMJ) in the tibialis muscle seven days
after injury in control mice; by day fourteen complete
reinnervation had occurred and NMJs appeared normal. In contrast,
muscles treated with either IGF-1 alone or VEGF/IGF-1 had
completely reformed NMJs and no damage to receptors or muscle
fibers was observed at 7 days. At this time point, VEGF delivery
also resulted in robust reinnervation of NMJs, although not to a
significantly greater extent than the blank hydrogel.
Dual Gel Delivery of VEGF&IGF-1 Enhances the Contraction Force
of Damaged Muscles
[0103] To test whether muscle changes induced by GF delivery would
correspond to increased function, the contractile force of the
muscles was analyzed. The weight normalized tetanic force of the
tibialis and gracilis muscles were measured after maximal tetanic
stimulation. Muscles treated with gel delivering both GFs showed a
significant increase above normal values in the tetanic force at 2
wks postsurgery (2.3 and 7.9 fold increase, respectively, for
tibialis and gracilis muscles, when compared with the blank)
followed by a decrease toward the normal value at 7 wks. Animals
receiving alginate delivering VEGF alone showed a similar trend,
but the increase in the force of contraction was less pronounced.
In particular, at 2 wks a 1.6 and 5.7 fold increase was measured,
respectively in tibialis and gracilis muscles compared with
alginate gel only. In contrast, the animal receiving alginate gel
without GFs had a markedly lower contractile function at all time
points.
[0104] The results from these studies demonstrate a beneficial
interplay between VEGF and IGF-1, when delivered appropriately, in
enhancing skeletal muscle regeneration, revascularization,
re-innervation and gain of function following ischemic injuries.
Past therapies to regenerate ischemic tissues typically relied on
bolus delivery or systemic administration of single growth factors.
Vascular endothelial growth factor (VEGF) specifically has been
widely used as a potent pro-angiogenic initiator in many strategies
to treat ischemic diseases. However, the impact on salvaging and
driving regeneration of ischemic muscle has not been addressed.
Moreover, an extensive body of literature supports a role for
insulin growth factor-1 (IGF-1) in regulating the establishment and
maintenance of the mature muscle phenotype in normal and
regenerating muscle tissue both in vitro and in vivo. In particular
IGF-1 has been implicated in early and late stages of muscle
developmental processes playing first a role in inducing myoblast
proliferation, and subsequently promoting myogenic differentiation.
Past approaches to exploit GF signalling in muscle regeneration
typically utilized bolus GF delivery, which leads to rapid
depletion of the factors in the target tissue. Supra-physiologic
concentrations of growth factors are used in an effort to offset
this issue, potentially leading to unwanted side-effects.
[0105] Sustained VEGF delivery alone from alginate gels had a
significant impact on angiogenesis, and tissue perfusion, but a
less pronounced effect on muscle regeneration. These results are in
accord to previous reports that the sustained and controlled
release of VEGF from both a PLG and the same injectable
alginate-based vehicle stimulated angiogenesis, returned perfusion
to normal levels, and prevented necrosis in ischemic hindlimbs.
VEGF has also recently been implicated in muscle regeneration and
muscle reinnervation via a direct neuro-protective and
neuro-directing effect. The contractile activity of skeletal
muscle, and hence its functionality, are regulated by the nervous
system and loss of innervation leads to a decrease in satellite
cell number and muscle atrophy. The results of this study suggest
delivery of VEGF alone has profound effects on muscle regeneration,
as increases in the diameter of regenerating fibers and the number
of centrally located nuclei in muscle fibers, both hallmarks of
regenerating myofibers, were found with gel-VEGF delivery. The
contractile properties of the injured muscle were also improved
with appropriate VEGF delivery.
[0106] IGF-1 delivery alone from alginate gels was found to have a
modest effect on muscle fiber regeneration and cell protection from
apoptosis. These data are consistent with data that increased
levels of IGF-1 augmented tissue DNA content (resulting from
activation of satellite cells) and muscle protein synthesis within
existing myofibers. Gel-IGF-1 delivery alone also induced
neo-angiogenesis in the tibialis muscle, and to a lesser effect in
the gracilis muscle. This effect was likely secondary to the
effects of IGF-1 on the muscle cells. The delivery approach used in
this study resulted in an initial burst delivery of this factor,
likely leading to a rapid diffusion of the factor from the site of
the injection. A more sustained delivery of IGF-1 increases muscle
regeneration.
[0107] Surprisingly, dual VEGF/IGF-I delivery from gels had a
synergetic effect on the regenerative parameters in both of the
analyzed muscles. In particular, both the mean fiber diameter and
the number of centrally located nuclei in the fibers were
significantly enhanced with alginate delivery of both GFs, showing
a more pronounced response in the muscle where the gel was injected
(gracilis). These results were qualitatively validated by an
increased number of myoblasts found in an active proliferative
state, the presence of myogenin positive cells, type IIC muscle
fibers, and decreased cell apoptosis. These results demonstrate an
enhancement in myoblast recruitment for neomuscle formation, which
is consistent with the larger size and mass of these muscles. The
enhanced myogenic regeneration in response to VEGF and VEGF/IGF
sustained delivery could also be explained by the existence of a
population of myoendothelial cells endowed with multilineage
potential, including high muscle regenerative potential.
Stimulation of angiogenesis may increase the pool of myogenic stem
cells which are available to drive muscle regeneration.
Furthermore, the combination of VEGF/IGF-1 was shown to alleviate
ischemia with a return to normal hemodynamic levels and a better
prevention of the necrosis associated with ischemia. Previous in
vivo studies, using this same animal model, confirmed that the
sustained delivery of bioactive growth factors (VEGF) from this gel
system led to long-term (>15 days) elevated muscle levels. This
contrasted with bolus delivery, as the factor concentration fell to
undetectable levels within hours following that delivery approach.
The sustained presence of factors enabled by alginate gel delivery
correlated with the long-term alterations in the vascular and
muscle tissue noted in the present study with gel delivery, as
contrasted to bolus delivery.
[0108] As the peripheral nervous system is also affected by
ischemic injury, the effects of sustained growth factor delivery on
innervation at the neuromuscular junction (NMJ) was also examined.
Ischemia is known to result in loss of NMJ innervation via
degeneration of the pre-synaptic axon, and this was observed in the
injury model used in this study. In the absence of growth factors,
axons required two weeks to fully regenerate. In contrast,
treatment with gels releasing either IGF-1 alone, VEGF alone or
IGF-1 and VEGF accelerated regeneration of damaged NMJs. IGF has
been shown to have neuroprotective effects in mouse models of ALS,
which is mediated by satellite cells and mature muscle fibers.
Upregulation of IGF in these models also leads to a decrease in
ubiquitin expression, suggesting that the mechanism of IGF
neuroprotection may be inhibition of Wallerian degeneration. The
reinnervation observed upon treatment with VEGF and IGF1 suggests
that gel delivery of factors is useful in treating the neurological
complications of chronic ischemia. Together these effects played
important roles in the early recovery of the mouse locomotive
skills.
[0109] Most strikingly, tetanic force measurements of the tibialis
and gracilis muscles demonstrated a significant increase to above
normal levels with dual delivery of GFs versus the untreated (blank
alginate) hindlimb, indicating functional muscle regeneration. In
particular, an increase in force above normal (non-injured) muscle
was noted at two weeks with these conditions, with a .about.2 and 8
fold increase in force for tibialis and gracilis, respectively,
compared to the blank. Conversely, a significant decrease toward
the normal value was observed, after 7 weeks, likely indicating an
adaptation to normal physiologic requirements for these muscles.
Increased muscle strength was also associated with a decrease in
fibrotic tissues. Previous studies have shown a role of IGF-1 in
finely modulating the balance between inflammation and
regeneration, which is crucial for accelerating the functional
recovery of injured muscle. After muscle injury, an inflammatory
response is activated, but prolonged accumulation of fibrotic
tissue limits muscle cell replacement, leading to less strength and
functional depletion compared with normal muscles. The increased
force observed in muscles with GFs delivery may also be related to
enhanced reinnervation, although the specific mechanisms by which
these GFs influence reinnervation remain to be defined.
[0110] In summary, the dual delivery of VEGF/IGF-1 from an
injectable biodegradable hydrogel leads to a complete functional
recovery of ischemic injured skeletal muscle. This strategy to
enhance skeletal muscle regeneration represents a new therapeutic
option for treatment of muscle damaged from a variety of causes.
Additional factors which play roles in regulating the proliferation
and differentiation of satellite cells and cells are optionally
incorporated and delivered with this system.
Example 2: Activation of Transplanted Cells by Dual Delivery of
VEGF and IGF-1 from a Macroporous Alginate Gel Leads to
Regeneration of a Functional Muscle
[0111] Prior to the invention, the two main existing strategies in
cell therapy consisted of the direct injection of cells into the
damaged tissue or their pre-culture and transplantion on scaffolds
that serve as a template for neo-tissue formation. However,
modulation of tissue regeneration subsequent to injury by cell
transplantation requires the survival of donor cells and their
stable incorporation into the host tissue. The improved strategy
described herein involves the transplantation of progenitor cells
on cell-instructive scaffolds designed to maintain cell viability,
promote cell activation (proliferation) and outward migration from
the scaffold in order to promote repopulation of the host damaged
tissue and regeneration of the myotoxin-injured skeletal muscle
ischemia. The goal was to direct the myogenic cells to bypass their
normal tendency to differentiate and remain in a proliferative
phase until a sufficient number of cells is attained to regenerate
the tissue.
[0112] Dual delivery of VEGF with IGF-1 from macro-porous
peptide-modified alginate scaffolds enhanced the engraftment of
transplanted myogenic stem cells participating in subsequent rounds
of injury repair, increased the proliferation of the satellite
cells, limited fibrosis and, accelerated the regenerative process
of injured skeletal muscle, resulting in increased muscle mass and
most importantly, improved contractile function. Together, these
results demonstrate the efficacy of finely controlled
differentiated state of myogenic stem cell transplant for treating
muscle degenerative disease.
Design of Cell Therapy/Drug Delivery System for Muscle
Generation
[0113] In normal/healthy muscle, highly specialized myofibers, the
basic contractile units of skeletal muscle, have the intrinsic
ability to contract and generate movement. In injured muscles, the
loss of myofibers' contractility can induce severe functional
deficiency. Among others cell populations found to be implicated in
muscle regeneration, such as muscle-resident side population (muSP)
multipotent adult progenitor cells (MAPC) bone marrow-derived
cells, the activation of the satellite cells, a quiescent
specialized sub-population of adult stem cells localized within the
basal lamina of the myofibers, is believed to be primarily
responsible in the physiologic muscle-regenerative potential. So
far, skeletal muscle regenerative efforts focused on cell therapies
or (single/multiple) drug delivery strategies. However, on one side
cell therapies, either the direct injection of cells into the
injured tissues and engineered tissue transplantion, are limited by
the massive death of the donor cells and by the poor integration of
the out of shelf tissues with the host/recipient. In the other side
the drug delivery strategies are limited by the rapidly depleted
local concentrations of growth factors (GFs) and by the loss of
bioactivity of the morphogens seriously impaired by the degradation
occurring by the fast enzymatic cleavage which takes place when
they are exposed to the in vivo environment. Furthermore, both
these approaches were found to induce a slight improvement in
tissue muscle regeneration.
[0114] Myoblast fate is finely regulated through biochemical and/or
biomechanical microenvironmental signals including both
extracellular matrix molecules and growth factors. To enhance
transplanted myoblast survival and proliferation and regulate the
extent of differentiation a arginine, glycine, aspartic acid
(RGD)-containing cell adhesion ligands and macroporous alginate
gels were used to encapsulate the cells and preserve/protect them
from apoptosis
[0115] Trophic factors regulate myoblast fate controlling the
proliferation and differentiation of satellite cells. In vitro and
in vivo studies have involved a number of factors, including both
inflammatory cytokines, and growth factors, insulin growth factors
acting as key modulatory role in muscle growth and regeneration.
The release of single or multiple GFs (e.g. HGF, FGF-2, VEGF,
IGF-1, PDGF-BB, etc) interspersed within natural or synthetic
matrices (alginate PLG) occurs with a kinetics that is controlled
by the physico-chemical properties of the scaffold material and
therefore is finely tunable. In particular, the dual delivery of
angiogenic (VEGF) and myogenic (IGF-1) factors from a biodegradable
injectable alginate were found to promote skeletal muscle
regeneration and induced a functional muscle regeneration of an
ischemic muscloskeletal muscle. Efforts were undertaken to further
improve the functional muscle recovery resulting from
myotoxin-injured skeletal muscle ischemia by combining satellite
cell transplantation and localized and sustained presentation of
factors, i.e., those that modulate the angiogenesis (VEGF) and the
myogenesis (IGF-1) processes.
[0116] The goal was to design a cell-instructive-scaffolds able to
preserve exogenous progenitor cells from apoptosis and instead be
activated and enter in the proliferative phase, migrate outward to
the site of injury, fuse and differentiate in order to enhance
repopulation of injured muscle from transplanted myoblasts and
increase regeneration.
[0117] Donor myoblasts were obtained from transgenic
Tg(ACTbEGFP)1Osb, constitutively expressing GFP in all the cells
and were seeded in scaffolds formed from arginine, glycine,
aspartic acid (RGD)-presenting polymer, which also provide a
sustained delivery of VEGF and IGF-1, and transplanted into
genetically matched normal mice to determine the engraftment and
hence the participation of host versus donor cells in regeneration.
The delivery of cells on scaffolds that preserve myoblast viability
and promote their activation and migration, led to a massive
engraftment and long-term contribution of the transplanted cells on
and in the host injured muscle tissue. The system was found to
accelerate the regenerative process of a severely injured skeletal
muscle, reduce degeneration, limit fibrosis, increase muscle mass,
and overall lead to a striking improvement of muscle contraction
function.
[0118] The following materials and methods were used to generate
the data described in Example 2.
Alginate Modification and Scaffold Fabrication
[0119] Ultrapure alginates were purchased from ProNova Biomedical
(Norway). MVG alginate, a high-G-containing alginate (M/G ratio of
40/60 as specified by the manufacturer) was used as the high
molecular weight (250 000 Da) component to prepare gels. Low
molecular weight (LMW) alginate (50 000 Da) was obtained by
.gamma.-irradiating high molecular weight alginate with a cobalt-60
source for 4 h at a .gamma.-dose of 5.0 Mrad (Phoenix Lab,
University of Michigan, Ann Arbor, Mich., USA). Both alginate
polymers were diluted to 1% w/v in double-distilled H.sub.2O, and
1% of the sugar residues in the polymer chains were oxidized with
sodium periodate (Aldrich, St Louis, Mo., USA) by maintaining
solutions in the dark for 17 h at room temperature. An equimolar
amount of ethylene glycol (Fisher, Pittsburgh, Pa., USA) was added
to stop the reaction, and the solution was subsequently dialyzed
(MWCO 1000, Spectra/Por.RTM.) over 3 days. The solution was
sterilized by filtration, lyophilized and stored at -20.degree. C.
Both alginates were modified with covalently conjugated
oligopeptides with a sequence of G.sub.4RGDSP (Commonwealth
Biotechnology, Richmond, Va.) at an average density of 3.4 mM
peptide/mole of alginate monomer using carbodiimide chemistry as
previously described. 2% irradiated alginate solutions were frozen
and lyophilized until completely dry. Lyophilized alginate was
added to IVIES buffer (Sigma-Aldrich, St. Louis, Mo.) to yield a 1%
w/v solution, and EDC, Sulfo-NHS, and RGDSP peptide were added to
the dissolved alginate and allowed to react for 20 h. The reaction
was quenched with hydroxylamine, and the solution was dialyzed with
decreasing concentrations of NaCl (7.5, 6.25, 5.0, 3.75, 2.5, 1.25,
and 0%) over 3 days. The solution was purified via the addition of
activated charcoal and subsequent sterile filtration. Sterile
filtered alginate was frozen and lyophilized and stored at
-20.degree. C. The modified alginates were reconstituted in
calcium-free DMEM (Invitrogen, Carlsbad, Calif.) to obtain 2% w/v
solution (50% LMW/50% MVG used in all experiments) prior to
gelation. Reconstituted alginate was stored at 4.degree. C. To
prepare gels, modified alginates were reconstituted in EBM-2
(Cambrex Corporation, Walkersville, Md., USA) to obtain a 2% w/v
solution (50% LMW, 50% MVG used in all experiments) prior to
gelation. The 2% w/v alginate solutions were cross-linked with
aqueous slurries of a calcium sulphate solution (0.21 g
CaSO.sub.4/mL distilled H.sub.2O) at a ratio of 25:1 (40 .mu.l of
CaSO.sub.4 per 1 mL of 2% w/v alginate solution) using a 1-mL
syringe. Alginates were first mixed with recombinant human VEGF165
protein (Biological Resources Branch of National Cancer Institute)
and/or with recombinant human IGF-1 (R&D system) by using two
syringes coupled by a syringe connector at a final concentration of
60 ug/mL for each protein. The calcium slurry (Sigma, St Louis,
Mo., USA) was then mixed with the resulting alginate/growth
factor/s solution using two syringes coupled by a syringe connector
to facilitate the mixing process and prevent entrapment of air
bubbles during mixing. The resulting solution was immediately
expressed into the molds 2 mm depth. A sterile glass plate was
placed over the mold and, after the alginate had completely gelled
for 30 min, square of 5 mm.times.5 mm were cut using a punch.
[0120] To produce macro-porous scaffolds with open interconnected
pores, the gels were cooled to -80.degree. C., and the gels were
lyophilized/freeze dried and stored at -20.degree. C. until cell
seeding. Fifty .mu.l (200.000 cells/gel) of a cell suspension
(4.times.10.sup.6 cells/ml) was gently poured onto modified
open-pore polymer scaffolds. The gel were incubated for about 20
min before adding a 500 .mu.l of complete culture medium, then
maintained at 4.degree. C. prior to animal implantation.
[0121] Scaffold manufacture, porosity, and characteristics are
further described in U.S. Ser. No. 11/638,796, U.S. Ser. No.
12/665,761, PCT/US2009/045856, PCT/US2009/000914, U.S. Ser. No.
61/168,909, and U.S. Ser. No. 61/281,663, hereby incorporated by
reference.
Myoblast Purification, Characterization and Cultures
[0122] Primary myoblasts were derived from 4-12 weeks-old wt
C57BL/6 and transgenic Tg(ACTbEGFP)1Osb, constitutively expressing
GFP in all the cells, mice skeletal musculature. After the
sacrifice, the satellite cells were isolated from hindlimbs using
standard methods. Under sterile conditions, hindlimb skeletal
musculature was surgically excised, finely minced, and
disassociated in 0.02% Trypsin (Gibco/Invitrogen) and 2%
collagenase type 4 (Worthington Biochemical, Lakewood, N.J.) for 60
min at 37.degree. C./5% CO.sub.2 while agitating on an orbital
shaker. Disassociated cells were strained through a 70 .mu.m sieve,
centrifuged at 1600 rpm (Eppendorf 5810R) for 5 min, and
re-suspended in high-glucose DMEM, with added pyruvate (Gibco). The
medium was further supplemented with 10% fetal bovine serum (FBS)
and 10% penicillin/streptomycin (P/S, Gibco) and this was used in
all cell culture studies (for both primary and cell line). Cells
were plated and cultured at 37.degree. C./5% CO.sub.2 for 72 h
before media change. After 72 h in culture, the media were changed
every 48 h until cells were 80% confluent (about 7 days). Cells
were collected via centrifugation and purified via Percoll
(Amersham Biosciences, Uppsala, Sweden) fractionation. To
characterize Percoll purified primary myoblast cultures, myogenic
differentiation was assessed by staining with desmin (1/100; Santa
Cruz Biotechnology, Santa Cruz, Calif.).
[0123] For clinical applications, as few as 10,000 cells,
1.times.10.sup.4, 1.times.10.sup.5, 1.times.10.sup.6,
1.times.10.sup.7 or 1.times.10.sup.8 cells are used to seed a
delivery scaffold. Sources and methods of obtaining myogenic cells
for seeding are further described in Saverio et al., 2010, J. Clin.
Invest. 120:11-19; hereby incorporated by reference.
Animals and Tissue Injury
[0124] GFP transgenic mice (C57BL/6-Tg(ACTbEGFP)1Osb) were used
only as a cell source, conversely six-seven weeks-old female wt
C57BL/6J mice (Jackson Laboratories, Bar Harbor, Me., USA), were
used for treatments. Mice were anesthetized with an intraperitoneal
injection of a mixture of ketamine 80 mg/kg and xylazine 5 mg/kg
prior to all surgical procedures. For myotoxin injuries, the
tibialis anterior muscles (TA) of the right legs of anesthetized
mice were injected with 10 ul of 10 ug/ml Notexin Np myotoxin from
Notechis Scutatus snake venom (Latexan) using a 5 ul Hamilton
syringe. After 6 days from notexin injection, hindlimb ischemia was
induced by unilateral external iliac and femoral artery and vein
ligation. After the vessel ligation, the middle part of tibialis
muscle was treated. The incision was surgically closed, and animals
monitored over time.
Ischemia and Perfusion
[0125] Measurements of the ischemic/normal limb blood flow ratio
were performed on anesthetized animals (n=10) using a LDPI analyzer
(Perimed AB, Stockholm, Sweden). Perfusion measurements were
obtained by scanning entire hindlimbs under basal conditions and
then weekly after surgery, and the ratio of perfusion between
ischemic to non-ischemic limb of the same animal was
calculated.
Histological Assessment of Skeletal Muscle
[0126] At 3 days, 2 weeks and 6 weeks following induction of
ischemic injury, anesthetized mice were sacrificed and hindlimb
muscle tissues (n=10 per time point per experimental condition)
were processed for histological analyses. For regeneration metrics,
the samples were stained with hematoxylin and eosin. Images were
captured at 20.times. magnification and merged in Adobe Photoshop
(Adobe systems, San Jose, Calif.) and then the number of centrally
located nuclei was manually measured and tallied. Vascular ECs were
identified by immunostaining for mouse CD31 (BD Biosciences
Pharmingen, San Diego, Calif., USA). For measurement of capillary
densities, histological analysis was performed in a blinded
fashion. All the merged tissue sections were randomly analyzed. The
number of positively stained blood vessels was manually counted and
normalized to the tissue area. Sections from each sample were
visualized at 200 and 400 with an Olympus IX81 light microscope
(Japan) connected to an Olympus DP70 digital image capture system
(Japan), and analyzed using IPLab 3.7 software (Scanalytics,
Rockville, Md., USA).
[0127] GFP expression was detected in both muscle cryo-section and
paraffin-section of cells engrafted muscles respectively by direct
GFP fluorescence and by anti-GFP immunofluorescence. In particular,
muscle paraffin-sections were permeabilized with 1% BSA-0.2%
Triton.times.100/PBS and 5% goat serum, and stained with 1:50
chicken anti-GFP (Molecular Probes) and, 1:200, 488 goat Alexa
Fluor anti chicken (Molecular Probes). Interstitial fibrosis was
morphometrically assessed in Masson Trichrome (Sigma Aldrich)
stained sections.
Mechanical Measurements
[0128] At 3 days, 2 and 6 weeks following the treatment, C57BJ6
mice (n=5/conditions) were anesthetized before muscle isolation and
then sacrificed by cervical dislocation. Intact Tibialis (T)
muscles for each conditions (blank alginate,
alginate+VEGF.sub.165/IGF-1, alginate+VEGF.sub.165/IGF-1 and cells,
bolus of VEGF.sub.165/IGF-1 and cells) and the uninjured
controlateral hindlimb were dissected for isolated muscle force
measurements. The muscle was mounted vertically midway between two
fine cylindrical parallel steel wire electrodes (1.6-mm diameter,
21 mm long), attached by its tendons to microclips connected to a
force transducer (FORT 25, WPII, Sarasota, Fla., USA) and bathed in
a physiological saline solution (in mM: 122.2 NaCl, 2.8 KCl, 1.2
KH.sub.2PO.sub.4, 25 NaHCO.sub.3, 1.2 MgSO.sub.4, 1.3 CaCl.sub.2,
and 5 D-glucose in a chamber oxygenated with 95% O.sub.2-5%
CO.sub.2. The experimental temperature (monitored in the bathing
solution) was maintained at 25.degree. C. The experimental protocol
involved adjustment of muscle length until maximum twitch force was
achieved (100-300 Hz). A wave pulse was initiated from a computer
using a custom-written LabVIEW program and delivered to the
stimulation electrodes via a purpose-built power amplifier (QSC USA
1310). A switch on the amplifier permitted stimulation via wire
electrodes. Contractions were continuously monitored on a LabView
chart recorder, and contractions saved on a PC. Contractions were
evoked every 5 min.
[0129] Tetani were usually evoked at 300 Hz-15-20 V with constant
pulse width and train duration of 2 ms and 1 s, respectively. These
stimulation frequencies and voltages were required to generate
maximum force but exceed the naturally occurring median firing
frequencies of 100-200 Hz in Tibialis. After force measurements
were completed the muscle were removed from the bath and weighed.
Peak tetanic force was determined as the difference between the
maximum force during a contraction and the baseline level, and
specific force calculated based on muscle weight.
Statistical Analyses
[0130] All results are expressed as mean.+-.standard deviation
(SD). Multivariate repeated-measures ANOVA was performed to test
for interaction between conditions. Differences between conditions
were considered significant if p value<0.05.
Enhancement of Skeletal Muscle Stem Cell Engraftment by Dual
Delivery of VEGF and IGF-1 from a Cell-Adhesive Macroporous
Alginate Gel
[0131] In this study, tibialis muscle of each recipient C57BJ6
mouse was preinjured by intramuscular injection of notexin six day
prior the transplantion in order to enhance the muscle regenerative
response After 6 days, when presynaptic activity, blocking the
release of acetylcholine, and the myotoxin effects exerted from
Notexin Np injection was markedly diffused into the middle part of
tibialis muscle, mice had undergone unilateral external iliac and
femoral artery and vein ligation to induce a more severe hindlimb
ischemia and treated. Analysis of the tissue sections at early time
revealed a largely necrotic defect with diffusely disorganized and
disrupted/broken myofibers in all the conditions analysed (FIG. 1
c-d). The combination of Notexin injection and ischemia injury was
selected as the most severe injury model, comparing the recovery of
the mechanical functionality of the tibialis muscles at 2 weeks
between muscles subjected to different type of injuries, including
partial laceration, cryoinjury, notexin injection alone or the
previous combined with the femoral artery and vein ligation. At the
time of vessel ligation the middle part of the tibialis muscle was
treated. A macroporous, degradable RGD-modified alginate gel with
open, interconnected pores was designed to deliver growth factors
and/or GFP-primary myoblasts.
[0132] In particular, one of the following four treatments were
used to heal the injury: (i) blank macroporous alginate vehicle,
(ii) alginate vehicle delivering VEGF (3 .mu.g) and IGF-1 (3
.mu.g), (iii) alginate gel delivering VEGF and IGF-1 (3 .mu.g each)
and GFP-satellite cells (200.000 cells/gel) and, (iv) bolus of
GFP-satellite cells (200.000 cells/gel) and VEGF and IGF-1 (3 .mu.g
each) in PBS. Even though no suture points, adhesive or glue were
used to maintain the scaffold at the site of implantation, at the
time of retrieval (3 days, 2 and, 6 weeks) the scaffolds were still
localized where it was implanted initially (FIG. 1-b). A complete
loss of locomotion of the injured hindlimb was immediately observed
in all the condition analysed (FIG. 1-e).
[0133] The capacity of donor GFP-primary myoblasts to engraft in
diseased muscle and to act as a regenerative precursor population
to repair muscle, was first analysed. Engraftment of donor-derived
myofibers in recipients was measured by direct epifluorescence for
GFP on transverse and longitudinal sections of muscle harvested at
6 weeks after transplant. However, GFP detection by epifluorescence
also was confirmed by immunofluorescence (FIG. 2). Analyses of
transplanted tibialis muscles of recipient mice revealed a robust
engraftment of donor-GFP myoblasts into the host regenerating
muscle when cells were transplanted on scaffolds releasing
VEGF/IGF-1 (FIG. 2). A more limited number of engrafted donor cells
was found in the conditions by using direct myoblast bolus
injection with GFs. No cells were noted in the other experimental
treatment (alginate gel VEGF/IGF-1) and control conditions (blank
alginate gel).
[0134] Furthermore, a significantly larger skeletal muscle mass was
noted at 3 days in injured muscles treated with alginate gel
containing/delivering both satellite cells and growth factors when
compared with the blank alginate gel at the same time (FIG. 3 B).
Quantification of the weight of these muscles confirmed a
pronounced changes with alginate releasing either cells and
VEGF/IGF-1, or VEGF/IGF-1 treatment with a statistically
significant increases of 28.5% and 20.4% (FIG. 3A) respectively,
compared with blank alginate gel at early time (3 days) with the
tendency to decrease gradually with time (showing respectively an
increase of 22.7% and 1.2% at 2 weeks and 23% and 3.1% at 6 weeks).
To directly analyze muscle regeneration, the number of postmitotic
centrally located nuclei per length of myofiber in the resolving
muscle tissue were quantified as index of newly regenerated
myofibers (FIG. 4A). At early time post-injury (3 days) the
tibialis muscle fibers in the injury group treated with VEGF and
IGF-1 without cell showed an approximately 60-50% increase in
centrally located nuclei as compared with the blank and bolus
factors delivery. The two factors in combination with cells
alginate delivery led to a 78% and a 45% increase in centrally
located nuclei per 100 um fiber length, as compared with the blank
alginate or with alginate delivering GFs (FIG. 4A). At 6 weeks
post-injury the tibialis muscle fibers treated with alginate
delivering cells and VEGF/IGF-1 showed an approximately 2.5
fold-increase in centrally located nuclei, while co-delivery of
both GFs without cells led to a 1.27 fold increase in the number of
centrally located nuclei, compared to the blank alginate gel, and a
3.1 fold increase compared to the bolus treatment (FIG. 4A).
[0135] Representative cross and longitudinal microsections of
tibialis tissue at postoperative 3 days and 6 weeks (FIG. 4 B),
stained with H&E highlight the increase in centrally located
myonuclei in the injured muscles treated with alginate delivering
cells and VEGF//IGF-1. In mice treated with gels delivering cells
and growth factors, multiple centrally located nuclei were observed
in tissue cross sections under high power magnification. The
capability of donor GFP-primary myoblasts transplantation to
improve blood vessel density and hemodynamic recovery (the
perfusion of ischemic injured tissues) was next analyzed.
Immunohistochemical analysis was carried out on tibialis tissue
sections for the presence of the endothelial marker CD31 (FIG. 5 A)
at early time (3 days) and at the late time (6 weeks)
post-treatment. In particular, at 3 days, it revealed that
VEGF/IGF-1-delivering alginate gels (FIG. 5A) increased muscle
blood vessel densities of 1.4 fold and 1.2 fold, as compared
respectively with injection of a blank vehicle or bolus delivery of
cells and VEGF/IGF. At 6 weeks, VEGF/IGF-1 delivery from the gels
resulted in an approximately 1.9 fold and 1.5 fold increase in
vessel density in tibialis muscle as compared to the ischemic
hindlimb treated with the blank alginate and bolus injection (FIG.
5A). Quantification revealed that gels delivering both myoblasts
and GFs induce an even greater increase in blood vessel density
leading to a 1.4-fold and 1.2-fold increase compared to gels
delivering only growth factors respectively at 3 days and 6 weeks
post treatment. Conversely, bolus delivery of cells and VEGF/IGF
had no significant effect on vascularization and a modest effect is
observed at early time as compared to the control (FIG. 5A).
Representative images of all the merged tissue sections at 6 weeks
post-treatment are shown in FIG. 5 B.
[0136] A Laser Doppler Perfusion Imaging (LDPI) system was used to
quantify perfusion of the hindlimbs (FIG. 6A-B). Images indicate
improved hemodynamic recovery of mice transplanted with alginate
gel delivering both cells and GFs (FIG. 6A). The GFs delivery alone
produced a milder improvement of the clinical outcome. In
particular quantification of the ischemic/non ischemic perfusion
ratio (FIG. 6 B) revealed, after an expected 20% reduction of the
blood flow immediately after surgery in all the conditions
analysed, a slow increase in reperfusion in mice treated with blank
alginate gel and bolus of cells and VEGF/IGF over time. In
contrast, dual VEGF/IGF delivery from the alginate gel led to a
gradually increase in tissue perfusion over time with a final
recovery of 75% of normal limbs at 6 weeks. Interestingly, animals
treated with alginate gels delivering both myoblasts and VEGF/IGF-1
showed a marked increase in blood flow starting from the first week
after the injury with a 78.6%, and reaching a 99% recovery at 5
weeks compared respectively with 64.9% recovery induced by gel
delivering VEGF/IGF-1 (FIG. 6 B).
[0137] To test whether muscle changes induced by GFs delivery and
engraftment/incorporation by satellite cells might correspond to
increased function and hence have a therapeutic benefit, the
contractile force of the muscles was measured. The weight
normalized tetanic force of the anterior tibialis (FIG. 7A) were
measured after maximal tetanic stimulation. At 3 days postsurgery,
all the muscle treatments induced about 1.2 fold loss of the
contractile force compared with the uninjured control. At 2 weeks
postsurgery, muscles treated with alginate gel delivering
VEGF/IGF-1 showed a significant increase above normal values in the
tetanic force (1.2 fold and 3.2 fold increase, respectively when
compared with the control and the blank alginate). A similar trend
was observed in animals receiving bolus treatment. A more
pronounced effect was measured with alginate gel delivering both
satellite cells and VEGF/IGF-1 (2 fold and 1.6 increase
respectively when compared with the control and the alginate gel
delivering VEGF/IGF-1). However, a decrease toward the normal value
was observed only in animals treated with alginate gel delivering
satellite cells and VEGF/IGF-1 at 6 wks postsurgery. In fact,
animals receiving alginate delivering only VEGF/TGF-1 or bolus
treated showed a similar trend, but the decrease in the contractile
function was markedly more pronounced.
[0138] Along with reduced recovery of the functional contractile
properties, a large content of fibrotic tissue was formed, as
imaged by Masson's trichrome staining (FIG. 7 B), in injured muscle
tissue treated with either blank alginate and bolus injection over
time, while control uninjured hindlimbs demonstrated little
fibrotic tissue, as expected. Conversely, limbs treated with
alginate gel delivering GFs alone and alginate gel delivering both
myoblasts and VEGF/IGF-1 exhibited a significant decrease in
fibrosis.
[0139] The strategy described herein involves the transplantation
of satellite cells on scaffolds appropriately designed to maintain
the viability of donor cells, promote their activation and their
afterward cell spreading and migration outside the scaffold and
their stable incorporation into the host tissue. This approach
showed to be effective in inducing both a repopulation of the host
damaged tissue and an enhancement of muscle repair.
[0140] Compared with standard approaches (cell therapy and drug
delivery), the scaffold does not serve as a tissue template, but it
has to mimic special tissue environment biochemical cues
immediately surrounding (the precursor/progenitor) cells, so called
"stem cell niche". The device comprising cells and at least 2
factors (VEGF and IGF, in this case) effectively mimics a
naturally-occurring stem cell niche. The niche is fundamental in
controlling the stem cell behavior, in particular, the quiescence,
self-renewal and cell fate commitment state of the implanted stem
cells. The viability and the ability of myoblasts to migrate from
vehicles are strongly regulated by four main factors, consisting of
the presentation of adhesion ligands by the material vehicle, the
material biodegradation, the pore structure/size and the release
kinetics of growth factors from the vehicle material.
[0141] Covalent modification of alginate with the adhesion
oligopeptides G.sub.4RGDSP prior to scaffold fabrication, compared
with scaffold lacking cell adhesion ligands, was demonstrated
diffusively to allow a controlled presentation of signals that
promote and regulate cell adhesion to this polymer, and hence the
viability and the proliferation of the primary myoblasts. In
addition, the feasibility to control the molecular weight
distribution of the polymer used to form gels allows to regulate
gel degradation, the pore size (nano, micro, macro-pores) and the
architectural structure (interconnected, aligned . . . ) of the
polymer and hence, to modulate the viability of alginate
encapsulated cells as well as their outward migration. In
particular, as compared to nano- and micro-porous (10-20 .mu.m
pores) peptide modified scaffolds, myoblasts seeded in macroporous
(.about.200-400 .mu.m) peptide modified scaffolds was demonstrated
to improve both the viability and outward migration. Similar
results were observed for smooth muscle cells, e.g., better
proliferation on macroporous scaffolds. The incorporation of
soluble factors significantly influences the
proliferative/differentiation state of the transplanted myoblasts.
The dual delivery of the pro-angiogenic regulator VEGF and the key
regulator of satellite cells activation and differentiation IGF-1
(VEGF/IGF-1 alginate gel) from an alginate gel was investigated in
both in vitro and in vivo (Example 1). The combination of these two
factors was demonstrated to enhance functional contractile skeletal
muscle regeneration, revascularization and re-innervation of muscle
tissue. In contrast to other combinations tested (e.g., FGF-2,
HGF), the VEGF/IGF combination was found to not only promote muscle
regeneration but also to profoundly improve the contractile
activity of the skeletal muscle. This surprising and significant
advantage is due to the synergic effect exerted by both VEGF and
IGF-1 on reinnervation.
[0142] These data indicate that controlled spatio-temporal release
of the two critical morphogens, VEGF.sub.165 and IGF-1, from a
macroporous RGD-modified alginate gel coupled with the
transplantation of donor satellite cells induce surprising and
remarkably more efficient functional muscle regeneration compared
to any other known method. This effect was demonstrated using a
severe ablating muscle injury model (myotoxin-ischemia induced
skeletal muscle injury). The results of this study indicate that
localized delivery of VEGF and IGF-1 from a macroporous scaffold
into injured myotoxin-ischemic muscles significantly enhances
muscle regeneration compared with the blank alginate treatment
(FIGS. 3A-4B). These data confirm and extend findings showing a
therapeutic benefit of the combined delivery of VEGF/IGF-1 from a
alginate gel on the overall muscle regeneration process (Example
1).
[0143] Transplanting the cells with a scaffold that simultaneously
delivers VEGF/IGF-1 dramatically enhanced the participation of
transplanted cells in muscle regeneration (FIG. 2), promoting both
cell viability and migration out of the scaffold in the surrounding
injured tissue. The robust transplanted cell engraftment was
maintained for extended period of time following the time of
treatment in mice (6 weeks). This capacity of enduring in time
indicated that the beneficial effect exerted by the dual delivery
of the GFs creates an appropriate micro-environmental niche for a
long-living progeny able to induce a lasting muscle regeneration
process. Conversely, the direct bolus injection of primary
satellite cells and GFs induced a significant lesser amount of
transplanted cells engrafted in the host muscle; this effect was
likely due to both the modality of delivery leading to a rapid lost
of the GFs' bioactivity on one side and, to a massive death of the
donor cells deprived of the adhesive initial polymeric support to
the other side. The enhancement in satellite cells engraftment in
the repair of severe ablative muscle injury is accompanied by a
higher recruitment of activated myoblasts as shown by the
quantitatively greater density of the centrally located nuclei per
length of myofibers in muscle treated with scaffold delivering
cells and growth factors. This result is also validated/consistent
with the increase in size and weight of these muscle as compared
with injured muscle treated with black alginate and bolus injection
from early time (FIGS. 3A and 3B). A lesser increase in muscle
regeneration parameters was observed with the implantation of
alginate delivering only GFs, but still detectable as compared with
blank alginate or bolus treatments.
[0144] The analysis of muscle injury section treated with localized
sustained delivery of GFs and cells revealed a better resolved
defect area at early time (FIG. 4 B) as compared with all the other
conditions where the larger injured area were characterized by
profoundly disorganized and necrotic myofibers at the same time
point. The data indicate that the methods lead to a faster, more
efficient, and more effective regenerative process due to a
contribution of satellite cells both by fusion with existing host
myofibers and/or by de novo myogenesis as a result of the
microenvironment created by the device and the presence and tuned
release of VEGF and IGF in concert. The synergistic effect was
demonstrated by a clinically relevant outcome of markedly earlier
recovery of the motility (walking ability) of the injured
hindlimb.
[0145] In addition to an improved and early effect on myogenesis,
the transplantation of cells from scaffolds delivering GFs promoted
pronounced angiogenesis and the return to the normal level tissue
perfusion as compared with all the other conditions, likely
activating pathways controlling the endogenous activity of these
cells. However, a slower but still significant pro-angiogenic
effect was quantified (FIG. 5A) in muscle injured treated with
alginate gel delivering GFs at late time from treatment as observed
in FIG. 5 B. Conversely, a modest increment in blood vessel density
was induced by blank alginate gel and bolus treatments.
[0146] Finally, the dual delivery of satellite cells and GFs from
macroporous alginate gel reduced muscle inflammation and fibrosis
(FIG. 7 B) and more importantly, improved strongly the muscle
contractile function (FIG. 7A). This result is related with the
synergic effect played by IGF-1 in modulating inflammation and
regeneration processes and in part with the proangiogenic and
neuro-protective effect of VEGF. After a 2.2 fold (54.32%) loss of
muscle strength immediately after the injury, a significant
increase of the muscle strength, above the normal level, was
observed after 2 weeks from the treatment, followed by a decrease
around the normal value at 6 week post-treatment. This trend is
likely associated to the two main phases of
activation/proliferation and late differentiation of the myoblasts
participating to the muscle regeneration process. These effects
were consistent with the modest increase in the fibrotic tissue
(FIG. 7 B). Conversely, the delivery of a combination of VEGF and
IGF-1 from scaffolds, in the absence of transplanted cells, had a
less pronounced effect on muscle regeneration.
[0147] The results provided startling evidence for the feasibility
of stem cell niches molecular mimicry in vivo and in a accepted and
clinically relevant animal model. In fact, the devices and methods
described herein demonstrate that the use of cell-instructive
scaffolds simultaneously function as a vehicle and a reservoir of
progenitor cells and growth factors. The myogenic response in vivo
and the transplanted cell fate was effectively modulated by the
synergic cooperation between structural ECM components associated
with angiogenic and myogenic growth factors.
Example 3: Muscle Regeneration and Revascularization in Aging
Subjects
[0148] In young subjects, a nominal level of muscle
regeneration/revascularization occurs after injury or disease to
the tissue. As is discussed above, delivery of VEGF and IGF in a
hydrogel matrix significantly enhances the regenerative effect.
However in older individuals, the naturally-occurring regenerative
response to injury/disease is greatly reduced or absent. In humans,
the total lean body mass (LBM) declines by about 18% in men and by
27% in women from the second to eighth decade of life; the decline
in LBM becomes detectable after the age of 45 years, and also
reflects a loss of regenerative capacity. Thus, the VEGF/IGF
devices and delivery methods are particularly useful for treatment
of such individuals.
[0149] Studies were undertaken to evaluate the effect of hydrogel
VEGF/IGF delivery to muscle tissue in young animals as compared to
old animals. Preparation and delivery of growth-factor loaded
hydrogels was carried out as described above. Rather than using
young mice (e.g., 6-8 weeks of age), old mice (approximately 2
years of age) were tested. FIG. 8 shows regional blood perfusion of
the hindlimb (ischemic vs nonischemic limb) of old mice. C57BL/6J
old animals (>2 years old) displayed little to no spontaneous
recover (gel), in terms of hemodynamic flow analysis, which
contrasts to the situation in young mice. Mice treated with gel
delivery of VEGF.sub.165 or IGF alone showed a low level of
recovery, while gel delivery of both VEGF and IGF alone led to a
much greater level of recovery. For example, the enhancement in
perfusion with delivery of IGF and VEGF is greater than 3-fold, as
compared to to untreated subjects.
[0150] A distribution and level of ischemic severity displayed in
the old animals is shown in FIG. 9. The ischemic grade used the
following score: 0--autoamputation of leg; 1--leg necrosis; 2--foot
necrosis; 3--two or more toe discoloration; 4--one toe
discoloration; 5--two or more nail discoloration; 6--One nail
discoloration; 7--No necrosis. The results indicated that gel
delivery of VEGF and IGF together led to a much less ischemic
injury than the factors alone, or control (gel with no
factors).
[0151] In addition to evaluation of blood perfusion and ischemic
severity, the functionality of the treated muscle tissue was
determined. The Tarlov score is a functional test that directly
evaluates the ability of animals to locomote and to bear their body
weight via the inferior limbs. The Tarlov grade use the following
score: 0--No movement; 1--Barely perceptible movement, no weight
bearing; 2--frequent and vigorous movement, no weight bearing;
3--supports weight, may take 1 or 2 steps; 4--walks with only mild
deficit; 5--normal but slow walking; 6--full and fast walking. Gel
delivery of VEGF and IGF together led to a significant improvement
(at least 1 and up to 2-3 units in Tarlov grade) in hindlimb
function. Muscle function of hindlimbs of old animals was also
evaluated using a force generation test at 12 weeks after surgery
and polymeric vehicle treatment. The force generation (normalized
to muscle mass) was measured by dissecting the muscle from the mice
at the 12 week time point. Gel delivery of VEGF and IGF was found
to lead to a significantly higher level of muscle regeneration and
function, as compared to injured, control muscles treated with
blank gel. Control, non-ischemic muscles are shown for
comparison.
[0152] These surprising results indicate that co-delivery of VEGF
and IGF to injured or diseased muscles is particularly efficacious
for treatment to regenerate muscle tissue of aged individuals.
Example 4: Minimally Invasive Repair of Injured Skeletal Muscle
with Biodegradable Scaffolds
[0153] The repair of damaged skeletal muscle may be enhanced by the
injection of muscle stem cells and/or recombinant growth factors,
but is currently limited by inefficient methods for their delivery.
A degradable covalently cross-linked alginate scaffold was
developed for delivery of progenitor muscle cells and growth
factors to treat skeletal muscle injuries. This scaffold was highly
porous and compressible, returning to its original shape when
rehydrated (i.e., the scaffold is characterized as having "shape
memory"). This composition allowed minimally invasive implantation
of the scaffold via a catheter and, since the scaffold is
degradable, there was no need for invasive follow-up surgery to
remove the implant once its repair function was completed.
[0154] The shape-memory alginate hydrogels are covalently
crosslinked and oxidized (to induce biodegradability). They are dry
and porous. Following lyophillization and compression, the material
is pliable (not brittle), e.g., it can be rolled up and put into a
syringe/needle assembly or angiocatheter (e.g., 10-14 gauge) for in
vivo delivery to the body. Once place in a desired location in the
body, a syringe/needle assembly or catheter is used to subsequently
deliver a cell suspension to the shape-memory device. Because the
device is hydrophilic, it then soaks up the cells. The cells are
then slowly released from the device and migrate out of the device
to bodily tissues.
[0155] Cultured muscle progenitor cells delivered alone (i.e., in
the absence of the hydrogel matrix/scaffold) are characterized by
poor survival and little or no proliferation post-delivery in vivo.
In contrast, muscle progenitor cells delivered within the scaffold
survived for several weeks (3-4 weeks and longer), proliferated,
and demonstrated active migration from the scaffold (i.e., out of
the scaffold and into muscle tissue of the treated subject). The
alginate scaffold was also capable of prolonged growth factor
release.
[0156] A severe muscle injury model was used to test the ability of
the growth factor-containing hydrogel scaffold to enhance the
muscle repair process. The scaffold was used to deliver
combinations of different treatments including primary murine
muscle cells, IGF-1 and/or VEGF.
[0157] The implanted scaffolds delivering cells and IGF-1 enhanced
cell survival and migration into the damaged muscle site compared
to cells and IGF-1 injected without scaffolds, resulting in
increased fusion of the injected cells with the regenerating host
muscle fibers. The addition of VEGF to the scaffold promoted
angiogenesis in the damaged muscle tissue, contributing to muscle
repair. The combined delivery of VEGF and IGF-1 from the scaffold
led to a significant reduction in fibrotic tissue and an increase
in muscle contractile function compared to their delivery without a
scaffold. The implanted scaffold did not stimulate an inflammatory
response. Thus, the shape-memory alginate scaffold is useful as a
synthetic matrix for use in a tissue repair to improve the
restoration of the structure and function of severely injured
skeletal muscle.
[0158] The 3-D degradable scaffold is highly compressible for in
vivo delivery by catheter yet returns to its original shape in vivo
(i.e. shape-memory characteristics), with the ability to deliver
growth factors and muscle precursor cells in vivo for skeletal
muscle repair. Transplanted cells in resorbable three-dimensional
(3-D) scaffolds, with the local release of growth factors
encapsulated in the scaffold, improve skeletal muscle regeneration
compared to current technologies. Insertion of such a repair
scaffold/cell/growth factor combination by minimally invasive
surgical techniques, (e.g., using a needle or catheter) has wide
clinical applicability.
[0159] The resorbable scaffold for delivery of growth factors and
muscle progenitor cells mediates the localized release of growth
factors and the enhancement of myoblast survival in the region of
injured muscle tissues. Porous and biocompatible matrices provide
space for cells to grow and survive and microenvironment for growth
factor/drug retention and release. The devices and methods
described represent a general approach to tissue regeneration that
is applicable to the transplantation of many different cell types
to enhance the regenerative response of multiple tissues.
Example 5: Neural Regeneration Using VEGF-Hydrogel Delivery
Compositions
[0160] Hydrogel compositions, e.g., alginate gels, loaded with VEGF
alone, i.e., in the absence of other growth factors, were found to
prevent degeneration at the neuromuscular junction. FIGS. 12A-B
show that innervation of neuromuscular junctions was significantly
decreased following nerve crush injury in sternomas-toid muscle.
Exogenous VEGF-gel delivery prevented complete degeneration and
accelerated re-iinervation. FIGS. 12C, D show that in the absence
of exogenous VEGF, Wallerian degeneration of the nerves was
observed within 24 h of the traumatic insult. Time-lapse imaging
showing retraction of the motor axons (white arrows) due to the
crush injury (FIG. 12E).
[0161] Neural regeneration by exogenous VEGF delivered in the
context of a hydrogel is time and dose dependent. FIGS. 13A-C show
dose-dependent neuromuscular joint innervations. Here, the optimal
dose of VEGF was 3 .mu.g. FIG. 13 D shows a timecourse of VEGF
release from alginate hydrogels in vitro, showing the bulk release
of VEGF within 7 days of incapsulation. The release kinetics are
similar, regardless of the VEGF dose, with a rapid release for the
first 5 days, followed by a slower, but continuing release for the
duration of the analysis.
[0162] Maturation of motor axons in motor endplates and
neuromuscular junction remodeling after the ischemic injury and
neural crush was observed after local delivery of VEGF. Seven days
after the injury, significant numbers of terminal axonal sprouts
were present in NMJ treated with blank hydrogels, whereas VEGF
delivery lowered the number of immature neuromuscular junctions
(FIGS. 14A, B). Multiple innervation was significantly increased in
blank-supplemented muscles, whereas VEFG delivery resulted in one
axon innervation of the motor endplate of injured muscle (FIGS.
14C,D). Both the percentage of neuromuscular junctions with
multiple axons and % of neuromuscular junctions with axons that did
not terminate at the motor endplate of the neuromuscular junctions
(both halmarks of immature neuromuscular junctions) was reduced
upon VEGF-gel delivery (FIG. 14E-F).
[0163] Injection of VEGF-loaded hydrogels into ischemic Tibialis
Anterior muscles elevates the expression of neurotrophic factors
within 7 days after the injury. Cryosections of the Tibilalis
Anterior (TA) muscle showing elevated expression of Netrin-1 upon
the delivery of VEGF (FIG. 15A). Panoramic images of the whole
cryosection showing increased levels of Netrin-1 in VEGF
supplemented ischemic TA muscle (FIG. 15B). Delivery of VEGF
further elevated the expression levels of Neural Growth Factor
(NGF) in ischemic TA muscles (FIG. 15C). The mechanism of VEGF-gel
influence on the motor endplate reinnervations involves endogenous
upregulation of these factors by endothelial cells.
[0164] Injection of alginate hydrogels supplemented with VEGF and
Netrin-1 significanity elevates levels of neuromuscular junction
innervation within 7 days of ischemic injury in TA muscle of mouse
hindlimb. FIG. 16A shows representative images of neuromuscular
junction innervation in ischemic TA muscles and increased
innervation following VEGF and Netrin-1 delivery. FIG. 16B shows
the results of a quantification of the TA innervation in ischemic
TA muscles and a synergistic effect of the combined VEGF and
Netrin-1 delivery. n=6 animals for each condition. Simultaneous
delivery of VEGF and Netrin-1 enhances reinnervation of the motor
endplates in a synergistic manner.
[0165] These data indicate that VEGF-containing hydrogels lead to a
neuroprotection in the anatomical vicinity (at or near) of the site
of administration. A therapeutic effect was noted at distances up
to several centimeters away from the injection site. To treat a
large tissue volume one would perform multiple gel injections,
appropriately spaced in order to impact the entire tissue
volume.
Example 6: Shape-Memory Polymers and Scaffolds
[0166] Shape memory polymers are characterized by their capacity to
be highly compressed and recover their original shape from a stored
packaged state in response to an environmental stimulus, e.g.,
administration into or onto a tissue of the body. Shape-memory
materials, such as the compressed hydrogels described herein, are
used to facilitate minimally invasive surgery by injection of a
compressed structure which is a fraction of its original volume,
but which then resumes its precompressed size and shape when
implanted in vivo. The advantages of this type of material are that
it combines the structure-defining property of implantable
materials with the minimally invasive method for implantation of
the material. This approach permits a less traumatic introduction
of the implant into the body and reduces pain and recovery time.
Macroporous alginate hydrogel scaffolds are prepared in predefined
geometries, dehydrated and compressed into smaller, temporary
forms. When rehydrated with a suspension of cells, e.g., by
dropping a suspension of cells onto the dried scaffold, the
scaffold returns to its original shape and was thus suitable for
minimally invasive surgery. Dehydrated scaffolds are delivered
through a needle or catheter and rehydrated in situ.
[0167] Macroporous scaffolds with shape memory are covalently
crosslinked. A scheme for the synthesis and cross-linking of
poly(aldehyde guluronate) is provided at FIG. 19 (Polymer, Bouhadir
K. H, Hausman D. S, Mooney D. J, Synthesis of cross-linked
poly(aldehyde guluronate) hydrogels. 1999. 40: p. 3575-3584).
[0168] The combination of shape-memory capability and
biodegradability increases the multi-functionality of a biomaterial
in medical devices used for minimally invasive surgery. Implant
materials are injected in a compressed state into the body through
a small incision or puncture hole (e.g., using a needle or
catheter). After implantation, the hydrogel becomes rapidly
rehydrated and restored to its previously-designed 3D shape. After
these 3D matrices have served their purposes (cell and growth
factor delivery), removal of the implants by follow-up surgery is
not necessary, as the implant degrades.
[0169] Polymer Degradation
[0170] Polymer degradation is controlled using a variety of
techniques such as irradiation, oxidation, and/or varying the
molecular weight distribution of the polymer chains. One way to
enhance degradation properties of alginate polymer is irradiation.
Polymer degradation results from changes in polymeric chemical
structure initiated by high energy electrons generated by gamma
irradiation. Low molecular weight (LMW) polymer can be generated as
the product of irradiated high molecular weight (HMW) polymer, and
is subject to faster degradation. Low molecular weight means
5000-50,000 daltons; High molecular weight means 100,000-500,000
daltons. The molecular weight of the LMW polymer generated is
determined by the irradiation dose and time of exposure. The
percent LMW polymer generated is determined by the strength of the
irradiation beam and time of exposure.
[0171] Exemplary compositions include 5% 1LMW:1HMW.
[0172] Oxidation
[0173] Another approach to control alginate gel degradation is
partial periodate oxidation which offers control over the
degradation rate--increasing the degree of oxidation accelerates
the rate of degradation. When alginate is oxidized by reacting with
sodium periodate (NaIO.sub.4), the carbon-carbon bond of the
cis-diol groups in the uronate residues are cleaved, and the
aldehyde groups of oxidized hexuronic-acid residues spontaneously
form six-membered hemiacetal rings with the closest hydroxyl groups
on two adjacent, unoxidized sugar residues in the chains. This
procedure alters the conformation of the molecules and creates
hydrolytically labile bonds that facilitate faster degradation.
[0174] Alginates are naturally derived long chain polysaccharide
copolymers formed by alternating or repeating the uronic functional
units: mannuronic acid (M) and guluronic acid (G) and thus its
chemical structure can be represented with MVG. Low molecular
weight alginate can be generated by irradiating high molecular
weight alginate at a dose of 5 Mrad for 4 hours. Both LMW and HMW
alginate are then be oxidized by reacting with NaIO.sub.4. The
extent of oxidation is dependent on the amount of NaIO.sub.4
reacted with the uronic acid function unit.
[0175] Molecular Weight Distribution
[0176] A third approach to regulate alginate gel degradation rate
involves control over the molecular weight distribution of the
polymer chains used to form scaffolds. Scaffolds formed from LMW
polymer chains rapidly degrade in vivo but are mechanically weak.
Therefore, the use of a bimodal molecular weight distribution
combining HMW: LMW polymer in different ratios may result in the
formation of mechanically stable gels with degradation rates that
can be controlled.
[0177] Modification of the Scaffold with ECM Components
[0178] Modification of the scaffold for cell delivery includes
binding of RGD peptides to encourage temporary cell adhesion.
Generally, the surface chemistry conveyed through the adsorbed
protein layer and macro-scale topographical features affect
cell-surface interactions and greatly influence the success of an
implant for tissue regeneration. Unmodified alginate does not
facilitate mammalian cell adhesion due to its poor binding of serum
proteins. Therefore, in order to allow the biomaterial to mimic the
physicochemical properties of natural tissues for cell survival and
proliferation, the surface and the bulk of the alginate needs
modification by adding cell-binding peptides.
[0179] RGD (arginine-glycine-aspartic acid) is a peptide sequence
that promotes cell adhesion and is present as the cell-binding
domain of many extracellular matrix proteins. With RGD, the cells
form attachments, which maintain and/or enhance cell survival and
proliferation. In addition, these cell-peptide interactions may
also promote other cell-specific functions, such as hormone
production or cell migration into a repair site
[0180] Scaffold Manufacture
[0181] LMW alginate was generated by gamma irradiation of HMW LF
20/40 alginate (FMC Biopolymer, Philadelphia, Pa., USA) at 5.0 Mrad
for 4 hours (h) with a cobalt-60 source for 4 h. To fabricate
oxidized alginates, both LMW and HMW alginate were diluted to 1%
w/v in ddH2O, and 1% and 5% and 10% of the sugar residues were
oxidized using different amount of sodium periodate (Sigma-Aldrich,
Saint Louis, Mo.) and maintaining solutions in the dark for 19 h at
room temperature. An equimolar amount of ethylene glycol (Fisher
scientific, Fair Lawn, N.J.) was added to quench the reaction, and
the solution was subsequently dialyzed with Spectra/Por dialysis
tubing (MWCO3500)(VWR International, Pittsburgh, Pa.), filtered and
lyophilized to generate 1% and 5% and 10% oxidized LMW and HMW
alginates. All alginate components were further modified with
linear RGD peptide (G.sub.4RGDSP-OH) (Commonwealth Biotechnology,
Inc.) using 1-ethyl-(dimethyl aminopropyl) carbodiimide (EDC
Sigma-Aldrich), N-hydroxysulfosuccinimide (sulfo-NHS, Pierce,
Rockford, Ill.), and the bifunctional cross-linker adipic acid
dihydrazide (AAD, Sigma-Aldrich).
[0182] To prepare covalently cross-linked alginate scaffolds,
sodium alginate 2% (w/v) was dissolved in IVIES buffer [0.1 M
2-(N-morpholino) ethanesulfonic acid (MES); 0.3 M NaCl], pH 6.0,
and covalently cross-linked hydrogels were formed by standard
carbodiimide chemistry using 1-ethyl-(dimethyl aminopropyl)
carbodiimide (EDC), 1-hydroxybenzotriazole, and the bifunctional
cross-linker adipic acid dihydrazide (AAD) (ratio of AAD: reactive
groups on polymer; 1:20) as depicted in FIG. 20. Scaffolds were
then placed in a large volume of distilled water, for a minimum of
24 hours, to attain equilibrium swelling and to remove residual
unpolymerized chemicals.
[0183] Three different scaffolds were fabricated as follows, each
with LMW and HMW alginates combined in a 1:1 weight ratio:
A. 1% binary group--1% oxidized HMW alginate X 1% oxidized LMW
alginate--(percent of oxidation refers to the percent of the uronic
groups that are oxidized) B. 5% binary group--5% oxidized HMW
alginate X 5% oxidized LMW alginate-- C. 10% binary group--10%
oxidized HMW alginate X 10% oxidized LMW alginate--
[0184] The resulting alginate materials were then frozen at
-20.degree. C. and lyophilized to generate macroporous scaffolds.
Scaffold porosity (void volume) and pore characteristics,
equilibrium swelling ratios (Qs), were all determined as described
below. Scaffold dimensions were measured with Vernier calipers,
lyophilized and then rehydrated with distilled water to determine
the ability of the scaffolds to return to their original
dimensions.
[0185] Swelling Ratio (Q) and Porosity Measurements
[0186] Scaffolds were equilibrated in distilled water at room
temperature. After removing excess liquid from the surfaces by
blotting, the scaffolds were weighed (WS) and diameter and
thickness measurements were taken using Vernier calipers. Scaffolds
were then frozen, lyophilized as described above, and the weight
measurements repeated (WD). The swelling ratio (QS) is defined as
the mass ratio of absorbed water to dry scaffold, calculated
from:
QS=(WS-WD)/WD.
[0187] To determine the porosity (void volume) of the dry
scaffolds, scaffolds were weighed (WS) and then reweighed after
freezing and lyophilization (WD). The porosity was calculated
from:
(WS-WD)/WS.times.100%
[0188] SEM Scaffold Surface Morphology
[0189] The lyophilized scaffolds were placed on the surface of
carbon adhesive paper and coated with gold nano-particles by a
sputter coater to make the surface conductive. Default settings
used for coating were: 4 min, 25 mA, 1 coating for each sample.
Images were taken by a HITACHI 2700 Scanning Electron Microscope
(Voltage: 6 KV/Beam current: 6*/Scanning speed: 160). The images
were collected with a Quartz PCI digital imaging system (Quartz
Imaging Corporation) and analyzed with ImageJ software (NIH).
[0190] Cell Distribution in Scaffolds
[0191] PMMGFP cells (GFP transduced primary mouse myoblasts) were
grown at low density in tissue culture plates, and harvested by
trypsinization. Five hundred thousand cells/scaffold were suspended
in 50 .mu.l PMMGM [PMMGM: 20% Fetal bovine serum (FBS), 39%
Dulbecco's Modified Eagle Medium (DMEM, Gibco), 39% Fibroblast
growth medium (FGM, Lonza), 1% ITS Liquid Media Supplement (Sigma)
and 1% penicillin/strepmyosin (Sigma)] and pipetted dropwise onto
the scaffolds in 35 mm diameter tissue culture dishes. The dishes
were placed for 30 min in a 5% CO.sup.2 humidified incubator at
37.degree. C. before being covered with 1 ml PMMGM to immerse the
scaffolds. The PMMGM was changed daily. Images were taken 2 weeks
after the cells were seeded onto the scaffolds with a Leica TCS SP2
AOBS spectral confocal microscope. Images were acquired and
analyzed with Leica confocal software (LCS) Version 2.5.
Shape Memory Properties of the Scaffold
[0192] Shape memory properties are important for delivery using a
minimally invasive method. To evaluate the shape memory capacity of
the 5% 1LMW:1HMW scaffold, the dehydrated and compressed scaffolds
were rehydrated in vitro and investigated two main shape memory
properties evaluated: porosity and swelling ratio. 1.2 mm-thick
scaffolds were compressed at 500 psi to a thin film with an average
thickness of 0.11 mm. Strikingly, the average swelling ratio was
approximately 11, indicating that the scaffold can swell to
.about.11 times its volume after rehydration.
TABLE-US-00006 Shape memory parameters Porosity Cross-link density
Swelling ratio % of volume recovered Original scaffold 98.1 + 0.1%
56.2 + 2.2 90.0% (compressed manually)[80] 5% LMW:HMW 90.7 + 0.3%
11.36 + 0.2 80.62% (compressed at 500 psi)
[0193] Average porosity and swelling ratio for 5% oxidized
covalently cross-linked scaffold. The lyophilized 5% oxidized
1LMW:1HMW scaffolds were compressed at 500 psi to a thin layer
(0.12 mm in depth, measured by Venier calipers) then rehydrated
with distilled water until equilibrium. The porosity and swelling
ratio are calculated from the weight before and after rehydration.
The original scaffold refers to an unmodified BMW covalently
cross-linked shape memory scaffold. The original scaffold was
compressed manually. Data represent mean.+-.SEM (n=4).
[0194] The porosity was measured at .about.90.7%, which implied
that the scaffolds are very porous and have high water content
(.about.90%). Collectively, these data indicate that the scaffolds
made with the 5% oxidized LMW and 5% oxidized HMW at the ratio 1:1
maintained good shape memory properties after physical and chemical
modifications to the original non-modified scaffolds. They also
appear to have good porosity for cell uptake.
[0195] Scaffold Surface Morphology and Cell Distribution
[0196] The large porosity of the 5% 1LMW:1HMW scaffolds should
offer a structural advantage for their use as a vehicle for
delivery of cells for various repair and bioengineering
applications. To further assess porosity of the scaffolds, their
surface was imaged using a scanning electronic microscope (SEM). As
illustrated in the FIG. 17a, the scaffolds were porous with the
average pore size of 412 p.m. This porosity feature facilitates
cell infiltration and migration out of the scaffold as well as
provides sufficient surface area for seeding significant cell
numbers. In addition, the open porous structure facilitates
exchange of nutrients and metabolites between seeded cells and the
neighboring microenvironment.
[0197] To assess cell distribution on these scaffolds, they were
RGD modified and loaded with PMMGFP cells as described above. The
cell suspension settled well into the pores of the scaffold (FIG.
17b), and after two weeks of incubation, cells appeared to be well
attached to the scaffold, growing in clusters (FIG. 17c). After 2
weeks in culture, the cells were viable and proliferating, as
determined by an increase in the number of viable cells inside the
scaffold (measured by GFP assay) and the cell clusters had an even
distribution pattern on the scaffold. (FIG. 17c)
Scaffold Geometries for Tibialis Muscle Regeneration and Design
Considerations
[0198] The shape-memory scaffolds are used as a delivery vehicle
for cells and growth factors to promote regeneration of injured
muscle. In addition to surgery, the scaffolds are administered
implantation using a minimally invasive technique to deposit it
next to an injured or diseased muscle site. Thus, the dimensions of
the scaffolds were designed to match geometries of target tissues.
A severe murine tibialis anterior injury model was used for in vivo
studies. In this case, the scaffolds were generated to be the
approximate length of a tibialis anterior (13.5 mm) and a width
equal to the circumference of a tibialis anterior (2.6 mm). The
size is varied to accommodate the target site in the subject and
the mode of administration (e.g., surgery, needle, or catheter). In
the mouse model, the scaffolds were approximately 1.1 mm thick
after rehydration. With these dimensions, the 5% 1LMW:1HMW was
processed and delivered through a 1.5 mm angiocath to lie next to
the tibialis anterior muscle (FIG. 18).
[0199] After rehydration, the covalently cross-linked scaffolds
made from the 5% oxidized low molecular weight and high molecular
weight alginate in a 1:1 ratio recovered more than 70% of their
original volume. The scaffolds are also capable of restoring to
their original dimensions after compression and rehydration. These
results show that the modified scaffolds possess good shape memory
properties.
[0200] The surface morphology shown by SEM imaging of a lyophilized
scaffold indicates that the scaffold has a porous and
interconnected structure. The porosity was also measured from the
same set of data that generates swelling ratio. The porosity
property confers to this covalently cross-linked scaffold an
appropriate structure for seeding cells, and facilitates exchange
of nutrients and metabolites within the surrounding in vivo
microenviroment. In addition, the porosity data indicates that the
rehydrated scaffolds have a high water content, which resembles
normal muscle tissues.
[0201] The shape memory properties permit delivery of the
dehydrated scaffolds to the site of the muscle damage with ease and
accuracy. The scaffold is injected in a compressed state into the
body through a small incision. After the injection of the scaffold,
a subsequent injection of aqueous solution containing growth
factors and cells efficiently rehydrates and restores the scaffold
geometries. Since the scaffold degrades within a pre-defined time
interval, surgical removal is not necessary after the scaffold has
served its purpose, i.e., improving the survival of delivered cells
and releasing growth factors for improved muscle regeneration.
[0202] Myoblasts Proliferate and Migrate Out to the Hydrogel
Scaffold into Muscle Tissue
[0203] Myoblasts proliferate and migrate out of the hydrogel
scaffold and into recipient's muscle tissue continuously for at
least 3 week period. To test cell proliferation on, and migration
out of the alginate scaffolds, 0.3 million GFP transduced primary
mouse myoblasts (PMMGFP) were seeded onto the scaffolds. The cells
absorbed well into the porous scaffold material. Over a 3-week
observation period, the cells proliferated and migrated from the
scaffold onto the collagen-coated tissue culture plates (surrogate
for muscle tissue of the subject). Cells migrated out of the
scaffold 2 weeks after they were initially seeded and continued to
grow on the plate surface. By the end of 3 weeks, there were
approximately 0.81 million viable cells in the scaffold based on
total GFP extraction and the cumulative number of cells that had
migrated off the scaffold was approximately 0.11 million cells
based on cell counts. These data indicate that myogenic cells
proliferate and migrate out of the candidate scaffold continuously
at a nearly constant rate during a 3-week period.
[0204] The efficiency of IGF-1 release from the scaffolds was also
tested. Approximately 90% of the IGF-1 was released at a constant
rate during the first three days, followed by sustained slower
release rate from Day 3 to Day 14. By Day 14, nearly 100% of IGF-1
was released. Since IGF-1 was largely released within the first
three days, IGF-1 has an effect on transplanted muscle progenitor
cells and host satellite cells in the early stage of the injury
recovery process. Satellite cells are activated immediately
following injury as a pulse lasting for only a few days, and since
IGF-1 stimulates proliferation and migration of satellite cells,
the early burst release of IGF-1 from the scaffold is spatially and
temporally synchronized with the activation of satellite cells.
Upregulation of satellite cell proliferation and migration by IGF-1
further enhances myogenic cell-mediated skeletal muscle
regeneration. The use of the scaffold to deliver growth factors
provides the advantage of localized delivery, as growth factors are
targeted to a small region near injury sites. By contrast, injected
growth factors (in the absence of the hydrogel delivery
vehicle/scaffold) are often either rapidly taken up by cells,
quickly degraded, or bound up by extracellular matrix molecules,
all of which cause a rapid decrease in their concentration. The
scaffolds/vehicles described herein function as a localized
delivery system for growth factors enhance the effects of the
growth factors locally while eliminating side effects at other
regions of the body, as would occur with systemic administration.
Localized delivery limits the global impact of growth factors by
minimizing their entry into the circulatory system. In addition,
localized delivery reduces the amount of growth factors needed to
achieve the desired effects. Thus, the biodegradable alginate
scaffold has significant advantages as a vehicle for delivering
cells and growth factors in vivo.
[0205] Growth Factor and Progenitor Cell Delivery from Scaffolds
Promotes Muscle Regeneration
[0206] The scaffold serves as a temporary delivery vehicle for
muscle progenitor cells and growth factors, while avoiding the
chronic problems associated with long term biomaterial
implantation. Enriched populations of myoblasts were seeded onto
the scaffold and the role of vehicle design in cell survival and
migration was examined. The data indicate that long-term survival
and migration of cells from the polymeric delivery vehicles and
into host muscle tissue was achieved. Muscle progenitor cells can
continuously proliferate and migrate out of the alginate scaffold
during a 3-week period and longer. The alginate scaffold is also
capable of prolonging IGF-1 release from the scaffold while
maintaining its high local concentration temporarily. These data
indicate that the alginate scaffold functions as a degradable ECM
and temporary delivery vehicle for muscle progenitor cells and
growth factors, which is useful to to restore the function and the
structure of the injured skeletal muscle.
[0207] Other embodiments are within the following claims.
Sequence CWU 1
1
91354PRTHomo sapiensmisc_featureHuman VEGF148 1Met Thr Asp Arg Gln
Thr Asp Thr Ala Pro Ser Pro Ser Tyr His Leu1 5 10 15Leu Pro Gly Arg
Arg Arg Thr Val Asp Ala Ala Ala Ser Arg Gly Gln 20 25 30Gly Pro Glu
Pro Ala Pro Gly Gly Gly Val Glu Gly Val Gly Ala Arg 35 40 45Gly Val
Ala Leu Lys Leu Phe Val Gln Leu Leu Gly Cys Ser Arg Phe 50 55 60Gly
Gly Ala Val Val Arg Ala Gly Glu Ala Glu Pro Ser Gly Ala Ala65 70 75
80Arg Ser Ala Ser Ser Gly Arg Glu Glu Pro Gln Pro Glu Glu Gly Glu
85 90 95Glu Glu Glu Glu Lys Glu Glu Glu Arg Gly Pro Gln Trp Arg Leu
Gly 100 105 110Ala Arg Lys Pro Gly Ser Trp Thr Gly Glu Ala Ala Val
Cys Ala Asp 115 120 125Ser Ala Pro Ala Ala Arg Ala Pro Gln Ala Leu
Ala Arg Ala Ser Gly 130 135 140Arg Gly Gly Arg Val Ala Arg Arg Gly
Ala Glu Glu Ser Gly Pro Pro145 150 155 160His Ser Pro Ser Arg Arg
Gly Ser Ala Ser Arg Ala Gly Pro Gly Arg 165 170 175Ala Ser Glu Thr
Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 180 185 190Ala Leu
Leu Leu Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro 195 200
205Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met
210 215 220Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu
Val Asp225 230 235 240Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr
Ile Phe Lys Pro Ser 245 250 255Cys Val Pro Leu Met Arg Cys Gly Gly
Cys Cys Asn Asp Glu Gly Leu 260 265 270Glu Cys Val Pro Thr Glu Glu
Ser Asn Ile Thr Met Gln Ile Met Arg 275 280 285Ile Lys Pro His Gln
Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln 290 295 300His Asn Lys
Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu305 310 315
320Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln
325 330 335Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser
Arg Cys 340 345 350Lys Met2371PRTHomo sapiensmisc_featureHuman
VEGF165 2Met Thr Asp Arg Gln Thr Asp Thr Ala Pro Ser Pro Ser Tyr
His Leu1 5 10 15Leu Pro Gly Arg Arg Arg Thr Val Asp Ala Ala Ala Ser
Arg Gly Gln 20 25 30Gly Pro Glu Pro Ala Pro Gly Gly Gly Val Glu Gly
Val Gly Ala Arg 35 40 45Gly Val Ala Leu Lys Leu Phe Val Gln Leu Leu
Gly Cys Ser Arg Phe 50 55 60Gly Gly Ala Val Val Arg Ala Gly Glu Ala
Glu Pro Ser Gly Ala Ala65 70 75 80Arg Ser Ala Ser Ser Gly Arg Glu
Glu Pro Gln Pro Glu Glu Gly Glu 85 90 95Glu Glu Glu Glu Lys Glu Glu
Glu Arg Gly Pro Gln Trp Arg Leu Gly 100 105 110Ala Arg Lys Pro Gly
Ser Trp Thr Gly Glu Ala Ala Val Cys Ala Asp 115 120 125Ser Ala Pro
Ala Ala Arg Ala Pro Gln Ala Leu Ala Arg Ala Ser Gly 130 135 140Arg
Gly Gly Arg Val Ala Arg Arg Gly Ala Glu Glu Ser Gly Pro Pro145 150
155 160His Ser Pro Ser Arg Arg Gly Ser Ala Ser Arg Ala Gly Pro Gly
Arg 165 170 175Ala Ser Glu Thr Met Asn Phe Leu Leu Ser Trp Val His
Trp Ser Leu 180 185 190Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp
Ser Gln Ala Ala Pro 195 200 205Met Ala Glu Gly Gly Gly Gln Asn His
His Glu Val Val Lys Phe Met 210 215 220Asp Val Tyr Gln Arg Ser Tyr
Cys His Pro Ile Glu Thr Leu Val Asp225 230 235 240Ile Phe Gln Glu
Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser 245 250 255Cys Val
Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu 260 265
270Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg
275 280 285Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe
Leu Gln 290 295 300His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg
Ala Arg Gln Glu305 310 315 320Asn Pro Cys Gly Pro Cys Ser Glu Arg
Arg Lys His Leu Phe Val Gln 325 330 335Asp Pro Gln Thr Cys Lys Cys
Ser Cys Lys Asn Thr Asp Ser Arg Cys 340 345 350Lys Ala Arg Gln Leu
Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys 355 360 365Pro Arg Arg
3703371PRTHomo sapiensmisc_featureHuman VEGF165b 3Met Thr Asp Arg
Gln Thr Asp Thr Ala Pro Ser Pro Ser Tyr His Leu1 5 10 15Leu Pro Gly
Arg Arg Arg Thr Val Asp Ala Ala Ala Ser Arg Gly Gln 20 25 30Gly Pro
Glu Pro Ala Pro Gly Gly Gly Val Glu Gly Val Gly Ala Arg 35 40 45Gly
Val Ala Leu Lys Leu Phe Val Gln Leu Leu Gly Cys Ser Arg Phe 50 55
60Gly Gly Ala Val Val Arg Ala Gly Glu Ala Glu Pro Ser Gly Ala Ala65
70 75 80Arg Ser Ala Ser Ser Gly Arg Glu Glu Pro Gln Pro Glu Glu Gly
Glu 85 90 95Glu Glu Glu Glu Lys Glu Glu Glu Arg Gly Pro Gln Trp Arg
Leu Gly 100 105 110Ala Arg Lys Pro Gly Ser Trp Thr Gly Glu Ala Ala
Val Cys Ala Asp 115 120 125Ser Ala Pro Ala Ala Arg Ala Pro Gln Ala
Leu Ala Arg Ala Ser Gly 130 135 140Arg Gly Gly Arg Val Ala Arg Arg
Gly Ala Glu Glu Ser Gly Pro Pro145 150 155 160His Ser Pro Ser Arg
Arg Gly Ser Ala Ser Arg Ala Gly Pro Gly Arg 165 170 175Ala Ser Glu
Thr Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 180 185 190Ala
Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro 195 200
205Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met
210 215 220Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu
Val Asp225 230 235 240Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr
Ile Phe Lys Pro Ser 245 250 255Cys Val Pro Leu Met Arg Cys Gly Gly
Cys Cys Asn Asp Glu Gly Leu 260 265 270Glu Cys Val Pro Thr Glu Glu
Ser Asn Ile Thr Met Gln Ile Met Arg 275 280 285Ile Lys Pro His Gln
Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln 290 295 300His Asn Lys
Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu305 310 315
320Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln
325 330 335Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser
Arg Cys 340 345 350Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys
Arg Ser Leu Thr 355 360 365Arg Lys Asp 3704389PRTHomo
sapiensmisc_featureHuman VEGF183 4Met Thr Asp Arg Gln Thr Asp Thr
Ala Pro Ser Pro Ser Tyr His Leu1 5 10 15Leu Pro Gly Arg Arg Arg Thr
Val Asp Ala Ala Ala Ser Arg Gly Gln 20 25 30Gly Pro Glu Pro Ala Pro
Gly Gly Gly Val Glu Gly Val Gly Ala Arg 35 40 45Gly Val Ala Leu Lys
Leu Phe Val Gln Leu Leu Gly Cys Ser Arg Phe 50 55 60Gly Gly Ala Val
Val Arg Ala Gly Glu Ala Glu Pro Ser Gly Ala Ala65 70 75 80Arg Ser
Ala Ser Ser Gly Arg Glu Glu Pro Gln Pro Glu Glu Gly Glu 85 90 95Glu
Glu Glu Glu Lys Glu Glu Glu Arg Gly Pro Gln Trp Arg Leu Gly 100 105
110Ala Arg Lys Pro Gly Ser Trp Thr Gly Glu Ala Ala Val Cys Ala Asp
115 120 125Ser Ala Pro Ala Ala Arg Ala Pro Gln Ala Leu Ala Arg Ala
Ser Gly 130 135 140Arg Gly Gly Arg Val Ala Arg Arg Gly Ala Glu Glu
Ser Gly Pro Pro145 150 155 160His Ser Pro Ser Arg Arg Gly Ser Ala
Ser Arg Ala Gly Pro Gly Arg 165 170 175Ala Ser Glu Thr Met Asn Phe
Leu Leu Ser Trp Val His Trp Ser Leu 180 185 190Ala Leu Leu Leu Tyr
Leu His His Ala Lys Trp Ser Gln Ala Ala Pro 195 200 205Met Ala Glu
Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met 210 215 220Asp
Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp225 230
235 240Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro
Ser 245 250 255Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp
Glu Gly Leu 260 265 270Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr
Met Gln Ile Met Arg 275 280 285Ile Lys Pro His Gln Gly Gln His Ile
Gly Glu Met Ser Phe Leu Gln 290 295 300His Asn Lys Cys Glu Cys Arg
Pro Lys Lys Asp Arg Ala Arg Gln Glu305 310 315 320Lys Lys Ser Val
Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys 325 330 335Lys Ser
Arg Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe 340 345
350Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser
355 360 365Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys
Arg Cys 370 375 380Asp Lys Pro Arg Arg3855395PRTHomo
sapiensmisc_featureHuman VEGF189 5Met Thr Asp Arg Gln Thr Asp Thr
Ala Pro Ser Pro Ser Tyr His Leu1 5 10 15Leu Pro Gly Arg Arg Arg Thr
Val Asp Ala Ala Ala Ser Arg Gly Gln 20 25 30Gly Pro Glu Pro Ala Pro
Gly Gly Gly Val Glu Gly Val Gly Ala Arg 35 40 45Gly Val Ala Leu Lys
Leu Phe Val Gln Leu Leu Gly Cys Ser Arg Phe 50 55 60Gly Gly Ala Val
Val Arg Ala Gly Glu Ala Glu Pro Ser Gly Ala Ala65 70 75 80Arg Ser
Ala Ser Ser Gly Arg Glu Glu Pro Gln Pro Glu Glu Gly Glu 85 90 95Glu
Glu Glu Glu Lys Glu Glu Glu Arg Gly Pro Gln Trp Arg Leu Gly 100 105
110Ala Arg Lys Pro Gly Ser Trp Thr Gly Glu Ala Ala Val Cys Ala Asp
115 120 125Ser Ala Pro Ala Ala Arg Ala Pro Gln Ala Leu Ala Arg Ala
Ser Gly 130 135 140Arg Gly Gly Arg Val Ala Arg Arg Gly Ala Glu Glu
Ser Gly Pro Pro145 150 155 160His Ser Pro Ser Arg Arg Gly Ser Ala
Ser Arg Ala Gly Pro Gly Arg 165 170 175Ala Ser Glu Thr Met Asn Phe
Leu Leu Ser Trp Val His Trp Ser Leu 180 185 190Ala Leu Leu Leu Tyr
Leu His His Ala Lys Trp Ser Gln Ala Ala Pro 195 200 205Met Ala Glu
Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met 210 215 220Asp
Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp225 230
235 240Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro
Ser 245 250 255Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp
Glu Gly Leu 260 265 270Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr
Met Gln Ile Met Arg 275 280 285Ile Lys Pro His Gln Gly Gln His Ile
Gly Glu Met Ser Phe Leu Gln 290 295 300His Asn Lys Cys Glu Cys Arg
Pro Lys Lys Asp Arg Ala Arg Gln Glu305 310 315 320Lys Lys Ser Val
Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys 325 330 335Lys Ser
Arg Tyr Lys Ser Trp Ser Val Pro Cys Gly Pro Cys Ser Glu 340 345
350Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser
355 360 365Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu
Leu Asn 370 375 380Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg385
390 3956412PRTHomo sapiensmisc_featureHuman VEGF206 6Met Thr Asp
Arg Gln Thr Asp Thr Ala Pro Ser Pro Ser Tyr His Leu1 5 10 15Leu Pro
Gly Arg Arg Arg Thr Val Asp Ala Ala Ala Ser Arg Gly Gln 20 25 30Gly
Pro Glu Pro Ala Pro Gly Gly Gly Val Glu Gly Val Gly Ala Arg 35 40
45Gly Val Ala Leu Lys Leu Phe Val Gln Leu Leu Gly Cys Ser Arg Phe
50 55 60Gly Gly Ala Val Val Arg Ala Gly Glu Ala Glu Pro Ser Gly Ala
Ala65 70 75 80Arg Ser Ala Ser Ser Gly Arg Glu Glu Pro Gln Pro Glu
Glu Gly Glu 85 90 95Glu Glu Glu Glu Lys Glu Glu Glu Arg Gly Pro Gln
Trp Arg Leu Gly 100 105 110Ala Arg Lys Pro Gly Ser Trp Thr Gly Glu
Ala Ala Val Cys Ala Asp 115 120 125Ser Ala Pro Ala Ala Arg Ala Pro
Gln Ala Leu Ala Arg Ala Ser Gly 130 135 140Arg Gly Gly Arg Val Ala
Arg Arg Gly Ala Glu Glu Ser Gly Pro Pro145 150 155 160His Ser Pro
Ser Arg Arg Gly Ser Ala Ser Arg Ala Gly Pro Gly Arg 165 170 175Ala
Ser Glu Thr Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 180 185
190Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro
195 200 205Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys
Phe Met 210 215 220Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu
Thr Leu Val Asp225 230 235 240Ile Phe Gln Glu Tyr Pro Asp Glu Ile
Glu Tyr Ile Phe Lys Pro Ser 245 250 255Cys Val Pro Leu Met Arg Cys
Gly Gly Cys Cys Asn Asp Glu Gly Leu 260 265 270Glu Cys Val Pro Thr
Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg 275 280 285Ile Lys Pro
His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln 290 295 300His
Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu305 310
315 320Lys Lys Ser Val Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg
Lys 325 330 335Lys Ser Arg Tyr Lys Ser Trp Ser Val Tyr Val Gly Ala
Arg Cys Cys 340 345 350Leu Met Pro Trp Ser Leu Pro Gly Pro His Pro
Cys Gly Pro Cys Ser 355 360 365Glu Arg Arg Lys His Leu Phe Val Gln
Asp Pro Gln Thr Cys Lys Cys 370 375 380Ser Cys Lys Asn Thr Asp Ser
Arg Cys Lys Ala Arg Gln Leu Glu Leu385 390 395 400Asn Glu Arg Thr
Cys Arg Cys Asp Lys Pro Arg Arg 405 410771PRTHomo
sapiensmisc_featureHuman IGF-1 7Met Gly Pro Glu Thr Leu Cys Gly Ala
Glu Leu Val Asp Ala Leu Gln1 5 10 15Phe Val Cys Gly Asp Arg Gly Phe
Tyr Phe Asn Lys Pro Thr Gly Tyr 20 25 30Gly Ser Ser Ser Arg Arg Ala
Pro Gln Thr Gly Met Val Asp Glu Cys 35 40 45Cys Phe Arg Ser Cys Asp
Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro 50 55 60Leu Lys Pro Ala Lys
Ser Ala65 708195PRTHomo sapiensmisc_featureHuman IGF-1B isoform
8Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe1 5
10 15Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His
Leu 20 25 30Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala
Thr Ala 35 40 45Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala
Leu Gln Phe 50 55 60Val Cys
Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly65 70 75
80Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys
85 90 95Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro
Leu 100 105 110Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg
His Thr Asp 115 120 125Met Pro Lys Thr Gln Lys Tyr Gln Pro Pro Ser
Thr Asn Lys Asn Thr 130 135 140Lys Ser Gln Arg Arg Lys Gly Trp Pro
Lys Thr His Pro Gly Gly Glu145 150 155 160Gln Lys Glu Gly Thr Glu
Ala Ser Leu Gln Ile Arg Gly Lys Lys Lys 165 170 175Glu Gln Arg Arg
Glu Ile Gly Ser Arg Asn Ala Glu Cys Arg Gly Lys 180 185 190Lys Gly
Lys 19599PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Gly Gly Gly Gly Arg Gly Asp Ser Pro1 5
* * * * *